Journal: Biomedicines

Article Title: RhoGDI1-Cdc42 Signaling Is Required for PDGF-BB-Induced Phenotypic Transformation of Vascular Smooth Muscle Cells and Neointima Formation

doi: 10.3390/biomedicines9091169

Figure Lengend Snippet: MG132 pretreatment blocks the reduced phenotypic transition of HA-VSMCs induced by crenolanib. HA-VSMCs were pretreated with crenolanib (50 nM) for 48 h or MG132 (5 μM) for 1 h and then exposed to 10 ng/mL PDGF-BB treatment for 24 h. Untreated cells were used as control. ( A ) Cell proliferation was checked by EdU assay (scale bar = 100 μm). Histogram showing the ratio of EdU-positive cells (red) to the total number of cells. **, p < 0.01 vs. the control group; ##, p < 0.01 vs. the group treated with PDGF-BB; $$, p < 0.01 vs. the PDGF-BB and crenolanib-treated group ( n = 3). ( B ) Cell migration was checked by wound-healing (scale bar = 200 μm) and transwell assays (scale bar = 100 μm). Histograms showing the quantification of the wound healing (migrating cells from the scratched boundary) and transwell assay (crystal violet stained cells migrating to the lower chamber) results. **, p < 0.01 vs. the control group; #, p < 0.05 and ##, p < 0.01 vs. the PDGF-BB-treated group; $, p < 0.05 vs. the PDGF-BB and crenolanib-treated group ( n = 3). These results show that MG132 pretreatment promoted the reduced cell proliferation and migration mediated by crenolanib. ( C ) Western blot analysis of MYH-11, SM22, and smoothelin. MG132 reduced the increased smoothelin expression induced by crenolanib. Histograms showing the ratios of target proteins to β-tubulin. *, p < 0.05 vs. the control group; #, p < 0.05 vs. the PDGF-BB-treated group; $, p < 0.05 vs. the PDGF-BB and crenolanib-treated group ( n = 3).

Article Snippet: Anti-RhoGDI1 (A1214), –Ubiquitin (A3207), –smoothelin (# 6745), –MYH11 (# 10827), -ki-67 (A11390), and -SM22α (# A6760) antibodies were purchased from ABclonal (Wuhan, China).

Techniques: Control, EdU Assay, Migration, Transwell Assay, Staining, Western Blot, Expressing

Journal: Biomedicines

Article Title: RhoGDI1-Cdc42 Signaling Is Required for PDGF-BB-Induced Phenotypic Transformation of Vascular Smooth Muscle Cells and Neointima Formation

doi: 10.3390/biomedicines9091169

Figure Lengend Snippet: RhoGDI1 suppression inhibits PDGF-BB-induced phenotypic transition in HA-VSMCs. Cells were transfected with RhoGDI1 siRNA for 48 h and then treated with 10 ng/mL of PDGF-BB for another 24 h. Untreated cells were used as control. ( A ) Confirmation of RhoGDI1 knockdown by western blotting. ( B ) EdU assay showed that RhoGDI1 suppression inhibited PDGF-BB-induced cell proliferation (scale bar = 100 μm). Histogram showing the ratio of EdU-positive cells (red) to total cells. **, p < 0.01 vs. the control group; ##, p < 0.01 vs. the group treated with PDGF-BB ( n = 3). ( C ) Wound-healing (scale bar = 200 μm) and transwell assays (scale bar = 100 μm) showed that RhoGDI1 knockdown reduced cell migration induced by PDGF-BB. The quantification method refers to . **, p < 0.01 vs. the control group; #, p < 0.05 vs. the PDGF-BB-treated group ( n = 3). ( D ) Western blot analysis of MYH-11, SM22, and smoothelin showed that RhoGDI1 suppression promoted the expression of smoothelin in PDGF-BB-treated cells. Histograms showing the ratios of target proteins to β-tubulin. *, p < 0.05 vs. the control group; #, p < 0.05 vs. the PDGF-BB-treated group ( n = 3).

Article Snippet: Anti-RhoGDI1 (A1214), –Ubiquitin (A3207), –smoothelin (# 6745), –MYH11 (# 10827), -ki-67 (A11390), and -SM22α (# A6760) antibodies were purchased from ABclonal (Wuhan, China).

Techniques: Transfection, Control, Knockdown, Western Blot, EdU Assay, Migration, Expressing

Journal: Biomedicines

Article Title: RhoGDI1-Cdc42 Signaling Is Required for PDGF-BB-Induced Phenotypic Transformation of Vascular Smooth Muscle Cells and Neointima Formation

doi: 10.3390/biomedicines9091169

Figure Lengend Snippet: Cdc42 inhibition reduces PDGF-BB-induced phenotypic transition in HA-VSMCs. HA-VSMCs were pretreated with ZCL278 (50 μM) for 30 min followed by treatment with 10 ng/mL of PDGF-BB for 24 h. Untreated cells were used as control. ( A ) Cell proliferation was analyzed by EdU assay (scale bar = 100 μm). Histogram showing the ratio of EdU-positive cells (red) to total cells. **, p < 0.01 vs. the control group; #, p < 0.05 vs. the PDGF-BB-treated group ( n = 3). ( B ) Cell migration was analyzed by wound-healing (scale bar = 200 μm) and transwell assays (scale bar = 100 μm). The quantification method refers to . **, p < 0.01 vs. the control group; #, p < 0.05 vs. the PDGF-BB-treated group ( n = 3). ZCL278 pretreatment significantly decreased cell proliferation and migration induced by PDGF-BB. ( C ) Western blot analysis of MYH-11, SM22, and smoothelin. ZCL278 pretreatment increased the expression of smoothelin in PDGF-BB-treated cells. Histograms showing the ratios of the target proteins to β-tubulin. *, p < 0.05 vs. the control group; #, p < 0.05 vs. the PDGF-BB-treated group ( n = 3).

Article Snippet: Anti-RhoGDI1 (A1214), –Ubiquitin (A3207), –smoothelin (# 6745), –MYH11 (# 10827), -ki-67 (A11390), and -SM22α (# A6760) antibodies were purchased from ABclonal (Wuhan, China).

Techniques: Inhibition, Control, EdU Assay, Migration, Western Blot, Expressing

Journal: Biomedicines

Article Title: RhoGDI1-Cdc42 Signaling Is Required for PDGF-BB-Induced Phenotypic Transformation of Vascular Smooth Muscle Cells and Neointima Formation

doi: 10.3390/biomedicines9091169

Figure Lengend Snippet: Both RhoGDI1 and Cdc42 suppression inhibit neointima formation and improve the expression of contractile proteins in a rat carotid injury model. For shRNA-mediated knockdown, the carotid artery was injected with approximately 0.2 mL of virus solution (titer: 1 × 10 10 pfu) after balloon injury operation. ( A ) H&E of arteries 14 days after balloon injury. Rats without balloon injury were used as the sham operation group. Histogram showing the area ratio of intima to media. Both RhoGDI1 and Cdc42 knockdown reduced intima/media area ratio. **, p < 0.01 vs. the sham operation group; ##, p < 0.01 vs. the injury model group ( n = 10). ( B ) Immunofluorescence staining for contractile proteins such as MYH-11, SM22, smoothelin (green), ki-67 (green), and α-SMA (green) and CD31 (red) as double staining (merge); nuclei were stained with DAPI (blue). Histograms showing the fluorescence intensity of the staining. RhoGDI1 and Cdc42 knockdown reduced the expression of ki-67 and co-localization of α-SMA with CD31 and increased the expression of the three contractile proteins. *, p < 0.05 vs. the sham operation group; #, p < 0.05 vs. the injury model group ( n = 10). ( C ) Western blots showing the expression of MYH-11, SM22, and smoothelin. Histograms showing the ratios of the contractile proteins to β-tubulin. The results are consistent with those from immunofluorescence analysis. *, p < 0.05 vs. the sham operation group; #, p < 0.05 and ##, p < 0.01 vs. the injury model group ( n = 10).

Article Snippet: Anti-RhoGDI1 (A1214), –Ubiquitin (A3207), –smoothelin (# 6745), –MYH11 (# 10827), -ki-67 (A11390), and -SM22α (# A6760) antibodies were purchased from ABclonal (Wuhan, China).

Techniques: Expressing, shRNA, Knockdown, Injection, Virus, Immunofluorescence, Staining, Double Staining, Fluorescence, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: RhoA/ROCK Pathway Activation is Regulated by AT1 Receptor and Participates in Smooth Muscle Migration and Dedifferentiation via Promoting Actin Cytoskeleton Polymerization

doi: 10.3390/ijms21155398

Figure Lengend Snippet: The effects of Ang II on cell migration and the expression of contractile marker proteins in human aortic vascular smooth muscle cells (HA-VSMCs). Cells were treated with Ang II at different concentrations (0, 10, 100, 1000 nM) or different time points (0, 3, 6, 12, 24 h). Untreated cells were used as the control. ( A and B ) Cell migration was detected by wound-healing and Transwell assays after Ang II treatment. Histograms show the quantification of the wound healing and Transwell assay results. The scar bar is 50 μm. * p < 0.05 and ** p < 0.01 vs. the control group ( n = 3). ( C and D ) Western blot analysis of MYH11, SM22α, and smoothelin. Histograms show the ratios of myosin heavy chain (MYH11) or SM22α or smoothelin to β-tubulin. * p < 0.05 and ** p < 0.01 vs. the control group; # p < 0.05 and ## p < 0.01 vs. the group treated with 10 nM Ang II; $ p < 0.05 vs. the group treated with 100 nM Ang II ( n = 3).

Article Snippet: Primary antibodies against smoothelin (# 6745), MYH11 (# 10827), SM22α (# A6760) were from ABclonal (Wuhan, China).

Techniques: Migration, Expressing, Marker, Control, Transwell Assay, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: RhoA/ROCK Pathway Activation is Regulated by AT1 Receptor and Participates in Smooth Muscle Migration and Dedifferentiation via Promoting Actin Cytoskeleton Polymerization

doi: 10.3390/ijms21155398

Figure Lengend Snippet: The effects of Ang II receptor inhibitors on cell migration and the expression of contractile marker proteins in HA-VSMCs. Cells were pretreated with candesartan (5 μM) for 6 h or PD123319 (5 μM) for 6 h and then exposed to 100 nM Ang II for 24 h. Untreated cells were used as the control. ( A ) Cell migration was detected by wound-healing and Transwell assays. Histograms show the quantification of the wound healing and Transwell assay results. The scar bar is 50 μm. ** p < 0.01 vs. the control group; # p < 0.05 vs. the Ang II-treated group ( n = 3). ( B ) Western blot analysis of MYH11, SM22α, and smoothelin. Histograms show the ratios of MYH11 or SM22α or smoothelin to β-tubulin. * p < 0.05 vs. the control group; # p < 0.05 vs. the Ang II-treated group ( n = 3).

Article Snippet: Primary antibodies against smoothelin (# 6745), MYH11 (# 10827), SM22α (# A6760) were from ABclonal (Wuhan, China).

Techniques: Migration, Expressing, Marker, Control, Transwell Assay, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: RhoA/ROCK Pathway Activation is Regulated by AT1 Receptor and Participates in Smooth Muscle Migration and Dedifferentiation via Promoting Actin Cytoskeleton Polymerization

doi: 10.3390/ijms21155398

Figure Lengend Snippet: The effects of RhoA inhibitor CCG-1423 on cell migration and the expression of contractile marker proteins in HA-VSMCs. Cells were pretreated with CCG-1423 (20 μM) for 18 h and then exposed to 100 nM Ang II for 24 h. Untreated cells were used as the control. ( A ) Cell migration was detected by wound-healing and Transwell assays. Histograms show the quantification of the wound healing and Transwell assay results. The scar bar is 50 μm. ** p < 0.01 vs. the control group; # p < 0.05 vs. the Ang II-treated group ( n = 3). ( B ) Western blot analysis of MYH11, SM22α, and smoothelin. Histograms show the ratios of MYH11 or SM22α or smoothelin to β-tubulin. * p < 0.05 and ** p < 0.01 vs. the control group; # p < 0.05 and ## p < 0.01 vs. the Ang II-treated group ( n = 3).

Article Snippet: Primary antibodies against smoothelin (# 6745), MYH11 (# 10827), SM22α (# A6760) were from ABclonal (Wuhan, China).

Techniques: Migration, Expressing, Marker, Control, Transwell Assay, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: RhoA/ROCK Pathway Activation is Regulated by AT1 Receptor and Participates in Smooth Muscle Migration and Dedifferentiation via Promoting Actin Cytoskeleton Polymerization

doi: 10.3390/ijms21155398

Figure Lengend Snippet: The effects of Rac1 inhibitor NSC23766 on cell migration and the expression of contractile marker proteins in HA-VSMCs. Cells were pretreated with NSC23766 (50 μM) for 30 min and then exposed to 100 nM Ang II for 24 h. Untreated cells were used as the control. ( A ) Cell migration was detected by wound-healing and Transwell assays. Histograms show the quantification of the wound healing and Transwell assay results. The scar bar is 50 μm. ** p < 0.01 vs. the control group ( n = 3). ( B ) Western blot analysis of MYH11, SM22α, and smoothelin. Histograms show the ratios of MYH11 or SM22α or smoothelin to β-tubulin. * p < 0.05 vs. the control group ( n = 3).

Article Snippet: Primary antibodies against smoothelin (# 6745), MYH11 (# 10827), SM22α (# A6760) were from ABclonal (Wuhan, China).

Techniques: Migration, Expressing, Marker, Control, Transwell Assay, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: RhoA/ROCK Pathway Activation is Regulated by AT1 Receptor and Participates in Smooth Muscle Migration and Dedifferentiation via Promoting Actin Cytoskeleton Polymerization

doi: 10.3390/ijms21155398

Figure Lengend Snippet: The effects of Cdc42 inhibitor ZCL278 on cell migration and the expression of contractile marker proteins in HA-VSMCs. Cells were pretreated with ZCL278 (50 μM) for 30 min and then exposed to 100 nM Ang II for 24 h. Untreated cells were used as the control. ( A ) Cell migration was detected by wound-healing and Transwell assays. Histograms show the quantification of the wound healing and Transwell assay results. The scar bar is 50 μm. ** p < 0.01 vs. the control group ( n = 3). ( B ) Western blot analysis of MYH11, SM22α, and smoothelin. Histograms show the ratios of MYH11 or SM22α or smoothelin to β-tubulin. * p < 0.05 vs. the control group ( n = 3).

Article Snippet: Primary antibodies against smoothelin (# 6745), MYH11 (# 10827), SM22α (# A6760) were from ABclonal (Wuhan, China).

Techniques: Migration, Expressing, Marker, Control, Transwell Assay, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: RhoA/ROCK Pathway Activation is Regulated by AT1 Receptor and Participates in Smooth Muscle Migration and Dedifferentiation via Promoting Actin Cytoskeleton Polymerization

doi: 10.3390/ijms21155398

Figure Lengend Snippet: The effects of ROCK inhibitor Y27632 on cell migration and the expression of contractile marker proteins in HA-VSMCs. Cells were pretreated with Y27632 (20 μM) for 30 min and then exposed to 100 nM Ang II for 24 h. Untreated cells were used as the control. ( A ) Cell migration was detected by wound-healing and Transwell assays. Histograms show the quantification of the wound healing and Transwell assay results. The scar bar is 50 μm. ** p < 0.01 vs. the control group; # p < 0.05 and ## p < 0.01 vs. the Ang II-treated group ( n = 3). ( B ) Western blot analysis of MYH11, SM22α, and smoothelin. Histograms show the ratios of MYH11 or SM22α or smoothelin to β-tubulin. * p < 0.05 vs. the control group; # p < 0.05 vs. the Ang II-treated group ( n = 3).

Article Snippet: Primary antibodies against smoothelin (# 6745), MYH11 (# 10827), SM22α (# A6760) were from ABclonal (Wuhan, China).

Techniques: Migration, Expressing, Marker, Control, Transwell Assay, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: RhoA/ROCK Pathway Activation is Regulated by AT1 Receptor and Participates in Smooth Muscle Migration and Dedifferentiation via Promoting Actin Cytoskeleton Polymerization

doi: 10.3390/ijms21155398

Figure Lengend Snippet: Immunofluorescence staining of MYH11, SM22α, and smoothelin (green). Nuclei were stained with DAPI (blue). Mice were treated the same way as in tablee 7. Histograms show the fluorescence intensity of the staining in the intima. * p < 0.05 vs. the saline-infused group; # p < 0.05 vs. the model group (Ang II infusion + Ad-GFP virus) ( n = 10). The scar bar is 50 μm.

Article Snippet: Primary antibodies against smoothelin (# 6745), MYH11 (# 10827), SM22α (# A6760) were from ABclonal (Wuhan, China).

Techniques: Immunofluorescence, Staining, Fluorescence, Saline, Virus

Journal: Renal Failure

Article Title: The m6A reader YTHDF2 protects vascular smooth muscle cells against the osteogenic differentiation through targeting Runx2

doi: 10.1080/0886022x.2025.2488876

Figure Lengend Snippet: Figure 1. Low levels of YTHDF2 expression were associated with an increase in the risk of vascular calcification. (A) Von kossa staining and the quantification of thoracic aortas from control rats and calcified rats. (n = 3 for control; n = 3 for CKD. ***p < 0.01 vs. control group. (n = 3 for control; n = 3 for CKD. Scale bar: 50 µm). (B) Representative immunohistochemistry images quantification of YTHDF2 and Runx2 in CKD rats aortic smooth muscle layer (n = 3 for control; n = 3 for CKD). Scale bars: 20 μm. (C) Alizarin red S staining revealed calcified nodules in VSMCs cultured in β-GP medium for 0, 7 or 14 days. Scale bar: 100 µm. (D–E) Calcium content and ALP activity were measure. (F) Protein levels of YTHDF2, Col I, and SM22a in VSMCs treated with β-GP medium for 0, 3,7,14 days. (G) Real-time qPCR analysis of YTHDF2 expression in VSMCs cultured with β-GP for 7 days compared with 0d. (H) Correlation analysis between YTHDF2 mRNA expression and calcium content of primary VSMCs (n = 3). All data are presented as mean ± SD. *p < 0.05, **p < 0.01, ***p = 0.0001, ****p < 0.0001.

Article Snippet: In this study, the following reagents and antibodies were used: β-glycerophosphate (β-GP) was purchased from Sigma Chemical Co. (St. Louis, Mo, uSa), trIzol reagent from Invitrogen Life technology (Grand Island, New york, uSa), PrimeScript rt reagent Kit with gdNa Eraser and tb Green Premix Ex taq II (tlirNaseH Plus) from takara Co. (otsu, Shiga, Japan), Lipo-fectamine 3000 from Invitrogen Co. (Carlsbad, Ca, uSa), SP-9001 and SP9002 immunohistochemistry kits from Zhongshan Golden bridge technology Co. (beijing, China). antibody against ytHdF2 (#80014, 1:500 for Western blot; 1:50 for immunohistochemistry) was bought from Cell Signaling technology. abcam (Cambridge, Ma, uSa, ab236639) provided the anti-runx2 antibody (1:500 for Wb and 1:100 for IHC). the following antibodies were acquired from affinity (biosciences, LNC, uSa, aF7001): CoL I (1:1000 for Wb), SM22α (abclonal, a6760).

Techniques: Expressing, Staining, Control, Immunohistochemistry, Cell Culture, Activity Assay

Journal: Renal Failure

Article Title: The m6A reader YTHDF2 protects vascular smooth muscle cells against the osteogenic differentiation through targeting Runx2

doi: 10.1080/0886022x.2025.2488876

Figure Lengend Snippet: Figure 2. YTHDF2 Regulates VSMCs osteogenic reprogramming. (A) YTHDF2 overexpression in VSMCs confirmed by RT-qPCR. (B) Alizarin red staining in all of the groups for detecting calcification formation. Quantitative analysis of alizarin red staining after transfection with YTHDF2. Mean ± SD. n = 3. ****p < 0.001 vs. N group; ***p < 0.01 vs. β-GP+NC-OE group. (C) Western blot analysis of osteogenic phenotype marker (Col I) and contractile phenotype marker (SM22α) expression in rat primary VSMCs with YTHDF2-OE transfection cultured in osteogenic medium for 7 days. (D) The mRNA levels of YTHDF2 were decreased after silencing of YTHDF2 with 2 small hairpin RNAs (sh-nU6-b and sh-nU6-b) in VSMCs. **p < 0.01 vs. sh-NC. (E) Representative images and quantitative analysis of alizarin red staining in rat primary VSMCs after transfection with shYTHDF2. Mean ± SD. n = 3. *p < 0.05 vs. N group, ***p < 0.01 vs. β-GP+shY THDF2 group. (F) Western blot analysis of osteogenic phenotype marker (Col I) and contractile phenotype marker (SM22α) expression in rat primary VSMCs with shYTHDF2 transfection cultured in osteogenic medium. All values are presented as mean ± SD. *p < 0.05, **p < 0.01.

Article Snippet: In this study, the following reagents and antibodies were used: β-glycerophosphate (β-GP) was purchased from Sigma Chemical Co. (St. Louis, Mo, uSa), trIzol reagent from Invitrogen Life technology (Grand Island, New york, uSa), PrimeScript rt reagent Kit with gdNa Eraser and tb Green Premix Ex taq II (tlirNaseH Plus) from takara Co. (otsu, Shiga, Japan), Lipo-fectamine 3000 from Invitrogen Co. (Carlsbad, Ca, uSa), SP-9001 and SP9002 immunohistochemistry kits from Zhongshan Golden bridge technology Co. (beijing, China). antibody against ytHdF2 (#80014, 1:500 for Western blot; 1:50 for immunohistochemistry) was bought from Cell Signaling technology. abcam (Cambridge, Ma, uSa, ab236639) provided the anti-runx2 antibody (1:500 for Wb and 1:100 for IHC). the following antibodies were acquired from affinity (biosciences, LNC, uSa, aF7001): CoL I (1:1000 for Wb), SM22α (abclonal, a6760).

Techniques: Over Expression, Quantitative RT-PCR, Staining, Transfection, Western Blot, Marker, Expressing, Cell Culture

Journal: Renal Failure

Article Title: The m6A reader YTHDF2 protects vascular smooth muscle cells against the osteogenic differentiation through targeting Runx2

doi: 10.1080/0886022x.2025.2488876

Figure Lengend Snippet: Figure 3. Through the regulation of Runx2, YTHDF2 suppresses osteoblast phenotypic transition of VSMCs. (A) Relative Runx2 mRNA levels after YTHDF2-OE in VSMCs were confirmed by RT-qPCR. (B) Western blot analysis of Runx2 expression in rat primary VSMCs with YTHDF2-OE transfection. (C) Relative Runx2 mRNA levels after shYTHDF2 in VSMCs were confirmed by RT-qPCR. (D) Western blot analysis of Runx2 expression in rat primary VSMCs with shYTHDF2 transfection. (E) Correlation between the Runx2 protein level and YTHDF2 protein level in VSMCs (Pearson’s correlation coefficient R value and P value are shown). *p < 0.05, ***p < 0.001.

Article Snippet: In this study, the following reagents and antibodies were used: β-glycerophosphate (β-GP) was purchased from Sigma Chemical Co. (St. Louis, Mo, uSa), trIzol reagent from Invitrogen Life technology (Grand Island, New york, uSa), PrimeScript rt reagent Kit with gdNa Eraser and tb Green Premix Ex taq II (tlirNaseH Plus) from takara Co. (otsu, Shiga, Japan), Lipo-fectamine 3000 from Invitrogen Co. (Carlsbad, Ca, uSa), SP-9001 and SP9002 immunohistochemistry kits from Zhongshan Golden bridge technology Co. (beijing, China). antibody against ytHdF2 (#80014, 1:500 for Western blot; 1:50 for immunohistochemistry) was bought from Cell Signaling technology. abcam (Cambridge, Ma, uSa, ab236639) provided the anti-runx2 antibody (1:500 for Wb and 1:100 for IHC). the following antibodies were acquired from affinity (biosciences, LNC, uSa, aF7001): CoL I (1:1000 for Wb), SM22α (abclonal, a6760).

Techniques: Quantitative RT-PCR, Western Blot, Expressing, Transfection

Journal: Renal Failure

Article Title: The m6A reader YTHDF2 protects vascular smooth muscle cells against the osteogenic differentiation through targeting Runx2

doi: 10.1080/0886022x.2025.2488876

Figure Lengend Snippet: Figure 4. Runx2 Expression is under the control of METTL3-mediated m6A modification in VSMCs. (A) (B)3-deazaadenosine (DAA) treatment strengthen Runx2 mRNA levels and protein in VSMCs. (C)Western blot was used to find the protein level of Runx2 increased significantly after METTL3 silencing. (D) 3-deazaadenosine (DAA) could partially reverse YTHDF2 overexpression-induced decrease in protein levels of Runx2. All values are presented as mean ± SD. *p < 0.05, **p < 0.01, ****p < 0.0001.

Article Snippet: In this study, the following reagents and antibodies were used: β-glycerophosphate (β-GP) was purchased from Sigma Chemical Co. (St. Louis, Mo, uSa), trIzol reagent from Invitrogen Life technology (Grand Island, New york, uSa), PrimeScript rt reagent Kit with gdNa Eraser and tb Green Premix Ex taq II (tlirNaseH Plus) from takara Co. (otsu, Shiga, Japan), Lipo-fectamine 3000 from Invitrogen Co. (Carlsbad, Ca, uSa), SP-9001 and SP9002 immunohistochemistry kits from Zhongshan Golden bridge technology Co. (beijing, China). antibody against ytHdF2 (#80014, 1:500 for Western blot; 1:50 for immunohistochemistry) was bought from Cell Signaling technology. abcam (Cambridge, Ma, uSa, ab236639) provided the anti-runx2 antibody (1:500 for Wb and 1:100 for IHC). the following antibodies were acquired from affinity (biosciences, LNC, uSa, aF7001): CoL I (1:1000 for Wb), SM22α (abclonal, a6760).

Techniques: Expressing, Control, Modification, Western Blot, Over Expression

Journal: Renal Failure

Article Title: The m6A reader YTHDF2 protects vascular smooth muscle cells against the osteogenic differentiation through targeting Runx2

doi: 10.1080/0886022x.2025.2488876

Figure Lengend Snippet: Figure 5. YTHDF2 Inhibits Runx2 expression through an N6-methyladenosine (m6A)-YTH-domain family member 2 (YTHDF2)-dependent manner. (A) RIP-RT-PCR was utilized to confirm the Runx2 mRNA enrichment by YTHDF2 in VSMCs. Data were analyzed with Student’s T test. (B) Several m6A modified sites on Runx2 mRNA with high confidence predicted by SRAMP. (C) Luciferase reporter assays indicated a specific inhibitory effect of YTHDF2 on Runx2. (D) Changes of half-life (t1/2) of Runx2 mRNA in VMSCs with or without YTHDF2 overexpression. (E) All the findings in this study were concluded as a schematic diagram. Data were analyzed with student’s T test. Error bars represent the SD obtained from at least three independent experiments; *p < 0.05, ***p < 0.001.

Article Snippet: In this study, the following reagents and antibodies were used: β-glycerophosphate (β-GP) was purchased from Sigma Chemical Co. (St. Louis, Mo, uSa), trIzol reagent from Invitrogen Life technology (Grand Island, New york, uSa), PrimeScript rt reagent Kit with gdNa Eraser and tb Green Premix Ex taq II (tlirNaseH Plus) from takara Co. (otsu, Shiga, Japan), Lipo-fectamine 3000 from Invitrogen Co. (Carlsbad, Ca, uSa), SP-9001 and SP9002 immunohistochemistry kits from Zhongshan Golden bridge technology Co. (beijing, China). antibody against ytHdF2 (#80014, 1:500 for Western blot; 1:50 for immunohistochemistry) was bought from Cell Signaling technology. abcam (Cambridge, Ma, uSa, ab236639) provided the anti-runx2 antibody (1:500 for Wb and 1:100 for IHC). the following antibodies were acquired from affinity (biosciences, LNC, uSa, aF7001): CoL I (1:1000 for Wb), SM22α (abclonal, a6760).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Modification, Luciferase, Over Expression