Additional file 1 of The phospholipid transporter PITPNC1 links KRAS to MYC to prevent autophagy in lung and pancreatic cancer
Creators: name: Entrialgo-Cadierno, Rodrigo
name: Cueto-Ureña, Cristina
name: Welch, Connor
name: Feliu, Iker
name: Macaya, Irati
name: Vera, Laura
name: Morales, Xabier
name: Michelina, Sandra Vietti
name: Scaparone, Pietro
name: Lopez, Ines
name: Darbo, Elodie
name: Erice, Oihane
name: Vallejo, Adrian
name: Moreno, Haritz
name: Goñi-Salaverri, Ainhoa
name: Lara-Astiaso, David
name: Halberg, Nils
name: Cortes-Dominguez, Ivan
name: Guruceaga, Elizabeth
name: Ambrogio, Chiara
name: Lecanda, Fernando
name: Vicent, Silve
Publisher: name: figshare
Year: 2024
Subjects: subject: Genetics
subject: FOS: Biological sciences

description: Additional file 1: Suppl. Figure 1. A. PITPNC1 expression levels (log2) in TCGA LUAD patient’s database. Mut EGFR: mutant EGFR, wt: wild type EGFR, N: Normal tissue. Mut vs wt (p=0.014), Mut vs N (p=0.021). B. PITPNC1 expression levels (log2) in TCGA LUAD patient’s database. Mut BRAF: mutant BRAF, wt: wild type BRAF, N: Normal tissue. Mut vs wt (p=0.275), Mut vs N (p=0.336). C. PITPNC1 expression levels (log2) in TCGA LUAD patient’s database with different KRAS point mutations. N: Normal tissue D. Western blot of PITPNC1 and KRAS expression in H1792 cells, expressing a control (GFPsh) or a tet-inducible KRAS shRNA (KRASsh) (activated by 1 μg/ml doxycycline). Twenty μg of protein were loaded per sample. β-TUBULIN was used as loading markes E. PITPNC1 mRNA expression in H2009 and H1792 cells expressing a control (GFPsh) or a tet-inducible KRAS shRNA (KRASsh) (activated by 1 μg/ml doxycycline) (Mann-Whitney or unpaired t-test). F. Western blot of pERK1/2, ERK1/2, pAKT, AKT, p-cJUN, cJUN, pERK5 and ERK5 in A549, H2009 and HPAFII treated with pharmacologic inhibitors: trametinib (MEKi, 0.5 μmol/L), BIX02189 (MEK5i, 10 μmol/L) or GSK2126458 (PI3Ki, 0.1 μmol/L) for 24 h, and SP600125 (JNKi, 10 μmol/L) for 2 h. Twenty μg of protein were loaded per sample. G. PITPNA, PITPNB, PITPNM1, PITPNM2, PITPNM3 mRNA expression in H2126 cells overexpressing a mutant (KRASG12D) or a wild type form (KRAS4B) of KRAS compared to the control (LacZ) (Dunnett’s multiple comparison test). H and I. PITPNA, PITPNB, PITPNM1, PITPNM2, PITPNM3 mRNA expression in H2009 (G) and H1792 (H) cells expressing a control (GFPsh) or an inducible KRAS shRNA (KRASsh) (activated by 1 μg/ml doxycycline) (Mann-Whitney or unpaired t-test). J. Western blot of PITPNC1 and KRAS expression in Kraslox/lox MEFs transduced with different human HA-tagged KRAS mutants (G12C, G12D, G12V, G12R, G12S, G13D and Q61H). K. PITPNC1 expression levels (log2) in TCGA LUAD patient’s database. Mut KRAS: mutant KRAS. Mut KRAS Amp: mutant KRAS amplification, N: Normal tissue. Mut KRAS vs Mut KRAS Amp (n.s). Suppl. Figure 2. A. PITPNC1 expression levels (log2) in TCGA LUAD patient’s database in presence of a panel of co-occurrence mutations. B. Western blot of LKB1 and PITPNC1 expression in H2009 cells expressing a control (Control) or LKB1 sgRNAs (LKB1 sgRNA1 or LKB1 sgRNA2). Twenty μg of protein were loaded per sample. β-TUBULIN was used as loading marker. C. Western blot of PITPNC1, p53, Keap1 or Lkb1 expression in KLA and LKR10 cells, expressing a control (Control) or p53, Lkb1 or Keap1 sgRNAs (p53 sgRNA, Lkb1 sgRNA or Keap sgRNA). Twenty μg of protein were loaded per sample. HSP90 and β-TUBULIN were used as loading markers. Suppl. Figure 3. A. Apoptosis analysis by Annexin V/7AAD labelling in the human LUAD A549, H2009, H1792 and H358 cell lines after PITPNC1 knockdown with a specific shRNA (sh6 or sh7) compared to the control (GFPsh). (Tukey’s multiple comparison test). B. Representative flow cytometry images depicting gating strategy in A. C. Representative images of A549- and PATU8902-derived xenografts (GFPsh, PITPNC1 sh6 and PITPNC1 sh7) stained for phospho-histone 3 (pH3) or cleaved caspase 3 (CC3). D. Western blot of PITPNC1 in A549 and H358 cells transfected with a control (pBabe) or PITPNC1 cDNA. Twenty μg of protein were loaded per sample. β-TUBULIN was used as loading marker E. Representative images of clonogenic ability of A549 and H358 PITPNC1-overexpressing cells compared with the control. F. Tumour volume (mm3) of A549 derived xenografts, (n=8), (Bonferroni’s multiple comparison test). G. Representative images of tumours from F. H. Tumor weight (g) of tumours in F (n=8) (Mann-Whitney or Unpaired T-test). I. Tumour volume (mm3) of H358 derived xenografts, (n=8), (Bonferroni’s multiple comparison test). J. Representative images of tumours from I. K. Tumour weight (g) of tumours in I (n=8) (Mann-Whitney or Unpaired T-test). L. Migration assay experiment in LacZ and PITPNC1-overexpressing A549 and H2009 cell lines. M. Representative bioluminescence images of a metastasis assay via intracardiac injection of LacZ and PITPNC1-overexpressing A549 cells. N. Ex vivo analysis of bioluminescence in lung, liver and kidney. Suppl. Figure 4. A. PHLDA2, GJB2, GPX2, BIRC5, PITPNC1, RASSF6 and ARK1B10 mRNA expression in A549 cells expressing a control (GFPsh) or a PITPNC1 shRNA (sh6 or sh7) (Dunnett´s multiple comparison test). B. Box plot comparing early (I-II) and advanced (III-IV) mut KRAS LUAD according to the expression of the dPITPNC1 gene signature (GS). C. Box plot comparing localized and advanced (locally advanced and metastatic) PDAC based on the expression of the dPITPNC1 gene signature (GS). D. Box plot comparing P53-mutated and LKB1-mutated LUAD patients with mut KRAS according to the expression of the dPITPNC1 gene signature (GS). E. Box plot comparing classical and basal PDAC patients based on the expression of the dPITPNC1 gene signature (GS). Suppl. Figure 5. A. Cell cycle analysis by EdU labelling in the human LUAD H1792 and H358 cell lines after PITPNC1 knockdown with a specific shRNA (sh6 or sh7) compared to the control (GFPsh) (Bonferroni´s multiple comparison test). B. Western blot of MYC expression in A549- and PATU8902-xenografts tumours. Twenty μg of protein were loaded per sample. HSP90 and GAPDH were used as loading markers. C. Cell cycle analysis by EdU labelling in A549 and Miapaca2 cell lines after PITPNC1 knockdown with a specific shRNA (sh6 or sh7) compared to the control (GFPsh) (Dunnet´s multiple comparison test). D. Western blot of MYC, E2F1 and P27 in A549, H2009, H1792 and Miapaca2 cells, expressing a control (GFPsh) or a shRNA against PITPNC1 (sh6 or sh7). Twenty μg of protein were loaded per sample. β-TUBULIN was used as loading control. E. mRNA analysis of RNAseq data of E2F1, P27 and P57 in A549 cells expressing a control (GFPsh) or a shRNA against PITPNC1 (sh6 or sh7). F and G. QPCR analysis of P27 (F) and P57 (G) mRNA expression in A549, H2009, H1792 and PATU8902 cells expressing a control (GFPsh) or a PITPNC1 shRNA (sh6 or sh7) (Dunnett´s multiple comparison test). H. Cell proliferation assay in A549 and H2009 cells expressing exogenous LacZ or MYC- and submitted to inhibition of PITPNC1 by specific shRNAs. I. Western blot of MYC, PITPNC1 in A549 and H2009 cells expressing exogenous LacZ or MYC- and submitted to inhibition of PITPNC1 by specific shRNAS. Twenty μg of protein were loaded per sample. HSP90 was used as loading control. J. RNAseq data of AURKA and PLK1 in A549 expressing a control (GFP) or two PITPNC1 shRNAs. Suppl. Figure 6. A. SESN1, SESN2 and SESN3 expression levels in H2009 cell line were measured by qPCR. Cells were virally infected to express a control (GFPsh) or a PITPCN1 shRNA (sh6 and sh7) (Dunnet´s multiple comparison test). GAPDH was used as housekeeping gene. B. mTOR/LAMP1 colocalization analysis by immunofluorescence in H2009 PITPNC1-depleted cells. C. Quantification of mTOR/LAMP1 Mander´s overlap coefficient (MOC) in H2009 of B (Dunnett’s multiple comparison test). D. Western blot of mTOR in A549, H2009 and H1792 cell lines expressing a control (GFP) or two PITPNC1 shRNAs. Twenty μg of protein were loaded per sample and HSP90 was used as loading control. E. Lysosomes per cell and average lysosomes size in H2009 of B (Dunn’s multiple comparison test). Lysosomes per cell and average lysosomes size in H2009 of B (Dunn’s multiple comparison test). F. Heatmap of autophagy and lysosome biogenesis genes upregulated upon PITPNC1 inhibition in A549 cells (data from RNAseq analysis). G and H. Western blot of protein level of LC3-I and LC3-II in A549, H2009 (G) and HPAFII (H) virally infected to express a shRNA control (C) or two PITPNC1 shRNAs (sh6 and sh7) and treated with or without hydroxychloroquine (CQ) (60 μM) for 6 h. Twenty μg of protein were loaded per sample and HSP90 was used as loading control. I. Western blot of mTOR signalling pathway (mTOR, 4EBP1 and S6K) in A549, H2009 and H1792 cell lines in which PITPNC1 was inhibited with specific shRNAs (sh6 and sh7). Twenty μg of protein were loaded per sample and HSP90 was used as loading control. Suppl. Figure 7. A. mTOR/LAMP1 colocalization analysis by immunofluorescence in A549 MYC-depleted cells. B. Quantification of mTOR/LAMP1 Mander´s overlap coefficient (MOC) in A549 of A (Dunnett’s multiple comparison test). C. mTOR/LAMP1 colocalization analysis by immunofluorescence in A549 MYC-depleted cells. D. Quantification of mTOR/LAMP1 Mander´s overlap coefficient (MOC) in MiaPaca2 MYC-depleted cells (unpaired t-test). E. Lysosomes per cell in A549 and MiaPaca2 of A and C respectively (Dunn’s multiple comparison test and unpaired t-test respectively). F. Average lysosomes size in A549 and MiaPaca2 of A and C respectively (Dunn’s multiple comparison test and unpaired t-test respectively). G. SESN1, SESN2 and SESN3 expression levels in A549 cell line were measured by qPCR. Cells were virally infected to express a control (GFPsh) or a MYC shRNA (sh42 and sh89) (Dunnet´s multiple comparison test). GAPDH was used as housekeeping gene. H. SESN1, SESN2 and SESN3 expression levels in Miapaca2 cell line were measured by qPCR. Cells were virally infected to express a control (GFPsh) or a MYC shRNA (sh42 and sh89) (Dunnet´s multiple comparison test). GAPDH was used as housekeeping gene. Suppl. Figure 8. A. Connectivity Map (CMap) analysis for dKRAS GS H358 transcriptomics. Perturbagen classes with mean connectivity scores &gt;90% are displayed. Each dot represents an individual drug included in the specific class. B. Representative image of crystal violet stained plates for drug combination experiment in G12C cell lines. C. Synergistic score (Bliss score) heatmaps of H1792, H2030, H358, H23 and MiaPaca2 treated for 5 days as indicated D. Representative image of 3D proliferation assay in H358 and H1792 cell lines treated with DMSO (Ctrl) Soto (60 nM) Fedra (1 μM) or both (Combo) for 5 days. E. Representative image of crystal violet stained plates for drug combination experiment in Soto-resistant (KR) G12C cell lines H358 and H23. Suppl. Figure 9. A and B. Representative images of H358- and Miapaca2-derived xenografts stained for phospho-histone 3 (pH3) or cleaved caspase 3 (CC3). C and D. Mouse weight change upon different treatments. S= start of the experiment; E= end of the experiment (Mann-Whitney or unpaired t-test).