Figure 7.
SIDT2 reduces mutant HTT levels through RNautophagy. (A) Neuro2a cells expressing HTTex1-Q22-EGFP or HTTex1-Q145-EGFP (48 h post-transfection) were subjected to filter-trap assay. (B, C) HTTex1-Q145-EGFP-expressing control or SIDT2-overexpressing Neuro2a cells (48 h post-transfection) were harvested with lysis buffer and cell lysates were subjected to western blotting using anti-GFP, anti-SIDT2 and anti-ACTB/β-actin antibodies (B) and to filter-trap assay (C). ****, P < 0.0001 (n = 3). (D) HTTex1-Q22-EGFP-expressing control or SIDT2-overexpressing Neuro2a cells (48 h post-transfection) were harvested with lysis buffer and cell lysates was subjected to western blotting using anti-GFP, anti-SIDT2 and anti-ACTB/β-actin antibodies. (E, F) HTTex1-Q145-EGFP-expressing control or WT, 2RS or 3RS SIDT2-overexpressing Neuro2a cells (48 h post-transfection) were harvested with lysis buffer and cell lysates were subjected to western blotting using anti-GFP, anti-SIDT2 and anti-β-actin antibodies (E) and to filter-trap assay (F). **, P < 0.01 vs Ctrl; #, P < 0.05 vs WT (n = 3). (G, H) HTTex1-Q145-EGFP-expressing control or WT or SIDT2F154T-overexpressing Neuro2a cells (48 h post-transfection) were harvested with lysis buffer and cell lysates were subjected to western blotting using anti-GFP, anti-SIDT2 and anti-ACTB/β-actin antibodies (G) and to filter-trap assay (H). **, P < 0.01 (n = 3). (I, J) HTTex1-Q145-EGFP-expressing control or WT or SIDT2S564A-overexpressing Neuro2a cells (48 h post-transfection) were harvested with lysis buffer and cell lysates were subjected to western blotting using anti-GFP, anti-SIDT2 and anti-ACTB/β-actin antibodies (I) and to filter-trap assay (J). **, P < 0.01 vs Ctrl; ##, P < 0.01 vs S564A (n = 3). (K, L) HTTex1-Q145-EGFP-expressing control or SIDT2-overexpressing Neuro2a cells (24 h post-transfection) were treated with 50 nM of epoxomicin for 24 h. Then, cells were harvested with lysis buffer and cell lysates were subjected to western blotting using anti-GFP, anti-SIDT2 and anti-ACTB/β-actin antibodies (K) and to filter-trap assay (L). ****, P < 0.0001 (n = 3). (M, N) HTTex1-Q145-EGFP-expressing control or SIDT2-overexpressing Neuro2a cells (24 h post-transfection) were treated with 50 μg/mL of E-64d and 50 μg/mL of pepstatin A for 24 h. Then, cells were harvested with lysis buffer and cell lysates were subjected to western blotting using anti-GFP, anti-SIDT2 and anti-ACTB/β-actin antibodies (M) and to filter-trap assay (N). ****, P < 0.0001 (n = 3). (O, P) HTTex1-Q145-EGFP-expressing control or SIDT2-overexpressing Neuro2a cells (24 h post-transfection) were treated with 5 mM of 3-methyladenine for 24 h. Then, cells were harvested with lysis buffer and cell lysates were subjected to western blotting using anti-GFP, anti-SIDT2 and anti-ACTB/β-actin antibodies (O) and to filter-trap assay (P). *, P < 0.05 (n = 3)