Journal: Nature Communications

Article Title: Mitophagy curtails cytosolic mtDNA-dependent activation of cGAS/STING inflammation during aging

doi: 10.1038/s41467-024-45044-1

Figure Lengend Snippet: a Representative images of ARPE-19 cells expressing the mito -QC reporter which subjected to PINK1/Parkin-depedent mitophagy ( PINK1; PARK2 knockdown) and/or mitochondrial biogenesis (100 μM Chloramphenicol) inhibition, and simultaneously treated with ABT-737 and/or UA. b Quantification of the number of mitolysosomes per cell as shown in Fig. 7a ( n = 4 independent experiments). c Quantification of mitochondrial mass in the presence or absence of Chloramphenicol to validate UA-induced mitochondrial biogenesis inhibition, as reported in Fig. ( n = 4 independent experiments). d Representative images showing immunostaining of ARPE-19 cells for DNA (green) and TOMM20 (magenta, mitochondria). e Quantification of the number of cytosolic DNA foci per cell as shown in Fig. 7d ( n = 4 independent experiments). f Quantification of PINK1 and PARK2 mRNA levels to validate siRNA-mediated knockdown efficiency ( n = 4 independent experiments). Scale bars, 25 μm. All data are expressed as the mean ± s.e.m. Dots represent independent experiments. P values were calculated using two-tailed Student’s t test ( b , c ) or two-tailed Mann–Whitney’s U -test ( b , Control:Control vs Control:ABT), 1-way ANOVA with Šídák’s post-hoc test ( e ) or 2-way ANOVA with Šídák’s post-hoc test ( f ). Source data are provided as a Source Data file.

Article Snippet: Knockdown efficiency was validated by RT-qPCR using TaqMan probes (Thermo Fisher) targeting PINK1 (Hs00260868_m1) or PARK2 (Hs01038318_m1), and ribosomal 18S (Hs99999901_s1) was used as reference gene.

Techniques: Expressing, Knockdown, Inhibition, Immunostaining, Two Tailed Test, Control

Journal: Cancer Research

Article Title: Parkin Deficiency Suppresses Antigen Presentation to Promote Tumor Immune Evasion and Immunotherapy Resistance

doi: 10.1158/0008-5472.CAN-22-2499

Figure Lengend Snippet: Parkin deficiency in human ccRCC is associated with poor clinical outcome, limited antigen presentation capabilities, and impacts immunotherapy efficacy. Parkin ( PRKN ) downregulation in patients with KIRC is significantly associated with overall survival and tumor stage. A, PRKN gene expression is significantly downregulated in patient tumor samples vs. matched controls ( N = 72 matches samples, P value = 3E−07 via paired Student t test; FPKMs mapped reads data from GDC HTSeq-FPKM pipeline was used). Dashed lines represent cutoffs used to bin patients with KIRC into low, mid, high PRKN gene expression levels. B, PRKN gene expression levels are significantly associated with overall survival ( N = 538 tumor samples, P < 0.0001 via log-rank test for trend). C, PRKN expression is significantly associated with overall survival in patients with stage 4 tumors (log-rank P < 0.007). D, mRNA expression levels of PRKN on HEK293, 786-O, CAKI-I, and A498 cells cultured overnight in growth media. E, mRNA and protein expression levels of A498 Parkin KO or EV cells. F, A498 Parkin KO or EV cells were stimulated with IFNγ (10 ng/mL) for 18 hours. Then, immunoblotting analysis was performed on Parkin, HLA-A, TAP1, and PSMB8. Data represent two to three independent experiments. Immunoblot data represent two to three independent experiments. G, IFNγ ELISA analysis was performed on donor-derived NY-ESO-1-specific T cells (5 × 10 4 ) cocultured in a 1:1 ratio overnight with A498 Parkin KO or EV cells. Tumor cells were prestimulated with IFNγ (10 ng/mL) or mock-treated for 18 hours before the assay. Data are represented as mean ± SD. ***, P < 0.001; unpaired, two-tailed t test.

Article Snippet: Housekeeping gene Rn18s (Mm03928990_g1), PRKN (Hs01038322_m1), Prkn (Mm01323528_m1), H2-k1 (Mm01612247_mH), Tap1 (Mm00443188_m1), Psmb8 (Mm00440207_m1), B2m (Mm00437762_m1), Ifng (Mm01168134_m1), Ifnb (Mm00439552_s1), Foxp3 (Mm00475162_m1), Il12b (Mm01288989_m1), and Gzmb (Mm00442837_m1).

Techniques: Immunopeptidomics, Gene Expression, Expressing, Cell Culture, Western Blot, Enzyme-linked Immunosorbent Assay, Derivative Assay, Two Tailed Test

Journal: Cancer Research

Article Title: Parkin Deficiency Suppresses Antigen Presentation to Promote Tumor Immune Evasion and Immunotherapy Resistance

doi: 10.1158/0008-5472.CAN-22-2499

Figure Lengend Snippet: Parkin regulates the MHC-I-associated tumor APM and fosters tumor progression. A and B, mRNA expression levels of Prkn , H2-k1 , Tap1 , Psmb8 , and B2m in RENCA cells stably transfected with sh Prkn _1 (sh#1), sh Prkn _3 (sh#3), and empty vector control (pLKO.1) lentiviral vectors. Data are represented as mean ± SD. ***, P < 0.001; unpaired, two-tailed t test. C, Protein expression analyses of Parkin, Tap1, Psmb8, and B2m (immunoblotting) and Parkin vs. MHC-I (intracellular staining, flow cytometry) of pLKO.1 controls, sh#1, and sh#3 RENCA cells. Data represent two to three independent experiments. D, IFNγ ELISPOT analysis was performed on T cells isolated from the spleens of pLKO.1 RENCA tumor-bearing mice at day 25 after challenge. T cells were cocultured overnight with stimulator Parkin-positive (pLKO.1) or -negative (sh#1, sh#3) RENCA cells (5:1 ratio). E, Parkin-positive (pLKO.1) or -negative (sh#1, sh#3) RENCA cells (10 6 ) were injected in the back of BALB/c mice and tumor growth was monitored. Data are represented as mean ± SEM. *, P < 0.05; ***, P < 0.001; two-way ANOVA.

Article Snippet: Housekeeping gene Rn18s (Mm03928990_g1), PRKN (Hs01038322_m1), Prkn (Mm01323528_m1), H2-k1 (Mm01612247_mH), Tap1 (Mm00443188_m1), Psmb8 (Mm00440207_m1), B2m (Mm00437762_m1), Ifng (Mm01168134_m1), Ifnb (Mm00439552_s1), Foxp3 (Mm00475162_m1), Il12b (Mm01288989_m1), and Gzmb (Mm00442837_m1).

Techniques: Expressing, Stable Transfection, Transfection, Plasmid Preparation, Control, Two Tailed Test, Western Blot, Staining, Flow Cytometry, Enzyme-linked Immunospot, Isolation, Injection

Journal: Cancer Research

Article Title: Parkin Deficiency Suppresses Antigen Presentation to Promote Tumor Immune Evasion and Immunotherapy Resistance

doi: 10.1158/0008-5472.CAN-22-2499

Figure Lengend Snippet: Parkin regulates cytosolic tumor antigen processing and presentation and facilitates effector CD8 + T-cell cancer immunity. A–C, Mouse melanoma B16-OVA Prkn knockout (KO, CRISPr/Cas9) or EV control cells were stimulated for 18 hours with IFNγ (10 ng/mL) or mock-stimulated before the analyses. A, mRNA expression levels of Prkn , H2-k1 , Psmb8 , and Tap1 in B16-OVA Parkin KO or EV cells. Data are represented as mean ± SD. ** P < 0.01; *** P < 0.001; unpaired, two-tailed t test. B, Protein expression analyses of Parkin, Tap1, and Psmb8 (immunoblotting) and SIINFEKL-bound to H-2Kb (MHCi-OVA; intracellular staining, flow cytometry) of B16-OVA KO and EV cells. Data represent two to three independent experiments. C, IFNγ ELISPOT analysis was performed on OT.1 T cells (10 5 ) cocultured overnight with B16-OVA KO and EV cells in a 5:1 ratio. D, B16-OVA Parkin KO or EV cells (10 6 ) were injected in the back of C57BL/6 mice and tumor growth was monitored. Five mice per group were treated with anti-CD8 blocking antibody (aCD8) or isotype (ISO) control. E, Percentage of CD45 − /MHC-OVA + cells present in B16-OVA Parkin KO or EV isotype-treated tumors analyzed by flow cytometry. F, SIINFEKL-specific CD8 + T-cell tumor infiltration present in B16-OVA Parkin KO or EV isotype-treated tumors. G, mRNA expression levels of IFNγ in B16-OVA Parkin KO or EV isotype-treated tumors. E–G, Data are represented as mean ± SEM. ns, not significant; *, P < 0.05; **, P < 0.01; unpaired, two-tailed t test. H, B16-OVA Parkin KO or EV cells (10 6 ) were injected in the back of C57BL/6-Tg(TcraTcrb)1100Mjb/J (OT.1) mice (5 per group) and tumor growth was monitored. I, Survival rate analysis (Kaplan–Meier, log-rank test) was performed by day 25 posttumor challenge, including five mice per group. D and H, Data are represented as mean ± SEM. ***, P < 0.001; two-way ANOVA.

Article Snippet: Housekeeping gene Rn18s (Mm03928990_g1), PRKN (Hs01038322_m1), Prkn (Mm01323528_m1), H2-k1 (Mm01612247_mH), Tap1 (Mm00443188_m1), Psmb8 (Mm00440207_m1), B2m (Mm00437762_m1), Ifng (Mm01168134_m1), Ifnb (Mm00439552_s1), Foxp3 (Mm00475162_m1), Il12b (Mm01288989_m1), and Gzmb (Mm00442837_m1).

Techniques: Knock-Out, CRISPR, Control, Expressing, Two Tailed Test, Western Blot, Staining, Flow Cytometry, Enzyme-linked Immunospot, Injection, Blocking Assay