Fig 2: Effects of the HFD-HF diet on the mRNA and protein expression of Beclin-1 (A,B), ATG5 (C,D), ATG12 (E,F), LC3-II (G,H), SQSTM1/p62 (I,J), and LAMP1 (K,L), respectively, in the ovaries of adult rats. Bars with n.s. denote statistically insignificant differences between the respective means, while those with asterisk (*, **, ***, ****) denote statistically significant differences (* p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.0001, **** p ≤ 0.0001 vs. control group) between the respective means.

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: TIGAR induces autophagy in cells with prolonged OGD. (A–C) SH-SY5Y cells were transfected with 3×Flag, 3×Flag-TIGAR for 24 h. (A) SH-SY5Y cells were subjected to 1 or 6 h of OGD followed by 3 h of reperfusion. The Flag, LC3B and p62 levels were determined by western blot. (B) Semi-quantitative analyses of LC3B-II and p62 are shown. (C) SH-SY5Y cells were pretreated with PBS as a vehicle control or 50 μM chloroquine (CQ) for 4 h and then subjected to 6 h of OGD followed by 3 h of reperfusion, the Flag, LC3B and p62 levels were detected by western blot. (D) SH-SY5Y cells were previously co-transfected with mCherry-LC3 and siRNA or siTIGAR and then subjected to 1 or 6 h of OGD followed by 3 h of reperfusion. Columns represent the number of mCherry-LC3–positive puncta (n = 40–60 cell for each group). (E) SH-SY5Y cells were previously transfected with siRNA or siTIGAR and then subjected to 1 or 6 h OGD followed by 3 h of reperfusion. The levels of TIGAR, LC3B, p62 were determined by western blot. (F) SH-SY5Y cells were transfected with 3×Flag, 3×Flag-TIGAR, or 3×Flag-TIGAR-Mutant (H11A/E102A/H198A) and then subjected to 1 or 6 h of OGD followed by 3 h of reperfusion. The expression of Flag, LC3B and p62 were determined by western blot. (G and H) SH-SY5Y cells were previously co-transfected with mCherry-LC3 and GFP, GFP-TIGAR or GFP-TIGAR-Mutant (H11A/E102A/H198A), then subjected to 1 or 6 h of OGD followed by 3 h of reperfusion. (G) Images shows representative examples of each group from three independent experiments. (H) Columns represent the number of mCherry-LC3–positive puncta (n = 30–80 cells for each group). (I and J) SH-SY5Y cells were pre-transfected with 3×Flag or 3×Flag-TIGAR and then subjected with 6 h of OGD followed by 3 h of reperfusion. (I) The levels of Beclin-1, Atg5 and Atg12 were determined by western blot. (J) Semi-quantitative analyses of Beclin-1, Atg5 and Atg12 are shown. All experiments are from at least three independent experiments. Data are expressed as mean ± SEM. Statistical comparisons were performed as one-way ANOVA with Turkey's multiple-comparisons test. *p < 0.05, **p < 0.01, ***p < 0.001 versus the indicated group. Ctrl, control; Num., number.

Fig 5: ATG12 conjugation to ATG5 enhances its affinity for ATG16L1 and stabilizes otherwise transient ATG5-ATG16L1 interactions.a DU145 cells, stably expressing empty vector (EV), wild-type ATG5 (WT), ATG5 tethered to an N-terminal fragment of ATG16L1 (16L1N-ATG5), wild-type ATG16L1, or an N-terminally deleted ATG16L1 (16L1ΔN), were treated with 10 μM MG132 for 8 h and immunoblotted for the indicated proteins. b DU145 cells, stably expressing EV, ATG5 WT, or a nonconjugatable ATG5 mutant (ATG5-K130R), were treated with 10 μM MG132 for 8 h and immunoblotted for the indicated proteins. c Wild-type and ATG7 CRISPR/Cas9 knockout (ATG7 KO) PC-3 cells were treated with 5 μg/mL cyclohexamide (CHX) for 4, 8, and 24 h and immunoblotted for the indicated proteins. Graphs were generated by normalizing each time point with its corresponding control (t = 0 h). d Lysates from wild-type (WT) and ATG7 KO PC-3 cells were mixed in a 1:3.5 ratio to equalize the amount of conjugated to unconjugated ATG5. GST pull downs were then performed using the lysate mixture and recombinant GST or GST-ATG16L1N at the indicated concentrations. EC50 values for ATG12–ATG5 and ATG5 were determined using a best-fit curve of plotted values (fraction bound) from a total of six experiments. GST-ATG16L1N bound to native ATG12–ATG5 conjugate in lysates with ~tenfold higher affinity than endogenous ATG5. See also Supplementary Fig. S4