Fig 2: Deficient autophagy blocks cell invasion into organotypic gels. (A) The knockdown efficiency of shRNAs for autophagy-related proteins, including ATG5, ATG6, ATG7 and SQSTM1, was assessed by Western blotting. (B) Representative H&E-stained sections of ATG5-, ATG6-, ATG7- and SQSTM1-depleted A549 cells in the 3D organotypic coculture invasion system with or without human CAFs. The experiment was performed in triplicate. Scale bars, 50 µm. (C) The viability of A549 cells was evaluated by a CCK8 assay after 72 h of treatment with Tre, CQ, DMSO (vehicle control), or CoCl2 at the indicated concentration (upper panel). Error bars, means ± SD of a representative set of triplicate experiments. Immunoblots of HIF1a, LC3B, and SQSTM1 with the indicated treatment for 10 h, ACTB used as a loading control (lower panel). (D) The invasion capability of A549 cells was evaluated in the 3D organotypic coculture invasion system on gels with or without CAFs under treatment with DMSO, Tre (50 mM), CQ (50 µM), or CoCl2 (150 µM) for 10 days. The experiment was performed in triplicate. Scale bars, 50 µm. The number of invasive strands was quantified in 5 H&E-stained sections of each organotypic coculture gels. Error bars, means ± SD of a representative set of triplicate experiments.

Fig 3: Increased SQSTM1-LC3B interaction at the tumor invasive front is correlated with poor outcome in NSCLC patients. (A) Endogenous physical interactions between SQSTM1 and LC3B in LUSC sections were detected by in situ PLA (indicated by red fluorescent puncta) and visualized by laser scan confocal microscopy. Membrane structures were counterstained with a DIO probe (green). The enlarged regions show positive PLA signals of SQSTM1-LC3B protein complexes at the tumor invasive front (a-b). Scale bars, 50 µm. The scatter diagram of the PLA results shows the number of red fluorescent puncta per cell at the tumor invasive front and inside the tumor body (P < 0.0001). Error bars, means ± SEM for 30 cells in a representative experiment. (B) Representative images of in situ bright-field PLA of SQSTM1-LC3B protein complexes in NSCLC specimens and tumor-adjacent tissues, counterstained by Hematoxylin. Scale bars, 50 µm. (C) in situ PLA of SQSTM1-LC3B protein interaction was performed on tumor-adjacent lung tissues (n = 10), LUSC (n = 39), and LUAD (n = 99) specimens. The frequency of patient with undetectable (0), low (< 4.5), or high (4.6–9.0) PLA signal scores stratified by IHC-defined lung cancer subtype (ANOVA, P = 0.003) (left panel) or by NSCLC grade (ANOVA, P = 0.0045) (right panel). (D) Survival rates in 64 of the 148 NSCLC patients determined by Kaplan-Meier analysis were compared between patients with undetectable and low (PLA signals score = 4.5, n = 48, blue line) and high (PLA signals score > 4.6, n = 16, red line) PLA signal scores for the SQSTM1-LC3B interaction. Three-year survival rates were 85% (undetectable and low PLA signal scores of SQSTM1-LC3B interaction) versus 33% (high PLA signal scores of SQSTM1-LC3B interaction; P = 0.028). (E and F), Representative IHC images of ATG7 and BECN1 in NSCLC specimens and tumor-adjacent tissues. Scale bars, 50 µm.

Fig 4: Expression of autophagy marker proteins in cells expressing different portions of the NS5A gene. (A) PK-15 cells were transfected with sequences expressing different portions of the NS5A gene or a mock plasmid. The expression of LC3, ATG5, BECN1, and GAPDH (loading control) was analyzed by immunoblotting with specific antibodies described in the Materials and Methods. The data represent the mean±SD of three independent experiments. Statistical comparisons were evaluated using ANOVA followed by Tukey’s HSD post hoc analysis. ***p<0.001, compared with the mock group.

Fig 5: Nonstructural protein 5A expression induces cellular autophagy. (A) PK-15 cells were transfected for NS5A expression, infected with CSFV (MOI=1) for 36h or treated with rapamycin (Rapa, 100nM) for 48h. (B-D) The expression of light chain 3 (LC3), ATG5, BECN1, and GAPDH (loading control) was analyzed by immunoblotting with specific antibodies described in the Materials and Methods. (E) PK-15 cells were transfected with NS5A or treated with Rapa (100nM) for 48h. The cells were then fixed and processed for indirect immunofluorescence using antibodies against LC3B, followed by the corresponding secondary antibodies conjugated to FITC as described in the Materials and Methods. The cell nuclei were counterstained with DAPI. The fluorescence signals were visualized by confocal immunofluorescence microscopy. In the images, nuclear staining is shown in blue, and LC3B staining is shown in green. (F) The average number of LC3B puncta in each cell was determined from at least 100 cells in each group. The data represent the mean±SD of three independent experiments. Statistical comparisons were evaluated using ANOVA followed by Tukey’s HSD post hoc analysis. ***p<0.001, **p<0.01, and compared with the mock group.