Fig. 1: Enhancement of KRAS protein stability by DX2.

a DX2 levels in H460 lung cancer cells were controlled by the introduction of Strep-DX2 and si-DX2. Protein and mRNA levels were determined by Western blotting (WB) and RT-PCR (RT), respectively. Phosphorylation of ERK and Akt was monitored for KRAS signaling. Actin was used as a loading control. Results are representative of at least three independent experiments. b KRAS level in doxycycline (Dox)-inducible DX2 transgenic mice (Ind-DX2). Levels of KRAS and DX2 in colorectal tissues from two controls (#1, 2) and two Dox-fed mice (#3, 4) were analyzed. c MEF isolated from Ind-DX2 were treated with Dox in a time-dependent manner and protein levels were determined. Results are representative of at least three independent experiments. d Heat map from the results showing DX2-dependent regulation of RAS isoforms. DX2-mediated changes in the levels, stability, and ubiquitination of isoforms were quantified from Supplementary Fig. 1c–e. Cell viability and anchorage-independent colony formation assay were quantified from Supplementary Fig. 1g, h. Maximum (max) and minimum (min) values from the indicated experiments were designated as different colors with the highest and lowest intensity, respectively, and the rests were graded according to their relative values. e Result of xenograft (n = 4) using H460 cells stably expressing the indicated combination of KRAS4B and sh-DX2. Representative images of mice and tumors for each group (left) and tumor sizes (right) were shown. Sh means short hairpin RNA. All error bars represent the standard deviation (S.D.). P value is from the two-sided t test. f Heat map representing the cellular levels of DX2 and KRAS in 18 lung, 12 colorectal and 8 pancreatic cell lines. The cellular levels of DX2 and KRAS in the tested cell lines were represented as the heat map of orange and blue colors, respectively. The maximum and minimum values quantitated as described in Methods were shown with the highest and lowest color intensity, respectively, and the rests were graded according to their relative values. g Analysis of the levels of DX2 and KRAS in lung and colorectal tumor tissues. Staining intensities of the two proteins were classified as low (L) and high (H) (Supplementary Fig. 2e). Number of the analyzed tissue samples is shown in brackets. h DX2 and KRAS levels in the tumor and matched normal (MN) tissues from 99 patients with colorectal cancer. Depending on the levels in tumor compared to MN, samples were classified as H and L as compared to MN levels (Supplementary Fig. 2f). Source data are provided as a Source data file.