Fig 2: Atg5/Atg7-independent alternative autophagy participates in the suppression of UVB-induced inflammasome activation in human keratinocytes.A, representative example showing the expression level of Atg5 (left) or Atg7 (right) evaluated by Western blotting in the cell lysates of keratinocytes, which were transfected with either control siRNA or Atg5 siRNA or Atg7 siRNA. B, representative example showing the expression level of LC3B determined by Western blotting in cell lysates of either Atg5- or Atg7-knockdown keratinocytes. C, quantification by ELISA of IL-1β in the supernatants of keratinocytes that were transfected with control siRNA or Atg5 siRNA for 48 h, then irradiated or not irradiated with UVB, and further cultured for 48 h (n = 4 per group). D, quantification by ELISA of IL-1β in the supernatants of keratinocytes that were transfected with control siRNA or Atg7 siRNA for 48 h, then irradiated or not irradiated with UVB, and further cultured for 48 h (n = 3 per group). E, representative example showing the expression level of p-Ulk1746 evaluated by immunoprecipitation-Western blotting in the cell lysates of keratinocytes, which were irradiated or not irradiated with UVB and further cultured for 6 h (top). Relative intensity of p-Ulk1746 is shown (n = 4 per group) (bottom). F, representative immunocytochemical staining of GS28 (green) and p-Ulk1746 (red) in keratinocytes, which were irradiated or not irradiated with UVB and further cultured for 6 h (left). The percentage of p-Ulk1746–positive keratinocytes. Cells were counted and averaged across 15 randomly selected images in each experiment. n = 15 in each group (right). Repeated three independent experiments were performed. G, representative example showing the expression level of Beclin1 or Rab9 determined by Western blotting in the cell lysates of keratinocytes transfected with either control siRNA, Beclin1 siRNA, or Rab9 siRNA. H, quantification by ELISA of IL-1β in the supernatants of keratinocytes that were transfected with control siRNA or Beclin1 siRNA for 48 h, then irradiated or not irradiated with UVB, and further cultured for 48 h (n = 3 per group). I, quantification by ELISA of IL-1β in the supernatants of keratinocytes that were transfected with control siRNA or Rab9 siRNA for 48 h, then irradiated or not irradiated with UVB, and further cultured for 48 h (n = 4 per group). Nuclei were stained with Hoechst (blue). All data are expressed as the mean ± SD. ∗ p < 0.05 and ∗∗∗ p < 0.001, ns: not significant. Scale bar represents 10 μm. IL, interleukin; UVB, ultraviolet B.

Fig 3: The effects of melatonin on mitochondrial and cytosolic ULK1 and Rab1. Hearts were subjected to 20 min global ischaemia and 30 min reperfusion with and without melatonin (0.3 μM) (Fig. 2) for subsequent preparation of cytosolic and mitochondrial fractions. Immunoblots and summary of the effects of melatonin on expression of myocardial cytosolic total ULK1 (tULK1), phospho ULK1 (pULK1) and phospho/total ULK1 (p/t ULK1) ratio (A); mitochondrial and cytosolic Rab9 (B) after Stb, isch and rep. For full non-adjusted images of gels see Supplementary file 1 Fig. 5A,B. Abbreviations: see Fig. 3. ULK1: unc-51 like kinase. *p < 0.05 vs corresponding control groups.

Fig 4: Atg9a regulates the effect of Rab9 on the Golgi apparatus.A. The protein-protein interaction network through the STRING database. B. Beas-2b cells were stimulated with IL-4 for 24 h and then transfected with siAtg9a and siRab9. The expression of Atg9a and Rab9 was observed through Western blotting. The original full gel blots are presented in the supplementary material. C. siControl and siRab9 were respectively introduced into ULK1-WT + HDM mice using nasal drip. Except WT group, mice in other groups were given 50 μg of HDM i.n. on days 0, 7, and 14, and 50 μg of HDM i.n. on days 21–27. For WT + HDM + siControl and WT + HDM + siRab9 mice, siControl and siRab9 were given i.n. every 3 days. D. Observation of Golgi apparatus in lung tissue using electron microscopy (scale: 10 μm). E. Measurement of GRASP65 expression in mouse lung tissue using Western blotting. F. Immunofluorescence analysis of Golgi apparatus and Rab9 expression in Beas-2b (scale: 100 μm). G. Measurement of GRASP65 expression in cells using Western blotting. **p < 0.01.

Fig 5: ER stress is required for autophagy initiation and EBV lytic reactivation. (a) HA cells were either untreated or treated with either 20 μM C7, 20 μM C7 precomplexed with iron or 20 μM FeCl3 alone for 2 h. The expression of PERK, IRE-1α, phosphorylated ERK1/2, Zta and Rab9 was detected by western blot analysis. Cellular α-tubulin was assessed as a loading control. (b) HA cells were treated with 20 μM C7 for 0, 1, 2, 4, 6, 8, 10, 12, 18, 24, 48 and 72 h. The expression of PERK, IRE-1α, BiP, XBP-1s, CHOP, Zta and LC3B was detected by western blot analysis. Cellular α-tubulin was used as a loading control. (c) C666-1 (d) NPC43 cells were either untreated or treated with 40 μM C7 for 24, 48 and 72 h. The expression of IRE-1α, Zta and lipidated or free LC3B (LC3B-II or LC3B-I, respectively) was detected by western blot analysis. Cellular α-tubulin was assessed as a loading control. (e) siRNA knockdown on PERK and IRE-1α was performed in HA cells. Scramble siRNA was performed as a control. The cells were treated with 20 C7 μM for 2 h. The expression of PERK, IRE-1α, KAP-1, phosphorylated ERK1/2, Zta, Rab9 and LC3B was detected by western blot analysis. Cellular β-actin was assessed as a loading control. Experiments were repeated and bands representing PERK, IRE-1α, Zta and LC3B were quantified and plotted as relative band intensity by ImageJ software and processed by GraphPad Prism 6 software. Statistics were calculated with Student’s T test. (f,g) siRNA knockdown on PERK and IRE-1α was performed in C666-1 and NPC 43 cells, respectively. Scramble siRNA was performed as a control. The cells were treated with 40 μM C7 for 72 h. The expression of PERK, IRE-1α, Zta, and LC3B was detected by western blot analysis. Cellular α-tubulin was assessed as a loading control.