Extended Data Fig. 9: Transcription factor KLF6 mediates KRAS-driven polyamine synthesis in PDA.
From: Ornithine aminotransferase supports polyamine synthesis in pancreatic cancer
a, b, Putative transcription factor (TF) binding sites identified and scored by FMatch tool in the promoter regions of all 4 ornithine and polyamine synthesis genes responsive to KRASG12D, i.e. OAT, ODC1, SRM and SMS. Predicted TFs are listed in descending order of statistical significance (p-value cut-off of 0.01), with matrix score similarity ranging from 0-1, where 1 is a perfect match. Eight are conserved in human (a) and mouse (b), out of which six (in blue) have binding sites present in the OAT promoter. c, mRNA levels of Myc, Oat, Odc1, Srm, Sms and Arg2 in 2 iKras cell lines with Scramble control (Scr) or Myc knockdown (2 distinct hairpins). n = 2 biological replicates. d, mRNA levels validating knockdown (2 or 3 hairpins per gene) of each of the 6 conserved TFs with binding sites for OAT (blue in a,b), and assessing their effects on expression of Oat, Odc1, Srm, Sms and Arg2 in iKras #1 cell line. n = 3 biological replicates. e, mRNA levels of ornithine and polyamine synthesis genes (Oat, Odc1, Srm, Sms and Arg2) in iKras #2 cells with Scr or Klf6 knockdown (2 distinct hairpins). n = 3 biological replicates. f, Levels of OAT, ODC1, SRM, SMS proteins in iKras cells with Klf6 or Scramble (Scr) knockdown, with or without Dox (72 h). β-Actin was used as loading control. g, Fold change in mRNA levels of Myc and each of the 6 TFs from d in iKras #1 cell line deprived of Dox for 48 h. n = 3 biological replicates. h, Relative abundance of total ornithine and putrescine in iKras#1 cells described in Fig. 3h. Cells were fed 15N-(amine)Gln or 15N4-Arg 24 h before harvest. n = 4 biological replicates. In c–e, g,h, data represent the mean ± s.d. p-values were obtained by one-way (d,e) or two-way (h) ANOVA followed by Tukey test, or paired two-tailed t-test (g). In d,e,g, statistical significance is for each TF knockdown vs. control Scramble (d,e), or for Off Dox vs. On Dox (g). Data represent the mean of biological replicates from 2 independent experiments (c) or mean ± s.d. of biological replicates from 3 independent experiments (d,e,g). In f,h, data are representative of 2 independent experiments.
