Journal: Cell Proliferation

Article Title: CircSMAD3 represses VSMC phenotype switching and neointima formation via promoting hnRNPA1 ubiquitination degradation

doi: 10.1111/cpr.13742

Figure Lengend Snippet: circSMAD3 interacts with hnRNPA1 and promotes its degradation through the protease‐ubiquitination pathway. (A) The 11 proteins associated with VSMC phenotype and interacting with circSMAD3. (B) RNA pull‐down to detect circSMAD3 interacted with hnRNPA1 and YBX1. (C) RIP assay showing the enrichment of circSMAD3 by hnRNPA1 and YBX1. Data are shown as mean ± SD (one‐way ANOVA). (D) Protein‐RNA program‐catRapid predicted the N‐terminal of hnRNPA1 interacting with circSMAD3. (E) HDOCK program for predicting the binding of hnRNPA1 to circSMAD3. (F) Truncated and RNA pull‐down assays demonstrating circSMAD3 interaction with the RGG‐M9 region. (G–J) The changes in hnRNPA1 in RNA (G) and protein levels (H–J) when circSMAD3 is overexpressed or silenced. Data are shown as mean ± SD (Student's t ‐test). (K–N) Cell fraction assay showing the detection of the changes in hnRNPA1 in response to circSMAD3 overexpression (K, L) and silencing (M, N) using Western blot. Data are shown as mean ± SD (Student's t ‐test). (O, P) Immunofluorescence assay showing the detection of the changes in hnRNPA1 when circSMAD3 is overexpressed (O) or silenced (P). Scale bar = 50 μm. (Q, R) Western blot showing the changes in hnRNPA1 protein in response to circSMAD3 overexpression and MG132 (20 ng/mL) or CHX (20 ng/mL). Data are shown as mean ± SD (two‐way ANOVA) in (Q) and mean ± SD (Student's t ‐test) in (R). (S) Ubiquitin assay to demonstrate that circSMAD3 promoted the level of ubiquitin on hnRNPA1. ns, no significance, *** p <0.001, **p<0.01, * p <0.05

Article Snippet: The primary antibody for western blot in this study included α‐SMA (A17910, Ablonal, China), TAGLN (ab10135, Abcam, USA), CNN1 (A3734, Abclonal, China), CDK1 (19532‐1‐AP, Proteintech, China), CDK2 (10122‐1‐AP, Proteintech, China), CCNE2 (11935‐1‐AP, Proteintech, China), CCND1 (60186‐1‐Ig, Abclonal, China), p21 (10355‐1‐AP, Proteintech, China), Puma (55120‐1‐AP, Proteintech, China), NOXA (A9801, Abclonal, China), hnRNPA1 (11176‐1‐AP, Proteintech, China), WDR76 (25528‐1‐AP, Proteintech, China), YBX1 (A3534, Abclonal, China), PHB2 (66424‐1‐Ig, Proteintech, China), PCBP1 (14523‐1‐AP, Proteintech, China), SMAD3 (ab40854, Abcam, USA), SMAD4 (ab40759, Abcam, USA), p53 ((DO‐1): sc‐126, Santa, USA), p53 (A10610, Abclonal, China), and p‐p53 (28961‐1‐AP, Abclonal, China).

Techniques: Ubiquitin Proteomics, Binding Assay, Over Expression, Western Blot, Immunofluorescence

Journal: Cell Proliferation

Article Title: CircSMAD3 represses VSMC phenotype switching and neointima formation via promoting hnRNPA1 ubiquitination degradation

doi: 10.1111/cpr.13742

Figure Lengend Snippet: circSMAD3 promoted the ubiquitination degradation of hnRNPA1 by E3 ligase WDR76. (A) RIP (upper) and RNA pull‐down (lower) assays showing the detection of the binding of hnRNPA1 to circSMAD3. Data are shown as mean ± SD (Student's t ‐test). (B, C) CatRapid and HDOCK predicted that the N‐terminus of hnRNPA1 interacts with circSMAD3. (D) Immunofluorescence assay showing the detection of the co‐localization of hnRNPA1 with WDR76 in HASMCs. Scale bar = 50 μm. (E, F) IP assay to detect the interaction between hnRNPA1 and WDR76 upon circSMAD3 silencing (E) and overexpression (F). (G) IP assay showing that circSMAD3 overexpression promoted the ubiquitination level of hnRNPA1 in HEK293T cells. (H) Western blot showing the ubiquitination levels of hnRNPA1 after Flag‐WDR76 overexpression and in response to MG132 treatment in HEK293T cells co‐transfected with hemaglutinin antigen (HA)‐tagged hnRNPA1 and Myc‐tagged ubiquitin (K63O, K48O, K33O, K29O, K27O, K11O, and K6O) constructs. K63O indicates ubiquitin, in which only lysines‐K63 were obtained. (I) Western blot showing the ubiquitination levels of hnRNPA1 after circSMAD3 overexpression and in response to MG132 treatment in HEK293T cells co‐transfected with HA‐tagged hnRNPA1, Flag‐WDR76, and Myc‐tagged ubiquitin (K63O, K48O, and K33O) constructs. *** p <0.001.

Article Snippet: The primary antibody for western blot in this study included α‐SMA (A17910, Ablonal, China), TAGLN (ab10135, Abcam, USA), CNN1 (A3734, Abclonal, China), CDK1 (19532‐1‐AP, Proteintech, China), CDK2 (10122‐1‐AP, Proteintech, China), CCNE2 (11935‐1‐AP, Proteintech, China), CCND1 (60186‐1‐Ig, Abclonal, China), p21 (10355‐1‐AP, Proteintech, China), Puma (55120‐1‐AP, Proteintech, China), NOXA (A9801, Abclonal, China), hnRNPA1 (11176‐1‐AP, Proteintech, China), WDR76 (25528‐1‐AP, Proteintech, China), YBX1 (A3534, Abclonal, China), PHB2 (66424‐1‐Ig, Proteintech, China), PCBP1 (14523‐1‐AP, Proteintech, China), SMAD3 (ab40854, Abcam, USA), SMAD4 (ab40759, Abcam, USA), p53 ((DO‐1): sc‐126, Santa, USA), p53 (A10610, Abclonal, China), and p‐p53 (28961‐1‐AP, Abclonal, China).

Techniques: Ubiquitin Proteomics, Binding Assay, Immunofluorescence, Over Expression, Western Blot, Transfection, Construct

Journal: Cell Proliferation

Article Title: CircSMAD3 represses VSMC phenotype switching and neointima formation via promoting hnRNPA1 ubiquitination degradation

doi: 10.1111/cpr.13742

Figure Lengend Snippet: circSMAD3 modulated p53 precursor RNA splicing by hnRNPA1. (A) Protein‐RNA program‐catRapid predicted the binding region of pre‐p53 with hnRNPA1. (B) RIP assay showing the enrichment of pre‐p53 by hnRNPA1 and the enhancement of this interaction by circSMAD3 silencing and overexpression. Data are shown as mean ± SD (two‐way ANOVA). (C) hnRNPA1 silencing significantly promoted the protein level expression of p53γ and its target genes. Data are shown as mean ± SD (Student's t ‐test). (D) hnRNPA1 overexpression significantly declined the protein level expression of p53γ and its target genes. Data are shown as mean ± SD (Student's t ‐test). (E) The silencing of hnRNPA1 reversed the downregulation of p53γ and its target genes induced by circSMAD3 silencing. Data are shown as mean ± SD (one‐way ANOVA). (F) The overexpression of hnRNPA1 reversed the upregulation of p53γ and its target genes induced by circSMAD3 overexpression. Data are shown as mean ± SD (one‐way ANOVA). *** p <0.001, ** p <0.01.

Article Snippet: The primary antibody for western blot in this study included α‐SMA (A17910, Ablonal, China), TAGLN (ab10135, Abcam, USA), CNN1 (A3734, Abclonal, China), CDK1 (19532‐1‐AP, Proteintech, China), CDK2 (10122‐1‐AP, Proteintech, China), CCNE2 (11935‐1‐AP, Proteintech, China), CCND1 (60186‐1‐Ig, Abclonal, China), p21 (10355‐1‐AP, Proteintech, China), Puma (55120‐1‐AP, Proteintech, China), NOXA (A9801, Abclonal, China), hnRNPA1 (11176‐1‐AP, Proteintech, China), WDR76 (25528‐1‐AP, Proteintech, China), YBX1 (A3534, Abclonal, China), PHB2 (66424‐1‐Ig, Proteintech, China), PCBP1 (14523‐1‐AP, Proteintech, China), SMAD3 (ab40854, Abcam, USA), SMAD4 (ab40759, Abcam, USA), p53 ((DO‐1): sc‐126, Santa, USA), p53 (A10610, Abclonal, China), and p‐p53 (28961‐1‐AP, Abclonal, China).

Techniques: Binding Assay, Over Expression, Expressing