Journal: Autophagy

Article Title: Copper-dependent autophagic degradation of GPX4 drives ferroptosis

doi: 10.1080/15548627.2023.2165323

Figure Lengend Snippet: Reagent sources.

Article Snippet: Human SQSTM1/p62 siRNA#2: 5′-TGAGGAAGATCGCCTTGGA-3′ , RiboBio , This paper.

Techniques: Ubiquitin Proteomics, Recombinant, Protease Inhibitor, Western Blot, Lysis, CCK-8 Assay, BIA-KA, Staining, Iron Assay, Enzyme-linked Immunosorbent Assay, SYBR Green Assay, Software

Journal: Science Advances

Article Title: The transmembrane endoplasmic reticulum–associated E3 ubiquitin ligase TRIM13 restrains the pathogenic-DNA–triggered inflammatory response

doi: 10.1126/sciadv.abh0496

Figure Lengend Snippet: ( A ) RAW264.7 cells were transfected with indicated vectors for 48 hours. After HSV-1 infection, STING levels in WCE were examined by Western blotting (A). ( B and C ) BMDMs (B) or MEFs (C) were transfected with or without mock or TRIM13-CA vectors for 48 hours. STING levels were examined by Western blotting. ( D and E ) BMDMs were examined for STING degradation in the presence of CHX (20 μM) (D) or after pretreatment with CHX for 1 hour (E) by Western blotting. ( F and G ) Trim13 +/+ or Trim13 −/− BMDMs (F) or RAW264.7 cells transfected with mock or TRIM13-Flag vectors for 48 hours (G) were infected with HSV-1 in the presence or absence of 20 μM ESI, and STING in WCE was examined by Western blotting. ( H to J ) RAW264.7 cells transfected with mock or TRIM13-Flag vectors for 48 hours (H and J) or Trim13 +/+ or Trim13 −/− BMDMs (H and I) were transfected with control (Ctrl) siRNAs or p62-specific siRNAs for 48 hours. The efficiency of p62 knockdown was evaluated by Western blotting (H). STING in WCE was examined by Western blotting (I and J). One representative experiment of three is shown.

Article Snippet: The siRNA duplexes specific for Sting (sc-154411) and p62 (sc-35233) were obtained from Santa Cruz Biotechnology (Dallas, TX). siRNA sequences specific for Rnf5 , Trim29 , and Trim30a are listed in table S2.

Techniques: Transfection, Infection, Western Blot, Control, Knockdown

Journal: Journal of cellular biochemistry

Article Title: IL-1β induces p62/SQSTM1 and represses androgen receptor expression in prostate cancer cells.

doi: 10.1002/jcb.24897

Figure Lengend Snippet: Fig. 2. IL-1b represses AR and induces p62 in C4-2 PCa cells. C4-2 cells were grown in vehicle control or 25 ng/ml recombinant human IL-1b protein for 2 days and analyzed for AR or p62 accumulation. A: QPCR demonstrated that IL-1b was sufficient to repress AR mRNA accumulation (P-value ¼ 0.01) and upregulate p62 mRNA levels (P-value ¼ 0.08) in C4-2 cells. Relative mRNA levels were normalized to control growth medium and error bars represent standard deviation of three biological replicates. B: Co-immunostaining for AR and p62 protein revealed that IL-1b repressed nuclear accumulation and induced p62 subcellular protein accumulation in C4-2 cells. Scale bar ¼ 50 mm. C: C4-2 cells were grown for 3 days in vehicle control or 25 ng/ml recombinant human IL-1b, in the absence or presence of 40 mM chloroquine (CQ), and analyzed for p62 protein by Western blot. CQ blocks autophagy-mediated degradation of p62 in the vehicle control and in the presence of IL-1b, indicating that IL-1b induction of p62 is not due to abrogated p62 degradation.

Article Snippet: Cells were transfected with a pool of four unique AR siRNA duplexes (M-003400-02-0005; Thermo Scientific) or a pool of three unique p62/SQSTM1 siRNA duplexes (SR305865; Origene) using siTran 1.0 transfection reagent (TT300001; Origene).

Techniques: Control, Recombinant, Standard Deviation, Immunostaining, Western Blot

Journal: Journal of cellular biochemistry

Article Title: IL-1β induces p62/SQSTM1 and represses androgen receptor expression in prostate cancer cells.

doi: 10.1002/jcb.24897

Figure Lengend Snippet: Fig. 3. HS-5 BMSC conditioned medium and IL-1b differently regulate p62 subcellular accumulation. C4-2 cells were grown in HS-5 BMSC conditioned medium or 25 ng/ml recombinant human IL-1b protein for 2 days and co-immunostained for AR and p62. HS-5 BMSC conditioned medium repressed AR nuclear accumulation and induced p62 speckle accumulation (inset). IL-1b repressed AR nuclear accumulation and induced diffuse p62 accumulation (representative cells indicated by arrows). Scale bar ¼ 50 mm.

Article Snippet: Cells were transfected with a pool of four unique AR siRNA duplexes (M-003400-02-0005; Thermo Scientific) or a pool of three unique p62/SQSTM1 siRNA duplexes (SR305865; Origene) using siTran 1.0 transfection reagent (TT300001; Origene).

Techniques: Recombinant

Journal: Journal of cellular biochemistry

Article Title: IL-1β induces p62/SQSTM1 and represses androgen receptor expression in prostate cancer cells.

doi: 10.1002/jcb.24897

Figure Lengend Snippet: Fig. 4. The C4-2 PCa cell population shows a heterogeneous response to IL-1b regulation of AR and p62. C4-2 cells were grown in HS-5 BMSC conditioned medium or 25 ng/ml recombinant human IL-1b protein for 2 days and co-immunostained for AR (red) and p62 (green). Image: Shown is a merged image of a representative responsive cell showing concomitant nuclear AR loss and diffuse cytoplasmic p62 upregulation (arrow). Graph: The percentage of cells showing the loss of nuclear AR, the upregulation of speckled/ diffuse p62, or both was graphed. Ninety-three percent of C4-2 cells grown in HS-5 BMSC conditioned medium showed concomitant loss of nuclear AR and induction of p62 speckles, while 9% of the C4-2 cells grown in IL-1b showed concomitant AR loss and diffuse p62 induction, indicating that the response of the C4-2 cell population to IL-1b is heterogeneous.

Article Snippet: Cells were transfected with a pool of four unique AR siRNA duplexes (M-003400-02-0005; Thermo Scientific) or a pool of three unique p62/SQSTM1 siRNA duplexes (SR305865; Origene) using siTran 1.0 transfection reagent (TT300001; Origene).

Techniques: Recombinant

Journal: Journal of cellular biochemistry

Article Title: IL-1β induces p62/SQSTM1 and represses androgen receptor expression in prostate cancer cells.

doi: 10.1002/jcb.24897

Figure Lengend Snippet: Fig. 5. IL-1b regulates AR and p62 through independent pathways. C4-2 cells were grown vehicle control or 25 ng/ml recombinant human IL-1b protein for 2 days in the presence of 40 mM control siRNA, AR siRNA, or p62 siRNA and then co-immunostained for AR and p62. Loss of AR protein did not induce p62 accumulation in vehicle control- treated or IL-1b-treated cells. Loss of p62 protein did not down regulate AR in vehicle control-treated cells or inhibit IL-1b-mediated AR repression. Arrows indicate representative cells.

Article Snippet: Cells were transfected with a pool of four unique AR siRNA duplexes (M-003400-02-0005; Thermo Scientific) or a pool of three unique p62/SQSTM1 siRNA duplexes (SR305865; Origene) using siTran 1.0 transfection reagent (TT300001; Origene).

Techniques: Control, Recombinant

Journal: Scientific Reports

Article Title: Diesel exhaust particles induce human umbilical vein endothelial cells apoptosis by accumulation of autophagosomes and caspase-8 activation

doi: 10.1038/s41598-022-21044-3

Figure Lengend Snippet: DEP initiate autophagy in HUVECs. ( a ) HUVECs were exposed to DEP (70 µg/ml) for the indicated times and phosphorylation of Beclin-1 at Ser93 was analyzed by immunoblotting. ( b ) Quantification of Beclin-1 phosphorylation normalized to total Beclin-1. Results are presented as means ± SD (n = 3). Statistical analysis was performed using one-way ANOVA. **P < 0.02 versus no treatment. ( c ) HUVECs were exposed to DEP (70 µg/ml) for the indicated times and protein levels of p62 and LC3B were measured by immunoblotting. ( d ) Quantification of p62 and LC3BII protein levels normalized to ⍺-tubulin. Results are presented as means ± SD ( n = 3). Statistical analysis was performed using one-way ANOVA. * P < 0.05 versus no treatment. **P < 0.02 versus no treatment. ( e ) HUVECs infected with GFP-LC3B-expressing adenovirus were exposed to DEP (70 µg/ml) for 18 h and formation of LC3B puncta was examined with fluorescence microscopy. ( f ) Quantification of LC3B puncta formation per cell. Results are presented as means ± SD ( n = 10). Statistical analysis was performed using two-tailed Student’s t test. **P < 0.02 versus control.

Article Snippet: Scrambled siRNA and human p62 siRNA were purchased from Santa Cruz Biotechnology Inc.

Techniques: Phospho-proteomics, Western Blot, Infection, Expressing, Fluorescence, Microscopy, Two Tailed Test, Control

Journal: Scientific Reports

Article Title: Diesel exhaust particles induce human umbilical vein endothelial cells apoptosis by accumulation of autophagosomes and caspase-8 activation

doi: 10.1038/s41598-022-21044-3

Figure Lengend Snippet: Caspase-8 is critical for DEP-induced apoptosis in HUVECs. ( a ) HUVECs were exposed to DEP (70 μg/ml) for 6 h. Cells were then fixed, permeabilized, and stained for LC3B, p62, and caspase-8. Arrows indicate co-localization between LC3B, p62, and caspase-8. ( b ) Intensity of each fluorescent signal along the dotted line shown in ( a ). S: Start, E: End. ( c ) HUVECs were exposed to DEP (70 µg/ml) for the indicated times and protein levels of cleaved caspase-8 and cleaved caspase-3 were analyzed by immunoblotting. ( d ) Quantification of cleaved caspase-8 and cleaved caspase-3 protein levels normalized to ⍺-tubulin. Results are presented as means ± SD ( n = 3). Statistical analysis was performed using one-way ANOVA. ** P < 0.02 versus no treatment. ( e ) HUVECs were pre-incubated with either Z-IETD-FMK (caspase-8 inhibitor, 5 µM) or vehicle control (DMSO) for 30 min and exposed to DEP (70 µg/ml) for 24 h. After performing WST-1 assay, cell viability relative to control is presented. Results are presented as means ± SD ( n = 8). Statistical analysis was performed using one-way ANOVA. * P < 0.02 versus control. **P < 0.02 versus DEP + DMSO. ( f ) HUVECs were pre-incubated either with Z-IETD-FMK (5 µM) or vehicle control (DMSO) for 30 min and exposed to DEP (70 µg/ml) for 24 h. Cells were then subjected to TUNEL staining. ( g ) Quantification of TUNEL-positive cells. TUNEL-positive apoptotic cells were counted and expressed as percentage of total nuclear counts. Results are presented as means ± SD from five randomly selected fields. Statistical analysis was performed using one-way ANOVA. * P < 0.02 versus control. **P < 0.02 versus DEP + DMSO.

Article Snippet: Scrambled siRNA and human p62 siRNA were purchased from Santa Cruz Biotechnology Inc.

Techniques: Staining, Western Blot, Incubation, Control, WST-1 Assay, TUNEL Assay

Journal: Scientific Reports

Article Title: Diesel exhaust particles induce human umbilical vein endothelial cells apoptosis by accumulation of autophagosomes and caspase-8 activation

doi: 10.1038/s41598-022-21044-3

Figure Lengend Snippet: p62 depletion suppresses DEP-induced apoptosis. ( a ) HUVECs were transfected with scrambled or p62 siRNA and exposed to DEP (70 μg/ml) for 18 h. Cleavage of caspase-8 and caspase-3 were analyzed by immunoblotting. ( b ) Quantification of cleaved caspase-8 and cleaved caspase-3 normalized to β-actin. Results are presented as means ± SD ( n = 3). Statistical analysis was performed using one-way ANOVA. * P < 0.02 versus scrambled siRNA with control. ** P < 0.05 versus scrambled siRNA with DEP. ( c ) HUVECs were transfected with either scrambled or p62 siRNA and exposed to DEP (70 μg/ml). After 24 h, cells were subjected to TUNEL staining. ( d ) Quantification of TUNEL-positive cells. TUNEL-positive apoptotic cells were counted and expressed as a percentage of total nuclear counts. Results are presented as means ± SD from five randomly selected fields. Statistical analysis was performed one-way ANOVA. * P < 0.02 versus scrambled siRNA with control. ** P < 0.02 versus scrambled siRNA with DEP.

Article Snippet: Scrambled siRNA and human p62 siRNA were purchased from Santa Cruz Biotechnology Inc.

Techniques: Transfection, Western Blot, Control, TUNEL Assay, Staining