Fig 2: Cisplatin treatment affects autophagy-related proteins and inhibits basal autophagy in Me21 cells.Me21 melanoma cells were untreated (C) or treated with 20 µM cisplatin; after 24 hrs, still viable adhering cells (Cis A) were harvested and analyzed separately from floating dead cells (Cis F). (A) Densitometric analysis of LC3-II of 8 experiments is presented as means ± S.E. *P<0.05. Western blot analysis of a typical experiment is shown. (B) Western blot analysis of LC3 performed in cells treated with cisplatin for 18 hours plus 30 µM chloroquine (CQ) for further 2 hours, in order to block the autophagic flux. In CQ-treated cells half protein has been loaded. A typical experiment out of 3 is shown, and densitometric analysis is reported. (C) Down-regulation of Beclin-1 in Me21 melanoma cells at different time-points of cisplatin treatment; densitometric analysis of Western blots is reported as means ± S.E. of 3 to 9 experiments; Beclin-1 level is expressed as percentage of control cells at 0 time. *P<0.05, compared to control cells at 0 time. A typical experiment is shown. (D) Beclin-1 proteolysis after 24 hours of cisplatin treatment, in absence or presence of the caspase-3/-7 inhibitor ac-DEVD-CHO (CHO); Western blot and densitometric analysis of a typical experiment out of 3 are reported. (E) Atg14 proteolysis after 24 hours of cisplatin treatment, in absence or presence of ac-DEVD-CHO; Western blot and densitometric analysis of a typical experiment out of 3 are reported. (F) LC3-II levels after 24 hours of cisplatin treatment, in absence or presence of ac-DEVD-CHO; Western blot and densitometric analysis of a typical experiment out of 3 are reported. Densitometric analyses were normalized to Ponceau S staining.

Fig 3: The NLRX1–Beclin 1–UVARAG complex regulates Group A Streptococcus (GAS) invasion by endocytosis. (A) NLRX1 interacts with the Beclin 1 complex. HeLa cells were transfected with EmGFP-empty vector or EmGFP-tagged Beclin 1 complex proteins (Beclin1, Vps34/PI3KC3, and Atg14) with FLAG-NLRX1. Similarly, cells were transfected with FLAG-empty vector or FLAG-tagged UVARG with EmGFP-NLRX1. Cell lysates were subjected to immunoprecipitations with anti-FLAG or—GFP (for UVRAG) antibody. The immunoprecipitated proteins and total cell lysates were analyzed by immunoblotting with anti-GFP or—FLAG (for UVRAG) antibody. (B,C) Invasion rate of GAS in HeLa wild-type, Beclin 1 KO, UVRAG KO, Rubicon KO, Vps34/PI3KC3 KD, and Atg14 KD cells. **P < 0.01. (D,F) Confocal microscopic images of EEA1 (D)- or Rab7 (F)-positive compartments containing GAS in HeLa wild-type, Beclin1, and UVRAG KO cells. Cells were transfected with EmGFP-Rab7, or immunostained with an anti-EEA1 antibody to visualize each endosomal marker. Cellular and bacterial DNA was stained with DAPI (blue). Scale bars, 10 µm. (E,G) The number of cells containing EEA1 (D)-, or Rab7 (F)-positive GAS were counted and presented as the percentage of the total number of GAS-infected cells. HeLa wild-type, Beclin 1, and UVRAG KO cells were infected with GAS for the indicated times. The data shown represent results from > 200 infected cells and indicate the mean value ± SD from three independent experiments. **P < 0.01.

Fig 4: UVRAG but not Atg14 regulates GAS internalization.(A) The number of cells containing GcAV were counted and presented as the percentage of the total number of GAS-infected cells. HeLa cells stably expressing GFP-LC3 were transfected with a control siRNA or Atg14 siRNA and infected with GAS (MOI = 100) for 4 h. Cellular and bacterial DNA was stained with DAPI. The data shown represent results from >200 infected cells in terms of the mean value ± SD from three independent experiments. (B) Confocal microscopic images of GcAV in Atg14L knockdown cells. White arrowheads show GcAVs. Scale bars, 10 µm. (C) Invasion rate of GAS in Atg14L knockdown cells. HeLa cells were transfected with a control siRNA or Atg14 siRNA and infected with GAS (MOI = 100). At 1 h post-infection, cells were disrupted with distilled water and serial dilutions of cellular extracts were plated on THY agar plates, and colony counting was performed. The data presents the invasion rate as the ratio of “total intracellular GAS at 2 h post-infection” to “total adherent GAS at 1 h post-infection”. Data are representative of = three independent experiments. (D) Intracellular survival rate of GAS in Atg14L knockdown cells. HeLa cells were transfected and infected as in (C). The data presents the survival rate as the ratio of “intracellular live GAS at 4 h post-infection” to “total intracellular GAS at 2 h post-infection”. Data are representative of = three independent experiments. (E) UVRAG interacts with Bcl-xL. HEK293T cells transfected with FLAG-control or -Bcl-xL and EmGFP-UVRAG and cultured under nutrient-rich and starvation conditions for 2 h, or were infected with GAS for 4 h, and then subjected to immunoprecipitations with an anti-FLAG antibody. The immunoprecipitated proteins and total cell lysates were analyzed by immunoblotting with anti-GFP antibody. (F) Invasion rate of GAS in UVRAG-overexpressing Beclin 1 KO cells. Wild-type HeLa cells transfected with FLAG-control and Beclin 1 KO cells transfected with FLAG-control or FLAG-UVRAG were infected with GAS (MOI = 100). At 1 h post-infection, cells were disrupted with distilled water and serial dilutions of cellular extracts were plated on THY agar plates, and colony counting was performed. The data presents the invasion rate as the ratio of “total intracellular GAS at 2 h post-infection” to “total adherent GAS at 1 h post-infection”. Data are representative of = three independent experiments. * P < 0.05. ** P < 0.01. (G) Intracellular survival rate of GAS in UVRAG-overexpressing Beclin 1 KO cells. Wild-type HeLa cells and Beclin 1 KO cells were transfected and infected as in (F). The data presents the survival rate as the ratio of “intracellular live GAS at 4 h post-infection” to “total intracellular GAS at 2 h post-infection”. Data are representative of = three independent experiments.