Fig 1: Loss of a single or double GEF is unable to rescue the Apcfl/fl phenotype.a H&E, BrdU incorporation and RNAscope for Lgr5 and Olfm4 on Vil-CreERT2 Apcfl/fl (APC), Vil-CreERT2 Apcfl/fl, Vav2-/-, Vav3-/- (APC V2V3) and Vil-CreERT2 Apcfl/fl Tiam1-/- (APC T). Scale bar represents 100 µm for H&E and BrdU and 50 µm for RNAscope images. b Quantification of BrdU positive cells. N = 6, 5 and 6 biologically independent animals for Vil-CreERT2 Apcfl/fl (APC), Vil-CreERT2 Apcfl/fl, Vav2-/-, Vav3-/- (APC V2V3; p = 0. 0303 as determined by a two-tailed Mann–Whitney) and Vil-CreERT2 Apcfl/fl Tiam1-/- (APC T; *p = 0.0260, as determined by a two-tailed Mann–Whitney) respectively. Data are presented as mean values ±SD. c Quantification of clonogenicity assay of intestinal organoids. N = 6, 3 and 3 biologically independent animals for Vil-CreERT2 Apcfl/fl (APC), Vil-CreERT2 Apcfl/fl, Vav2-/-, Vav3-/- (APC V2V3; p = 0.5476, as determined by a two-tailed Mann–Whitney) and Vil-CreERT2 Apcfl/fl Tiam1-/- (APC T) respectively (p = 0.5476, as determined by a two-tailed Mann–Whitney). Data are presented as mean values ±SD. d, e Survival of Lgr5-EGFP-IRES-creERT2 Apcfl/fl (Lgr5 APC), Lgr5-EGFP-IRES-creERT2 Apcfl/fl Vav2-/-, Vav3-/- (Lgr5 APC V2V3) and Lgr5-EGFP-IRES-creERT2 Apcfl/fl Tiam1-/- (Lgr5 APC T). d N = 19 and 18 biologically independent animals for Lgr5 APC and Lgr5 APC V2V3 respectively (p = 0.7142, as determined by Log-rank (Mantel-Cox) test). e N = 15 and 10 biologically independent animals for Lgr5 APC and Lgr5 APC T respectively (under the control of Lgr5-EGFP-IRES-creERT2). (p = 0.1982, as determined by Log-rank (Two-tailed Mantel-Cox test). The same Lgr5 APC control cohort was used in both d and e as well as in Fig. 4f and Supplementary Fig. 6D. f Quantification of intestinal tumour burden following APC loss in the Lgr5 stem cell compartment. Tumour burden is determined as percentage of intestine which is covered by lesion or adenomas. Lgr5-EGFP-IRES-creERT2 Apcfl/fl (APC; n = 9 biologically independent animals) vs Lgr5-EGFP-IRES-creERT2 Apcfl/fl Vav2-/-, Vav3-/- (APC V2V3; n = 6 biologically independent animals) p = >0.9999 as determined by a two-tailed Kruskal–Wallis with Dunn’s multiple comparisons test. Lgr5-EGFP-IRES-creERT2 Apcfl/fl (APC) vs Lgr5-EGFP-IRES-creERT2 Apcfl/fl Tiam1-/- (APC T; n = 7 biologically independent animals), p = 0.7532 as determined by a two-tailed Kruskal–Wallis with Dunn’s multiple comparisons test. Data are presented as mean values ±SD. g Solid adenomas (H&E) were observed in each genotype, indicated by the arrow. Scale bar represents 500 µm.
Fig 2: Loss of three GEFs is able to suppress the loss of Apc phenotype.a RNAscope staining for Vav2 in intestine from Vil-CreERT2 Apcfl/fl (APC) and Vil-CreERT2 Apcfl/fl Tiam1-/- (APC T) intestines. Scale bar represents 100 µm. b Quantification of Vav2 RNAscope in Vil-CreERT2 Apcfl/fl (APC) and Vil-CreERT2 Apcfl/fl Tiam1-/- (APC T) intestines. N = 5 biologically independent animals for each genotype *p = 0.0159 as determined by a two-tailed Mann–Whitney test. Data are presented as mean values ±SD. c Images for H&E and BrdU incorporation and RNAscope for Lgr5 and Olfm4 on wild-type (WT), Vav2-/-, Vav3-/ Tiam1-/- (V2V3T), Vil-CreERT2 Apcfl/fl (APC) and Vil-CreERT2 Apcfl/fl, Vav2-/- and Vav3-/-Tiam1-/- (APC V2V3T). Scale bar represents 100 µm for H&E and BrdU and 50 µm for RNAscope images. d Quantification of BrdU positive cells. N = 7, 6 and 8 biologically independent animals for wild-type, Vil-CreERT2 Apcfl/fl (APC) and Vil-CreERT2 Apcfl/fl, Vav2-/- and Vav3-/- Tiam1-/- (APC V2V3T) respectively. WT vs APC ***p = 0.0006, APC vs APC V2V3T *p = 0.0013 as determined by a one-tailed Mann–Whitney test. Control data (APC) as in Fig. 3c. Data are presented as mean values ±SD. e Quantification of clonogenicity assay of intestinal organoids. N = 6 biologically independent cell lines for both Vil-CreERT2 Apcfl/fl (APC) and Vil-CreERT2 Apcfl/fl, Vav2-/-, Vav3-/- Tiam1-/- (APC V2V3T). *p = 0.0260 as determined by a two-tailed Mann–Whitney test. f Compared to Lgr5-EGFP-IRES-creERT2 Apcfl/fl (Lgr5 APC), Lgr5-EGFP-IRES-creERT2 Apcfl/fl Vav2-/-, Vav3-/- Tiam1-/- (Lgr5 APC V2V3T) mice have a significant survival advantage. N = 19 and 15 biologically independent animals for Lgr5 APC and Lgr5 APC V2V3T respectively. **p = 0.0091 as determined by a two-tailed Log-rank (Mantel-Cox) test. Lgr5 APC control cohort is the same cohort as used in Fig. 3d, e as well as Supplementary Fig. 6D. Data are presented as mean values ±SD. g Quantification of intestinal tumour burden at clinical endpoint following APC loss in the Lgr5 stem cell compartment. N = 8 and 9 biologically independent animals for Lgr5-EGFP-IRES-creERT2 Apcfl/fl (Lgr5 APC) and Lgr5-EGFP-IRES-creERT2 Apcfl/fl Vav2-/-, Vav3-/- Tiam1-/- (Lgr5 APC V2V3T) respectively. p = 0.9522 as determined by a two-tailed Mann–Whitney test. Data are presented as mean values ±SD. h Solid adenomas (asterisk) were observed in the Lgr5-EGFP-IRES-creERT2 Apcfl/fl (Lgr5 APC) cohort, whereas cystic adenomas were observed (H&E) in Lgr5-EGFP-IRES-creERT2 Apcfl/fl Vav2-/-, Vav3-/- Tiam1-/- (Lgr5 APC V2V3T) and Lgr5-EGFP-IRES-creERT2 Apcfl/fl, Rac1fl/fl (Lgr5 APC Rac1), as indicated by arrows. Scale bar represents 100 µm. i Intestinal tumour number in an AOM-DSS colitis-associated model of tumourigenesis. Study was carried out on wild-type mice or Vav2-/-, Vav3-/- Tiam1-/- mice. N = 9 and 4 biologically independent animals for wild-type or Vav2-/-, Vav3-/- Tiam1-/- respectively. **p = 0.0042 as determined by a one-tailed Mann–Whitney Test. Data are presented as mean values ±SD.
Fig 3: VAV3 and TIAM1 are upregulated following APC loss.a Heatmap derived from RNA-seq analysis comparing whole tissue from wild-type (Vil-CreERT2) and APC intestines (Vil-CreERT2 Apcfl/fl) n = 3 biologically independent animals for both APC and WT intestinal tissue. Log2FC of GEF expression displayed on the right, genes significantly deregulated displayed in bold (FDR < 0.05) displayed in bold. b RNAscope of Vav2 in the intestinal epithelium of Vil-CreERT2 Apcfl/fl (APC) and Vil-CreERT2 Apcfl/fl, Vav3-/- (APC V3) mice, Scale bar represents 100 µm. c Quantification of Vav2 RNAscope from Vil-CreERT2 Apcfl/fl and Vil-CreERT2 Apcfl/fl Vav3-/- intestines. N = 5 biologically independent animals for each genotype. P = 0.0079 as determined by a two-tailed Mann–Whitney test. Data are presented as mean values ±SD.
Fig 4: Loss of the triple GEFs results in a downregulation of junctional RAC1 activity.a Immunoprecipitation of Active RAC1 from intestinal epithelium, blotted with total RAC1 and the corresponding scoring. n = 3 biologically independent samples for Vil-CreERT2 Apcfl/fl (APC) and Vil-CreERT2 Apcfl/fl, Vav2-/-, Vav3-/- Tiam1-/- (APC V2V3T). *p = 0.00799 as determined by a one-tailed Mann–Whitney test. Data are presented as mean values ±SD. b FLIM-FRET analysis of organoids day 3 post isolation from Vil-CreERT2 Apcfl/fl (APC) and Vil-CreERT2 Apcfl/fl Vav2-/-, Vav3-/-, Tiam1-/- (APC V2V3T) Scale bar represents 20 µm. c Quantification of FLIM-FRET analysis at cell–cell contacts. N = 3 biologically independent cell lines derived from independent mice for each genotype. *p = 0.0426 as determined a two-tailed unpaired T-test. Data are presented as mean values ±SD.
Fig 5: Oncogenic mutation of KRAS drives resistance to RAC-GEF depletion.a Quantification of BrdU positive cells. N = 5, 5 and 10 biologically independent animals for Vil-CreERT2 Apcfl/fl KRasG12D (APC KRAS), Vil-CreERT2 Apcfl/fl, KRasG12D, Rac1fl/fl (APC KRAS Rac1fl/fl) and Vil-CreERT2 Apcfl/fl, KRasG12D, Vav2-/-, and Vav3-/- Tiam1-/- (APC KRAS V2V3T) respectively. APC KRAS vs APC KRAS Rac1fl/fl p = 0.0461 APC KRAS vs APC KRAS V2V3 p = 0.9363 as determined by a two-tailed Kruskal–Wallis statistical analysis with Dunn’s mutltiple comparisons test (under the control of Vil-CreERT2). Data are presented as mean values ±SD. b Representative images for BrdU incorporation of Vil-CreERT2 Apcfl/fl KRasG12D (APC KRAS), Vil-CreERT2 Apcfl/fl, KRasG12D, Rac1fl/fl (APC KRas Rac1fl/fl) and Vil-CreERT2 Apcfl/fl, KRasG12D, Vav2-/- and Vav3-/- Tiam1-/- (APC KRAS V2V3T). Scale bar represents 100 µm. c Representative images of RNAscope for Lgr5 and Olfn4 and IHC for CD44 and SOX9 Vil-CreERT2 Apcfl/fl KRasG12D (APC KRAS), Vil-CreERT2 Apcfl/fl, KRasG12D, Rac1fl/fl (APC KRAS Rac1fl/fl) and Vil-CreERT2 Apcfl/fl, KRasG12D, Vav2-/- and Vav3-/- Tiam1-/- (APC KRAS V2V3T). Scale bar represents 100 µm. d Quantification of Lgr5 RNAscope. Vil-CreERT2 Apcfl/fl KRasG12D (APC KRAS vs Vil-CreERT2 Apcfl/fl, KRasG12D, Rac1fl/fl (APC KRAS Rac1fl/fl) p = 0.1105 APC KRAS vs Vil-CreERT2 Apcfl/fl, KRasG12D, Vav2-/-, Vav3-/- Tiam1-/- (APC KRAS V2V3T). n = 6, 5 and 8 biologically independent animals for APC KRAS, APC KRAS Rac1fl/fl, and APC KRAS V2V3T respectively. p = >0.999 as determined by a two-tailed Kruskal–Wallis statistical analysis with Dunn’s mutltiple comparisons test (under the control of Vil-CreERT2). Data are presented as mean values ±SD. e Quantification of Olfm4 RNAscope. Vil-CreERT2 Apcfl/fl KRasG12D (APC KRAS) vs Vil-CreERT2 Apcfl/fl, KRasG12D, Rac1fl/fl (APC KRAS Rac1fl/fl) p = 0.356 APC KRAS vs Vil-CreERT2 Apcfl/fl, KRasG12D, Vav2-/-, Vav3-/- Tiam1-/- (APC KRAS V2V3T). n = 6, 4 and 9 biologically independent animals for APC KRAS, APC KRAS Rac1fl/fl and APC KRAS V2V3T respectively. p = 0.9381 as determined by a two-tailed Kruskal–Wallis statistical analysis with Dunn’s mutltiple comparisons test (under the control of Vil-CreERT2). Data are presented as mean values ±SD. f Quantification of CD44 IHC. Vil-CreERT2 Apcfl/fl KRasG12D (APC KRAS) vs Vil-CreERT2 Apcfl/fl, KRasG12D, Rac1fl/fl (APC KRAS Rac1fl/fl) p = 0.2061 APC KRAS vs Vil-CreERT2 Apcfl/fl, KRasG12D, Vav2-/-, Vav3-/- Tiam1-/- (APC KRAS V2V3T). n = 7, 4 and 8 biologically independent animals for APC KRAS, APC KRAS Rac1fl/fl and APC KRAS V2V3T respectively. p = >0.999 as determined by a two-tailed Kruskal–Wallis statistical analysis with Dunn’s mutltiple comparisons test (under the control of Vil-CreERT2). Data are presented as mean values ±SD. g Quantification of SOX9 IHC. Vil-CreERT2 Apcfl/fl KRasG12D (APC KRAS) vs Vil-CreERT2 Apcfl/fl, KRasG12D, Rac1fl/fl (APC KRAS Rac1fl/fl) p = >0.999 APC KRAS vs Vil-CreERT2 Apcfl/fl, KRasG12D, Vav2-/-, Vav3-/- Tiam1-/- (APC KRAS V2V3T). n = 7, 5 and 9 biologically independent animals for APC KRAS, APC KRAS Rac1fl/fl and APC KRAS V2V3T respectively p = 0.4088 as determined by a two-tailed Kruskal–Wallis statistical analysis with Dunn’s mutltiple comparisons test (under the control of Vil-CreERT2). Data are presented as mean values ±SD.
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