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. 2019 Aug 29;20(10):e45986. doi: 10.15252/embr.201845986

Figure EV4. Treatment of H2S induces PC3 cell invasion through NF‐κB‐mediated signaling pathways.

Figure EV4

  • A
    22Rv1 and C4‐2 cells were incubated for invasion assay for 16–18 h. DMEM/10% FBS, together with 1 μM NaHS served as a chemoattractant.
  • B
    PC3 cells were incubated for invasion assay for 24 h. DMEM/10% FBS, together with 1 or 10 μM GYY4137, served as a chemoattractant.
  • C
    Top: PC3 cells treated with 1 nM–1 mM NaHS for 1 h were subjected to the modified biotin switch assay with the antibody against p65 to detect S‐sulfhydration. Bottom: Quantitative analysis of SSH‐p65 protein level, and normalized with total p65 level.
  • D
    PC3 cells were treated with 1 μM NaHS for 24 h in the absence of serum. Subcellular localization of p65 was detected by immunocytochemistry. Nuclei were counterstained with DAPI. Scale bar: 25 μm.
  • E
    Nuclear translocation of p65 was scored by counting the number of nuclear positive stained cells of p65 to the total number of cells in random microscopic fields.
  • F
    PC3 cells were incubated for invasion assay for 24 h. DMEM/10% FBS, together with 1 μM GYY4137 and NF‐κB inhibitors (50 μg/ml SN50, or 100 nM QNZ), served as a chemoattractant.
  • G
    Real‐time RT–PCR analysis for IL‐1β, MMP‐13, VEGF, and RPS3 mRNA level in PC3 cells with control or RPS3 knockdown.
  • H
    Left: PC3 cells with p65 knockout with p65 C38S mutant and PC3 parental cells were treated with 100 μM NaHS for 1 h and subjected to the modified biotin switch assay with the antibody against p65 to detect S‐sulfhydration. Right: Quantitative analysis of SSH‐p65 protein level, and normalized with total p65 level.
  • I
    PC3 cells with p65 knockout were transfected with vector, p65 wild‐type, or p65 C38S mutant and then exposed to IL‐1β (20 ng/ml) for 1 h. Subcellular localization of p65 was detected by immunocytochemistry. Nuclei were counterstained with DAPI. The representative images are shown. Scale bars: 10 μm.
  • J
    Nuclear translocation of p65 was scored by counting the nuclear positive stained cells and the total number of cells quantified in random microscopic fields.
Data information: (A–C, E–H, J) Data shown represent the means ± SD (n = 3 biological replicates). Student's t‐test (A, E, G) or ANOVA followed by Tukey's post hoc test (B, F, H, I) was used for statistical analysis (*P < 0.05; **P < 0.01; ***P < 0.001).Source data are available online for this figure.