RAW264.7 cell line was obtained from ATCC (Manassas, VA, United States). Control and GBF1 siRNA (E11) were designed and synthesized by GenePharma (Suzhou, China). Dulbecco’s modified Eagle’s medium, fetal bovine serum (FBS), Opti-MEM^® I Reduced-Serum Medium, penicillin, and streptomycin were obtained from Gibco (Rockville, MD, United States). Soluble recombinant mouse RANKL, Golgicide A (GCA), and TRAP staining kit were purchased separately from PeproTech (Rocky Hill, NJ, United States), Selleck (Shanghai, China), and Sigma-Aldrich (St. Louis, MO, United States). Antibodies against NFATc1, V-ATPase, EIF2α, p-EIF2α, PERK, p-PERK, Atg5, LC3A/B, GAPDH, Cleaved Caspase-3, Caspase-3, Bax, Bcl-xL, Cytochrome C, and LAMP1 were obtained from Cell signaling Technology. Antibodies against c-Fos, MMP9, Beclin1, and p62 were obtained from Abcam. Antibodies against GBF1, BiP, and EEA1 were obtained from BD Transduction Laboratories^TM. Antibodies against FAM129A, Rab7, Bcl-2, and β-tubulin were obtained from Proteintech.

RAW264.7 cells were cultured in DMEM containing 10% FBS at 37°C with 5% CO₂. For the formal experiments, cells were cultured in the presence of RANKL (50 ng/ml) with or without different concentrations of GCA (0, 1.5, 2, and 2.5 μM) in a six-well plate (3 × 10⁴ cells/well) for 56 days, with the medium being changed every other day.

According to the manufacturer’s instructions, the siRNA sequence targeting GBF1 (NM_178930.3) or the negative control was transfected into RAW264.7 cells using Lipofectamine^TM 2000 (Invitrogen). The gene ID number of GBF1 is 8729. Cells were collected at the logarithmic growth phase and the cell suspension was adjusted to 1 × 10⁴/ml, inoculated in a six-well plate, and placed in a 5% CO₂ incubator at 37°C for 12 h. When the cell density reached 70–80% confluence, 5 μl of lipo2000 was added to 250 μl of Opti-MEM and mixed well, followed by the addition of 250 μl of Opti-MEM mixed with 100 pmol siRNA, which was incubated for a further 5 min at room temperature (RT). The lipo2000 was mixed with the siRNAs and incubated for 20 min at RT. The serum-containing medium in the six-well plate was removed, before 1.5 ml of serum-free medium containing siRNAs was added and cultured in a 5% CO₂ incubator at 37°C. The solution was changed after 6 h to a normal medium (Xu et al., 2021).

RAW264.7 cells were cultured in a 96-well plate (7 × 10⁴/ml) and treated with or without GCA (0, 0.5, 1, 1.5, 2, and 2.5 μM) for 72 h. Thereafter, CCK8 kit solution was added to each well. Absorbance was measured at 450 nm using an ELISA microplate reader after 3 h of incubation at 37°C in 5% CO₂ (BioTek Synergy H1, MA, United States). Cell viability was presented as the percentage of control.

RAW264.7 cells were inoculated in the Osteo Assay Surface 96-well plate (Corning, St. Lowell, MA, United States) and OC differentiation was induced according to the aforementioned methods. Subsequently, the cells were washed with 10% bleach solution and air dried for 3–5 h at RT. The resorption pits were captured under a light microscope (Nikon, Tokyo, Japan). The bone absorption area of each group was calculated by Image-Pro-Plus 6 software.

The staining reagents were prepared according to the manufacturer’s instructions. RAW264.7 cells were fixed with 10% formalin at RT for 10 min and incubated in staining solution at 37°C for 1.5 h in the dark. After washing three times with deionized water, hematoxylin was added to redyeing nucleus for 2–3 min. Using a light microscope, positive multinucleated OCs (nuclei ≧ 3) were observed and counted (Nikon, Tokyo, Japan).

RAW264.7 cells were inoculated in a 96-well plate, and after GBF1 knockout, the cells were induced to OCs and treated with 10 μg/ml acridine orange. After incubating at 37°C for 15 min, the cells were washed with PBS to remove any remaining dye. The fluorescence value was measured with a fluorescence microplate reader using 485-nm excitation wavelength and 588-nm emission wavelength (BioTek, Winooski, VT, United States). The intensity of fluorescence was directly proportional to the level of intracellular acidification.

RAW264.7 cells were inoculated in a 24-well plate according to the aforementioned transfection and induction methods. After 5–7 days of culture, the medium was collected and centrifuged at 4°C and 3000 rpm for 20 min. The level of MMP9 was determined using ELISA kit according to the manufacturer’s instructions (Invitrogen, Carlsbad, CA, United States). Cytokine-specific antibody pre-coated plates were blocked with a blocking buffer for 1 h at RT, followed by incubation with the supernatant at 1:2 dilution. For detection, biotin-conjugated anti-mouse MMP9 antibodies, extravidin-horseradish peroxidase (HRP) conjugate, and TMB solution were used. The optical density at 450 nm was measured. The concentrations of MMP9 were calculated using a standard curve prepared according to the manufacturer’s instructions.

RAW264.7 cells were cultured on glass cover slips according to the aforementioned description. The cells were harvested and fixed with 4% paraformaldehyde, permeabilized in 0.5% Triton X-100, and blocked with 10% normal goat serum in PBS. Subsequently, the cells were incubated with appropriate diluted primary antibodies (GBF1, FAM129A, and GM130, 1:100 in primary antibody diluent) for 1 h at RT, followed by incubation with Rhodamine Phalloidin (Invitrogen, Carlsbad, CA, United States) and Alexa Fluor-labeled secondary antibodies (1:1000 in blocking buffer) for 1 h in the dark. After washing with PBS, prolong^® Gold anti-fade reagent with DAPI (Molecular Probes, Waltham, MA, United States) was employed to counterstain the nuclei and the cells were inspected using a confocal microscope (Nikon, Tokyo, Japan) (Zhou et al., 2016).

The total RNA from cells was isolated with TRIzol reagent (Invitrogen Carlsbad, CA, United States). The RNA samples (0.5 pg–1 μg) were reverse transcribed into cDNA using Bestar^TM qPCR RT Kit (DBI^® Bioscience, Germany Quality). Quantitative real-time PCR analysis was performed in triplicate with a QuantiNova^TM SYBR^® Green PCR kit (QIAGEN) and detected by a LightCycler^® 480 Real-Time PCR System (Roche and Swiss). The thermal cycling conditions were as follows: 2 min at 95°C, followed by 40 cycles at 95°C for 5 s, 60°C for 10 s, and then 1 cycle at 95°C for 5 s, 65°C for 1 min, and finally slowly cooled down to 40°C and kept for 1 s. All the PCR reactions were performed using specific primers following the mouse sequences (Supplementary Table 1). To calculate the relative expression of each target gene, the comparative 2^–ΔΔCt method was used. The mRNA levels were normalized to levels of the endogenous housekeeping gene β-actin.

RAW264.7 cells were homogenized on ice in radioimmune precipitation assay (RIPA) buffer containing protease inhibitors and phosphatase inhibitors for 30 min. The lysates were centrifuged at 12,000 × g for 15 min at 4°C, and the supernatant was collected to determine the protein concentration using a Pierce^TM BCA protein assay kit (Thermo Scientific^TM). Isolated proteins (10 μl and 30–35 μg) were fractioned using 10% SDS-PAGE and electro-transferred to polyvinylidene fluoride membranes (Bio-Rad, Hercules, CA, United States). After blocking with 5% skim milk dissolved in TBST buffer for 1 h at RT, membranes were incubated with primary antibodies (1:500 for MMP9, GM130; 1:1,000 for GBF1, c-Fos, NFATc1, V-ATPase, CtsK, COPGI, FAM129A, EIF2α/p-EIF2α, PERK/p-PERK, Beclin1, p62, Atg5, LC3A/B, GAPDH, Cleaved Caspase-3, Caspase-3, Bax, Bcl-2, Bcl-xL, Cytochrome C, EEA1, Rab7, and LAMP1; 1:5,000 for BiP, β-tubulin) overnight at 4°C and then probed with horseradish peroxidase-conjugated secondary anti-rabbit and anti-mouse antibodies for 1 h at RT. Signals were detected with enhanced chemiluminescence (ECL) and visualized using ChemiDoc^TM Touch Imaging System (Bio-Rad, Hercules, CA, United States). The band density was analyzed using the Image J software. Data were presented with reference to control intensities of GAPDH or β-tubulin.

All data were expressed as mean ± SD (n = 3, each with duplicates). The results were assessed with one-way ANOVA followed by Dunnett’s post hoc test, with differences of p-value < 0.05 being considered statistically significant.

The cell membranes of differentiated and matured OCs are highly polarized. The proton pumps localized on the ruffled border secrete acids, degrade organic bone matrix with releasing hydrolase, and form resorption lacunae on the bone surface (Boyle et al., 2003). siRNA sequence specifically targeting GBF1 and the specific inhibitor GCA thereof in RAW 264.7 cells was used to knock down the expression of GBF1, so as to explore the role of GBF1 on OC differentiation and activation. Compared with negative control, suppression of GBF1 significantly decreased the number and size of TRAP⁺ multinucleated OCs (Figures 1A–D). Additionally, upon stimulation of RANKL, GBF1-mediated bone absorptive activity significantly improved (Figure 1E). Correspondingly, the area and depth of bone erosion were more intuitively represented in the stack 3D surface plot (Figures 1F,G). Subsequently, impaired acid secretion in GBF1 knocking down OCs using acridine orange staining was found, and the extracellular MMP9 content was significantly reduced (Figures 1H,I). The aforementioned data indicate that GBF1 was vital to the activation and functions of OCs in vitro.

To further investigate the effect of GBF1 on osteoclastogenesis, the expression of OC-related transcription factors was subsequently investigated. The present results reveal that transfection with GBF1 siRNA reduced the expression of early OC transcription factor NFATc1 and c-Fos (Figures 2A,D,E). Subsequently, compared with the negative control, the expression of the downstream target genes (TRAP, CtsK, MMP9, and V-ATPase) also decreased (Figures 2A,D,E). Recent studies have provided strong support for GCA as a potent, highly effective, and rapidly reversible small inhibitor likely targeting GBF1 (Pan et al., 2008). In the present study, further verification on whether GCA could impair osteoclastogenesis by inhibiting the expression of OC-specific genes was provided. RAW264.7 cells were first treated with different concentrations of GCA to measure the survival rate. There was no significant difference in cell viability under GCA treatment (Figure 2B). Hence, 1.5, 2, and 2.5 μM were chosen for subsequent experiments. Consistent with previous data, the protein levels of V-ATPase, NFATc1 and MMP9, but not GBF1, were inhibited by 2 μM GCA (Figures 2C,F,G). Unexpectedly, the increase in mRNA levels of these genes was highly variable (Figures 2C,H), indicating that GCA mediated a negative feedback loop, compensatively increased the level of OC activating factors, and maintained cell viability.

The COPI coat complex subunit gamma 1 (COPGI) is a cytoplasmic protein complex that reversibly binds to non-clathrin-coated vesicles budding from the Golgi membrane, and further mediates biosynthetic protein transport between the ER and the Golgi apparatus (Deng et al., 2009). Tumor promotor FAM129A has recently been reported to activate FAK signaling pathway and upregulate MMP2 and MMP9 in tumor tissues (Feng et al., 2019; Zhang et al., 2019). A significant increase in the protein levels of c-Fos, NFATc1, and FAM129A during osteoclastogenesis was detected, while FAM129A GI expression exhibited no difference (Figures 3A–C). To confirm the influential function on COPGI and FAM129A caused by GBF1, different concentrations of GCA were selected to verify the increased protein levels of COPGI and FAM129A in a time-dependent manner, among which 2.5 μM GCA upregulated the expression of FAM129A more strikingly (Figures 3A,D,F). Furthermore, as evidenced by immunofluorescence, higher amounts of endogenous FAM129A were captured in the OC nuclei on the third day within supplementation of 2 μM GCA (Figure 3E). The aforementioned results indicate that GBF1 promoted COPI-mediated vesicle trafficking in OCs and FAM129A might be a downstream effector.

In consideration of GBF1 being partly located at ER exit sites, the relationship between GBF1 and ER in OCs was further determined. Western blot analysis data from both knocking down of GBF1 and GCA treatment reveals that the destruction of the GBF1 function upregulated the levels of ERS response-related protein BiP, p-PERK, and p-EIF2α, but not the total expression of PERK and EIF2α (Figures 4A,B,E,F). In addition, the stress level was dosage-dependent, and especially increased after 6 h treatment (Figures 4C,D). Similar to the consequences caused by ERS, the pro-apoptotic protein Bax, Cytochrome C, and Cleaved Caspase-3 were upregulated, while the anti-apoptotic protein Bcl-2 was downregulated (Figures 4G–L). Taken together, the aforementioned results and the time course of the changes suggest that the specific inhibition of GBF1 in OCs had significant effects on the ERS and apoptosis of OCs.

A complex interaction exists between autophagy and apoptosis (Song et al., 2017). In previous research by the present authors, the effect of GBF1 on ERS and apoptosis was confirmed, and further hypothesis was made in terms of whether GBF1 regulated the autophagy process of OCs. As expected, suppression of GBF1 in OCs increased the protein levels of Beclin1, Atg5, and LC3, and the expression of p62 was reduced in a dosage-dependent manner in vitro (Figures 5A,B,E,F), in line with the increased autophagy activity at 6 h (Figures 5C,D). Subsequently, the levels of several proteins involved in endosomes were measured, such as early endosomal marker (EEA1), secondary endosomal marker (Rab7), and late endosomal marker (LAMP1). The expression levels of Rab7 and LAMP1 were significantly increased in OCs, which was contrary to the level of EEA1 (Figures 5H–K). Notably, GM130 staining was found to be widely scattered and the Golgi apparatus was extensively dispersed in GBF1-deficient OCs (Figure 5G). The aforementioned results further validated that GBF1 deficiency enhanced autophagy activity, possibly by affecting endosome maturation and the Golgi apparatus.