Figure 8. IBMPFD mutant expression or... | Rockefeller University Press

Figure 8.

$Figure 8. IBMPFD mutant expression or autophagic inhibition redistributes TDP-43 to the cytosol in cells. (A) Quantitation of mCherry–TDP-43 distribution (nuclear only or cytoplasmic) in control U20S, VCP-WT–, VCP-RH–, VCP-AE–, or VCP-EQ–expressing cells, or control U20S cells treated with 10 µM Baf or 50 µM chloroquine (Chlq) for 4 h from 10 different fields from two independent experiments. Error bars represent the standard error from 20 fields in two independent experiments. *, P < 0.02 when compared with VCP-WT–expressing cells. (B) Epifluorescent images for mCherry–TDP-43 (red) and DAPI (blue) in control U20S, VCP-WT–, VCP-RH–, or VCP-AE–expressing cells, or control U20S cells treated with Baf or chloroquine for 4 h. Arrows denote cytosolic TDP-43 and perinuclear TDP-43 inclusions. (C, top) Immunoblot for TDP-43, lamin A/C, and actin from nuclear or cytosolic lysate fractions of mCherry–TDP-43–transfected VCP-WT–, VCP-RH–, VCP-AE–, or VCP-EQ–expressing cells. Note the increase in cytosolic TDP-43 from IBMPFD mutant– and EQ-expressing cells. (bottom) Immunoblot for TDP-43, lamin A/C, and actin from nuclear or cytosolic lysate fractions of untreated U20S cells transfected with mCherry–TDP-43 or similarly transfected cells treated with 30 µg/ml (cq1) or 120 µg/ml (cq2) chloroquine diphosphate or 200 ng/ml Baf. Data are representative of three independent experiments. Bar, 15 µm.$

IBMPFD mutant expression or autophagic inhibition redistributes TDP-43 to the cytosol in cells. (A) Quantitation of mCherry–TDP-43 distribution (nuclear only or cytoplasmic) in control U20S, VCP-WT–, VCP-RH–, VCP-AE–, or VCP-EQ–expressing cells, or control U20S cells treated with 10 µM Baf or 50 µM chloroquine (Chlq) for 4 h from 10 different fields from two independent experiments. Error bars represent the standard error from 20 fields in two independent experiments. *, P < 0.02 when compared with VCP-WT–expressing cells. (B) Epifluorescent images for mCherry–TDP-43 (red) and DAPI (blue) in control U20S, VCP-WT–, VCP-RH–, or VCP-AE–expressing cells, or control U20S cells treated with Baf or chloroquine for 4 h. Arrows denote cytosolic TDP-43 and perinuclear TDP-43 inclusions. (C, top) Immunoblot for TDP-43, lamin A/C, and actin from nuclear or cytosolic lysate fractions of mCherry–TDP-43–transfected VCP-WT–, VCP-RH–, VCP-AE–, or VCP-EQ–expressing cells. Note the increase in cytosolic TDP-43 from IBMPFD mutant– and EQ-expressing cells. (bottom) Immunoblot for TDP-43, lamin A/C, and actin from nuclear or cytosolic lysate fractions of untreated U20S cells transfected with mCherry–TDP-43 or similarly transfected cells treated with 30 µg/ml (cq1) or 120 µg/ml (cq2) chloroquine diphosphate or 200 ng/ml Baf. Data are representative of three independent experiments. Bar, 15 µm.

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