hnRNP A1 downregulation promotes MP20 expression. (A) WB analysis confirming the expression of FLAG-MPs in HEK293T cells. The anti–TDP-43 antibody detected endogenous TDP-FL (FL-endo) and FLAG-MPs. (B) Subcellular localization of FLAG-MPs in HeLaS3 cells. FLAG (green) and Hoechst (blue). Magnified images of FLAG are shown in the right panels. Scale bars: 20 μm (left) and 10 μm (right). (C) WB analysis of TDP-43 protein levels in HEK293T with TDP-FL KD (FL KD). Data are normalized to β-actin. Statistical analysis was performed using Welch’s t test. (D) RT-qPCR analysis of TDP-FL, GPSM2 CE, and ATG4B CE mRNA levels in HEK293T with FL KD. Data are normalized to ACTB. Statistical analysis was performed using Welch’s t test (n = 3 for each group). (E) WB analysis of FLAG-MPs with Rmt’s (see Fig. 2 E). (F) Schematic representation of the sMP13 isoform of TARDBP, induced by hnRNP A1 and E1. (G) Percentage of cytoplasmic leakage in FLAG-hnRNPs–positive cells. Data present 564 cells per group. (H) RT-qPCR analysis of hnRNP A1, TDP-FL, and MP20 (127) mRNA levels in HEK293T cells with hnRNP A1 KD. Data are normalized to ACTB. Statistical analyses were performed using Welch’s t test (n = 3 for each group). (I) WB analysis of TDP-43 protein levels in HEK293T cells with hnRNP A1 KD. Quantification of FL-endo, SVs-endo, and MP20-endo protein levels is normalized to β-actin. Statistical analyses were performed using Welch’s t test. (J) Immunocytochemistry of HeLaS3 cells with hnRNP A1 KD. hnRNP A1 (magenta), MP20 Ab (green), and Hoechst (blue). Scale bar: 50 μm. (K) Quantification of nuclear hnRNP A1 or MP20 intensity. Data present 100 cells per group. Statistical analyses were performed using Welch’s t test. All graphs show the mean ± SEM. *P < 0.05, **P < 0.01, ***<0.001, and ****P < 0.0001. Ab, antibody. Source data are available for this figure: SourceData FS1.
Figure S1.

hnRNP A1 downregulation promotes MP20 expression. (A) WB analysis confirming the expression of FLAG-MPs in HEK293T cells. The anti–TDP-43 antibody detected endogenous TDP-FL (FL-endo) and FLAG-MPs. (B) Subcellular localization of FLAG-MPs in HeLaS3 cells. FLAG (green) and Hoechst (blue). Magnified images of FLAG are shown in the right panels. Scale bars: 20 μm (left) and 10 μm (right). (C) WB analysis of TDP-43 protein levels in HEK293T with TDP-FL KD (FL KD). Data are normalized to β-actin. Statistical analysis was performed using Welch’s t test. (D) RT-qPCR analysis of TDP-FL, GPSM2 CE, and ATG4B CE mRNA levels in HEK293T with FL KD. Data are normalized to ACTB. Statistical analysis was performed using Welch’s t test (n = 3 for each group). (E) WB analysis of FLAG-MPs with Rmt’s (see Fig. 2 E). (F) Schematic representation of the sMP13 isoform of TARDBP, induced by hnRNP A1 and E1. (G) Percentage of cytoplasmic leakage in FLAG-hnRNPs–positive cells. Data present 564 cells per group. (H) RT-qPCR analysis of hnRNP A1, TDP-FL, and MP20 (127) mRNA levels in HEK293T cells with hnRNP A1 KD. Data are normalized to ACTB. Statistical analyses were performed using Welch’s t test (n = 3 for each group). (I) WB analysis of TDP-43 protein levels in HEK293T cells with hnRNP A1 KD. Quantification of FL-endo, SVs-endo, and MP20-endo protein levels is normalized to β-actin. Statistical analyses were performed using Welch’s t test. (J) Immunocytochemistry of HeLaS3 cells with hnRNP A1 KD. hnRNP A1 (magenta), MP20 Ab (green), and Hoechst (blue). Scale bar: 50 μm. (K) Quantification of nuclear hnRNP A1 or MP20 intensity. Data present 100 cells per group. Statistical analyses were performed using Welch’s t test. All graphs show the mean ± SEM. *P < 0.05, **P < 0.01, ***<0.001, and ****P < 0.0001. Ab, antibody. Source data are available for this figure: SourceData FS1.

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