Figure 1.
Protein aggregation is increased in human cells with SETX deficiency. (A) Protein levels of SETX in human U2OS cells with depletion of endogenous SETX and expression of recombinant wild-type (WT), without N-terminal domain (∆N-term, 1 a.a.–665 a.a.) or without helicase domain (∆HD, 1931 a.a.–2456 a.a.) by Western blotting with anti-SETX antibody; β-actin monitored for normalization. (B) Flow cytometric analysis of protein aggregates in U2OS cells with control or SETX shRNA using Proteostat (Enzo). Proteasome inhibitor MG132 (10 μM) treated cells were employed as positive control. At least 10,000 cells were measured in each replicate; three replicates are shown with box plot, the center line indicates the median, the box bounds indicate the first and third quartiles. (C) Schematic diagram of SETX showing known features, including N-terminal protein interaction domain (yellow) and helicase domain (blue). P413L and L1976R are reported SETX mutations causing AOA2 disease. (D) Proteostat intensities per cell were quantified by FACS in U2OS cells as in B with SETX shRNA and expression of recombinant SETX WT, ∆N-term, or ∆HD as indicated. (E) Levels of SETX in U2OS cells with SETX shRNA and expression of recombinant WT, P413L or L1976R alleles by Western blotting; β-actin monitored for normalization. (F) Proteostat intensities as in B from U2OS cells with SETX depletion and expression of recombinant SETX WT, P413L, or L1976R as indicated. (G) Levels of SETX in control and SETX knock-out (KO) U2OS cells by Western blotting; Tubulin serving as an internal control. (H) Proteostat intensities as in B from wild-type or SETX KO U2OS cells compared to wild-type cells with MG132 (10 μM) treatment. (I) Degradation of a UbL-YFP-eRR model proteasome substrate was measured relative to an mCherry expression control. Ratio of YFP fluorescence per cell normalized by mCherry signal as measured in at least 10,000 cells per condition. YFP/mCherry fluorescence ratios per cell were quantified by FACS in U2OS cells with control or SETX shRNA and expression of recombinant SETX WT, or MG132 (10 μM) as indicated. (J) Proteasome activity was measured as in I, comparing wild-type and SETX KO cells to MG132 (10 μM)-treated cells. (K) Activation of the unfolded protein response and ER stress was measured with a CHOP-mCherry reporter (Oh et al., 2012) by FACS with at least 10,000 cells per condition. mCherry fluorescence levels per cell were quantified by FACS in U2OS cells with control or SETX shRNA and expression of recombinant SETX WT, or tunicamycin (1 μg/ml) as indicated. (L) ER stress in U2OS wild-type and SETX KO cells as in WT and SETX KO data also shown in Fig. S8. All P values are derived using a two-tailed t test assuming unequal variance, using the mean of the fluorescence values from three biological replicates (n = 3). ** indicates P < 0.005, *** indicates P < 0.0005. Box plots show all measurements from all three replicates. Source data are available for this figure: SourceData F1.

Protein aggregation is increased in human cells with SETX deficiency. (A) Protein levels of SETX in human U2OS cells with depletion of endogenous SETX and expression of recombinant wild-type (WT), without N-terminal domain (∆N-term, 1 a.a.–665 a.a.) or without helicase domain (∆HD, 1931 a.a.–2456 a.a.) by Western blotting with anti-SETX antibody; β-actin monitored for normalization. (B) Flow cytometric analysis of protein aggregates in U2OS cells with control or SETX shRNA using Proteostat (Enzo). Proteasome inhibitor MG132 (10 μM) treated cells were employed as positive control. At least 10,000 cells were measured in each replicate; three replicates are shown with box plot, the center line indicates the median, the box bounds indicate the first and third quartiles. (C) Schematic diagram of SETX showing known features, including N-terminal protein interaction domain (yellow) and helicase domain (blue). P413L and L1976R are reported SETX mutations causing AOA2 disease. (D) Proteostat intensities per cell were quantified by FACS in U2OS cells as in B with SETX shRNA and expression of recombinant SETX WT, ∆N-term, or ∆HD as indicated. (E) Levels of SETX in U2OS cells with SETX shRNA and expression of recombinant WT, P413L or L1976R alleles by Western blotting; β-actin monitored for normalization. (F) Proteostat intensities as in B from U2OS cells with SETX depletion and expression of recombinant SETX WT, P413L, or L1976R as indicated. (G) Levels of SETX in control and SETX knock-out (KO) U2OS cells by Western blotting; Tubulin serving as an internal control. (H) Proteostat intensities as in B from wild-type or SETX KO U2OS cells compared to wild-type cells with MG132 (10 μM) treatment. (I) Degradation of a UbL-YFP-eRR model proteasome substrate was measured relative to an mCherry expression control. Ratio of YFP fluorescence per cell normalized by mCherry signal as measured in at least 10,000 cells per condition. YFP/mCherry fluorescence ratios per cell were quantified by FACS in U2OS cells with control or SETX shRNA and expression of recombinant SETX WT, or MG132 (10 μM) as indicated. (J) Proteasome activity was measured as in I, comparing wild-type and SETX KO cells to MG132 (10 μM)-treated cells. (K) Activation of the unfolded protein response and ER stress was measured with a CHOP-mCherry reporter (Oh et al., 2012) by FACS with at least 10,000 cells per condition. mCherry fluorescence levels per cell were quantified by FACS in U2OS cells with control or SETX shRNA and expression of recombinant SETX WT, or tunicamycin (1 μg/ml) as indicated. (L) ER stress in U2OS wild-type and SETX KO cells as in WT and SETX KO data also shown in Fig. S8. All P values are derived using a two-tailed t test assuming unequal variance, using the mean of the fluorescence values from three biological replicates (n = 3). ** indicates P < 0.005, *** indicates P < 0.0005. Box plots show all measurements from all three replicates. Source data are available for this figure: SourceData F1.

This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal