Enhanced cellular stress pathways and hyperinflammatory cytokine responses in PARK7-deficient neuronal SH-SY5Y cells. (A) Human pulmonary A594 cells deficient in PARK7 (red) and control AAVS1^KO (grey) were mock treated or infected with RSV (RSV-A strain Long, MOI 1) for 24 and 48 h, and cells were collected for RNA isolation and analysis of RSV RNA by RT-PCR. Bars indicate mean ± SD values of two experiments, performed in duplicate. Repeats of the experiments are shown in Fig. S3 E. (B and C) Whole cell lysates from SH-SY5Y with PARK7^KO (red) or AAVS1^KO (control, grey) were analyzed for phosphorylation of ASK1/2 (pASK1/2) and expression of total ASK1 and vinculin (VCL) as loading control by immunoblotting (B). Graph depicting the densitometric quantification of pASK1/2 relative to total ASK1 (C). Bars indicate the mean ± SD values of three experiments. (D–G) Neuronal SH-SY5Y cells deficient in PARK7 or control AAVS1^KO were mock transfected (transf ctrl) or transfected with PolyIC (tPolyIC, 500 ng/ml). Cells were collected after 6 h, and RNA was isolated, and IFNB1 (D), MX1 (E), CXCL10 (F), and IL6 (G) gene transcription quantification by RT-PCR. Bars indicate mean ± SD values of a representative experiment performed in triplicate. Repeats of the experiment are shown in Fig. S3, N–Q and R–U. (H–J) Neuronal SH-SY5Y cells deficient in PARK7 or AAVS^KO (control) were left untransduced (−) or were transduced with lentiviral vectors to express PARK7 or GFP (control). Cells were collected after 48 h for RNA isolation to determine PARK7 mRNA expression (H), and whole cell lysates were analyzed by immunoblotting for GFP and PARK7 expression with VCL as loading control (I). (J) Cells were transfected with PolyIC (tPolyIC, 500 ng/ml) and collected after 6 h for quantification of IFNB1 transcription by RT-PCR. Bars indicate mean ± SD values of a representative experiment performed in duplicate. (K–M) Neuronal SH-SY5Y cells deficient for PARK7 were transduced with lentiviral vectors to express PARK7 WT, the patient PARK7 variant R28*, the R98Q variant frequent in the healthy population (MAF>10⁻³), and the L166P variant associated with Parkinson’s disease. Cells were collected for RNA isolation to determine PARK7 mRNA expression (K), and whole cell lysates were analyzed by immunoblotting for PARK7 expression with VCL as loading control (L). (M) Cells were transfected with PolyIC (tPolyIC, 500 ng/ml) and collected after 6 h for quantification of IFNB1 transcription by RT-PCR. Bars indicate mean ± SD values of a representative experiment performed in duplicate. (N–P) Primary fibroblasts from the patient (P) were left untransduced (−) or were transduced with lentiviral vectors to express GFP, PARK7WT, PARK7 R28*, PARK7 R98Q, or PARK7 L166P. As control (C), primary fibroblasts from a healthy donor were left untransduced or were transduced to express GFP. Cells were collected for RNA isolation to determine PARK7 mRNA expression (N), and whole cell lysates were analyzed by immunoblotting for GFP and PARK7 expression with VCL as loading control (O). Cells were infected with mock virus (mock) or RSV (RSV-A strain Long, MOI 3, 24 h) and stained with Annexin-V (apoptosis) and 7-AAD (viability dye) to analyze induction of noninflammatory apoptotic cell death by flow cytometry (P). Bars show mean ± SD values of a single experiment performed in duplicate and triplicate. Statistics were calculated using the multiple paired t test (A), the unpaired t test (C), the one-way ANOVA (H and K), and ordinary two-way ANOVA with Šídák’s multiple comparisons (J, M, and P). * = P < 0.05, ** = P < 0.01, *** = P < 0.001, and ns = not significant. SD, standard deviation. Source data are available for this figure: SourceData F4.