Journal: Arteriosclerosis, Thrombosis, and Vascular Biology

Article Title: m ⁶ A Modification of Profilin-1 in Vascular Smooth Muscle Cells Drives Phenotype Switching and Neointimal Hyperplasia via Activation of the p-ANXA2/STAT3 Pathway

doi: 10.1161/atvbaha.124.321399

Figure Lengend Snippet: Figure 1. Single-cell RNA sequencing reveals that PFN1 (profilin-1) plays a critical role in neointimal hyperplasia. A, Schematic diagram of balloon-injury procedure in rat right carotid arteries and flow chart of single-cell suspension. B, Distribution of balloon- injured and sham groups in vascular smooth muscle cells (VSMCs) and 6 clusters based on gene expression similarity. C, Expression and distribution of ACTA2, S100A4 (S100 calcium-binding protein A4), and PFN1 in each single cell of VSMCs. D, Violin plot visualizing the distribution of ACTA2, S100A4, and PFN1 mRNA expression and its probability density. E, Representative immunoblots and relative quantification analysis of PFN1 protein in rat carotid arteries (n=6 per group). F, Representative images showing double immunostaining for PFN1 (red) and α-SMA (actin alpha 2, smooth muscle, aorta; green) of rat carotid arteries in sham group and balloon-injured group (n=6 per group). G, Representative images showing immunohistochemistry for PFN1 of rat carotid arteries in sham group and balloon-injured group (n=6 per group). Scale bar, 50 µm. ACTA2 indicates actin alpha 2, smooth muscle, aorta; GEM, gel beads in emulsions; and tSNE, t-distributed stochastic neighbor embedding.

Article Snippet: The floxed PFN1 mice (with LoxP [locus of X-over P1] sites flanking exon 2; CKOCMP-18643-PFN1-B6J-VA) and YTHDF3 (YTH N6-methyladenosine RNA binding protein F3)-global knockout mice (with LoxP sites flanking exon 3; KOCMP-229096YTHDF3-B6N-VA) were obtained from Cyagen Biosciences.

Techniques: RNA Sequencing, Suspension, Gene Expression, Expressing, Binding Assay, Western Blot, Quantitative Proteomics, Double Immunostaining, Immunohistochemistry

Journal: Arteriosclerosis, Thrombosis, and Vascular Biology

Article Title: m ⁶ A Modification of Profilin-1 in Vascular Smooth Muscle Cells Drives Phenotype Switching and Neointimal Hyperplasia via Activation of the p-ANXA2/STAT3 Pathway

doi: 10.1161/atvbaha.124.321399

Figure Lengend Snippet: Figure 2. Multiple in vivo models confirm the participation of PFN1 (profilin-1) in neointimal hyperplasia. A, Representative images showing immunohistochemistry for α-SMA (actin alpha 2, smooth muscle, aorta) and PFN1 in in-stent restenotic (ISR) area in swine (n=6). Scale bar, 1 mm. B, Serum quantification of PFN1 protein concentration from no-ISR patients (n=35) and ISR patients (n=35). Data are presented as box-and-whisker plots, with 75th and 25th percentiles; bars represent maximal and minimal values. Statistical comparison: unpaired t test. C, Correlation between serum PFN1 level and lumen area loss in 35 ISR patients; statistical comparison: Pearson correlation analysis. D, Representative immunoblots of PFN1 protein in murine carotid arteries in sham group and wire-injured group (n=6 per group). E, Representative images showing double immunostaining for PFN1 (red) and α-SMA (green) in uninjured and wire-injured murine carotid arteries (n=6 per group). Scale bar, 50 µm. F, Representative images showing immunohistochemistry for PFN1 in uninjured and (Continued )

Article Snippet: The floxed PFN1 mice (with LoxP [locus of X-over P1] sites flanking exon 2; CKOCMP-18643-PFN1-B6J-VA) and YTHDF3 (YTH N6-methyladenosine RNA binding protein F3)-global knockout mice (with LoxP sites flanking exon 3; KOCMP-229096YTHDF3-B6N-VA) were obtained from Cyagen Biosciences.

Techniques: In Vivo, Immunohistochemistry, Protein Concentration, Whisker Assay, Comparison, Western Blot, Double Immunostaining

Journal: Arteriosclerosis, Thrombosis, and Vascular Biology

Article Title: m ⁶ A Modification of Profilin-1 in Vascular Smooth Muscle Cells Drives Phenotype Switching and Neointimal Hyperplasia via Activation of the p-ANXA2/STAT3 Pathway

doi: 10.1161/atvbaha.124.321399

Figure Lengend Snippet: Figure 3. PFN1 (profilin-1) deletion suppresses vascular smooth muscle cell (VSMC) proliferation, migration, and phenotype switching. A, Representative immunofluorescence staining showing the distribution of SMMHC (myosin, heavy polypeptide 11, smooth muscle) protein (green) and S100A4 (S100 calcium-binding protein A4) protein (red) in wire-injured carotid arteries from PFN1F/F (PFN1flox/flox) and PFN1−/− (PFN1SMC-IKO [SMC-specific PFN1 knockout]) mice (n=6 per group). B, Representative immunoblots and relative quantification analysis of PFN1 protein in primary mice aorta smooth muscle cells (PASMCs) stimulated with or without PDGF-BB (platelet-derived growth factor-BB; n=4). Statistical comparison: 1-way ANOVA with Tukey post hoc tests. C, Cell viability measured in PASMCs from PFN1F/F and PFN1−/− mice stimulated with PDGF-BB or not (n=4). Statistical comparison: 1-way ANOVA with Tukey post hoc tests. D, Representative immunofluorescence staining showing 5-ethynyl-20-deoxyuridine (EdU)–positive (Continued )

Article Snippet: The floxed PFN1 mice (with LoxP [locus of X-over P1] sites flanking exon 2; CKOCMP-18643-PFN1-B6J-VA) and YTHDF3 (YTH N6-methyladenosine RNA binding protein F3)-global knockout mice (with LoxP sites flanking exon 3; KOCMP-229096YTHDF3-B6N-VA) were obtained from Cyagen Biosciences.

Techniques: Migration, Immunofluorescence, Staining, Binding Assay, Knock-Out, Western Blot, Quantitative Proteomics, Derivative Assay, Comparison

Journal: Arteriosclerosis, Thrombosis, and Vascular Biology

Article Title: m ⁶ A Modification of Profilin-1 in Vascular Smooth Muscle Cells Drives Phenotype Switching and Neointimal Hyperplasia via Activation of the p-ANXA2/STAT3 Pathway

doi: 10.1161/atvbaha.124.321399

Figure Lengend Snippet: Figure 4. N6-methyladenosine (m6A) methylation regulates the translation efficiency of PFN1 (profilin-1) mRNA. A, RT-qPCR (quantitative real-time PCR) assay assessing the mRNA level of PFN1 in primary mice aorta smooth muscle cells (PASMCs) stimulated with or without PDGF-BB (platelet-derived growth factor-BB; n=5). Statistical comparison: unpaired t test. B, Representative immunoblots and quantification analysis of PFN1 protein in cycloheximide (CHX)-treated PASMCs stimulated with PDGF-BB or not (n=4). C, RT-qPCR assay assessing the mRNA level of PFN1 in actinomycin-D (Act-D)–treated PASMCs stimulated with PDGF-BB or not (n=4). D, Representative immunoblots and quantification analysis of PFN1 protein in doxorubicin (DOX)-treated PASMCs stimulated with PDGF-BB or not (n=4). E, Polysome profiling of PASMCs stimulated with PDGF-BB or not. PFN1 mRNA in different ribosome fractions was extracted and subjected to qPCR analysis. F, PASMCs were transected with si-NC or si-METTL3 (N6-methyladenosine methyltransferase), followed by stimulation with PDGF-BB or not. Representative immunoblots and quantification analysis of PFN1 protein and PFN1 mRNA (n=4). Statistical comparison: unpaired t test. G, PASMCs were transected with si-NC or si-ALKBH5, followed by stimulation with PDGF-BB or not. Representative immunoblots and quantification analysis of PFN1 protein and PFN1 mRNA (n=4). Statistical comparison: unpaired t test. All quantitative data are expressed as mean±SEM. Con indicates control group.

Article Snippet: The floxed PFN1 mice (with LoxP [locus of X-over P1] sites flanking exon 2; CKOCMP-18643-PFN1-B6J-VA) and YTHDF3 (YTH N6-methyladenosine RNA binding protein F3)-global knockout mice (with LoxP sites flanking exon 3; KOCMP-229096YTHDF3-B6N-VA) were obtained from Cyagen Biosciences.

Techniques: Methylation, Quantitative RT-PCR, Real-time Polymerase Chain Reaction, Derivative Assay, Comparison, Western Blot, Control

Journal: Arteriosclerosis, Thrombosis, and Vascular Biology

Article Title: m ⁶ A Modification of Profilin-1 in Vascular Smooth Muscle Cells Drives Phenotype Switching and Neointimal Hyperplasia via Activation of the p-ANXA2/STAT3 Pathway

doi: 10.1161/atvbaha.124.321399

Figure Lengend Snippet: Figure 5. METTL3 (N6-methyladenosine methyltransferase) facilitates the translation of N6-methyladenosine (m6A)–modified PFN1 (profilin-1) mRNA. A, RNA m6A dot-blot assays assessing m6A levels of total mRNA in primary mice aorta smooth muscle cells (PASMCs) stimulated with PDGF- BB (platelet-derived growth factor-BB) or not (n=3). B, Representative immunoblots and relative quantification analysis of METTL3 protein in PASMCs stimulated with or without PDGF-BB (n=4). Statistical comparison: 1-way ANOVA with Tukey post hoc tests. C, Representative immunohistochemistry of METTL3 in sham group, balloon-injured rat carotid arteries (left), and wire-injured murine carotid arteries (right; n=6). D, PASMCs were transfected with si-NC or si-METTL3, followed by stimulation with PDGF-BB or not. Representative immunoblots and quantification analysis of PFN1 protein (n=3). Statistical comparison: 1-way ANOVA with Tukey post hoc tests. E, RNA (Continued )

Article Snippet: The floxed PFN1 mice (with LoxP [locus of X-over P1] sites flanking exon 2; CKOCMP-18643-PFN1-B6J-VA) and YTHDF3 (YTH N6-methyladenosine RNA binding protein F3)-global knockout mice (with LoxP sites flanking exon 3; KOCMP-229096YTHDF3-B6N-VA) were obtained from Cyagen Biosciences.

Techniques: Modification, Dot Blot, Derivative Assay, Western Blot, Quantitative Proteomics, Comparison, Immunohistochemistry, Transfection

Journal: Arteriosclerosis, Thrombosis, and Vascular Biology

Article Title: m ⁶ A Modification of Profilin-1 in Vascular Smooth Muscle Cells Drives Phenotype Switching and Neointimal Hyperplasia via Activation of the p-ANXA2/STAT3 Pathway

doi: 10.1161/atvbaha.124.321399

Figure Lengend Snippet: Figure 6. YTHDF3 (YTH N6-methyladenosine RNA binding protein F3) promotes PFN1 (profilin-1) translation efficiency in an N6-methyladenosine (m6A)–dependent manner. A, RT-qPCR (quantitative real-time PCR) assay assessing mRNA expression in primary mice aorta smooth muscle cells (PASMCs) stimulated with PDGF-BB (platelet-derived growth factor-BB) or not (n=4). Statistical comparison: unpaired t test. B, Expression and distribution of YTHDF3 mRNA in every single cell of vascular smooth muscle cells (VSMCs) in single-cell RNA sequencing database. C, PASMCs were transfected with si-NC, si-YTHDF1, or si-YTHDF3. Representative immunoblots and quantification analysis of PFN1 protein (n=4). Statistical comparison: 1-way ANOVA with Tukey post hoc tests. D, Representative immunohistochemistry for YTHDF3 in uninjured and wire-injured murine carotid arteries (n=6). E, m6A-IP analysis of PASMCs stimulated with PDGF-BB or not for 24 hours using antibodies of m6A or IgG heavy chain, respectively (n=3). Statistical comparison: 1-way ANOVA with Tukey post hoc tests. F, RNA immunoprecipitation (RIP)-qPCR analysis of PASMCs stimulated with PDGF-BB or not for 24 hours using the antibody of YTHDF3 or IgG heavy chain (n=3). Statistical comparison: 1-way ANOVA with Tukey post hoc tests. G, Polysome profiling of PDGF-BB–pretreated/PDGF-BB–untreated PASMCs transfected with si-NC, si-METTL3 (m6A methyltransferase), and si-YTHDF3. PFN1 mRNA in different ribosome fractions was extracted and subjected to qPCR analysis. H, Representative images showing immunohistochemistry for PFN1 in carotid arteries from YTHDF3+/− and YTHDF3−/− mice (n=6). Scale bar, 50 µm. All quantitative data are expressed as mean±SEM. Con indicates control group; and IP, immunocoprecipitation.

Article Snippet: The floxed PFN1 mice (with LoxP [locus of X-over P1] sites flanking exon 2; CKOCMP-18643-PFN1-B6J-VA) and YTHDF3 (YTH N6-methyladenosine RNA binding protein F3)-global knockout mice (with LoxP sites flanking exon 3; KOCMP-229096YTHDF3-B6N-VA) were obtained from Cyagen Biosciences.

Techniques: RNA Binding Assay, Quantitative RT-PCR, Real-time Polymerase Chain Reaction, Expressing, Derivative Assay, Comparison, RNA Sequencing, Transfection, Western Blot, Immunohistochemistry, RNA Immunoprecipitation, Control

Journal: Arteriosclerosis, Thrombosis, and Vascular Biology

Article Title: m ⁶ A Modification of Profilin-1 in Vascular Smooth Muscle Cells Drives Phenotype Switching and Neointimal Hyperplasia via Activation of the p-ANXA2/STAT3 Pathway

doi: 10.1161/atvbaha.124.321399

Figure Lengend Snippet: Figure 7. PFN1 (profilin-1) activates ANXA2 (annexin A2)/STAT3 (signal transducer and activator of transcription 3) signaling pathway to promote vascular smooth muscle cell (VSMC) phenotype switching. A, Primary mice aorta smooth muscle cells (PASMCs) were stimulated with PDGF-BB (platelet-derived growth factor-BB) for 24 hours. The graph below shows the secondary mass spectrum of ANXA2 identified by mass spectrometry. B, Pull-down assays to pull down proteins interacting with PFN1. C, HEK293T cells were transfected with Myc-PFN1 and Flag-ANXA2 plasmids, and CO-IP (co-immunoprecipitation) analysis using Myc-PFN1 and IgG as the bait. D, HEK293T cells were transfected with Myc-PFN1 and Flag-ANXA2 plasmids, and CO-IP analysis using Flag- ANXA2 and IgG as the bait. E, Representative images showing double immunostaining for PFN1 (red) and ANXA2 (green) in PDGF-BB–treated PASMCs (n=3) and wire-injured murine carotid arteries (n=6). F, Representative immunoblots and relative quantification analysis of PFN1 and p-ANXA2 (phospho-ANXA2)/t-ANXA2 protein in PASMCs from PFN1F/F and PFN1−/− mice (n=4). Statistical comparison: (Continued )

Article Snippet: The floxed PFN1 mice (with LoxP [locus of X-over P1] sites flanking exon 2; CKOCMP-18643-PFN1-B6J-VA) and YTHDF3 (YTH N6-methyladenosine RNA binding protein F3)-global knockout mice (with LoxP sites flanking exon 3; KOCMP-229096YTHDF3-B6N-VA) were obtained from Cyagen Biosciences.

Techniques: Derivative Assay, Mass Spectrometry, Transfection, Co-Immunoprecipitation Assay, Immunoprecipitation, Double Immunostaining, Western Blot, Quantitative Proteomics, Comparison

Journal: Arteriosclerosis, Thrombosis, and Vascular Biology

Article Title: m ⁶ A Modification of Profilin-1 in Vascular Smooth Muscle Cells Drives Phenotype Switching and Neointimal Hyperplasia via Activation of the p-ANXA2/STAT3 Pathway

doi: 10.1161/atvbaha.124.321399

Figure Lengend Snippet: Figure 8. Proposed working model of PFN1 (profilin-1) in vascular smooth muscle cell (VSMC) in the context of atherosclerosis. Upon stimulation, VSMCs exhibit enhanced PFN1 translation efficiency via N6-methyladenosine (m6A) methyltransferase METTL3 (m6A methyltransferase) and the m6A-specific reader protein YTHDF3 (YTH N6-methyladenosine RNA binding protein F3) in an m6A-dependent manner. It has been mechanistically determined that the upregulation of PFN1 protein expression leads to the recruitment of Src (SRC proto-oncogene, nonreceptor tyrosine kinase), which, in turn, facilitates the phosphorylation of ANXA2 (annexin A2). This promotes the phosphorylation and nuclear translocation of STAT3 (signal transducer and activator of transcription 3), thereby inducing phenotypic switching in VSMCs. Src indicates SRC proto-oncogene, nonreceptor tyrosine kinase.

Article Snippet: The floxed PFN1 mice (with LoxP [locus of X-over P1] sites flanking exon 2; CKOCMP-18643-PFN1-B6J-VA) and YTHDF3 (YTH N6-methyladenosine RNA binding protein F3)-global knockout mice (with LoxP sites flanking exon 3; KOCMP-229096YTHDF3-B6N-VA) were obtained from Cyagen Biosciences.

Techniques: RNA Binding Assay, Expressing, Phospho-proteomics, Translocation Assay

Journal: Frontiers in bioscience (Landmark edition)

Article Title: Shared Gene Expression Dysregulation Across Subtypes of Sanfilippo and Morquio Diseases: The Role of PFN1 in Regulating Glycosaminoglycan Levels.

doi: 10.31083/j.fbl2912415

Figure Lengend Snippet: Fig. 3. Levels of PFN1, MFAP-5 and MMP12 in different subtypes of MPS III and IV cells relative to control cells. Representative

Article Snippet: For PFN1 silencing, the Profilin 1 (PFN1) Human small interferring RNA (siRNA) Oligo Duplex kit (OriGene, Newark, DE, USA; #SR303470) was used according to the manufacturer’s instructions.

Techniques: Control

Journal: Frontiers in bioscience (Landmark edition)

Article Title: Shared Gene Expression Dysregulation Across Subtypes of Sanfilippo and Morquio Diseases: The Role of PFN1 in Regulating Glycosaminoglycan Levels.

doi: 10.31083/j.fbl2912415

Figure Lengend Snippet: Fig. 4. Levels of the PFN1 protein (Profilin 1) in different sub- types of MPS III and IV cells relative to control cells. Rep-

Article Snippet: For PFN1 silencing, the Profilin 1 (PFN1) Human small interferring RNA (siRNA) Oligo Duplex kit (OriGene, Newark, DE, USA; #SR303470) was used according to the manufacturer’s instructions.

Techniques: Control

Journal: Frontiers in bioscience (Landmark edition)

Article Title: Shared Gene Expression Dysregulation Across Subtypes of Sanfilippo and Morquio Diseases: The Role of PFN1 in Regulating Glycosaminoglycan Levels.

doi: 10.31083/j.fbl2912415

Figure Lengend Snippet: Fig. 7. Levels of PFN1, MFAP-5 and MMP12 in different subtypes of MPS III and IV as well as control cells treated with DMSO

Article Snippet: For PFN1 silencing, the Profilin 1 (PFN1) Human small interferring RNA (siRNA) Oligo Duplex kit (OriGene, Newark, DE, USA; #SR303470) was used according to the manufacturer’s instructions.

Techniques: Control

Journal: Frontiers in bioscience (Landmark edition)

Article Title: Shared Gene Expression Dysregulation Across Subtypes of Sanfilippo and Morquio Diseases: The Role of PFN1 in Regulating Glycosaminoglycan Levels.

doi: 10.31083/j.fbl2912415

Figure Lengend Snippet: Fig. 8. Levels of the PFN1 protein in different subtypes of MPS III and IV cells as well as control cells treated with DMSO

Article Snippet: For PFN1 silencing, the Profilin 1 (PFN1) Human small interferring RNA (siRNA) Oligo Duplex kit (OriGene, Newark, DE, USA; #SR303470) was used according to the manufacturer’s instructions.

Techniques: Control

Journal: Frontiers in bioscience (Landmark edition)

Article Title: Shared Gene Expression Dysregulation Across Subtypes of Sanfilippo and Morquio Diseases: The Role of PFN1 in Regulating Glycosaminoglycan Levels.

doi: 10.31083/j.fbl2912415

Figure Lengend Snippet: Fig. 11. Levels of the PFN1 protein and GAG concentrations in MPS IIIC, IVB and control cells treated with PFN1 siRNA or

Article Snippet: For PFN1 silencing, the Profilin 1 (PFN1) Human small interferring RNA (siRNA) Oligo Duplex kit (OriGene, Newark, DE, USA; #SR303470) was used according to the manufacturer’s instructions.

Techniques: Control