Fig 2: Abolished ATG3 protein expression but comparable LC3–PE lipidation in thioglycolate-elicited atg7−/- macrophages. Representative immunoblotting of LC3, ATG5, ATG7, and ß-actin in whole-cell lysates isolated from thioglycolate-elicited WT, (Ai) atg5−/-, and (Aii) atg7−/- peritoneal macrophages. Each lane represents a macrophage sample from an individual mouse. (Aiii) Quantification of LC3-I and LC3-II in atg5−/-, atg7−/-, and corresponding control macrophages normalized to ACTB/β-actin (n = 6). Statistics were calculated using Student’s t-test (two-tailed) with Welch correction for unequal variances; ** p < 0.01, *** p < 0.001. LC3 protein abundance in cellular fractions of WT, (Bi) atg5−/-, and (Bii) atg7−/- thioglycolate-elicited peritoneal macrophages with GAPDH as loading control. Each lane represents a sample pool from three mice. (C) mRNA expression of Atg5, Atg7, Atg3, and Atg10 in WT, atg5−/-, and atg7−/- macrophages relative to Hprt (n = 12). (Ci) Representative immunoblotting of (ATG12–)ATG5, ATG3 (upper blot), and ATG10 (lower blot), using the same samples obtained from three individual mice. (Dii) Protein quantification relative to ACTB/β-actin (n = 5–6). Data represent mean values + SD. Statistics in (C and Dii) were calculated using one-way ANOVA with Bonferroni correction; * relative to WT; # atg5−/- relative to atg7−/-; *, # p < 0.05, ** p < 0.01, ***/### p < 0.001. (E) Schematic presentation of the summarized results

Fig 3: miR-495 modulates autophagy. (A) RT-qPCR analysis was performed to determine the miR-495 expression levels in CAL-62 and SW579 cells transfected with sh-NC, sh-LGMN-1 or sh-LGMN-2. ***P<0.001. (B) RT-qPCR analysis of miR-495 expression levels in CAL-62 and SW579 cells transfected with the indicated amounts of pcDNA3.1-LGMN. ***P<0.001. (C) RT-qPCR was performed to establish the miR-495 expression levels in normal thyroid epithelial cells (Nthy-ori3-1) and thyroid carcinoma cells (CAL-62 and SW579). ***P<0.001. (D) RT-qPCR analysis of miR-495 expression levels in CAL-62 cells transfected with NC mimics, miR-495 mimics, NC inhibitor and miR-495 inhibitor. ***P<0.001. (E) Colony formation assay to determine the colony number of CAL-62 cells transfected with EV (pcDNA3.1), pcDNA3.1-LGMN, or pcDNA3.1-LGMN plus miR-495 mimics. (F-J) Western blot analysis was performed to measure the protein expression levels of ATG3 and p62 in CAL-62 cells transfected with NC or miR-495 mimics, and SW579 cells transfected with NC or miR-495 inhibitors. ***P<0.001. (K-M) Western blot analysis was carried out to determine the protein expression levels of ATG3 and p62 in SW579 cells transfected with EV (pcDNA3.1) or pcDNA3.1-LGMN. ***P<0.001. Experiments were performed for three biological replicates; western blotting was performed once (n=1). miR-495, microRNA-495; LGMN, pseudogene legumain; RT-q, reverse transcription-quantitative; sh-, short hairpin RNA; NC, negative control; ATG3, autophagy-related gene 3; EV, empty vector.