Fig 1: Knockdown of GOLPH3 inhibits cell proliferation. (A) GOLPH3 siRNA inhibited proliferation of PDAC cells as detected using CCK-8 assays at different time points. (B) GOLPH3 siRNA inhibited colony formation in pancreatic cancer cells. (C) Cell cycle assay to determine the role of GOLPH3 in cell cycle progression after downregulating GOLPH3. (D) EdU staining assay to determine the effect of GOLPH3 downregulation on cell proliferation. *p < 0.05, **p < 0.01, and ***p < 0.001.
Fig 2: GOLPH3 enhances angiogenesis through exosomes in HUVEC model. a Representative images of exosomes obtained by transmission electron microscopy (TEM). b Size distribution of exosomes analyzed by NanoSight. c Expression levels of exosome markers: CD63 and Alix as determined by western blot analysis. d, e Tube formation assay of HUVECs treated with exosomes derived from GOLPH3-downregulated HCC cells compared with vector cells. (f and g) Capillary tube formation assay of HUVECs treated with exosomes derived from GOLPH3-overexpression HCC cells compared with control cells. h, i Migration rate of HUVECs treated with exosomes as determined by transwell migration assay. j, k Migration rate of HUVECs treated with exosomes as determined by transwell migration assay. Data are expressed as mean ± SD of 3 independent experiments and analyzed by one-way ANOVA with Dunnett’s multiple comparisons test (d, e, h, i), or t test (f, g, j, k). ***p < 0.001; Exo exosome
Fig 3: Exosomal miR-494-3p regulates sorafenib resistance in HCC cells. a, b IC50 value were decreased in miR-494-3p-silenced, and increased in miR-494-3p-overexpressing HCC cells. c SNU-449-NC, SNU-449-miR-494-3p, SNU-449-anti-NC and SNU-449-anti-miR494-3p cells were treated with sorafenib (10 µmol/L) for 48 h, then, treated cells were harvested to detect Bcl-2 and Bax levels. GAPDH was used as the loading control. d MHCC-97H cells transfected with NC, miR-494-3p mimics, anti-NC and miR-494-3p inhibitor, respectively were exposed to 20 µmol/L sorafenib for 48 h, and Bcl-2 and Bax levels determined by WB. e, f Histogram for the ratio of relative gray values of Bcl-2 and Bax proteins in c and d. g The decrease in IC50 value in SNU-449 cells transfected with the miR-494-3p inhibitor was partly reversed by exosomes derived from GOLPH3-overexpressed SNU-449 cells. h For SNU-449 cells transfected with the anti-NC or miR-494-3p inhibitor incubated with indicated exosomes and treated with sorafenib (10 µmol/L) for 48 h, Bcl-2 and Bax levels in indicated cells were determined by western blot, with GAPDH as the normalized control. i The histogram describes the Bcl-2/Bax ratios in h by gray value analysis. Data are expressed as mean ± SD from 3 independent experiments and analyzed by t test (a, b, g) or two-way ANOVA with Tukey’s multiple comparisons test (e, f, i). *p < 0.05; **p < 0.01; ***p < 0.001
Fig 4: GOLPH3 correlates positively with STIP1 in PDAC tissues. (A) Immunohistochemical staining for GOLPH3 and STIP1 in PDAC tissues (T) and paired normal pancreatic tissues (ANT). (B) Co-expression of GOLPH3 and STIP1 in PDAC tissues as assessed using immunofluorescence. (C) Expression of GOLPH3 analyzed using qRT-PCR in paired normal and PDAC tissues. (D) STIP1 levels were measured in paired PDAC tissues and adjacent normal tissues using qRT-PCR. The data were normalized to GAPDH levels as a control. (E) Correlation of GOLPH3 and STIP1 expression in PDAC tissues. (F) GOLPH3 and STIP1 protein levels were measured in paired PDAC tissues using western blotting. (G) The expression of GOLPH3 and STIP1 in different PDAC cell lines compared with that in pancreatic cyst tissue as a control.
Fig 5: Exosomes from GOLPH3-overexpressed cells enhanced sorafenib resistance in HCC cells. a IC50 value of SNU-449 treated with indicated exosomes from 3 independent experiments. b IC50 value of MHCC-97H treated with indicated exosomes from 3 independent experiments. c For SNU-449 cells incubated with indicated exosomes and thereafter treated with sorafenib (10 µmol/L) for 48 h, Bcl-2 and Bax levels were determined by western blot, with GAPDH as the normalized control. d For MHCC-97H cells incubated with indicated exosomes and exposed to 20 µmol/L sorafenib for 48 h, levels of Bcl-2 and Bax were detected via western blotting. e, f Grayscale value analysis of Bcl-2/Bax ratio by Image J. Error bars represent mean ± SD from 3 independent experiments. Data are analyzed by t test (a, b) or two-way ANOVA with Tukey’s multiple comparisons test (e, f). *p < 0.05; **p < 0.01
Supplier Page from Abcam for Anti-GOLPH3/MIDAS antibody