Table 1.
RT-qPCR Primer sequences.
Table 2.
shRNA constructs.
Figure 1.
Osteoclast formation potential of H10 cells vs. C8 cells.
Photomicrograph of TRACP and DAPI stained osteoclasts derived from H10 and C8 cells after 4 days in culture with sRANKL. H10 cells were able to fuse formed large TRACP positive multinucleated cells (FI>60%) while C8 cells did not fuse at all (FI = 0%).
Figure 2.
Heatmap of microarray analysis.
Total RNA was extracted from H10 and C8 cells undergoing day 2 of RANKL-induced OCG. Microarray analysis was performed with an Affymetrix array and genes with a FDR of 0.01 and fold change over 5 were selected. Heatmap showing 8 randomly selected genes for RT-qPCR confirmation, that have not been reported to be involved in OCG.
Figure 3.
RT-qPCR confirming microarray results.
Real-time qPCR analysis was used to quantify gene expression of osteoclast cultures at days 0, 2, 4, 6, 8 for BMMs (A); days 0, 1, 2, 3 and 4 for RAW264.7 cells (B) and day 0, 1, 2, 3 and 4 for H10 and C8 cells (C and D). Day 0 was used as the control in BMMs and RAW264.7 RT-qPCR. CD109 mRNA level in H10 and C8 cells, as well as in unstimulated RAW264.7 cells were expressed as normalized 2-△△CT. C8 cells were used as the control for H10 cells. There was a statistically significant increase of CD109 mRNA expression over the days of OCG in RAW264.7 and bone marrow monocytes as well as when comparing mRNA expression of CD109 in H10 cells vs. both C8 cells and unstimulated RAW264.7 cells at day 1, 2, 3 and 4 of OCG (n = 3, P<0.05). Note that CD109 mRNA expression level in C8 cells not only less than that of the H10 cells, but also less than that of the original RAW264.7 cells, regardless of RANKL stimulation (day 1–4 with RANKL, day 0 without RANKL stimulation).
Figure 4.
CD109 Protein expression over the days of OCG.
Western blot analysis showing that CD109 protein expression is increased in the critical days of osteoclastogenesis in H10 cells, RAW264.7 cells and BMMs (n = 3, P<0.05). Band intensity of CD109 expression was normalized with β-actin. Band intensity of C8 cells was used as the baseline for 4A. Day 1 for 4B, and Day 0 for 4C.
Figure 5.
Western blot analysis was used to reveal the percentage of protein knockdown per construct used. β-actin was used as the internal loading control. CD109 protein expression in cells expressing a scrambled shRNA (shRNA construct 13) was used as the control. Quantification of the Western blot showed that the higher knockdown efficiency was observed in constructs 97 and 99 when compared to scramble control. Note that 07 is RAW264.7 cells stably transfected with the empty shRNA vector pGFP-V-RS, which was used to harbor gene specific shRNA to knockdown CD109.
Figure 6.
In vitro OCG with CD109 knockdown cell lines.
RANKL-induced OCG in vitro experiments showed decreased fusion efficiency in CD109 KD cells when compared to the negative shRNA control. (n = 3, P<0.05).
Figure 7.
Schematic model of CD109 down-regulation of TGF-β.
CD109 down-regulation of TGF-β signaling and inhibition Smad3 signaling, would cause a decrease in the TRAF6-TAB1-TAK1 complex formation resulting in a decrease in osteoclast formation. Up-regulation of CD109 showed an increase in OC formation suggesting that CD109 plays a role in OCG and it might be independent of TGF-β in osteoclasts.