Fig. 2: Wild-type Kras deletion increases proliferation, active KRAS and MAPK signalling. | Nature Communications

Fig. 2: Wild-type Kras deletion increases proliferation, active KRAS and MAPK signalling.

From: KRAS allelic imbalance drives tumour initiation yet suppresses metastasis in colorectal cancer in vivo

Fig. 2

a Representative H&E and BrdU IHC of Apcfl/fl Kras+/G12D and Apcfl/fl Krasfl/G12D mice sampled 3 days post Cre-induction. Arrowheads indicate de-differentiating cells in the villi. Scale, 100 μm. b Boxplots showing BrdU-positive cells from at least 25 half-crypts in SI in Apcfl/fl, Apcfl/fl Kras+/G12D and Apcfl/fl Krasfl/G12D mice. Boxes depict interquartile range, central line indicates median and whiskers indicate minimum/maximum values (Apcfl/fl, n = 6, 4M, 2F; Apcfl/fl Kras+/G12D, n = 6, 2M, 4F; and Apcfl/fl Krasfl/G12D, n = 5, 1M, 4F mice). **P = 0.0022, **P = 0.0043. c RAF-Ras Binding Domain (RBD) agarose affinity purification assay of three biologically independent samples per condition from Apcfl/fl Kras+/G12D and Apcfl/fl Krasfl/G12D intestinal organoids. Pulldown of RAS-GTP with RAF-RBD agarose beads. Top: Precipitates were immunoblotted using a pan-RAS antibody and input pan-RAS served as loading control. RAS-GTP activation levels were quantified and normalised to pan-Ras loading control. Apcfl/fl Kras+/G12D, n = 3, 2M, 1F; and Apcfl/fl Krasfl/G12D, n = 3, 1M, 2F. Bottom: Precipitates were immunoblotted using a KRASG12D antibody and input KRASG12D served as loading control. KRASG12D-RAF-RBD levels were quantified and normalised to KRASG12D loading control. Apcfl/fl Kras+/G12D, n = 3, 3M; and Apcfl/fl Krasfl/G12D, n = 3, 1M, 2F. d Quantification of RAF-RBD assay (left) and KRASG12D immunoblot (right from c), **P = 0.0045 (pan-RAS), **P = 0.0044 (KRASG12D). e qRT-PCR analysis of Etv5, Etv4, Dusp4, Dusp5 and Spry3 in Apcfl/fl Kras+/G12D (n = 6, 3M, 3F) and Apcfl/fl Krasfl/G12D (n = 5, 1M, 4F) intestinal organoids. Data are mean ± s.e.m. Transcript levels were normalised to Gapdh. *P = 0.0260 (Spry3) *P = 0.0411 (Dusp5). f Left: Immunoblots showing PTEN, pAKT (Ser473), AKT, pERK1/2, ERK1/2, pMEK1/2, MEK1/2 and ß-actin in Apcfl/fl Kras+/G12D and Apcfl/fl Krasfl/G12D intestinal organoids. 6 Apcfl/fl Kras+/G12D and 5 Apcfl/fl Krasfl/G12D biological replicates per group, each lane represents organoids generated from individual mice from genotype indicated. Right: Quantification of immunoblots, phosphorylated proteins were normalised to total protein levels. Data are mean ± s.e.m. **P = 0.0043 (pERK/ERK), *P = 0.0152 (pMEK/MEK), **P = 0.0087 (pAKT/AKT), *P = 0.015 (PTEN/ß-actin). b, d, e, f using one-way Mann–Whitney U test. Source data are provided as a Source Data file.

Back to article page