To elucidate the role of Cx43 in osteocytes under oxidative stress, we treated osteocyte MLO-Y4 cells with H₂O₂ and investigated the cellular distribution of Cx43. We found that the mitochondrial localization of Cx43 was increased by its fluorescence signal overlap with MitoTracker, a mitochondrial specific dye (Figure 1A). Manders’ overlap coefficiency analysis indicated a significant increase in co-localization between Cx43 and MitoTracker with H₂O₂ (Figure 1B). For further confirmation, succinate dehydrogenase (SDHA), a component of mitochondrial complex II, was also used as a marker for mitochondria as well. High-magnification images of isolated mitochondria further confirmed a clear overlap of SDHA and Cx43 fluorescence signals (yellow area; Figure 1C). Western blot analysis further showed the accumulation of Cx43 protein in mitochondria (Figure 1D), with a significant increase after 2 hr of treatment, peaking after 3 hr (Figure 1E). Purify of mitochondria preparation was examined using different organelle markers (Figure 1—figure supplement 1). To assess if the mtCx43 formed functional HCs, a dye-uptake assay was performed. Lucifer yellow (LY) dye (425 Da), which can permeate Cx43 HCs, was used as an indicator of active HCs, while tetramethylrhodamine (TRITC)-dextran (10 kDa), which is too large to pass through HCs, was used as a control to exclude non-specific dye uptake. The HC function was indicated by the ratio of LY/TRITC-dextran. Under standard conditions (Veh), the dye uptake in isolated mitochondria, following H₂O₂, was elevated (Figure 1F). To further confirm that functional mtCx43 HCs caused the increase in dye uptake induced by H₂O₂, carbenoxolone (CBX), a conventional Cx channel blocker, and Cx43 C-terminal (CT) antibody, which specifically binds to Cx43 C-terminus, were used. We used Cx43(CT) antibody to assess mtCx43 HCs because the C-terminus of mtCx43 faces the mitochondrial intermembrane space (Boengler et al., 2009; Miro-Casas et al., 2009). Cx43(E2) antibody, a specific Cx43 HC inhibitor targeting the extracellular loop of Cx43, and rabbit IgG, were used as controls (Siller-Jackson et al., 2008). We found that the LY uptake increment was inhibited by CBX and the Cx43(CT) antibody in mitochondria isolated from H₂O₂-treated cells, while Cx43(E2) and IgG antibodies had no effect on mtCx43 HCs (Figure 1F). Further confirmation was done with another cell line, osteoblastic cell, MC3T3-E1. We observed the similar translocation phenotype in response to H₂O₂ (Figure 1—figure supplement 2). These results suggest that Cx43 migrates to mitochondria, and mtCx43 HCs open in response to oxidative stress.

By using the CRISPR-Cas9 lentivirus system, we KD Gja1 in MLO-Y4 cells with Rosa26 KD as a control. Whole-cell membrane and mitochondrial membrane proteins were extracted to evaluate the knockdown efficiency. An approximate 75% reduction in Cx43 protein level was seen from the total cell membrane (Figure 2A), and mitochondrial membrane protein (Figure 2B) extracts compared to the control group. Immunostaining of the isolated mitochondria from control and Cx43KD MLO-Y4 cells showed an overlap of SDHA and Cx43 proteins (orange signals), and the mtCx43 signal was much less in the Cx43KD group (Figure 2C). Meanwhile, the significant increase in dye uptake induced by H₂O₂ in control mitochondria was not seen in Cx43KD mitochondria (Figure 2D).

Mitochondrial electric membrane potential (ΔΨm), the main driver of oxidative phosphorylation (OXPHOS), is a key indicator of normal mitochondrial function. Tetramethyl rhodamine and ethyl ester (TMRE) was used to measure ΔΨm, and its depolarization indicated a damaged mitochondrial inner membrane stabilization (Wang and Youle, 2009). The TMRE intensity was significantly reduced in Cx43KD cells, indicating the depolarization and impairment of ΔΨm (Figure 2E–F). To mitigate the possible effects of the cytosol, we used mitochondrial complex specific inhibitors antimycin A (AA) and rotenone (Rot) to stimulate the generation of mitochondrial ROS (mtROS). We found that the basal level of mtROS was already decreased after Cx43 KD compared with its parallel control. AA and Rot were capable of inducing ROS generation in both control and Cx43KD mitochondria. However, the Cx43KD group generated less mitochondrial superoxide with the presence of inhibitors in intact cells, detected by mitoSOX (Figure 2G), and less ROS in isolated mitochondria, detected by DCF staining (Figure 2H). Together, the data suggest that Cx43 KD osteocytes had attenuated mitochondrial inner membrane potential and lower ROS generation.

Mitochondrial electron transport chain (ETC) coupling with OXPHOS is a fundamental mechanism for ATP generation by utilizing oxygen (Nolfi-Donegan et al., 2020). The decreased ΔΨm is likely to impede mitochondrial OXPHOS. We performed an ETC coupling study. With the application of plasma membrane permeabilizer (PMP) in the assay medium, we conducted measurements in sequential order: basal respiration (state 2), after the injections of ADP (state 3), oligomycin (OLIGO) (state 4o), p-trifluoromethoxy carbonyl cyanide phenylhydrazine (FCCP) uncoupler (state 3u), and AA (Figure 3A–B). Oxygen consumption rate (OCR) in state 3 induced by ADP was significantly lower in Cx43KD cells compared with the control group. The maximal respiration rate by FCCP uncoupling in state 3u was also lower in Cx43KD cells compared to control. The non-mitochondrial OCR measured after injection of AA, a complex III inhibitor, was very low, almost to the same level as the control group. Both basal ATP production and ADP-induced ATP production were reduced in Cx43KD cells. Proton leak, measured by subtracting OCR after AA injection from state 4o, also showed a significant decrease in the Cx43KD group (Figure 3C). To demonstrate the ATP production level more directly, we extracted mitochondria and detected the ATP amount in the entire cell, mitochondria, and cytosol. A bioluminescence assay based on ATP recombinant luciferase and the substrate luciferin were used for ATP detection. ATP concentration was much less in the whole-cell, mitochondria, and cytosol of Cx43KD compared with control cells (Figure 3D). The consistent data generated from mitochondrial coupling and ATP detection assays indicated that ATP production in Cx43KD MLO-Y4 cells was decreased remarkably. In addition, quantitative PCR (qPCR) was conducted to further confirm if complexes in the mitochondrial respiration chain were affected at the mRNA level. We chose the marker proteins SDHA for complex II, ATP synthase subunit f (coded by Atp5j2) for complex V, and ADP/ATP translocase 2 (coded by Slc25a5) as its regulation of mitochondrial permeability transition pore (mPTP). The result showed the mRNA level of Sdha and Atp5j2 was decreased in Cx43KD cells compared to the control group, while that of Slc5a5 did not show a significant difference (Figure 3E).

mtCx43 is reported to modulate K⁺ influx into the mitochondrial matrix, and increased K⁺ in the matrix promotes the respiration and efflux of protons from the matrix to intermembrane space (Boengler et al., 2012; Heinen et al., 2007). The proton gradient between the intermembrane space and matrix is a key factor for ATP generation. To determine the roles of mtCx43 HCs in the proton gradient under oxidative stress, we used pHluorin, a green fluorescent protein (GFP)-based fluorescent protein that is sensitive to H⁺ signals, and live-cell imaging to detect proton changes. The process works by pH-insensitive blue fluorescent protein mTagBFP being fused before the N-terminus of pHluorin as a calibration, and the signal ratio of GFP/BFP increasing during the increment of pH (Wu et al., 2019). mTagBFP-pHluorin was not specifically localized, primarily in the cytosol (Figure 4A). After H₂O₂ stimulation, a decrease in pHluorin signal was observed, while the signal change was significantly less in Cx43KD cells (Figure 4B). ATP5J2, acting as a subunit of ATP synthase, is localized on the mitochondrial inner membrane. We observed most of the ATP5J2-pHluorin signal was merged with MitoTracker Red (Figure 4—figure supplement 1). Therefore, the ATP5J2-pHluorin signal indicates the pH changes in the mitochondrial area. After inserting the ATP5j2 sequence before the NH2-terminal of pHluorin, we monitored the pH signal (GFP/BFP) from Atp5j2-mTagBFP-pHluorin transfected MLO-Y4 cells (Figure 4C). H₂O₂ stimulation evoked a rise in the pHluorin signal in transfected cells (Figure 4D). This phenomenon was probably caused by the influx of protons from the mitochondrial intermembrane space to the mitochondrial matrix. The amplitude of the elevation was significantly lower in Cx43KD cells (Figure 4D). When plotting signal changes around the mitochondrial area, an oscillation of the mitochondrial pHluorin signal was observed. The amplitude of the oscillation was greatly tapered in Cx43KD mitochondria. Representative plots and the statistical analysis of the maximal amplitude of ATP5J2-pHluorin oscillation are shown in Figure 4E. Live-cell imaging results demonstrated that mtCx43 is critical for mitochondrial H⁺ flux in response to H₂O₂ stimuli.

The membrane tropology of Cx43 on the mitochondrial membrane is important, with its C-terminus facing the intermembrane space. For, as we showed Cx43(CT) antibody, not Cx43(E2) antibody, has access to inhibit mtCx43 HCs (Figure 1F). For this reason, we used the Cx43(CT) antibody as a specific mtCx43 HC inhibitor in the mitochondrial seahorse coupling assay after the addition of a PMP. Cx43(CT) antibody treatment inhibited the increase of ATP production, similar to CBX, while the rabbit IgG control group did not show a reduction in oxygen consumption (Figure 5A). Respiratory control ratios (RCRs, state 3/state 4o) and uncoupling control ratios (UCRs, state 3 u/state 4o) were similar among the groups without any significant difference (Figure 5B). The basal respiration (state 2) had no significant difference detected. State 3 after ADP stimulation and state 3u after FCCP uncoupling demonstrated lower OCR in the presence of CT antibody and CBX (Figure 5C). We further examined complex I activity using pyruvate and malate as substrates. CT antibody and CBX treated groups showed an inhibitory role compared with Vehicle (Veh) and IgG control groups (Figure 5D). CT antibody, and CBX showed lower OCR, indicating reduced complex II activity (Figure 5E). The inhibitory role that CT and CBX played was generally observed in Cx43KD MLO-Y4 cells in state 3. ADP-stimulated ATP production significantly decreased in CBX treated group; the CT antibody-treated group displayed a trend of inhibition while no significance was detected (Figure 5—figure supplement 1). We also confirmed whether the antibodies did reach mitochondria. After incubating antibodies in the presence of PMP, we stained the cells with a fluorescent anti-rabbit IgG antibody. Cx43(CT) antibody displayed localization on mitochondria (Figure 5—figure supplement 2). The signal of CT Ab rather than Cx43(E2) Ab in mitochondria suggested the orientation of mtCx43 CT facing the intermembrane space/cytosolic side, consistent with the mtCx43 orientation reported previously (Boengler et al., 2009; Miro-Casas et al., 2009).

To further validate the involvement of mtCx43 in proton changes, we generated a Cx43-pHluorin plasmid and expressed it in MLO-Y4 cells (Figure 6A). The Cx43-pHluorin signal in live imaging was quantified in the whole cell as well as in the local mitochondrial area after H₂O₂ treatment. A decrease in whole-cell Cx43-pHluorin signals calibrated with BFP was observed after adding H₂O₂ (Figure 6B). In the mitochondrial area, indicated by mitochondrial marker MitoTracker Red, a similar oscillation of pHluorin signal was demonstrated (Figure 6C). Since Cx43 was expressed in different subcellular regions, the whole-cell and mitochondrial signals show consistent pHluorin signals in the cytosol and ATP5J2-pHluorin signals in mitochondria. PMP was used to permeate the plasma membrane with intact mitochondria. With a permeable plasma membrane, the Cx43(CT) antibody could reach and function in mitochondria. Our results showed the CT antibody caused a reduction of pHluorin signal oscillation when compared with IgG treatment (Figure 6D–E). Although there was a minor effect, the CT antibody did not show significant inhibition of ATP5J2-pHluorin signals (Figure 6—figure supplement 1). The results showed that the inhibition of mtCx43 HCs by Cx43(CT) antibody caused a decrease in pHluorin signal, indicating a reduced proton gradient across the mitochondrial inner membrane. The increased pH gradient by mtCx43 HCs may be one of the underlying mechanisms that impacts mitochondrial function in response to oxidative stress. Considering that the proton gradient across the mitochondrial inner membrane is essential for ATP synthesis, we investigated the connection between mtCx43 and mitochondrial ATP synthase.

We showed that mtCx43 HCs are functionally involved in ATP generation. We then determined the spatial relationship between mtCx43 and ATP generation machinery mitochondrial complex V. In complex V, ATP5J2 is the subunit that circles the ATP synthase peripheral stalk with the NH2-terminus facing the matrix and a membrane inserted helix (Leithe et al., 2018) and was identified to be associated with Cx43 in human chondrocytes (Gago-Fuentes et al., 2015). To investigate if there is physical interaction between the mtCx43 and ATP synthesis protein, we employed the Förster resonance energy transfer (FRET) approach. Cx43-cyan fluorescent protein (CFP) and ATP5J2-enhanced yellow fluorescent protein (EYFP) were co-transfected into MLO-Y4 cells. CFP and YFP form a FRET pair in which CFP works as the donor and YFP as the acceptor (Miyawaki et al., 1997). The FRET signal was detected when the two fluorescent proteins were in proximity of less than 10 nm to each other. ATP5J2-EYFP signal was primarily detected in the mitochondria region, while the Cx43-CFP signal was also detected in other parts of the cell. However, the FRET signal was detected (Figure 7A), suggesting that Cx43 and ATP5J2 physically interacted with each other in the mitochondria. An immunoprecipitation assay was also conducted using the GFP or ATP5J2-GFP transfected MLO-Y4 cells (Figure 7—figure supplement 1A). Cx43 band was detected when an anti-GFP antibody against ATP5J2-GFP was used as an immunoprecipitating antibody (Figure 7B). To further investigate the binding between Cx43 and ATP5J2 in mitochondria, we performed a protein pull-down assay with ATP5J2-GFP transfected MLO-Y4 cell extracts using Glutathione s-transferase tag (GST)-Cx43 CT immobilized on glutathione-agarose beads (Figure 7—figure supplement 1B). The results showed that GST-Cx43 CT, not GST control, was able to pull down ATP5J2-GFP, further demonstrating a direct interaction of Cx43 CT with ATP5J2 in intermembrane space (Figure 7C). Together, these findings suggested a direct binding of complex ATPase subunit ATP5J2 with Cx43 CT in mitochondria.

To assess whether Cx43 migrates and accumulates in mitochondria in response to oxidative stress in vivo, we used a transgenic mouse model with increased oxidative stress in osteocytes. Our previous study suggested that deficiency of macrophage-colony stimulating factor 1 (CSF1), a key factor involved in cell signaling and remodeling, increases Nox4 oxidase expression in primary osteocytes, resulting in elevated oxidative stress (Werner et al., 2020). We isolated the primary osteocytes from Csf1^± and measured the ROS level by DCF staining. We observed a significantly increased ROS production in primary osteocytes with decreased CSF1 (Figure 8A). Furthermore, the significant increase of co-localization of Cx43 and SDHA suggested the increased migration of Cx43 to the mitochondrial in Csf1^± compared with the wild-type (WT) (Csf1^+/+) cells (Figure 8B). The result recapitulated the mitochondria migration and accumulation of Cx43 in oxidized osteocytes in vivo.

MLO-Y4 osteocyte cells established from primary osteocytes were cultured on type-Ⅰ rat tail collagen-coated dishes in α-modified essential medium with 2.5% fetal bovine serum (FBS) and 2.5% bovine calf serum (BCS) with penicillin-streptomycin (PS; Kato et al., 1997). The authenticity of the MLO-Y4 cell line was confirmed by examining cell morphology with dendritic processes and maker expression, including high amount of osteocalcin, Cx43 and CD44, and low amount of alkaline phosphates. No mycoplasma was detected. The KD of Gja1 by CRISPR-Cas9 system in MLO-Y4 cells with Rosa26 KD as a control was done as previously described (Hua et al., 2022). Transfected MLO-Y4 cells were cultured without PS. Cx43(E2) antibody and Cx43(CT) antibody were both developed in the Jiang laboratory (He et al., 1999; Siller-Jackson et al., 2008). Cx43(E2) antibody has been used as a Cx43 HC inhibitor as previously described (Kar et al., 2013; Sáez et al., 2013; Siller-Jackson et al., 2008). Mouse anti-SDHA antibody was purchased from Invitrogen (#459200, Invitrogen, Waltham, MA, USA).

Cells were seeded in cell culture plates or 12 mm glass coverslips for 2 days. Cells were then stained in 37°C for 20 min using MitoTracker Deep Red (200 nM, Invitrogen, Waltham, MA, USA), if needed, before fixation with 4% paraformaldehyde for 10 min at room temperature (RT). After washing in PBS buffer for three times, cells were incubated with affinity-purified antibodies against Cx43(E2) (1:300 dilution) or SDHA (1:1000 dilution) in blocking buffer (0.25% Triton X-100, 2% gelatin, 2% donkey serum, and 1% bovine serum albumin) for 1 hr at RT, followed by incubation with fluorescein-conjugated secondary antibody for 1 hr at RT. After incubation with 4, 6-diamidino-2-phenylindole for 5 min, the cells were rinsed with PBST (phosphate-buffered saline with 0.1% Tween-20) for three times. After mounting, the slides were imaged using a fluorescence microscope (Keyence BZ-X710, Tokyo, Japan) or a confocal microscope (Carl Zeiss, Thornwood, NY, USA).

Membrane protein extracts from MLO-Y4 cells were prepared for western blot and GST pull-down assays. Cells were lysed and homogenized by a 20 G needle with ice-cold lysis buffer (5 mM EDTA, 5 mM EGTA, and 5 mM Tris, pH 8.0). Proteinase inhibitors (2 mM phenylmethylsulfonyl fluoride (PMSF), 5 mM N-ethylmaleimide (NEM), 1 mM Na₃VO₄, and 0.2 mM leupeptin) were added. The cell lysates were then centrifuged at 600×g for 5 min, removing intact cells and nuclei. The supernatant was then centrifugated (Beckman Coulter, Brea, CA, USA) at 125,000×g for 30 min at 4°C. The pellets were then resuspended in lysis buffer with 1% SDS for western blot or in 0.5% Triton X-100 buffer for GST pull-down assay.

Protein concentration was quantified using the micro-BCA assay (Pierce, Rockford, IL, USA), and equal amounts of proteins were loaded on a SDS-polyacrylamide gel electrophoresis (PAGE) and electroblotted onto a nitrocellulose membrane. Membranes were incubated first with primary antibodies, anti-Cx43(CT) (1:300 dilution), anti-SDHA (1:1000, Invitrogen), and anti-β actin antibodies (1:5000) overnight at 4℃, followed with the incubation with secondary antibodies, goat anti-rabbit IgG conjugated IRDye 800CW, or goat anti-mouse IgG conjugated IRDye 680RD (1:15,000 dilution), 1 hr at RT. The images were captured using a Licor Odyssey Infrared Imager (Lincoln, NE, USA), and protein band intensity was quantified using NIH Image J software.

The coding region of mouse Cx43 (Gja1) was amplified by PCR, and DNA fragments were inserted into the EGFP-N1 or CFP-N1 vector to generate Cx43 (Gja1)-EGFP or Cx43 (Gja1)-CFP, respectively. Atp5j2 cDNA was inserted into the EGFP-N1and EYFP-N1 vector to generate Atp5j2-EGFP or Atp5j2-EYFP, respectively. The mTagBFP-pHluorin plasmid was used to generate Cx43-mTagBFP-pHluorin and Atp5j2-mTagBFP-pHluorin constructs. For live-cell imaging, MLO-Y4 cells were transfected with Gja1-mTagBFP-pHluorin or Atp5j2-mTagBFP-pHluorin using the Neon transfection system with parameters set as 1200 mV with a duration of 20 ms, 1 pulse. The ratio of cells and transfected plasmids was 10⁶ cells with 5 µg plasmids. The expression of endogenous plasmids was determined by fluorescence imaging intensity.

MLO-Y4 cells were collected, washed with ice-cold PBS twice, and centrifuged for at 1000×g for 5 min at 4°C. The pellets were resuspended in 1 mL of mannitol buffer (220  mM mannitol, 70  mM sucrose, 20  mM HEPES, 1  mM EGTA, 0.1% BSA, and adjusted to pH 7.2 with KOH) and then homogenized. The homogenate was centrifuged at 1000×g for 10 min to remove intact cells and nuclei, and the supernatant was then centrifuged at 9000×g for 15 min to precipitate down mitochondria. The pellet containing mitochondria was washed once with mannitol buffer and was resuspended in mannitol buffer with 1% BSA for further usage.

Mitochondrial purity validation. Isolated mitochondria extracts were loaded as well as the whole cell lysates on 12% SDS-PAGE and immunoblotted with SDHA antibody (1:1000, Invitrogen), protein disulphide isomerase antibody (1:1000, #3501, CST, Boston, MA, USA), syntaxin 6 (STX6) antibody (1:1000, #2869, CST, Boston, MA,USA) at 4℃ overnight. Isolated mitochondria were incubated with fluorescent dyes specific to different organelles; MitoTracker Deepred (1 μM, # M22426, Invitrogen, Waltham, MA, USA), ER-Tracker Blue-White (1 μM, # E12353, Invitrogen, Waltham, MA, USA) and Hochest 33,342 (100 nM, # H21492, Invitrogen, Waltham, MA, USA) for 30 min at 37℃. Isolated mitochondria were resuspended in 1× isotonic mitochondrial assay solution (220  mM mannitol, 70  mM sucrose, 10 mM KH₂PO₄, 2 mM HEPES, 5 mM MgCl₂, 1  mM EGTA, 0.2% BSA, and adjusted pH 7.2 with KOH), and after rinsing two times, confocal images were captured under a 63× objective lens.

Intracellular ROS was determined using mitoSOX (#M36008, Invitrogen, Waltham, MA, USA) in intact MLO-Y4 cells or using fluorescence-based probes Carboxy-H₂DCFDA (#C2938, Invitrogen, Waltham, MA, USA) in isolated mitochondria. Cells were rinsed with Hanks’ balanced salt solution (HBSS) three times and incubated with 100 nM mitoSOX for 20 min at 37°C. After three washes with HBSS, fluorescence images were immediately captured using a Keyence BZ-X710 microscope. Isolated mitochondria were stained with 10 μM Carboxy-H2DCFDA for 10 min at 37°C, and the data were collected using a microplate reader (Bio-Tek Synergy HT, Winooski, VT, USA).

Mitochondria dye uptake assay was performed based on published studies (Miro-Casas et al., 2009; Soetkamp et al., 2014). Isolated mitochondria from MLO-Y4 cells were resuspended in isosmotic succinate buffer (150 mM KCl, 7 mM NaCl, 2 mM KH₂PO₄, 1 mM MgCl₂, 6 mM MOPS, 6 mM succinate, 0.25 mM ADP, 0.5 μM Rot, and 1 nM PMP with pH 7.2) at 0.4 mg/mL. Mitochondria were then aliquoted into five groups, with the addition of either vehicles or Cx43 HC blockers. After a 20 min incubation period at RT and 200×g centrifugation for 3 min, 50 μM of the Cx43 HC-permeable dye LY CH dilithium salt (LY, #L453, Invitrogen, Waltham, MA, USA) and 25 μg/mL of the HC impermeable dye Tetramethylrhodamine-dextran 10,000 MW (TRITC-dextran, #D1816, Invitrogen, Waltham, MA, USA) were added. Samples were incubated for 25 min and centrifuged at 200×g at RT. Mitochondria were subsequently washed for two times and gently resuspended in 200 μL succinate buffer. Fluorescence of LY (λ_ex 430 nm/λ_em 535 nm) and TRITC- dextran (λ_ex 545 nm/λ_em 600 nm) was measured by a microplate reader (Bio-Tek Synergy HT, Winooski, VT).

Mitochondrial OCRs were measured using a Seahorse XFe96 analyzer (Seahorse Bioscience, North Billerica, MA, USA), which was equilibrated at 37°C overnight. 5×10³ MLO-Y4 cells were seeded in a pre-coated XF96 plate and cultured overnight for adherence. On the same day of the experiment, 50 μL containing 1× mitochondrial assay solution (220  mM mannitol, 70  mM sucrose, 10 mM KH₂PO₄, 2 mM HEPES, 5 mM MgCl₂, 1  mM EGTA, 0.2% BSA, and adjusted pH 7.2 with KOH) with 10 mM succinate and 2 μM Rot as substrates for the coupling assay was added to each well of the XF96 plate and followed by addition of PMP (Invitrogen) with a final concentration 2 nM. To assess various parameters of mitochondrial function, OCRs were measured after injecting ADP, OLIGO (a complex V inhibitor), FCCP (a protonophore and mitochondrial uncoupler), and AA (a complex III inhibitor). The final concentrations of drugs in the well were: ADP, 5 mM; OLIGO, 5 μM; FCCP, 5 μM; AA, 10 μM; and Rot, 2 μM. The OCR was measured using the Seahorse XFe96 extracellular flux analyzer (Andersen et al., 2019; Au - Traba et al., 2016).

Transfected MLO-Y4 - pHluorin plasmid cells were used for live-cell imaging. Cells were maintained in a recording medium, and images were captured with a Zeiss LSM-810 confocal laser scanning microscope with ZEN imaging software. Transfected cells with good pHluorin signal were first located under a 10× objective, and the images were then acquired using a 40× water objective. Images were acquired at a frame size of 512×512 pixels with a time interval of 5 s between each frame.

GST fusion protein containing Cx43 CT and GST control without fusion protein was individually expressed in Escherichia coli and purified by binding to glutathione-coupled agarose beads as described in published procedures (Jiang et al., 1994). By using purified GST and recombinant GST-CT fusion protein, we performed the GST pull-down assay (Sambrook and Russell, 2006). MLO-Y4 cells transfected with Atp5j2-EGFP were cultured in a 10 cm culture plate, and cells were collected after 2 days of culturing. Crude cell membrane was prepared and resuspended in a buffer assay medium (20 mM Tris, 50 mM NaCl, and pH 7.5) containing nonionic (NP-40, Triton X-100) and zwitterionic (CHAPS). The crude membrane extracts were used in the pull-down experiments. The glutathione agarose beads (Piece, IL) were washed, equilibrated with PBS, and aliquoted to 50 μL of the beads into two microcentrifuge tubes. The beads were incubated with 20 μg GST-CT or the same amount of GST control for 1 hr at 4°C. An equal amount (1 mg) of crude membrane extracts was then added to the tubes containing GST-CT and the GST vials. The mixture was incubated overnight at 4°C. The glutathione agarose beads were washed with PBS five times for 5 min each. An elution buffer (20 mM Tris, pH 8.0) containing 10 mM reduced glutathione was used to elute the proteins bound to the beads. Together with the input samples, the eluted fractions were analyzed using 10% SDS–PAGE, and immunoblotting was done using an anti-GFP antibody (1:2000, #ab290, Abcam, Cambridge, MA, USA).

For FRET microscopy, MLO-Y4 cells were transfected with ATP5J2-YFP and Cx43-CFP plasmids. After 48 hr of culturing, cells were imaged with an LSM-810 confocal microscope and an argon laser (Carl Zeiss, Thornwood, NY, USA) at excitation wavelength 458 nm for CFP and 514 nm for YFP, and emission wavelength 470–500 nm for CFP and 530–600 nm for YFP and FRET channels. Confocal images were acquired sequentially in CFP and YFP channels. Identical parameters were used within each channel as well in all test groups. Images were processed using the FRET analysis plugin in NIH ImageJ software.

Primary osteocytes from Csf1^+/+ (WT) to Csf1^± were prepared as previously described (Werner et al., 2020). Briefly, femurs and tibias were isolated from 21-day-old male mice and bone marrow removed. The soft tissue attached on the bone surface was also carefully removed. The cortical bone was cut into 1 mm pieces and then incubated with 300 U/mL collagenase type 1 (Sigma, St. Louis, MO, USA) solution for three times, 5 min each, and digests were discarded. Then, bone pieces were incubated with either 4 mM EDTA or collagenase at 37°C for 30 min twice to remove other cell types. The remaining cortical bone was then incubated with 4 mM EDTA and then with collagenase, collecting the fractions after fraction 7. These later digests were filtered through a 100 µm cell strainer and were enriched osteocytes. The primary osteocytes were cultured on coated dishes with α-MEM (#12561056, Life Technologies, Carlsbad, CA, USA) supplemented with 5% FBS and 5% BCS (Hyclone, Logan, UT, USA). All animal protocols were performed following the National Institutes of Health guidelines for care and use of laboratory animals and approved by the UT Health San Antonio Institutional Animal Care and Use Committee.

Total RNA was isolated from MLO-Y4 cells using TRIzol reagent (#15596026, Invitrogen, Waltham, MA, USA). cDNA libraby was established by a high-capacity cDNA reverse transcription kit (#4388950, Applied Biosystems, Carlsbad, CA, USA) according to the manufacturer’s instructions. mRNA level was analyzed with a two-step amplification (94°C for 10 s and 60°C for 30 s, 40 cycles) using an ABI 7900 PCR device (Applied Biosystems, Bedford, MA, USA). The primers for RT-qPCR are: Sdha, Forward 5’-GAGATACGCACCTGTTGCCAAG-3’, Reverse 5’-GGTAGACGTGATCTTTCTCAGGG-3’; Atp5j2, Forward 5’-CGAGCT GGATAATGATGCGGGA-3’, Reverse 5’-GCAGTAGCTGAAAACCACGTAGG-3’; Slc25a5, Forward 5’-ACACGGTTCGCCGTCGTATGAT-3’, Reverse 5’-AAAGCC TTGCTCCCTTCATCGC-3’; Gapdh, Forward 5’-CTTCAACAGCAACTCCCAC TCTTC-3’, Reverse 5’-TCTTACTCCTTGGAGGCCATGT-3’.

Statistical analysis was conducted using GraphPad Prism 9 software (GraphPad Software, La Jolla, CA, USA). Two-tailed t-test was conducted to the comparison between two groups. Multiple group comparisons were done using one-way ANOVA. Two-way ANOVA and Tukey multiple comparisons were used to compare the mean differences between two independent variable groups. The data were presented as mean ± SEM of at least three measurements. p<0.05 was designated as a statistically significant difference. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001. Images were analyzed using NIH Image J software.

The newly created plasmids generated in this study are available from the lead contact with a completed materials transfer agreement without restriction.All data were included in the manuscript. Source data has been provided.