Journal: Advanced Science

Article Title: WTAP Transcriptional Suppression by KLF9 Drives Osteoclastogenesis via M 6 A‐Mediated Regulation of CSF1R Signaling in Estrogen‐Deficient Osteoporosis

doi: 10.1002/advs.77227

Figure Lengend Snippet: WTAP expression was decreased during osteoclastogenesis in OP patients and OVX mice. (A) tSNE plot of single‐cell RNA sequencing data ( GSE147287 ) from OP patient bone marrow, annotated for major cell types. (B) Expression levels of WTAP, Ctsk, and Nfatc1 mapped onto the tSNE plot. (C) Pseudotemporal trajectory analysis showing the relative expression of WTAP, Ctsk, and Nfatc1 during osteoclast differentiation. (D) Representative H&E, TRAP, and WTAP immunohistochemical staining images of bone tissue sections from non‐OP and OP patients. Scale bars: 500 µm (H&E, TRAP) and 100 µm (WTAP). (E) Quantification of WTAP‐positive cells in bone tissue sections from non‐OP and OP patients ( n = 8 per group). (F) Serum CTX‐1 levels in non‐OP and OP patients ( n = 8 per group). (G) Correlation analysis between WTAP expression in bone tissue and serum CTX‐1 levels. (H) Representative H&E, TRAP, and WTAP immunohistochemical staining images of bone tissue sections from sham‐operated (Ctrl) and OVX mice. Scale bars: 200 µm (H&E, TRAP) and 50 µm (WTAP). (I) Representative immunofluorescence images showing co‐localization of WTAP (green) and C‐fos (red) in bone tissue sections from Ctrl and OVX mice. Scale bars, 50 µm. (J) Quantification of WTAP‐positive area in bone tissue sections from Ctrl and OVX mice ( n = 5 per group). (K) Heatmap showing the expression of m 6 A‐related genes during RANKL‐induced osteoclast differentiation ( GSE246769 ). (L) qRT‐PCR analysis of Wtap expression in BMDMs at different time points after RANKL induction. (M) Western blot of WTAP, CTSK, MMP9, and ACTB protein levels in BMDMs at different time points after RANKL induction. (N) Representative immunofluorescence images showing WTAP (red) expression in BMDMs at different time points after RANKL stimulation. F‐actin was stained with phalloidin (green). Scale bars, 20 µm. Data are presented as mean ± SEM. Statistical significance was determined by unpaired two‐tailed Student's t ‐test (E, F, J) or one‐way ANOVA with Tukey's post hoc test (L). * p < 0.05, *** p < 0.001.

Article Snippet: After blocking, membranes were incubated overnight at 4°C with primary antibodies against ACTB (AC026, Abclonal, Wuhan, China), CTSK (A1782, Abclonal, Wuhan, China), MMP9, WTAP, KLF9, YTHDF2, and CSF1R.

Techniques: Expressing, Single Cell, RNA Sequencing, Immunohistochemical staining, Staining, Immunofluorescence, Quantitative RT-PCR, Western Blot, Two Tailed Test

Journal: Advanced Science

Article Title: WTAP Transcriptional Suppression by KLF9 Drives Osteoclastogenesis via M 6 A‐Mediated Regulation of CSF1R Signaling in Estrogen‐Deficient Osteoporosis

doi: 10.1002/advs.77227

Figure Lengend Snippet: Intervention of WTAP inversely regulated osteoclastogenesis and OP pathogenesis in vitro and in vivo. (A) Schematic diagram of the experimental design for Wtap knockdown in BMDMs using siRNA. (B) qRT‐PCR analysis showing the relative expression of WTAP, Nfatc1, Ctsk, and Mmp9 in BMDMs transfected with NC or Wtap siRNAs. (C) Western blot analysis confirming the protein levels of WTAP, CTSK, and MMP9 in BMDMs after Wtap knockdown. (D) Representative TRAP staining images and corresponding microscopic views showing osteoclast formation in BMDMs under siWtap intervention. Scale bars, 20 µm. (E) Representative immunofluorescence images showing F‐actin ring formation (green) in BMDMs. Scale bars, 20 µm. (F) Schematic diagram of the experimental design for Wtap overexpression in BMDMs using plasmid transfection. (G) qRT‐PCR analysis showing the relative expression of WTAP, Nfatc1, Ctsk, and Mmp9 in BMDMs transfected with a vector control or WTAP overexpression plasmid. (H) Western blot analysis confirming the protein levels of WTAP, CTSK, and MMP9 in BMDMs after Wtap overexpression. (I) Representative TRAP staining images and corresponding microscopic views showing osteoclast formation in BMDMs under oeWtap intervention. Scale bars, 20 µm. (J) Representative immunofluorescence images showing F‐actin ring formation (green) in BMDMs. Scale bars, 20 µm. (K) Schematic diagram of the generation of myeloid lineage‐specific Wtap conditional knockout (WTAP CKO) mice and the experimental timeline for OVX and tamoxifen induction. (L) Representative H&E staining images of femur sections from WTAP Ctrl and WTAP CKO mice treated with sham or OVX surgery. Scale bars, 100 µm. (M) Representative micro‐CT images of the distal femur from WTAP Ctrl and WTAP CKO mice treated with sham or OVX surgery. (N) Quantification of bone mineral density (BMD) in the distal femur of WTAP Ctrl and WTAP CKO mice subjected to sham or OVX surgery. Data are presented as mean ± SEM. Statistical significance was determined by one‐way ANOVA with Tukey's post hoc test (B, G, N). * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: After blocking, membranes were incubated overnight at 4°C with primary antibodies against ACTB (AC026, Abclonal, Wuhan, China), CTSK (A1782, Abclonal, Wuhan, China), MMP9, WTAP, KLF9, YTHDF2, and CSF1R.

Techniques: In Vitro, In Vivo, Knockdown, Quantitative RT-PCR, Expressing, Transfection, Western Blot, Staining, Immunofluorescence, Over Expression, Plasmid Preparation, Control, Knock-Out, Micro-CT

Journal: Advanced Science

Article Title: WTAP Transcriptional Suppression by KLF9 Drives Osteoclastogenesis via M 6 A‐Mediated Regulation of CSF1R Signaling in Estrogen‐Deficient Osteoporosis

doi: 10.1002/advs.77227

Figure Lengend Snippet: KLF9 regulated osteoclastogenesis and OP progression via a WTAP‐dependent pathway. (A) Schematic diagram of the experimental design for generating a double‐intervention mouse model. Tamoxifen‐induced WTAP CKO and WTAP Ctrl mice underwent OVX and were treated with both tail vein and intraosseous injections of AAV‐shKLF9 or AAV‐NC. (B) Representative H&E staining images of bone tissue sections from the indicated groups. Scale bars: 200 µm and 100 µm. (C) Representative TRAP and MMP9 staining images of bone tissue sections from the indicated groups. Scale bars, 100 µm. (D) Quantification of MMP9‐positive area in bone tissue sections ( n = 5 per group). (E) Serum levels of CTX‐1 in the indicated groups ( n = 5 per group). (F) Representative micro‐CT images of the distal femur from the indicated groups. (G) Quantification of bone mineral density (BMD) from micro‐CT analysis ( n = 5 per group). (H) Western blot analysis of CTSK and MMP9 protein levels in BMDMs under siRNA interventions. (I) Representative TRAP staining and immunofluorescence images of osteoclasts formed under siRNA interventions. Scale bars, 20 µm. (J) Western blot analysis of CSF1R and KLF9 protein levels in BMDMs under overexpression or knockdown interventions. (K) Representative immunohistochemical staining images of CSF1R in bone tissue sections from sham, OVX, and OVX + AAV‐shKLF9 mice. Scale bars, 100 µm. L. Western blot analysis of CSF1R protein levels in BMDMs under siRNA interventions. (M) Western blot analysis of CSF1R, WTAP, and YTHDF2 protein levels in BMDMs under siRNA interventions. Data are presented as mean ± SEM. Statistical significance was determined by one‐way ANOVA with Tukey's post hoc test (D, E, G). ** p < 0.01, *** p < 0.001, ns, not significant.

Article Snippet: After blocking, membranes were incubated overnight at 4°C with primary antibodies against ACTB (AC026, Abclonal, Wuhan, China), CTSK (A1782, Abclonal, Wuhan, China), MMP9, WTAP, KLF9, YTHDF2, and CSF1R.

Techniques: Staining, Micro-CT, Western Blot, Immunofluorescence, Over Expression, Knockdown, Immunohistochemical staining