Figure S2.
VAMP4 deficiency causes increases in intracellular (pro)insulin and insulin release. (A) Schematic diagram of strategies for the generation of VAMP4 gene-edited INS-1 cells using the CRISPR/Cas9 technique. An sgRNA was designed after the start codon of the Vamp4 gene. A VAMP4 KD cell line in which a single allele was edited and a VAMP4 KO cell line in which double alleles were edited were then obtained. (B) Representative Western blot images showing the expression levels of VAMP4 in WT, KD, and KO INS-1 cells (n = 3 biological independent experiments). GAPDH was used as a loading control. (C) Representative confocal images of VAMP4 staining (green) in WT, KD, and KO INS-1 cells (n = 3 biological independent experiments). The nuclei of cells were stained with DAPI (blue). Scale bars, 5 μm. (D and E) Quantification of the proinsulin levels (D) and insulin levels (E) in WT, KD, and KO INS-1 cells by ELISA (n = 4 biological independent experiments). (F) Analysis of the ratio of proinsulin to total insulin (proinsulin and insulin) in WT, KD, and KO INS-1 cells based on data shown in D and E (n = 4 biological independent experiments). (G) Representative Western blot images showing the expression levels of VAMP4, proinsulin, and insulin in WT, KD, KO, and rescued INS-1 cells. Actin was used as a loading control (n = 3 biological independent experiments). (H) RT–PCR analysis of the Ins mRNA levels in WT, KD, KO, and rescued INS-1 cells. 18S ribosomal RNA was used as an internal control (n = 3 biological independent experiments). (I) Measurement of insulin secretion from WT, KD, KO, and rescued INS-1 cells incubated for 1 h in the presence of 2.8 or 16.7 mM glucose (n = 6 biological independent samples). (J and K) Representative Western blot images (upper panel) showing the expression levels of STX7 (J) and STX8 (K) in INS-1 cells 72 h after transfection with siRNAs (siCtrl, siSTX7, and siSTX8). The bar graphs (lower panel) show the knockdown efficiency of STX7 and STX8 quantified from three independent Western blot results. Actin was used as the loading control. (L) Measurement of insulin secretion from INS-1 cells at 72 h after transfection with siCtrl, siSTX7, or siSTX8. The cells were incubated for 1 h in 2.8 or 16.7 mM glucose. The data are shown as the mean ± SEM (n = 3 biological independent experiments). The data are presented as the mean ± SEM. *, P < 0.05 and **, P < 0.01. The statistical analyses were performed with two-tailed unpaired Student’s t test (J and K) and one-way ANOVA (D–F, H, I and L). Source data are available for this figure: SourceData FS2.

VAMP4 deficiency causes increases in intracellular (pro)insulin and insulin release. (A) Schematic diagram of strategies for the generation of VAMP4 gene-edited INS-1 cells using the CRISPR/Cas9 technique. An sgRNA was designed after the start codon of the Vamp4 gene. A VAMP4 KD cell line in which a single allele was edited and a VAMP4 KO cell line in which double alleles were edited were then obtained. (B) Representative Western blot images showing the expression levels of VAMP4 in WT, KD, and KO INS-1 cells (n = 3 biological independent experiments). GAPDH was used as a loading control. (C) Representative confocal images of VAMP4 staining (green) in WT, KD, and KO INS-1 cells (n = 3 biological independent experiments). The nuclei of cells were stained with DAPI (blue). Scale bars, 5 μm. (D and E) Quantification of the proinsulin levels (D) and insulin levels (E) in WT, KD, and KO INS-1 cells by ELISA (n = 4 biological independent experiments). (F) Analysis of the ratio of proinsulin to total insulin (proinsulin and insulin) in WT, KD, and KO INS-1 cells based on data shown in D and E (n = 4 biological independent experiments). (G) Representative Western blot images showing the expression levels of VAMP4, proinsulin, and insulin in WT, KD, KO, and rescued INS-1 cells. Actin was used as a loading control (n = 3 biological independent experiments). (H) RT–PCR analysis of the Ins mRNA levels in WT, KD, KO, and rescued INS-1 cells. 18S ribosomal RNA was used as an internal control (n = 3 biological independent experiments). (I) Measurement of insulin secretion from WT, KD, KO, and rescued INS-1 cells incubated for 1 h in the presence of 2.8 or 16.7 mM glucose (n = 6 biological independent samples). (J and K) Representative Western blot images (upper panel) showing the expression levels of STX7 (J) and STX8 (K) in INS-1 cells 72 h after transfection with siRNAs (siCtrl, siSTX7, and siSTX8). The bar graphs (lower panel) show the knockdown efficiency of STX7 and STX8 quantified from three independent Western blot results. Actin was used as the loading control. (L) Measurement of insulin secretion from INS-1 cells at 72 h after transfection with siCtrl, siSTX7, or siSTX8. The cells were incubated for 1 h in 2.8 or 16.7 mM glucose. The data are shown as the mean ± SEM (n = 3 biological independent experiments). The data are presented as the mean ± SEM. *, P < 0.05 and **, P < 0.01. The statistical analyses were performed with two-tailed unpaired Student’s t test (J and K) and one-way ANOVA (D–F, H, I and L). Source data are available for this figure: SourceData FS2.

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