Fig 2: N450Y mutant MYOC activates autophagy. (A) Western blot detection of mTOR, p-mTOR, LC3B, P62 and GAPDH (B,C). Band intensity analysis in (A) using ImageJ software. *, p < 0.05; **, p < 0.01; ns, no significance. (D) HTM cells were infected with lentiviruses expressing MYOC-WT or MYOC-N450Y for 48 h and transduced with EGFP-LC3 for 24 h **, p < 0.01. (E) Number of autophagosomes in (D). **, p < 0.01. (F,G) Cell viability was detected using CCK8 after rapamycin treatment. *, p < 0.05. Empty: empty vector lentiviruses; MYOC-WT: lentiviruses expressing wild type myocilin gene; MYOC-N450Y: Lentiviruses expressing N450Y mutant myocilin gene.

Fig 3: DEK accelerates the phenotype of GC. (A) Lung metastases were observed after luciferase-labeled DEK-knockdown (sh-DEK) or no-knockdown (sh-NC) SGC7901 cells were injected into nude mice via tail vein. (B) The luciferase bioluminescence values of mouse lung metastases (n = 3). (C) HE staining analysis of lung metastasis in nude mice. (D) Xenografts were obtained by subcutaneous injection of DEK-knockdown (sh-DEK) or no-knockdown (sh-NC) SGC7901 cells. (E,F) The weight and volume of subcutaneous xenografts were counted and presented in the form of histograms (**: p < 0.01, ***: p < 0.001). (G,H) Western Blot analysis in subcutaneous xenograft tissue LC3B expression, results were normalized with Actin, and LC3BII/I ratios were analyzed. (H,I) Western Blot analysis of Cleaved-caspase-3 protein in subcutaneous xenograft tissue and normalized with Actin (**: p < 0.01, ***: p < 0.001).

Fig 4: Autophagy is involved in SOX2-induced chemoresistance, CSCs properties, and EMT in CRC.A, B Western blot analysis of LC3II/LC3I and P62 expression in SW480 and SW620 cells (A) as well as in SW480 and SW620 transfected with SOX2 clone or SOX2 shRNA (B). C SOX2-overexpressing SW480 cells or SOX2-deficient SW620 cells were transiently transfected with mRFP-GFP-LC3B adenovirus for 6 h, then the number of LC3B puncta was analyzed by confocal microscopy. D Transmission electron microscopy was utilized to assess the number of autophagic vesicles in SW480 and SW620 cells transfected with SOX2 clone or SOX2 shRNA. Red arrows indicated autophagic vesicles. E SW480 or SW620 cells transfected with SOX2 clone or SOX2 shRNA were treated with 3-MA (10 mM) or Rapamycin (50 nM) for 24 h, then western blot was performed with indicated antibodies. F–J The ability of chemoresistance (F), proliferation (G), stemness (H), migration (I), and invasion (J) in SOX2-overexpressing SW480 cells treated with 3-MA was examined by drug sensitivity assay, cell viability assay, tumor sphere formation assay, wound healing assay, and transwell invasion assay. Experiments were conducted in triplicate. Data are shown as mean ± SEM. *P < 0.05, **P < 0.01, and ***P < 0.001.

Fig 5: LC3B lipidation and punctate formation in cortical neurons due to high glucose and PGRN. (A) Western blot analysis of LC3 lipidation (i.e., LC3-II:I ratio) in primary cortical neurons after 72 h of treatment. The LC3-II:I ratio decreased from control to high glucose (1.000 ± 0.208 AU to 0.464 ± 0.092 AU), an effect that appeared unaffected when treated with PGRN alongside high glucose (0.474 ± 0.076 AU). No difference was observed between control and PGRN-treated samples (0.938 ± 0.309 AU). N = 9 samples. (B) Immunofluorescence analysis of LC3B puncta expression in neurons after 72 h of treatment. The amount of punctate expression trended toward an increase due to PGRN (from 1.000 ± 0.069 AU, to 1.183 ± 0.077 AU). This difference was significant under high-glucose conditions (from 0.916 ± 0.045 AU, to 1.190 ± 0.097 AU). N = 11-29 cells. (C) Representative immunofluorescence images of primary neurons after 72 h of treatment, with blue as DAPI, green as MAP2, and red as LC3B. Scale bar, 10 µm. (D) Immunofluorescence of LC3B puncta expression in astrocytes after 72 h of treatment increased significantly due to high glucose as well as PGRN treatment (from 1.000 ± 0.0374 AU to 1.512 ± 0.087 AU and 2.792 ± 0.099 AU, respectively). This decreased to control levels under HG + PGRN treatment, to 0.899 ± 0.049 AU. N = 12-29 cells. (E) Representative immunofluorescence images of primary astrocytes after 72 h of treatment, with blue as DAPI, green as GFAP, and red as LC3B. Scale bar, 10 µm. **p < 0.01; ***p < 0.001.