Figure S3.
Generation of PARK7-deficient A549 and SH-SY5Y cells and cytokine responses to RSV infection. (A and B) Immunoblot and RT-PCR demonstrating absence of RSV replication in primary fibroblasts. Primary fibroblasts from a healthy donor were infected with RSV (RSV-A strain Long, MOI 1), and cells were collected at indicated time points. Whole cell lysates were analyzed by immunoblotting for expression of RSV matrix protein (RSV-M2) and compared to vinculin (VCL) as loading control (A), and cells were collected for RNA extraction and quantification of RSV RNA by RT-PCR (B). (C and D) Immunoblot demonstrating KO of PARK7 in A549 pulmonary cells (C) and PARK7 KO in neuronal SH-SY5Y cells (D). Vinculin (VCL, C) and GAPDH (D) were used as loading control. (E) Repeat of experiment shown in Fig. 4 A, showing additional replicates of two experiments performed in duplicate of human pulmonary A594 cells deficient in PARK7 (red) and control AAVS1KO (grey) 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. (F–M) PARK7KO (red) and control AAVS1KO (grey) A549 cells were mock treated or infected with RSV (RSV-A, strain Long, MOI 1) for 24 h. (F–I) Cells were collected for RNA isolation, and quantification of IFNL1 (F), CXCL10 (G), IL6 (H), and TNF (I) gene transcription was performed by RT-PCR. (J–M) Cell culture supernatants were collected for IFNλ1 (J), CXLC10 (K), IL6 (L), and TNF (M) protein quantification by ELISA. (N–U) Repeats of experiment shown in Fig. 4, D–G, showing an additional two repeat experiments performed in triplicate (N–Q shows the second experiment and R–U shows the third experiment) where neuronal SH-SY5Y cells deficient in PARK7 or control AAVS1KO were mock transfected (transf ctrl) or transfected with PolyIC (500 ng/ml). Cells were collected after 6 h, and RNA was isolated, and IFNB1 (N and R), CXCL10 (O and S), IL6 (P and T), and MX1 (Q and U) gene transcription quantification by RT-qPCR. Bars indicate mean ± SD values. Statistics were calculated using the two-way ANOVA with multiple comparisons. * = P < 0.05, ** = P < 0.01, *** = P < 0.001, and ns = not significant. SD, standard deviation. Source data are available for this figure: SourceData SF3.

Generation of PARK7-deficient A549 and SH-SY5Y cells and cytokine responses to RSV infection. (A and B) Immunoblot and RT-PCR demonstrating absence of RSV replication in primary fibroblasts. Primary fibroblasts from a healthy donor were infected with RSV (RSV-A strain Long, MOI 1), and cells were collected at indicated time points. Whole cell lysates were analyzed by immunoblotting for expression of RSV matrix protein (RSV-M2) and compared to vinculin (VCL) as loading control (A), and cells were collected for RNA extraction and quantification of RSV RNA by RT-PCR (B). (C and D) Immunoblot demonstrating KO of PARK7 in A549 pulmonary cells (C) and PARK7 KO in neuronal SH-SY5Y cells (D). Vinculin (VCL, C) and GAPDH (D) were used as loading control. (E) Repeat of experiment shown in Fig. 4 A, showing additional replicates of two experiments performed in duplicate of human pulmonary A594 cells deficient in PARK7 (red) and control AAVS1KO (grey) 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. (F–M) PARK7KO (red) and control AAVS1KO (grey) A549 cells were mock treated or infected with RSV (RSV-A, strain Long, MOI 1) for 24 h. (F–I) Cells were collected for RNA isolation, and quantification of IFNL1 (F), CXCL10 (G), IL6 (H), and TNF (I) gene transcription was performed by RT-PCR. (J–M) Cell culture supernatants were collected for IFNλ1 (J), CXLC10 (K), IL6 (L), and TNF (M) protein quantification by ELISA. (N–U) Repeats of experiment shown in Fig. 4, D–G, showing an additional two repeat experiments performed in triplicate (N–Q shows the second experiment and R–U shows the third experiment) where neuronal SH-SY5Y cells deficient in PARK7 or control AAVS1KO were mock transfected (transf ctrl) or transfected with PolyIC (500 ng/ml). Cells were collected after 6 h, and RNA was isolated, and IFNB1 (N and R), CXCL10 (O and S), IL6 (P and T), and MX1 (Q and U) gene transcription quantification by RT-qPCR. Bars indicate mean ± SD values. Statistics were calculated using the two-way ANOVA with multiple comparisons. * = P < 0.05, ** = P < 0.01, *** = P < 0.001, and ns = not significant. SD, standard deviation. Source data are available for this figure: SourceData SF3.

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