Fig 1.
Validation of ATG7KD as an in vitro model of autophagy-deficient human LSCs.
(A) Knockdown efficiency of ATG7 verified by qPCR analysis. (B) Western blot analysis of ATG7 expression in SCR and ATG7KD LSC colonies. (C) ATG7 expression in SCR and ATG7KD LSCs assessed by immunofluorescence. Scale bar, 50 μm. (D) Representative micrographs of autophagosome staining by CYTO-ID assay. Arrowheads indicate autophagosomal vesicles, asterisks denote diffuse pattern of autophagic components. Scale bar, 50 μm. (E) Quantification of autophagosomes in SCR and ATG7KD LSCs in response to rapamycin treatment. *p < .05, **p < .01.
Fig 2.
Autophagy is activated during LSC’s stress response to UVA.
(A) Representative images of autophagosomes in ATG7KD LSCs under UVA stress. Arrowheads show autophagic cells, asterisks indicate absence of autophagosomes. Scale bar, 50 μm. (B) Quantification of cells with autophagic activity in response to UVA. (C) Representative western blot image of autophagic flux in UVA-irradiated LSC colonies in absence and presence of BafA1, with or without UVA. LC3B-I and II were detected by immunoblotting at indicated time points. (D) Densitometric analysis of LC3B-II expression normalized to GAPDH. n = 3, *p < .05.
Fig 3.
Autophagy regulates intracellular ROS levels in LSCs under UVA stress.
(A) Intracellular ROS determined by CM-H2DCFDA staining of SCR and ATG7KD LSCs, with or without UVA and antioxidant pretreatment. Scale bar, 100 μm. (B) Quantification of ROS levels by mean fluorescence intensity. *p < .05, **p < .01.
Fig 4.
Autophagy mediates nuclear export of PAX6 in response to UVA-elicited oxidative stress.
(A) Representative immunofluorescent images depicting subcellular localization of PAX6 in controls and H2O2-treated LSCs. Scale bar, 50 μm. (B) Quantification of cells expressing nuclear PAX6 after H2O2 treatment. (C) Representative micrographs of PAX6 subcellular localization in LSCs exposed to UVA with or without antioxidant pretreatment. Arrowheads indicate nuclear localization of PAX6, asterisks denote cytoplasmic distribution of PAX6. Scale bar, 100 μm. (D) Percentage of LSCs expressing nuclear PAX6. *p < .05, **p < .01.
Fig 5.
Autophagy mediates PCNA expression and PAX6 cyto-localization in UVA-irradiated LSC colonies.
(A) Dual immunofluorescent staining against PCNA and PAX6 in UVA-irradiated LSC colonies with or without adenoviral overexpression of PAX6. Scale bar, 100 μm. (B) Quantification of PAX6+PCNA+ cells. *p < .05, **p < .01, ***p < .001.
Fig 6.
PAX7 overexpression in nuclear PAX6-depleted LSCs does not rescue UVA-induced PCNA downregulation.
(A) Representative micrographs of PCNA immunofluorescence in irradiated SCR and ATG7KD colonies with or without adenoviral overexpression of PAX7. Scale bar, 100 μm. (B) Quantitative analysis of nuclear PCNA expression. *p < .05, **p < .01.
Fig 7.
PAX6KD attenuates the uncurbed cell cycle response in ATG7KD LSCs following UVA irradiation.
(A) Representative images of PCNA immunofluorescence in ATG7KD, PAX6KD and ATG7/PAX6 KD colonies following UVA irradiation. Scale bar, 100 μm. (B) Quantification of cells expressing PCNA in ATG7KD, PAX6KD or double KD LSCs. *p < .05, **p < .01.
Fig 8.
Autophagy mediates p21 induction in response to UVA via nuclear export of PAX6.
(A) Micrographs of p21 and PAX6 cyto-localization in irradiated SCR and ATG7KD LSCs with or without PAX6 overexpression. Arrowheads denote PAX6+ p21- nuclei, asterisks indicate cells expressing nuclear p21 and cytoplasmic PAX6. Scale bar, 50 μm. (B) Quantification of p21+ nuclei. *p < .05, ***p < .001.
Fig 9.
PAX6KD restores UVA-induced, p21-mediated cell cycle arrest in ATG7KD LSC.
(A) Immunofluorescence of p21 in LSC colonies with KD of ATG7, PAX6 or both. Scale bar, 100 μm. (B) Quantification of p21-expressing cells in ATG7KD, PAX6KD or ATG7/PAX6 double KD LSCs. *p < .05, **p < .01.
Fig 10.
Model of autophagy-mediated regulation of PAX6 cyto-localization and cell cycle in UVA-stressed LSCs.
In autophagy-competent LSCs, UVA activates autophagy by inducing ROS. In return, autophagic activity stabilizes intracellular ROS at physiological level. Furthermore, autophagy regulates cell cycle by facilitating cytoplasmic export of nuclear PAX6, which normally represses p21 in PCNA+, proliferative LSCs. In lack of functional autophagy, UVA induces excess levels of ROS, PAX6 is retained in the nucleus and the p21-mediated cell cycle response is hampered.