Fig 2: MMP13 enzyme activity, subcellular distribution, DCLK1-L and DCLK1-S promoter-reporter assays.RKO cells were treated with PMA or PMA plus selective and potent MMP13 inhibitor WAY 170523 at varying doses as indicated for 30 min followed by measurement of enzymatic activity. A. Reference curve showing relative fluorescence units (RFUs). B, C. Dose-dependent decrease in MMP13 enzymatic activity (n = 3 independent experiments; *p<0.05). D. Western blots showing relative protein abundance in three colon cancer cell lines. Boxed area represents levels of the indicated proteins in RKO cells. E. Subcellular compartmentalization of proteins from RKO cells. 1: Cytosolic Fraction, 2: Nuclear Fraction, 3: Membrane Fraction, 4: Cytoskeletal fraction (n = 3 independent experiments). F. Promoters for DCLK1-L and DCLK1-S isoforms were cloned and transfected in HEK293 cells and promoter-reporter activity assays were performed using Dual-Luciferase Reporter Assay System (E1910, Promega, Madison, WI). Luminescence was measured using a BioTek Synergy Neo luminometer. P values as indicated; n = 3 independent experiments.

Fig 3: Co-localization of MMP13 with DCLK1 in various models of colitis.A. Paraffin sections prepared from the colons of Control, CR or CR+DBZ group of Dclk1ΔIEC mice were subjected to IMC. MMP13 (green) is overlayed with DCLK1 (red) and DNA (blue). Boxed areas in CR group indicates significant co-localization of MMP13 with DCLK1. Scale bars as indicated (100 μm); 8-10 mice /group. B. Box plots of DCLK1 and MMP13 counts based on IMC data set in the Control, CR, CR+DBZ groups. C. DCLK1 and MMP13 staining from IMC in control and Dclk1-SOE group after DSS-induced colitis. Lane 1 DCLK1(red) staining is overlayed with DNA (blue), Lane 2 MMP13 (red) staining is overlayed with DNA (blue), Lane 3 DCLK1(red)/MMP13(green)/DNA(Blue) colocalization in the Dclk1-SOE mice. P values as indicated. D. MMP13 promoter-reporter activity (*, **p<0.05; n = 3 independent experiments). Ei. Western blot data of HCT116 colon cancer cells treated with PMA and PMA+DBZ. Eii. In silico molecular docking studies to predict MMP13 and DCLK1 binding. CR: Citrobacter rodentium, CR+DBZ: Citrobacter rodentium + Dibenzazepine (DBZ), PMA: Phorbol 12-Myristate 13-Acetate.

Fig 4: DCLK1-S persists during colon inflammation and displays an inverse correlation with FOXD3.A. Using the IMC data sets from Dclk1ΔIEC mice, DCLK1 (red) is overlayed with FoxD3 (green) in the indicated groups. B. Development of DCLK1-SOE mice. Mouse genomic fragments containing homology arms (HAs) were amplified using high fidelity Taq and were subsequently assembled into a targeting vector together with recombination sites and selection markers shown. Dclk1-Short+19-floxed mice were bred with Villin-Cre mice to generate Dclk1-Short+19-floxed; Villin-Cre DCLK1-SOE mice. Mice were subsequently genotyped. Ci. WT littermates and DCLK1-SOE mice received 4% DSS in drinking water for 7 days and euthanized after 2 days of normal water. Representative H&E staining (n = 6 mice/genotype) to visualize crypt architecture. Scale bar = 150 μm. Cii. Bar graph showing histological scores. D. IMC showing DCLK1(red) staining overlayed with DNA (blue), FoxD3 (red) staining overlayed with DNA (blue) and DCLK1 (red)/FoxD3 (green) and DNA (blue) colocalization in the Control or DSS-treated DCLK1-SOE mice. Scale bars as indicated; n = 6 mice/genotype. E. Percent fold change based on staining intensity in DCLK1+ cells, fold change of DCLK1 is in blue, fold change of FoxD3 is in red, and the ratio of both DCLK1/FoxD3 is in orange.

Fig 5: Molecular docking and in vitro kinase assays.A-D. Molecular docking study showing the interaction of proMMP13 (A) and mature MMP13 (C) with DCLK1-S. B. Ser 73 and Ser 114 highlighted in green in proMMP13, represent the phosphorylation sites for DCLK1-S. D. Arg207 and Gln211 are donor sites from MMP13 in mature protein close to Thr197 (circled). E. Both rhMMP13 and rhDCLK1 were incubated in kinase buffer in presence or absence of ATP at 30 °C for 1 hr. rhMMP13 didn’t show any autophosphorylation in the same condition. Phosphorylation was detected by pan phospho-Serine/Threonine antibody. F. rhMMP13 was incubated with rhDCLK1 in presence of ATP at 30 °C for 1 hr. Phosphorylated rhMMP13 was detected with immunoblotting using pan phospho-Serine/Threonine antibody. Representative immunoblots showing the levels of phosphorylation of rhMMP13. Right panel represents the Coomassie staining (n = 3 independent experiments). G, H. Protein band intensity ratios, pMMP13/pDCLK1 and pMMP13/Total MMP13, showing rhMMP13 phosphorylation by rhDCLK1.