Fig 2: Inhibition of GPCRs-IP3-IP3R-Ca2+ and MAPK pathways in MTEP treated SMB-S15 cells. (A) Left : Western blot for level of mGluR5 in 100 and 200 µM of MTEP treated SMB-S15 cells. Right: Densitometric analyses of the average gray values of the signals of the mGluR5 after being equilibrated with that of ß-actin. (B) ELISA for the levels of IP3. (C) Immunocytochemical assays for the levels of Ca2+ concentration. (D-F) Left: Western blots for the levels of p-ERK (D), p-JNK (E), and p-p38 (F) after MTEP treatment. Relative molecular weights are marked on the left. SMB cell lines are indicated above. (1, 2, 3) indicate the number of replicates. Right: Densitometric analyses of the average gray values of the signals of the target proteins after being equilibrated with that of ß-actin. The quantitative results are presented as mean + SEM. The IOD values of p-p38, p-ERK, and p-JNK in SMB-S15 cells is set to 1 and Y-axis represents the fold changes in MTEP-treated SMB-S15 versus SMB-S15. (G,H) Cell viability of SMB-S15 cell treated with L-glutamate only or L-glutamate and MTEP alternatively.

Fig 3: Quantal responses are not mediated by heterogenous stores(A) Ca2+ content of the ER of populations of permeabilized DT40-IP3R1 cells after addition of ATP, CPA (10 µM), and then the indicated concentrations of 2. Results are typical of three experiments, each with duplicate determinations.(B) Responses to 2 shown after subtraction of the basal Ca2+ leak.(C) Effects of a submaximal concentration of 2 (1 µM) added directly or by incremental additions. Results show mean ± SD of three replicates.(D) Summary results (individual values, mean ± SEM, n = 5, each with three replicates) show the final Ca2+ content of the stores of permeabilized DT40-IP3R1 cells (determined at 150 s). A maximal concentration of 2 (30 µM) released 52% ± 3% of the Ca2+ stores (red symbols). ***p < 0.001, one-way repeated ANOVA with Bonferroni’s test relative to all other values.(E) Ca2+ release from wild-type HEK cells evoked by 2 (30 µM) alone or followed by a supra-maximal concentration of IP3 (100 µM). Mean of duplicate determinations.(F) Summary results (individual values, mean ± SEM, n = 5) show Ca2+ release (determined at 500 s) evoked by the indicated concentrations of 2 followed by IP3 in wild-type HEK cells. **p < 0.01, ***p < 0.001,****p < 0.0001; ns, not significant (for indicated comparisons, ANOVA with Bonferroni’s test).(G) Maximal IP3 concentrations fully empty the IP3-sensitive Ca2+ stores, but it was unclear whether quantal responses to submaximal IP3 concentrations arise from incomplete emptying of the entire ER (i) or complete emptying of a fraction of the ER (ii). Results with a maximally effective concentration of 2, which partially activates all IP3Rs leaving Ca2+ trapped within the ER, indicate that quantal responses are not due to all-or-nothing emptying of heterogenous stores (ii).

Fig 4: Summary of the proposed molecular mechanisms of osteodifferentiation. Potential biomarkers are bold and italic denoted. Abbreviations: ALB (albumin), ACSBG2 (acyl-CoA synthetase bubblegum family member 2), ACSL1, ACSL3 (acyl-CoA synthetase long chain family members 1 and 3), ANGPTL4 (angiopoietin like 4), ACOX1, ACOX3 (acyl-CoA oxidases 1 and 3), APOA2, APOC3 (apolipoproteins A2 and C3), CXCL1, CXCL5, CXCL6 (C-X-C motif chemokine ligands 1, 5, and 6), FHL1 (four and a half LIM domains 1), FTH1 (ferritin heavy chain 1), GFAP (glial fibrillary acidic protein), GK (glycerol kinase), HSP90 (heat shock protein 90), HSPA5 (heat shock protein family A (Hsp70) member 5), INS (insulin), IP3R (inositol 1,4,5-trisphosphate receptor type 2), MMP1, MMP3 (matrix metallopeptidases 1 and 3), MSMO1 (methylsterol monooxygenase 1), MT1E (metallothionein 1E), NSDHL (NAD(P) dependent steroid dehydrogenase-like), PDIA4 (protein disulfide isomerase family A member 4), PLIN2 (perilipin 2), PRMT1 (protein arginine methyltransferase 1), SOD2 (superoxide dismutase 2), STAT1, STAT3 (signal transducers and activators of transcription 1 and 3), STEAP2 (STEAP2 metalloreductase), and TTR (transthyretin).

Fig 5: Activation of the GPCRs-IP3-IP3R-Ca2+ pathway in two SMB cell lines. (A) Left: Western blots for the levels of Gq/11a. Relative molecular weights are marked on the left. Two SMB cell lines are indicated above. (1, 2, 3) indicate the number of replicates. Right: Densitometric analyses of the average gray values of the signals of the Gq/11a after being equilibrated with that of ß-actin. (B) ELISA for the levels of IP3. (C) Top: Western blots for the levels of IP3R. Relative molecular weights are marked on the left. Two SMB cell lines are indicated above. Bottom: Densitometric analyses of the average gray values of the signals of the IP3R after being equilibrated with that of ß-actin. (D) Top: Immunocytochemical assays for the levels of Ca2+ concentration. Bottom: The IOD analysis for the signals of Ca2+ in the images of two SMB cell lines. The quantitative results are presented as mean + SEM.