Fig 2: AR12 and neratinib in combination reduce the expression of TDP-43 via autophagy. (A) HCT116 ATG16L1 T300 cells were transfected to express TDP-43. Twenty-four h later, cells were treated with vehicle control, neratinib (50 nM), AR12 (2 μM), fingolimod (FTY, 100 nM), MMF (5 μM) or the drugs in combination as indicated for 6h or 12h. Cells were fixed in place and the expression of each protein plus ERK2 as a loading control determined by in-cell immuno-staining. (n = 3 +/-SD). * p < 0.05 less than vehicle control. (B) HCT116 ATG16L1 T300 cells were transfected to express TDP-43 and co-transfected with a scrambled siRNA or with siRNA molecules to knock down either Beclin1 or ATG5. Twenty-four h later, cells were treated with vehicle control, neratinib (50 nM), AR12 (2 μM), fingolimod (FTY, 100 nM), MMF (5 μM) or the drugs in combination as indicated for 12h. Cells were fixed in place and the expression of each protein plus total ERK2 as a loading control determined by in-cell immuno-staining. (n = 3 +/-SD). * p < 0.05 less than vehicle control.

Fig 3: The effect of chloroquine, spautin-1 and MG132 on ATG protein levels in A549 cells. (A) A549 cells were treated with 50 μM chloroquine, 10 μM spautin-1 or 5 μM MG132 in the presence or absence of 250 pM TGFβ1 for 24 h. Cells were lysed and subjected to SDS-PAGE and immunoblotting anti-ATG3, anti-ATG5, anti-ATG7, anti-ATG9, anti-ATG12, anti-ATG12-ATG5 complex, anti-ATG16L1, anti-BECN1, anti-ULK1, anti-LC3B, anti-P-Smad2, anti-Smad2 and anti-GAPDH (loading control) antibodies. (B) The steady state levels of ATG3, ATG5, ATG7, ATG9, ATG12, ATG12-ATG5, ATG16L1, BECN1, ULK1, and LC3B were quantitated using QuantityOne software and graphed (n=3, mean±s.e.m.). Significance is indicated as *=P<0.05, **P<0.01 and ****=P<0.0001.

Fig 4: EDEM1 accelerates the degradation of misfolded polypeptides even when ERAD is not functional. (A) HEK293T cells were transfected with siRNA targeting a non-specific sequence (CTRL), SEL1L, OS-9, XTP3-B, and HDR1 for 72 h; during the last 24 h, another transfection was made to overexpress an empty vector (CTRL), EDEM1 (E1), and ∆-EDEM1 (∆-E1), along with α-1AT. Cells were harvested, lysed in Triton-X 100-containing buffer, and an equal amount of protein from each sample was separated by SDS-PAGE in reducing conditions. Protein expression was estimated by Western blotting using antibodies against α-1AT, EDEM1, SEL1L, OS-9, XTP3-B, HRD1, and CNX as an internal control. (B): The same experiment was performed for NHK and its expression, and the above-mentioned proteins were detected by Western blotting. (C,D) Band densitometry plot of α-1AT and NHK presented in (A) and (B), respectively (mean n = 3 ± SEM), and two-way ANOVA comparison with Bonferroni correction was applied for statistical analysis (* p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.001). For simplicity only statistically significant samples are indicated. (E,F) During the last 24 h of siRNA transfection, BACE-476 and Ri-332 were co-expressed with an empty vector (CTRL), EDEM1 (E1), and ∆-EDEM1 (∆-E1) in HEK293T, in a similar experiment to that in (A). The protein expression was estimated by Western blotting using antibodies against BACE1 (E) and Ribophorin I (F) alongside EDEM1, SEL1L, OS-9, XTP3-B, HRD1, and CNX as an internal control. (G,H) Band densitometry plot of BACE-476 and Ri-332 presented in (E) and (F), respectively (mean n = 3 ± SEM), and two-way ANOVA comparison with Bonferroni correction was applied for statistical analysis (*p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.001). For simplicity of representation, only statistically significant samples are indicated. (I): HEK293T cells were transfected with siRNA that targeted a non-specific sequence (CTRL), SEL1L, or p97/VCP for 72 h; during the last 24 h, the cells were transfected to co-express Ri-332 alongside an empty vector (CTRL), EDEM1(E1), and ∆-EDEM1 (∆-E1). Samples were processed as in (A), and the membranes were probed with antibodies for Ribophorin-I, EDEM1, SEL1L, VCP, LC3, ATG5, CNX, and GAPDH. (J): Band densitometry plot of Ri-332 is presented in (I) (mean n = 2 ± SEM).

Fig 5: JQ1 enhances autophagy and restores autophagic flux after SCI. (A–D) Primary cortical neurons were treated with 200 nM JQ1 for 6 h, the level of LC3, Atg5, and Beclin-1 in each group of neuron were evaluated by western blotting and statistically analyzed, n = 5. (E,F) Primary cortical neurons were treated with Baf-A1 (100 nM) and JQ1 (200 nM) for 6 h, the level of LC3 in each group of neuron were detected by western blotting and statistically analyzed, n = 5. (G–I) Western blotting and quantification of p62 and LC3 expression in each group of mice at 3 days after SCI, n = 5. (J,K) Representative co-staining for LC3 (red) and NeuN (green) in each group of mice at 3 days after SCI, n = 5. Scale bar = 50 μm, scale bar (enlarged) = 10 μm. (L,M) Primary cortical neurons were treated with TBHP (50 μM) and JQ1 (200 nM) for 6 h, the co-localization of LC3 (green) and Lamp2 (red) were captured by immunofluorescence staining and quantitatively analyzed, n = 10. Scale bar = 20 μm, scale bar (enlarged) = 5 μm. GAPDH was the loading control. *P < 0.05, **P < 0.01, ***P < 0.001. Data were presented as means ± SD.