Figure S6.
Analysis of the post-transcriptional regulation of fibronectin by Atg7 and the identification of CREB-dependent fibronectin expression. (A) The Atg7 KO HBMECs and control cells were treated with cycloheximide (CHX, 20 μg/ml) for indicated of times. The cell lysates were subjected to Western blot analysis to detect the expression of fibronectin (FN) and Atg7 (left). β-Actin was used as an internal loading control. The band densities were quantified by ImageJ software and normalized to the vehicle control. The rate of decay of fibronectin expression was plotted (right). Data were shown as the mean ± SD (n = 3). (B) The HBMECs were treated with MG132 (10 μM), with the cells treated with vehicle as control. The cell lysates were subjected to Western blot analysis to detect the expression of fibronectin and Atg7 (top). β-Actin was used as an internal loading control. The band densities were quantified by ImageJ software and normalized to the Cas9 group (bottom). Data were shown as the mean ± SD (n = 3). ***, P < 0.001. The ns represents no statistical significance. Statistics were calculated by one-way ANOVA coupled with Dunnett’s post hoc test. (C) The HBMEC lysates were subjected to the ChIP assay using the EST1, GATA2, and HOXB13 antibody, respectively. The immunoprecipitated DNA fragments were amplified by qPCR using the primers flanking the promoter regions of fibronectin gene. Data were shown as the mean ± SD (n = 3). The ns represents no statistical significance. Unpaired two-tailed Student’s t test for comparison of two groups. (D) The HBMECs were seeded on coverslips and immunofluorescence was performed with antibody against CREB (red). DAPI (blue) was used for counterstaining. The representative images were presented (left). The fluorescence intensity of CREB was quantified (right). A total of 40 cells were analyzed per group. Data were shown as the mean ± SD. The ns represents no statistical significance. Unpaired two-tailed Student’s t test for comparison of two groups. (E and F) HBMECs were transfected with siRNAs against CREB or non-silencing control siRNA (NC). 48 h after transfection, (E) cell lysates were subjected to Western blot analysis to detect the expression of fibronectin, CREB, and p-CREB (ser133), respectively (left). β-Actin was used as an internal loading control. The band densities were quantified by ImageJ software and normalized to the NC group (right). Data were shown as the mean ± SD. **P < 0.01. Statistics were calculated by one-way ANOVA coupled with Dunnett’s post hoc test. (F) Immunofluorescence was performed to analyze the cellular expression of fibronectin (green). DAPI (blue) was used for counterstaining (left). The fluorescence intensity of fibronectin was quantified (right). Data were shown as the mean ± SD. ***, P < 0.001. Statistics were calculated by one-way ANOVA coupled with Dunnett’s post hoc test. Source data are available for this figure: SourceData FS6.

Analysis of the post-transcriptional regulation of fibronectin by Atg7 and the identification of CREB-dependent fibronectin expression. (A) The Atg7 KO HBMECs and control cells were treated with cycloheximide (CHX, 20 μg/ml) for indicated of times. The cell lysates were subjected to Western blot analysis to detect the expression of fibronectin (FN) and Atg7 (left). β-Actin was used as an internal loading control. The band densities were quantified by ImageJ software and normalized to the vehicle control. The rate of decay of fibronectin expression was plotted (right). Data were shown as the mean ± SD (n = 3). (B) The HBMECs were treated with MG132 (10 μM), with the cells treated with vehicle as control. The cell lysates were subjected to Western blot analysis to detect the expression of fibronectin and Atg7 (top). β-Actin was used as an internal loading control. The band densities were quantified by ImageJ software and normalized to the Cas9 group (bottom). Data were shown as the mean ± SD (n = 3). ***, P < 0.001. The ns represents no statistical significance. Statistics were calculated by one-way ANOVA coupled with Dunnett’s post hoc test. (C) The HBMEC lysates were subjected to the ChIP assay using the EST1, GATA2, and HOXB13 antibody, respectively. The immunoprecipitated DNA fragments were amplified by qPCR using the primers flanking the promoter regions of fibronectin gene. Data were shown as the mean ± SD (n = 3). The ns represents no statistical significance. Unpaired two-tailed Student’s t test for comparison of two groups. (D) The HBMECs were seeded on coverslips and immunofluorescence was performed with antibody against CREB (red). DAPI (blue) was used for counterstaining. The representative images were presented (left). The fluorescence intensity of CREB was quantified (right). A total of 40 cells were analyzed per group. Data were shown as the mean ± SD. The ns represents no statistical significance. Unpaired two-tailed Student’s t test for comparison of two groups. (E and F) HBMECs were transfected with siRNAs against CREB or non-silencing control siRNA (NC). 48 h after transfection, (E) cell lysates were subjected to Western blot analysis to detect the expression of fibronectin, CREB, and p-CREB (ser133), respectively (left). β-Actin was used as an internal loading control. The band densities were quantified by ImageJ software and normalized to the NC group (right). Data were shown as the mean ± SD. **P < 0.01. Statistics were calculated by one-way ANOVA coupled with Dunnett’s post hoc test. (F) Immunofluorescence was performed to analyze the cellular expression of fibronectin (green). DAPI (blue) was used for counterstaining (left). The fluorescence intensity of fibronectin was quantified (right). Data were shown as the mean ± SD. ***, P < 0.001. Statistics were calculated by one-way ANOVA coupled with Dunnett’s post hoc test. Source data are available for this figure: SourceData FS6.

This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal