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: AR12 and neratinib in combination reduce the expression of SOD1 G93A and mutant CAG repeat Huntingtin via autophagy. (A) HCT116 ATG16L1 T300 cells were transfected to express SOD1 G93A or CAG 145 repeat Huntingtin. 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 SOD1 G93A 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 4: The Kaplan–Meier plots of the relapse-free survival (RFS)-based survival probability of patients with ERBB2-positive and -negative breast tumors. The plots were downloaded from the Kaplan–Meier Plotter website (http://kmplot.com/analysis/index.php?p=service&cancer=breast (accessed on 19 November 2019)). (A) Data of patients with ATG12 expression (ATG12 mRNA) in breast tumors. (B) Data of patients with MAP1LC3B (LC3B) expression (MAP1LC3B mRNA) in breast tumors. (C) Data of patients with BECN1 expression (BECN1 mRNA) in breast tumors.

Fig 5: TRIM5 actions as a pattern-recognition receptor require autophagy factors.(A) Effect of TRIM5 knockout on the ability of HIV-1 CA P90A and WT HIV-1 to infect THP-1 macrophages. Control or TRIM5 KO cells (lines 1 and 2) were PMA-differentiated and then infected with single-cycle fluorescent protein-expressing pseudovirions for two days (CrFK MOI = 1). The percentage of cells showing red fluorescence (P90A) or green fluorescence (HIV WT) was determined by high content imaging 2 days post infection. Each data point represents the results from an individual well of a 96-well plate (>2000 cells analyzed per well). TRIM5 KO line 2 was used for subsequent experiments. (B) The effect of HIV-1 P90A infection on the expression of inflammatory gene IFNB1 in THP-1 macrophages expressing or not autophagy factors and TRIM5. THP-1 cell lines knocked out for the autophagy factors ULK1, BECN1, ATG7 or TRIM5 (or control) were PMA-differentiated and then exposed to HIV-1 CA P90A for two hours (CrFK MOI = 3) prior to RNA isolation and qPCR analysis. 18S ribosomal RNA was used as a normalization control. Data, each data point represents an independent biological replicate; **, P < 0.01; ***, P < 0.001, ****, P < 0.0001; †, not significant by ANOVA.