Fig 1: The ULTR-p38i 2015 inhibits CRC liver metastases.a, Generation of an organoid-based CRC liver metastasis mouse model by splenic injection of KAP organoids into WT mice. b, Representative pictures of KAP metastases stained for p38α, P-p38αT180/Y182, MK2 and P-MK2T334 after 4 days of treatment with 2015 or carrier (n = 4 mice per group). Scale bars, 100 µm. c, Representative pictures of KAP metastases stained for Ki67, yH2A.XS139, cleaved caspase 3, HMGA2 and SA-β-Gal activity after 4 days of treatment with 2015 or carrier (n = 4 mice per group). Scale bars, 100 µm. d,e, Representative pictures of livers (d) and survival analysis of mice (e) with KAP liver metastasis that were treated with 2015, 1639, SKL or carrier (Kaplan–Meier curve; n = 7 (p38α inhibitor groups) or n = 12 (carrier) mice). Statistical significance was calculated using a log-rank test (P < 0.0001). Treatment was started 5 weeks after organoid transplantation. Scale bar, 1 cm. f, Quantification of CD4+ and CD8+ T cells in the CD3+ cell fraction in blood, spleen and tumors of mice with KAP metastases upon 4 days of treatment with 2015 or carrier (determined by FACS measurements; gating strategy in Extended Data Fig. 8a; n = 4 (tissues) or n = 3 (2015-treated tumors); data are presented as the mean ± s.d.). Statistical significance was calculated using a two-tailed Student’s t-test (P < 0.05). g, Quantification of CD69+ cells in tumor-free livers or KAP tumors of mice upon 4 days of treatment with 2015 or carrier (determined by FACS measurements; gating strategy in Extended Data Fig. 8a; n = 4 (tissues) or n = 3 (2015-treated tumors); data are presented as the mean ± s.d.). Statistical significance was calculated using an ANOVA and Tukey’s multiple-comparisons test (P < 0.05). h, Generation of a KAP liver metastasis model in Rag2−/− mice. i, Survival analysis of Rag2−/− mice with KAP liver metastasis that were treated with 2015 or carrier (Kaplan–Meier curve; n = 7 mice). Statistical significance was calculated using a log-rank test (P = 0.0001). The stainings in b and c were independently performed twice, with similar results.Source data
Fig 2: Generation and treatment of CRC cultures.a, Representative pictures of NT-I colonoids after 0, 1 and 5 d of isolation. Scale bar, 100 µm. b, Representative pictures of NT-I colonoids stained for CK20 and CDX2 (n = 2 biologically independent experiments). Scale bars, 100 µm. c, Representative pictures of NT-I and KAP organoids upon withdrawal of R-Spondin-1 (RSPO), Wnt and EGF. Scale bar, 100 µm. d, Representative PCR analysis of cre recombinase-mediated modifications of Trp53 in KAP organoids (in comparison to NT-I, n = 2 biologically independent experiments). 370 bp = floxed (fl), 612 bp = recombined (rb) allele. e, Representative PCR analysis of cre-mediated modifications of LSL-KrasG12D in KAP organoids (in comparison to NT-I, n = 2 biologically independent experiments). 500 bp = floxed (fl), 622 bp = WT and 650 bp = recombined (rb) allele. f, Representative western blot analysis of Apc in NT-I and KAP organoids (cropped blot images, n = 3 biologically independent experiments). g, Representative pictures of KAP organoids stained for CK20 and CDX2 (n = 2 biologically independent experiments). Scale bars, 50 µm. h, Representative pictures of KAP2D cells stained for CK20 and CDX2 (n = 2 biologically independent experiments). Scale bars, 100 µm. i, Schematic outline of medium exchange experiment in KAP2D. j, Representative western blot analysis in KAP2D donor- and KAP2D- recipient cells (cropped blot images, n = 3 biologically independent experiments). k, Cell viability analysis in KAP organoids upon 4 d of treatment with BIRB-796 or DMSO (n = 3 biologically independent experiments, data are presented as mean values +/- SD). l, Representative western blot analysis of p38α, P-p38αT180/Y182, MK2 and P-MK2T334 in KAP2D upon 1 d of treatment with 5 µM SKL, BIRB-796 or DMSO (cropped blot images, n = 3 biologically independent experiments). m, Representative western blot analysis of p38α, P-p38αT180/Y182, MK2 and P-MK2T334 in KAP2D cells upon 1 d of treatment with 1 µM SKL, 1639 or DMSO (cropped blot images, n = 3 biologically independent experiments). The experiments in Extended Data Fig. 1b, d, e, g, h were independently performed twice and the experiments in Extended Data Fig. 1f, j, k, l, m were independently performed three times, all with similar results. Source data
Fig 3: Generation of Myc- and BRAFV600E-driven, murine CRC organoids.a, Representative pictures of KMP organoids stained for CK20 and CDX2 (n = 2 biologically independent experiments). Scale bars, 100 µm. b, Representative western blot analysis in NT-I and KMP organoids (cropped blot images, n = 3 biologically independent experiments). c, Representative pictures of BAP organoids stained for CK20 and CDX2 (n = 2 biologically independent experiments). Scale bars, 100 µm. d, Representative western blot analysis in NT-II and BAP organoids (cropped blot images, n = 3 biologically independent experiments). e, Representative pictures of KMP organoids stained for p38α, P-p38αT180/Y182, MK2 and P-MK2T334 after 1 d of treatment with 1 µM SKL, 1639, 2015, 2545 or DMSO (n = 3 cultures per condition). Scale bar, 50 µm. The experiments in Extended Data Fig. 3a, c, e were independently performed twice and the experiments in Extended Data Fig. 3b, d were independently performed three times, all with similar results.
Fig 4: Analyzing p38α-MK2 signaling in subcutaneous CRCs upon treatment with p38i.Representative pictures of KAP subcutaneous tumors stained for p38α, P-p38αT180/Y182, MK2, P-MK2T334, HSP27, P-HSP27S82, Atf2, P-Atf2T71, Elk1, P-Elk1S383 after 12 d of treatment with SKL, 1639, 2015 or carrier (n = 4 tumors per group). Scale bars, 100 µm. The stainings were independently performed twice with similar results.
Fig 5: Analyzing an ULTRi-p38i therapy in CRC organoids.Representative pictures of KAP organoids stained for p38α, P-p38αT180/Y182, MK2 and P-MK2T334 after 1 d of treatment with 1 µM SKL, 1639, 2015, 2545 or DMSO (n = 3 cultures per condition). Scale bar, 50 µm. The experiments were independently performed twice with similar results.
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