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Figure 2
The Oxidoreductase Activity of PDI Is Protective against Inclusion Formation and Protein Unfolding in Mutant TDP-43 Expressing Cells.
(A) Immunoblotting was performed to confirm that similar transfection efficiencies were present and that co-expression of PDI-WT or PDI-QUAD did not alter the expression of TDP-43 EGFP. An anti-TDP-43 antibody was used to detect the presence of wild-type TDP-43 (TDP-WT) or mutant TDP-43M337V (TDP-M337V), in cells co-expressing either empty vector pcDNA3.1 or PDI-V5 (WT or QUAD), β-actin was used as a loading control.
(B) Immunofluorescence detection of EGFP in cells expressing EGFP-tagged TDP-WT (row 1), TDP-M337V with empty vector alone (row 2) or co-expressing PDI-WT or PDI-QUAD, or administered with BMC (rows 3, 4, 5).
(C) Significantly fewer cells formed inclusions when PDI-WT was co-expressed with TDP-M337V or treated with BMC (∗∗p < 0.01). Significant differences were observed between TDP-M337V cells co-expressing PDI-WT or PDI-QUAD, and TDP-M337V cells co-expressing PDI-QUAD or BMC (∗∗p < 0.01).
(D) Neurons expressing EGFP only (row 1), TDP-WT (row 2), TDP-M337V alone (row 3), or co-expressing PDI-WT or PDI-QUAD, or BMC-treated (rows 4, 5, 6).
(E) Significantly fewer cells formed inclusions when PDI-WT was co-expressed with TDP-M337V (∗∗p < 0.01) and treated with BMC (∗p < 0.05). Significant differences were observed between TDP-M337V cells co-expressing PDI-WT or PDI-QUAD (∗p < 0.05).
(F) TPE-MI fluorescence in Neuro-2a cells expressing TDP-WT (row 1), TDP-M337V with empty vector alone (row 2), or co-expressing PDI-WT or PDI-QUAD, or treated with BMC (rows 3, 4, 5), arrows represent TPE-MI fluorescence.
(G) Significantly fewer cells displayed TPE-MI fluorescence (representing the cellular load of unfolded proteins, blue) when PDI-WT was co-expressed with TDP-M337V or cells were treated with BMC (∗∗p < 0.01) compared to controls.
Scale bars: 8 μm in (B), 5 μm in (D), 12 μm in (F).