Extended Data Fig. 2: ADAM9 maintains KRAS stability by reducing PAI-1 interaction.
(A) Effects of stress on the KRAS protein levels, activity (KRAS-GTP), and its downstream signals were determined in indicated PDAC cells. Cells were challenged with either normal (10% FBS), serum-starved (no FBS), or nutrient deprivation (EBSS) conditions for 24 hours. Lysates were probed with the indicated antibodies. (B) The effect of KRAS on ADAM9 protein was determined. PANC-1 cells were transfected with a serial dose of human KRAS (0, 1, and 2 μg) for 16 hours and treated with serum deprivation for another 8 hours. Cell lysates were probed with the indicated antibodies. (C) Kras/KRAS transcripts in ADAM9 WT and KO PDAC cells. Cells were treated with serum starvation for 24 hours before mRNA extraction. Bars are the mean ± SD from three independent experiments (n = 3 repeats). Significance was analyzed by two-sided unpaired Student’s t-test. (D) Cells were treated with 50 μg/mL CHX for 24 hours before the MTT assay and the morphology of treated cells was pictured after using crystal violet stain. Bars are the mean ± SD from three independent experiments (n = 5 repeats). Significance was analyzed by two-sided unpaired Student’s t-test. Scale bar = 400 μm. (E) PANC-1 cells were treated with 5 μM CQ and 10 μM MG132 under serum deprivation for 24 hours. Ubiquitin (Ub) was immunoprecipitated from cell lysates, and interacting proteins were probed with the indicated antibodies. The red arrows indicate the increasing ubiquitination of KRAS in ADAM9 knockout cells, and the black arrows indicate the unmodified KRAS. (F) Indicated cells were treated with either 10 μM CQ or 20 μM MG132 under serum deprivation for 24 hours. Lysates were probed with the indicated antibodies. (G) PANC-1 cells were treated with 5 μM CQ and 10 μM MG132 under serum deprivation for 24 hours. The RAS was immunoprecipitated from cell lysates, and interacting proteins were probed with ADAM9 antibody. (H) Indicated cells were treated with or without 10% FBS for 24 hours. Lysates were probed with the indicated antibodies. (I) Pan18 cells were treated with serum deprivation for 24 hours and collected. The subcellular fractions of cells were extracted to perform the immunoblot. Lysates were probed with the indicated antibodies. (J) ADAM9 WT and KO PANC-1 cells were used for ChIP-qPCR enrichment analysis with primer sets (Q to L) in the SERPINE1 promoter region. Data are mean ± SD from three independent experiments (n = 3 repeats). *P < 0.05, **P < 0.01, and ***P < 0.001 by two-sided unpaired Student’s t-test (p = 0.0052 in site P; p = 0.0012 in site J; p = 0.0005 in site M; p = 0.007 in site I; p = 0.0146 in site L). (K) Pan18 cells were treated with 5 μM CQ and 10 μM MG132 under serum deprivation for 24 hours. RAS and PAI-1 were immunoprecipitated from cell lysates, and interacting proteins were probed with the indicated antibodies. (L) Wild-type KRAS cells (BxPC-3) were transfected with Myc-KRAS and FLAG-PAI-1 for 16 hours and treated with serum deprivation for another 8 hours before the pull-down assay. The Myc and FLAG tags were immunoprecipitated from cell lysates, and interacting proteins were probed with indicated antibodies. (M) Representative images (left) and quantification (right) of PLA validated the interaction of KRAS with PAI-1 (red immunofluorescence). Pan18 cells were treated with 5 μM CQ and 10 μM MG132 under serum deprivation for 24 hours before PLA. Scatter plot shows the mean ± SD (n = 3 fields from one experiments). **P < 0.01 by two-sided unpaired Student’s t-test (p = 0.0052). (N) Pan18 cells were transfected with shPAI-1 (two different clones are indicated with superscript numbers) for 16 hours and subjected to serum deprivation for another 8 hours. Cell lysates were probed with the indicated antibodies. (O) Indicated cells were transfected with serial doses of FLAG-PAI-1 (0, 0.5, 1, and 2 μg) for 16 hours and treated with serum deprivation for another 8 hours before immunoblots. An additional 50 μg/mL CHX was added to PANC-1 cells during serum deprivation. Cell lysates were probed with the indicated antibodies. (P) HEK293T cells were transfected with HRAS-HA and FLAG-PAI-1 for 16 hours before the pull-down assay. The HA and FLAG tags were immunoprecipitated from cell lysates, and interacting proteins were probed with indicated antibodies. (Q) Indicated cells were transfected with HRAS (1 μg) and serial doses of PAI-1 (0, 0.5, and 1 μg) for 16 hours and treated with serum deprivation for another 8 hours before immunoblots. An additional 50 μg/mL CHX was added to PANC-1 cells during serum deprivation. Cell lysates were probed with the indicated antibodies.
