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. 2023 Feb 16;31:568–585. doi: 10.1016/j.omtn.2023.02.014

Figure 4.

Figure 4

BMAL1 targeted the circadian-controlled gene TTK to regulate H2Bub1 levels to affect the osteogenic capacity of MSCs

(A and B) Relative mRNA (A) and protein (B) expression of TTK in the MSCs infected with Sh-NC, Sh-BMAL1, OE-NC, or OE-BMAL1 lentiviruses on the 10th day of osteogenic differentiation. (C) The putative E-boxes in the TTK promoter region. (D) CUT&Tag-qPCR showed the percentage of BMAL1 occupancy on the TTK promoter. Data are shown as the proportion of input level and normalized to the IgG control. (E) CUT&Tag-qPCR analysis showing the H2Bub1 occupancy on RUNX2 and OSX in the MSCs infected with Sh-NC and OE-NC, Sh-BMAL1 and OE-NC or Sh-BMAL1 and OE-TTK lentiviruses that were undergoing osteogenic differentiation. (F and G) Relative mRNA expression (F) and protein expression (G) of RUNX2 and OSX in MSCs infected with Sh-NC and OE-NC, Sh-BMAL1 and OE-NC, or Sh-BMAL1 and OE-TTK lentiviruses that were undergoing osteogenic differentiation. Bar graphs showing the relative expression. (H) ARS and ALP staining of the MSCs infected with Sh-NC and OE-NC, Sh-BMAL1 and OE-NC, or Sh-BMAL1 and OE-TTK lentiviruses on the 14th day of osteogenic differentiation. (I) HE and Masson staining and Col I immunohistochemistry of transplanted HA/TCP embedded with the MSCs infected with Sh-NC and OE-NC, Sh-BMAL1 and OE-NC, or Sh-BMAL1 and OE-TTK lentiviruses. All data are presented as mean ± SD; n = 3; ∗p < 0.05.