Fig 1: Roles of splicing repressors in the splicing switching of Itgb1 isoforms. (A) Western blotting examination of hnRNP A1 and PTB proteins during myodifferentiation of C2C12 myoblasts. DM, differentiation medium. Bottom: pooled data. *P < .05 compared with day 0. (B) Schematic of hnRNP A1 and PTB-binding sites in the flanking introns of alternative exon D in the splicing reporter of Itgb1. (C) Effects of hnRNP A1 on the protein expression of ITGB1D in myocytes. (D) Effects of LRP6 on the protein expression of hnRNP A1 and PTBP1. The proteins were extracted from neonatal cardiomyocytes subject to hnRNP A1- or Lrp6-siRNAs for 48 h. Right: pooled data. *P < .05 compared with si-Ctrl. (E-F) Effects of the splice repressor PTBP1 on the splicing of Itgb1D in the presence and absence of LRP6 in culture neonatal cardiomyocytes. Western blotting examination of LRP6, PTBP1 and ITGB1D proteins (E). E-right: pooled data. *P < .05 compared with si-Ctrl. (F-top) Gel electrophoresis analysis of Itgb1 RNAs; (F-bottom) gel electrophoresis analysis of the specific activity of PTBP1 on Itgb1 pre-mRNA using the minigene. (G) Model of the LRP6-mediated splicing of Itgb1D in striated muscle cells. Representative blots from five independent experiments with similar results are shown
Fig 2: Correlations between tumor immunity and the expression of PTBP1 in pan-cancer. (a) Correlations of the expression of PTBP1 with immune checkpoint genes. (b) Correlations of the expression of PTBP1 with neoantigens count. (c) Correlation between the expression of PTBP1 and ESTIMATEScore. (d) Correlations of the expression of PTBP1 with immune cell infiltration. *P < 0.05, **P < 0.01, and ***P < 0.001.
Fig 3: PTBP1 facilitates chemoresistance of osteosarcoma cells to DXR in vitro. DXR-resistant osteosarcoma cell lines (U2OS/DXR, MG63/DXR, HOS/DXR) were treated with expression vector containing PTBP1 gene and their parental cells with siRNA targeting PTBP1. (A) The knockdown efficiency of si-PTBP1 was verified using RT-qPCR in U2OS/DXR cells. (B) Western blot analysis determined the protein level of PTBP1 (normalized to GAPDH) in DXR-resistant osteosarcoma cells and their parental cells. CCK-8 assays were performed to examine cell viability and IC50 of DXR in U2OS/DXR cells (C) and their parental cells (D). (E) EdU labeling assays were used to reflect the proliferation of DXR-resistant U2OS and MG63 cells and their parental cells. Values obtained from three independent experiments in triplicate are analyzed by unpaired t test between two groups and by ANOVA with Tukey's test among three or more groups. Values at different time points were tested by repeated measurement ANOVA followed by Bonferroni test. *p < 0.05 compared with parental cells treated with oe-NC or DXR-resistant osteosarcoma cells treated with si-NC.
Fig 4: The mechanism graph of transcription factor ELK1 in chemoresistance in osteosarcoma. ELK1 promotes aerobic glycolysis by inhibiting miR-134 and upregulating PTBP1 expression, thus enhancing chemotherapeutic resistance in osteosarcoma.
Fig 5: Overexpressing PTBP1 abolished the in vivo preventive benefits of silencing circEXOC5 on acute lung injury.Mice were injected with Lv-shcircEXOC5 or Lv-shcircEXOC5 + OE-PTBP1 before the CLP procedure. (A) Histology of lung tissues from indicated groups was examined by HE staining and scored for lung injury. Scale bar: 200 µm. (B) Numbers of total cells, neutrophils, and macrophages in BALF were quantified. (C) Production of TNF-a, IL-6, and IL-1ß in BALF was measured by ELISA. (D) Autophagosomes in lung tissues were observed under a transmission electron microscope, with the number counted from indicated groups. Scale bar: 2 or 1 µm. (E) Runx2 expression was detected by immunohistochemical analysis in indicated lung tissues. Scale bar: 200 µm. *P < 0.05, **P < 0.01, and ***P < 0.001.
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