Journal: Translational Andrology and Urology

Article Title: Temporal relationship of the orphan receptor TR3 translocation and expression with zinc-induced apoptosis in prostate cancer cells

doi: 10.21037/tau-23-61

Figure Lengend Snippet: Quantitative real time-polymerase chain reaction was used to detect the TR3 mRNA expression levels in prostate cancer cells treated with zinc. The results show that there was no significant change in the TR3 mRNA expression levels in the PC-3 or LNCaP cells after different zinc treatment times (0, 4, 8, 12, and 24 h; P >0.05). mRNA, messenger RNA.

Article Snippet: To identify TR3 protein expression, cells were first incubated with an anti-TR3 immunoglobin G (IgG) antibody (sc365113; Santa Cruz Biotechnology Inc., Dallas, TX, USA) and then with a corresponding FITC-conjugated anti-IgG antibody (AS10115; Agrisera, Vasterbotten, Sweden) as the secondary antibody.

Techniques: Real-time Polymerase Chain Reaction, Expressing

Journal: Translational Andrology and Urology

Article Title: Temporal relationship of the orphan receptor TR3 translocation and expression with zinc-induced apoptosis in prostate cancer cells

doi: 10.21037/tau-23-61

Figure Lengend Snippet: Immunofluorescence costaining results were observed under a confocal microscope. (A) The TR3-FITC protein was localized in the nuclei of PC-3 and LNCaP cells at 0 h and then started to diffusely distribute into the mitochondria at 8 h of zinc treatment. (B) Cytochrome c was present in the mitochondria of PC-3 and LNCaP cells at 0 h of zinc treatment and then dispersed into the cytosol at 8 h. Cyto c, cytochrome c; FITC, fluorescein isothiocyanate; HSP60, heat shock protein 60.

Article Snippet: To identify TR3 protein expression, cells were first incubated with an anti-TR3 immunoglobin G (IgG) antibody (sc365113; Santa Cruz Biotechnology Inc., Dallas, TX, USA) and then with a corresponding FITC-conjugated anti-IgG antibody (AS10115; Agrisera, Vasterbotten, Sweden) as the secondary antibody.

Techniques: Immunofluorescence, Microscopy

Journal: Aging (Albany NY)

Article Title: Cx32 inhibits the autophagic effect of Nur77 in SH-SY5Y cells and rat brain with ischemic stroke

doi: 10.18632/aging.203526

Figure Lengend Snippet: OGD/R induced up-regulation of Cx32 expression and activation of autophagy. ( A ) Double labeling of Cx32 and β-tubulin in the Ctrl or OGD/R (2h OGD following 24 h recovery) group. Scale bars, 50 μm. ( B ) The mRNA level of Cx32 was detected by qPCR. ( C – D ) Representative bands of Cx32 protein in the cells after OGD/R. ( E ) The autophagosome was labeled with LC3 (green), autophagolysosome was marked by LysoTracker probes (red), and cell nuclei were counterstained by DAPI (blue). Scale bars, 20 μm. ( F – G ) Representative bands of Beclin1 and LC3 protein in SH-SY5Y cells after OGD/R. Variation in protein loading was determined by blotting with an anti-β-actin antibody. Densitometric scanning of band intensities were calculated as means ± SD ( n = 3). * p < 0.05 vs. Ctrl group, ** p < 0.01 vs. Ctrl group.

Article Snippet: Primary Abs used were Cx32 (Abcam, ab66613), Nur77, LC3 (Santa Cruz Biotechnology, sc365113, sc271625), Rac1 (Movus, NBP2-67091), β-tubulin (CST4466).

Techniques: Expressing, Activation Assay, Labeling

Journal: Aging (Albany NY)

Article Title: Cx32 inhibits the autophagic effect of Nur77 in SH-SY5Y cells and rat brain with ischemic stroke

doi: 10.18632/aging.203526

Figure Lengend Snippet: Inhibition of Cx32 activated autophagy and promoted Nur77 translocating from nucleus to mitochondria after OGD/R injury. ( A ) After transfection with control siRNA or Cx32 siRNA for 24 h, the cells were collected for Western blots of Cx32. Cells were transfected with Cx32 siRNA for 24 h followed by OGD/R (2 h OGD following by 24 h recovery) before harvested. ( B ) A MTT assay was used for cell viability. ( C ) Images acquired by transmission electron microscope. The arrow marks autophagic vacuoles. Scale bars, 2 μm. ( D – E ) Images of Monodansylcadaverine (MDC) staining and acridine orange (AO) staining under fluorescence microscope. Scale bars, 100 μm. ( F ) Representative bands of Beclin1, LC3 and p62 protein in siCx32 cells after OGD/R. ( G ) Representative bands of Nur77 protein in the cells after OGD/R. Variation in protein loading was determined by blotting with an anti-β-actin antibody ( n = 3). ( H ) Mitochondrial translocation of Nur77 was examined after OGD/R. Mitochondria Nur77 expression was normalized against COX IV. Nucleus Nur77 expression was normalized against α-tubulin expression. ( I ) A MTT assay was applied for the cell viability. Data are expressed as means ± SD ( n = 6). ** p < 0.01, * p < 0.05.

Article Snippet: Primary Abs used were Cx32 (Abcam, ab66613), Nur77, LC3 (Santa Cruz Biotechnology, sc365113, sc271625), Rac1 (Movus, NBP2-67091), β-tubulin (CST4466).

Techniques: Inhibition, Transfection, Control, Western Blot, MTT Assay, Transmission Assay, Microscopy, Staining, Fluorescence, Translocation Assay, Expressing

Journal: Aging (Albany NY)

Article Title: Cx32 inhibits the autophagic effect of Nur77 in SH-SY5Y cells and rat brain with ischemic stroke

doi: 10.18632/aging.203526

Figure Lengend Snippet: Nur77 activated mitophagy after the inhibition of Cx32 following OGD/R injury. Cells were transfected with Cx32 siRNA or Nur77 siRNA for 24 h followed by OGD/R (2 h OGD following by 24 h recovery) before harvested. ( A ) The autophagosome was marked with LC3 (red); mitochondria was marked by TOMM20 (green). Scale bars, 25 μm. ( B – C ) Representative bands of BNIP3L/NIX, BNIP3, PINK, Parkin, and GABARAP protein in the cells after OGD/R. ( D – E ) Representative bands of Rac1 protein after OGD/R. Variation in protein loading was determined by blotting with an anti-β-actin antibody. Densitometric scanning of band intensities were calculated as means ± SD ( n = 3). ** p < 0.01 vs. Ctrl group, ## p < 0.01 vs. OGD/R. ( F ) Double labeling of Nur77 and Rac1 using fixed cells of Control, OGD/R or siCx32 group. Scale bars, 50 μm.

Article Snippet: Primary Abs used were Cx32 (Abcam, ab66613), Nur77, LC3 (Santa Cruz Biotechnology, sc365113, sc271625), Rac1 (Movus, NBP2-67091), β-tubulin (CST4466).

Techniques: Inhibition, Transfection, Labeling, Control