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: For immunohistochemistry, sections were subjected to antigen retrieval, blocked, and incubated with primary antibodies against WTAP (A22751, Abclonal, Wuhan, China), MMP9 (10375‐2‐AP, Proteintech, Wuhan, China), KLF9 (A7196, Abclonal, Wuhan, China), or CSF1R (A3019, Abclonal, Wuhan, China) overnight at 4°C.

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: For immunohistochemistry, sections were subjected to antigen retrieval, blocked, and incubated with primary antibodies against WTAP (A22751, Abclonal, Wuhan, China), MMP9 (10375‐2‐AP, Proteintech, Wuhan, China), KLF9 (A7196, Abclonal, Wuhan, China), or CSF1R (A3019, Abclonal, Wuhan, China) overnight at 4°C.

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: WTAP governed osteoclastogenesis via YTHDF2‐mediated regulation of Csf1r mRNA stability. (A) Heatmap showing the expression patterns of m 6 A reader proteins (YTHDC, YTHDF, IGF2BP, HNRNP families) in BMDMs from WTAP CKO mice compared to WTAP Ctrl mice and in BMDMs overexpressing Wtap compared to control. (B) Representative immunofluorescence images showing the colocalization of YTHDF2 (green) and WTAP (red) in bone tissue sections from WTAP Ctrl and WTAP CKO mice. Scale bars: 50 µm. (C) Western blot analysis showing the expression levels of YTHDF2 protein in BMDMs under Wtap knockdown. (D) qRT‐PCR analysis showing the expression levels of YTHDF2 mRNA in BMDMs under Wtap knockdown and overexpression. (E, F) Western blot and qRT‐PCR analysis validating the efficiency of Ythdf2 knockdown using siRNA in BMDMs. (G) RIP‐PCR assay showing the binding of YTHDF2 to CSF1R mRNA in BMDMs under WTAP knockdown or overexpression. (H) mRNA stability analysis using Actinomycin D chase assay, showing the decay kinetics of CSF1R mRNA in BMDMs overexpressing Wtap with or without Ythdf2 knockdown. (I, J) Western blot and qRT‐PCR analysis showing the expression levels of CSF1R, MMP9, and YTHDF2 in BMDMs under specific interventions. (K) Representative TRAP staining and immunofluorescence images of osteoclasts formed under specific interventions. Scale bars, 20 µm. (L, M) Quantification of MNC rate and cell fusion area under specific interventions. Data are presented as mean ± SEM. Statistical significance was determined by one‐way ANOVA with Tukey's post hoc test (D, F, G, J, L, M). * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: For immunohistochemistry, sections were subjected to antigen retrieval, blocked, and incubated with primary antibodies against WTAP (A22751, Abclonal, Wuhan, China), MMP9 (10375‐2‐AP, Proteintech, Wuhan, China), KLF9 (A7196, Abclonal, Wuhan, China), or CSF1R (A3019, Abclonal, Wuhan, China) overnight at 4°C.

Techniques: Expressing, Control, Immunofluorescence, Western Blot, Knockdown, Quantitative RT-PCR, Over Expression, Binding Assay, Staining

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 acted as an upstream transcriptional repressor of WTAP to regulate osteoclastogenesis and OP progression via direct DNA binding after nuclear translocation. (A) Venn diagram of the intersection of DEGs from RNA‐seq of osteoclast differentiation (OC), transcription factor (TFs) binding sites predicted by the UCSC Genome Browser, and DEGs from scRNA‐seq of osteoclast precursors (Mφ). (B) Representative immunohistochemical staining images of KLF9 in bone tissue sections from non‐OP and OP patients. Scale bars, 50 µm. (C) qRT‐PCR analysis showing the relative expression of Klf9 mRNA in BMDMs at different time points after RANKL induction. (D) Representative immunofluorescence images showing the co‐localization of KLF9 (red) and F‐actin (green) in BMDMs treated with or without RANKL. Scale bars, 20 µm. (E) Schematic diagram of the experimental design for in vivo knockdown of Klf9 using AAV‐shKLF9 in OVX mice. (F) Representative H&E and TRAP staining images of bone tissue sections from sham, OVX, OVX + AAV‐NC, and OVX + AAV‐shKLF9 mice. Scale bars, 100 µm. (G) Representative micro‐CT images of the distal femur microstructure from the specific groups. (H) Quantification of bone mineral density (BMD) from micro‐CT analysis. (I) Schematic representation of the Wtap promoter region showing the predicted KLF9 binding motifs (Site 1: −1669 to −1657 bp; Site 2: −528 to −516 bp). (J) ChIP‐PCR showing the enrichment of KLF9 binding to the WTAP promoter at Site 1 and Site 2 in BMDMs overexpressing WTAP. (K) Dual‐luciferase reporter assay showing the relative luciferase activity of the Wtap promoter with wild‐type (WT) or mutated (mut1, mut2, mut1+2) KLF9 binding sites in the presence of Klf9 overexpression. (L) qRT‐PCR analysis of Wtap expression in BMDMs under KLF9 knockdown or overexpression. (M) Western blot analysis of WTAP and KLF9 protein levels in BMDMs under specific interventions. (N) Representative immunofluorescence images showing the subcellular localization of WTAP (red) and KLF9 (green) in BMDMs treated with or without RANKL. Scale bars, 20 µm. (O) Western blot analysis of KLF9 protein levels in cytoplasmic (Cyt) and nuclear (Nuc) fractions of BMDMs treated with or without RANKL. Data are presented as mean ± SEM. Statistical significance was determined by unpaired two‐tailed Student's t ‐test (J) or one‐way ANOVA with Tukey's post hoc test (H, K, L). ** p < 0.01, *** p < 0.001, ns, not significant.

Article Snippet: For immunohistochemistry, sections were subjected to antigen retrieval, blocked, and incubated with primary antibodies against WTAP (A22751, Abclonal, Wuhan, China), MMP9 (10375‐2‐AP, Proteintech, Wuhan, China), KLF9 (A7196, Abclonal, Wuhan, China), or CSF1R (A3019, Abclonal, Wuhan, China) overnight at 4°C.

Techniques: Binding Assay, Translocation Assay, RNA Sequencing, Immunohistochemical staining, Staining, Quantitative RT-PCR, Expressing, Immunofluorescence, In Vivo, Knockdown, Micro-CT, Luciferase, Reporter Assay, Activity Assay, Over Expression, 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: 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: For immunohistochemistry, sections were subjected to antigen retrieval, blocked, and incubated with primary antibodies against WTAP (A22751, Abclonal, Wuhan, China), MMP9 (10375‐2‐AP, Proteintech, Wuhan, China), KLF9 (A7196, Abclonal, Wuhan, China), or CSF1R (A3019, Abclonal, Wuhan, China) overnight at 4°C.

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

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: Targeting the WTAP‐CSF1R axis in osteoclast precursors ameliorated OVX‐induced osteoporosis. (A) Schematic diagram of the experimental design for the AAV‐mediated intervention. OVX C57 mice were injected with AAV‐oeWtap or AAV‐NC on day 7 post‐surgery and harvested on day 60. 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) Schematic diagram of the experimental design for the pharmacological intervention with PLX3397. OVX C57 mice were administered PLX3397 or vehicle on day 7 post‐surgery and harvested on day 60. (I) Representative H&E staining images of bone tissue sections from the indicated groups. Scale bars: 200 µm and 100 µm. (J) Representative micro‐CT images of the distal femur from the indicated groups. (K) Quantification of bone mineral density (BMD) from micro‐CT analysis ( n = 5 per group). Data are presented as mean ± SEM. Statistical significance was determined by one‐way ANOVA with Tukey's post hoc test (D, E, G, K). * p < 0.05, *** p < 0.001.

Article Snippet: For immunohistochemistry, sections were subjected to antigen retrieval, blocked, and incubated with primary antibodies against WTAP (A22751, Abclonal, Wuhan, China), MMP9 (10375‐2‐AP, Proteintech, Wuhan, China), KLF9 (A7196, Abclonal, Wuhan, China), or CSF1R (A3019, Abclonal, Wuhan, China) overnight at 4°C.

Techniques: Injection, Staining, 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: Scheme of the KLF9/WTAP/YTHDF2/m 6 A/CSF1R regulatory axis in osteoclastogenesis and estrogen‐deficient osteoporosis. WTAP‐mediated m 6 A modification of CSF1R mRNA governs osteoclastogenesis via a YTHDF2‐mediated mechanism. Pathological upregulation of KLF9 drives Wtap transcription, leading to increased m 6 A deposition on the 3’‐UTR of Csf1r mRNA. This reduces YTHDF2 binding affinity, destabilizing CSF1R mRNA and elevating CSF1R protein levels upon M‐CSF stimulation, thereby enhancing osteoclastogenesis and promoting bone resorption. Conversely, therapeutic inhibition of KLF9 or WTAP decreases m6A modification on CSF1R, restores YTHDF2 binding, stabilizes CSF1R mRNA, and reduces CSF1R expression, ultimately suppressing osteoclast differentiation. Pharmacological blockade of CSF1R with PLX3397 further attenuates osteoclastogenesis, providing a strategy to alleviate estrogen‐deficient osteoporosis.

Article Snippet: For immunohistochemistry, sections were subjected to antigen retrieval, blocked, and incubated with primary antibodies against WTAP (A22751, Abclonal, Wuhan, China), MMP9 (10375‐2‐AP, Proteintech, Wuhan, China), KLF9 (A7196, Abclonal, Wuhan, China), or CSF1R (A3019, Abclonal, Wuhan, China) overnight at 4°C.

Techniques: Modification, Binding Assay, Inhibition, Expressing