Fig 2: ATDC up-regulates Cyclin D1 and c-Myc via activation of NF-?B signaling pathway.A. ATDC overexpression up-regulated NF-?B reporter luciferase activity in HBE cells and ATDC depletion inhibited NF-?B reporter luciferase activity in both A549 and H1299 cells. B. ATDC transfection increased p-I?B expression in HBE cells and ATDC depletion decreased the level of p-I?B in A549 and H1299 cells. C. NF-?B inhibitor Bay 11-7082 completely blocked NF-?B reporter luciferase activity and reversed the effect of ATDC on cyclin D1, c-Myc and p-Rb up-regulation.

Fig 3: ATDC does not seem to be involved in regulating Wnt or p53 activity in lung cancer cell lines.A. There was no significant change of Topflash luciferase activity after ATDC transfection in HBE cells and after siRNA treatment in A549 and H1299 cells. B. There was no significant change of ß-catenin and active ß-catenin protein levels after ATDC transfection in HBE cells and after siRNA treatment in A549 and H1299 cells. C. ATDC transfection in HBE cells or its depletion in A549 cells did not change the level of p53 luciferase activity. D. ATDC transfection in HBE or its depletion in A549 cells did not change the protein level of p53 target gene p21.

Fig 4: Gal-9 enhances STING interaction with and ubiquitination by TRIM29.a Upper panel: TW03 cells stably expressing Flag-Gal-9 or EV were treated with cycloheximide (CHX, 20 µg/ml) for indicated time points (in hours) and then harvested for immunoblot. Lower panel: CD33+ cells transfected with lenti-flag-tagged-EV or lenti-flagged-Gal-9 vector for 36 h, then treated with CHX (20 µg/ml) for indicated time points (in hours) and harvested for immunoblot. b Upper panel: Immunoblot analysis of protein extracts of TW03 cells stably expressing Flag-Gal-9 or EV, and treated with DMSO, MG132 (10 µM), 3-Methyladenine (3-MA, 2.5 mM) or Chloroquine phosphate (CQ, 20 mM) for 6 h. Lower panel: CD33+ cells transfected with lenti-flag-tagged-EV or lenti-flagged-Gal-9 vector for 36 h, then treated with DMSO, MG132 (10 µM), 3-Methyladenine (3-MA, 2.5 mM) or Chloroquine phosphate (CQ, 20 mM) for 6 h and harvested for immunoblot. c Protein extracts of HEK293T cells transfected with a combination of plasmids coding for Myc-STING and HA-UB, in the presence or absence of Flag-Gal-9 and subjected to immunoprecipitation with anti-Myc-beads and immunoblot analysis with anti-HA antibody. d Protein extracts of HEK293T cells transfected with various combinations of plasmids coding for Flag-STING, Myc-Gal-9, HA-UB-K48, or HA-UB-K48R (arginine instead of lysine 48) and subjected to immunoprecipitation with anti-Flag-beads and immunoblot analysis with indicated antibodies. e Control (Ctrl) and TRIM29-KO TW03 cells were transfected with Flag-Gal-9 or EV for 24 h. The cell protein extracts were immunoblotted with the indicated antibodies. f Control (Ctrl) and TRIM29-KO TW03 cells were transfected with a combination of plasmids coding for Flag-STING, HA-K48, Myc-Gal-9 for 36 h, and treated with MG132 (10 µM) for 6 h. The extracts from TW03 cells were subjected to IP with an anti-Flag-beads and immunoblot analysis with anti-HA antibody. g Immunofluorescence assay demonstrating the partial colocalization of endogenous Gal-9 (green) and TRIM29 (red) in CD33+ cells. Cell nuclei were stained with DAPI (blue). Scar bar: 2 µm. h HEK293T cells were transfected with Flag-STING and HA-TRIM29 followed by transfection with increasing amounts of Myc-Gal-9 for 24 h. The protein extracts were subjected to IP with anti-Flag-beads and immunoblot analysis with an anti-HA antibody. i C666-1 cells were transfected with shRNA targeting Gal-9 (C666-1-shGal-9) or corresponding control vectors (C666-1-shCtrl) for 24 h, then transfected with HA-TRIM29 and Flag-STING for 24 h, finally subjected to IP with anti-Flag-beads and immunoblot with indicated antibodies. All experiments were performed at least three times. Ctrl Control, KO knock out, WCL whole cell lysis, IP Immunoprecipitation.

Fig 5: Epithelial cells harbour a subset of cancer stem cells and show heterogeneity of DNA copy number variations. (A) Differentiation status of malignant cells analysed by CytoTRACE. (B) RNA velocity of malignant cells. (C) Differentiation trajectory of malignant cells, with each colour coded for clusters (left) and pseudo-time (right). (D) Expression of FOSL1 in malignant cells was shown in the tSNE view. (E) The expression of EPCAM, TRIM29 and DKK1 was shown with violin plots. (F) Multiplex immunofluorescence staining of LC tissue. (G) Candidate drugs that target AREG and F3 in C3 or G6PD in C8 of malignant cells. (H) Bubble plots showed the expression of genes related to CIN was shown. (I) Expression of genes in CIN cells. (J) KEGG pathway of total epithelial cells enriched between PDL1 high compared with PDL1 low expression group. Circle means downregulated pathways and triangle means upregulated pathways. The dot represents the enriched gene number. (K) Expression of GATA6 was shown with violin plots and tSNE view. (L) Kaplan–Meier plots of overall survival (OS) for NSCLC patients with CTLs with the top and bottom TIDE prediction scores. The P value was calculated by testing the association between TIDE prediction scores and overall survival with the two-sided Wald test in a Cox-PH regression. (M) Expression of genes was shown with violin plots and tSNE view.