Figure S3.
Knockdown of FAM43Aupregulates autophagy and mitophagy flux. (A) Representative spinning disk imaging of FAM43A (green) and Hoechst (blue) in WT (sgCtrl) and FAM43A knockout (FKO) H292 cells verifying loss of FAM43A. Scale bar: 10 µm. (B) Relative mtDNA copy number of WT and FKO H292 cells measured by qPCR using ND1 primers normalized to nuclear B2M (n = 3 technical triplicates). Error bars represent mean ± SEM, Unpaired Student’s t test; ****, P < 0.0001. (C) Representative immunoblots of LC3 and ACTIN (loading control) in IMR90 cells transfected with the indicated siRNA. 96 h after transfection, cells were treated with chloroquine (CQ) (50 µM) for 4 h. (D) Quantification of the ratio between the optical density (OD) of LC3-II and ACTIN from four independent experiments treated as in C. Error bars represent mean ± SD, multiple Unpaired Student’s t test; *, P < 0.05. ****, P < 0.0001. (E) Representative immunoblots of TFAM, LC3, and ACTIN (loading control) of IMR90 cells treated with ddC (20 µM) for 48 or 96 h. Cells were treated with CQ 4 h prior to collection. (F) Quantification of the ratio between the optical density (OD) of LC3-II and ACTIN from three independent experiments treated as in E. Error bars represent mean ± SD, multiple Unpaired Student’s t test; ns, P > 0.05; *, P < 0.05. ***, P < 0.001. (G) Representative immunoblots of TFAM, LC3, and ACTIN (loading control). IMR90 cells were transfected with either control (siCtrl) or siRNA against FAM43A (siFAM43A #1). Cells were treated with either vehicle (UNT) or ddC 48 h after transfection. Then, 96 h after transfection, cells were treated with CQ for the indicated time. (H) Quantification of the ratio between the optical density (OD) of LC3-II and ACTIN from three independent experiments treated as in G. Error bars represent mean ± SD, multiple Unpaired Student’s t test; ns, P > 0.05; *, P < 0.05; **, P < 0.01; ****, P > 0.0001. (I) Quantification of mitophagy via ratiometric flow cytometry performed in IMR90 cells transfected with the indicated siRNA. After 72 h of transfection, siCtrl cells were treated with vehicle (UNT), CCCP (20 µM), Deferiprone (DFP 1 mM), or Chloroquine (CQ 50 µM) for 24 h before all samples were collected for flow cytometry. The data are shown as the ratio of PE-CF594/BV605 (n = 3–5 biological replicates/condition). Error bars represent mean ± SD, multiple Unpaired Student’s t test; *, P < 0.05; **, P < 0.01; ****, P < 0.0001. (J) Representative immunoblots of ATG5, LC3, and ACTIN (loading control) in IMR90 cells stably expressing the indicated shRNA and transfected with the indicated siRNA for 96 h. (K) Relative mtDNA copy number of IMR90 cells stably expressing the indicated shRNA and transfected with the indicated siRNA for 96 h as measured by qPCR using ND1 primers and normalized to nuclear B2M. Data points represent the average of technical triplicates from three biological replicates. Error bars represent mean ± SD, one-way ANOVA with Dunnett’s multiple comparisons; ****, P < 0.0001. (L) Relative FAM43A mRNA expression of wild-type (WT) and ATG7 knockout H292 cells transfected with the indicated siRNA and collected 96 h after transfection. Data points represent the average of technical triplicates from three biological replicates. Error bars represent mean ± SD, multiple Unpaired Student’s t test; **, P < 0.01: ***, P < 0.001. (M) Relative mtDNA copy number of WT and ATG7 knockout H292 cells transfected with the indicated shRNA and collected 96 h after transfection as measured by qPCR using ND1 primers normalized to nuclear B2M (n = 3 technical replicates). Error bars represent mean ± SD, multiple Unpaired Student’s t test; ns, P > 0.05: ***, P < 0.001. Source data are available for this figure: SourceData FS3.

Knockdown of FAM43A upregulates autophagy and mitophagy flux. (A) Representative spinning disk imaging of FAM43A (green) and Hoechst (blue) in WT (sgCtrl) and FAM43A knockout (FKO) H292 cells verifying loss of FAM43A. Scale bar: 10 µm. (B) Relative mtDNA copy number of WT and FKO H292 cells measured by qPCR using ND1 primers normalized to nuclear B2M (n = 3 technical triplicates). Error bars represent mean ± SEM, Unpaired Student’s t test; ****, P < 0.0001. (C) Representative immunoblots of LC3 and ACTIN (loading control) in IMR90 cells transfected with the indicated siRNA. 96 h after transfection, cells were treated with chloroquine (CQ) (50 µM) for 4 h. (D) Quantification of the ratio between the optical density (OD) of LC3-II and ACTIN from four independent experiments treated as in C. Error bars represent mean ± SD, multiple Unpaired Student’s t test; *, P < 0.05. ****, P < 0.0001. (E) Representative immunoblots of TFAM, LC3, and ACTIN (loading control) of IMR90 cells treated with ddC (20 µM) for 48 or 96 h. Cells were treated with CQ 4 h prior to collection. (F) Quantification of the ratio between the optical density (OD) of LC3-II and ACTIN from three independent experiments treated as in E. Error bars represent mean ± SD, multiple Unpaired Student’s t test; ns, P > 0.05; *, P < 0.05. ***, P < 0.001. (G) Representative immunoblots of TFAM, LC3, and ACTIN (loading control). IMR90 cells were transfected with either control (siCtrl) or siRNA against FAM43A (siFAM43A #1). Cells were treated with either vehicle (UNT) or ddC 48 h after transfection. Then, 96 h after transfection, cells were treated with CQ for the indicated time. (H) Quantification of the ratio between the optical density (OD) of LC3-II and ACTIN from three independent experiments treated as in G. Error bars represent mean ± SD, multiple Unpaired Student’s t test; ns, P > 0.05; *, P < 0.05; **, P < 0.01; ****, P > 0.0001. (I) Quantification of mitophagy via ratiometric flow cytometry performed in IMR90 cells transfected with the indicated siRNA. After 72 h of transfection, siCtrl cells were treated with vehicle (UNT), CCCP (20 µM), Deferiprone (DFP 1 mM), or Chloroquine (CQ 50 µM) for 24 h before all samples were collected for flow cytometry. The data are shown as the ratio of PE-CF594/BV605 (n = 3–5 biological replicates/condition). Error bars represent mean ± SD, multiple Unpaired Student’s t test; *, P < 0.05; **, P < 0.01; ****, P < 0.0001. (J) Representative immunoblots of ATG5, LC3, and ACTIN (loading control) in IMR90 cells stably expressing the indicated shRNA and transfected with the indicated siRNA for 96 h. (K) Relative mtDNA copy number of IMR90 cells stably expressing the indicated shRNA and transfected with the indicated siRNA for 96 h as measured by qPCR using ND1 primers and normalized to nuclear B2M. Data points represent the average of technical triplicates from three biological replicates. Error bars represent mean ± SD, one-way ANOVA with Dunnett’s multiple comparisons; ****, P < 0.0001. (L) Relative FAM43A mRNA expression of wild-type (WT) and ATG7 knockout H292 cells transfected with the indicated siRNA and collected 96 h after transfection. Data points represent the average of technical triplicates from three biological replicates. Error bars represent mean ± SD, multiple Unpaired Student’s t test; **, P < 0.01: ***, P < 0.001. (M) Relative mtDNA copy number of WT and ATG7 knockout H292 cells transfected with the indicated shRNA and collected 96 h after transfection as measured by qPCR using ND1 primers normalized to nuclear B2M (n = 3 technical replicates). Error bars represent mean ± SD, multiple Unpaired Student’s t test; ns, P > 0.05: ***, P < 0.001. Source data are available for this figure: SourceData FS3.

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