Fig 1: Yoda1 and TRAIL induce mitochondrial dysfunction.a Representative flow plots of JC-1 assay after Yoda1 or DMSO and TRAIL treatment. b Percent of cells with depolarized mitochondria after DMSO or Yoda1 and TRAIL treatment (n = 3). c Flow plots of MOMP due to DMSO or Yoda1 and TRAIL treatment. d Average MOMP of PC3 cells after treatment with DMSO or Yoda1 and TRAIL (n = 3). e MOMP of PC3 cells treated with DMSO or Yoda1 and TRAIL at 1, 4, 8, 12, and 24 h timepoints (n = 3). f Representative images of Bax activation of PC3 cells treated with DMSO or Yoda1 and TRAIL. The red channel is actin, green is active Bax, and blue is DAPI. Scale bars = 20 µm. g Fluorescent intensity of active Bax in PC3 cells treated with DMSO and TRAIL (n = 57) or Yoda1 and TRAIL (n = 40). h TRAIL sensitization of PC3 cells when treated with Yoda1 after scrambled siRNA, cytochrome c (CYCS) and Smac knockdown. a, c, f One representative experiment of three independent experiments. b, d, e, g, h Means and SD of three independent experiments. Statistical analysis was done using one-tailed ANOVA (b, d) and two-tailed unpaired t-test (g, h). *p < 0.05, ***p < 0.005, ****p < 0.001
Fig 2: Impaired mitochondrial respiratory chain complexes in the salivary glands from pSS. (A) A heatmap shows significant down regulation in the bulk respiratory chain complex (I–V) genes in pSS from the validation cohort, particularly those encoded in the mitochondria in pSS-high-infiltration group. (B) Heatmap of the Spearman correlation values between respiratory chain complex (I–V) genes and immune responses in pSS from the validation cohort. (C) Immunohistochemical staining for cytochrome c of paraffin-embedded labial salivary gland specimens from our own cohort. Scale bar = 100 μm. (D) Heatmap of the Spearman correlation values between respiratory chain complex (I–V) genes and 4 hub genes (CD38, CMPK2, TBC1D9, and PYCR1) in pSS from the validation cohort. (E–H) Scatter plot depicted the correlation between interested respiratory chain complex genes (MT-CYB, MT-ATP6, COX7A1, NUBPL, COX4I1, CYCS) and CD38 (E), CMPK2 (F), TBC1D9 (G), and PYCR1 (H) based on the data from Figure 6D , respectively.
Fig 3: Regulation of apoptotic factors by BRG1 depends on p53. Rheumatoid fibroblast-like synoviocyte MH7A cells were transfected with pcDNA3.1-BRG1 vector to overexpress BRG1, or the short hairpin RNA for p53 (sh-p53) to knockdown p53. Western blot was performed at 48 h post-transfection. (A) Western blot showing that apoptotic factors cytochrome c (CYCS), cleaved caspase 3 (cleaved-CASP3), cleaved-CASP9, and B-cell CLL/lymphoma 2 (BCL2) were regulated by BRG1, but the regulation was suppressed when p53 was knocked down. (B) Relative protein levels calculated based on the band density in Western blot results. P values are indicated. BCL2 = B-cell chronic lymphocytic leukemia/lymphoma 2, BRG1 = Brahma-related gene 1, cleaved-CASP3 = cleaved caspase 3, CYCS = cytochrome c.
Fig 4: Simulation of altered initial conditions of intrinsic-apoptotic pathway proteins.a Simulation of apoptosis and MOMP of cancer cells with differential expressions of XIAP. b Apoptosis and MOMP of cancer cells with variable initial expression of cytosolic Bcl-2. c The effect of Bax expression on apoptosis and MOMP. d The role of reduced expression of cytochrome c (CYCS) on apoptosis. e The effect of Smac expression on apoptosis
Fig 5: Hepatic SMPD3 retains anti-cancer properties of LR-derived exosomes by maintaining their pro-apoptotic and anti-invasive activities in vitro. (A) Schematic diagram depicting experimental design of exosome extraction. Exosomes were isolated from serum of mice at POD3 after PH (LR-Exo). Exosomes isolated from mice that received sham surgery were used as the vehicle control (sham-Exo). (B–D) Verification of isolated exosomes. (B) Ultrastructure of exosomes observed under transmission electron microscope. (C) NTA analysis of exosome size. (D) Expression of exosome surface markers HSP90, HSP70, CD63, and CD9 measured by Western blot analysis. (E) PKH67-labeled sham-Exo and LR-Exo were uptaked by MC38 cells. (F) Effects of sham-Exo and LR-Exo on migration of invasion of MC38 cells. (G) Schematic diagram depicting experimental design of exosome extraction. Exosomes were isolated from serum of shSmpd3 and shCON mice at POD3 after PH or sham surgery. (H and I) Effects of shSmpd3-Exo and shCON-Exo on migration of invasion of MC38 cells. (J) Examination on apoptosis in MC38 cells with treatment of shSmpd3-Exo and shCON-Exo. Immunoblotting of BCL-2, BAX, CYC, cCASP3, and cPARP on proteins extracted from MC38 cells. Images in B, C, and D represent results from 1 of 5 pairs of mice in each group. Images in E, F, H, I, and J represent results from 3 independent experiments. Data in F, H, and I were expressed as mean ± standard deviation, n = 5 in each group. ∗∗P < .01, ∗∗∗P < .001.
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