EMBO/SourceData huggingface dataset:
Figure 6(A) HeLa cells were subjected to IFNγ induction and reinduction regime as outlined in Figure 1A with 2 days and 7 days recovery time (primed state) after IFNγ washout. Cell extracts were prepared at indicated timepoints, processed for western blotting and probed for STAT1, pSTAT1-Y701 and pSTAT1-S727. α-Tubulin (Tubulin) as a loading control.(C) (H) STAT1KO::STAT1-S727E and STAT1KO::STAT1-S727D expressing cells or their parental controls (WT) were subjected to IFNγ induction and reinduction regime as outlined in Figure 1A, RNA was isolated and GBP5 mRNA level was determined by RT-qPCR and normalized to ACTB mRNA level. Statistical significance was determined using Ordinary one-way ANOVA. Data are shown as mean (error bars, SD; n=3 biological replicates).(E) Characterization of dTAG-induced destruction and recovery. STAT1-EGFP-dTAG expressing cells were induced with IFNγ for 24h, then treated with dTAG13 (100 nM) for DMSO vehicle control (I) or STAT1 depletion (II) for 24h. To determine recovery of STAT1 expression, cells were washed three times with medium and harvested 48h after IFNγ washout (120 hour timepoint, lane 4). In Parallel, (III) cells were treated with dTAG13 (100 nM) for 48h during 24h induction with IFNγ. Immunoblot of STAT1-GFP-dTAG at indicated treatments confirm STAT1 depletion and recovery, respectively. α-Tubulin (Tubulin) was used as a loading control.(G) RNA was isolated and GBP5 mRNA level as indicated in (F) was determined by RT-qPCR, normalized to ACTB mRNA level. Statistical significance was determined using two-way ANOVA. Data are shown as mean (error bars, SD; n=3 biological replicates)
Figure 1Western blot analysis (B) of DCLK1 in aortas of LFD and HFD-fed ApoE-/- mice. β-actin was used as the loading control (n=10 biological replicates).(E) Representative immunofluorescence staining of CD31 (green) and DCLK1 (red) in aortic roots (scale bar=25 μm). (F) Representative immunofluorescence staining of α-SMA (green) and DCLK1 (red) in aortic roots (scale bar=25 μm). (G) Representative immunofluorescence staining of F4/80 (green) and DCLK1 (red) in aortic roots (scale bar=25 μm). White arrows indicate co-location of F4/80 and DCLK1.(H-I) Time-course of DCLK1 induction in response to oxLDL in mouse primary peritoneal macrophages (MPMs). MPMs were exposed to oxLDL (50 μg/mL) for indicated time. Western blot analysis (H) and densitometric quantification (I) of DCLK1 were shown. GAPDH was used as the loading control (n=3 biological replicates). Data information: Data were shown as mean ± SEM; *, P < 0.05; ns, not significant, two-tailed unpaired Student's t-test.
Figure 2Representative en face Oil Red O staining (A) of Oil Red O-positive plaque lesion area in aortas. Plaque area was defined as percentage of total surface area of the aorta (n=10 biological replicates).Representative images (C) of plaque lesion area in aortic arches (n=10 biological replicates).(E-F) Representative images of Oil Red O staining (E) and quantification (F) of atherosclerotic lesion in aortic roots (scale bar=250 μm, n=10 biological replicates). Plaque area was quantified by the proportion of plaque area to aortic root area. Data information: Data were shown as mean ± SEM; *, P < 0.05; ns, not significant, two-tailed unpaired Student's t-test.(G-H) Representative images of Masson's Trichrome staining (G) and quantification (H) for collagen deposition in aortic roots (scale bar=25 μm, n=10 biological replicates). Data information: Data were shown as mean ± SEM; *, P < 0.05; ns, not significant, two-tailed unpaired Student's t-test.
Figure 3Representative immunofluorescence staining images (A) of F4/80 (green) in aortic roots. Tissues were counterstained with DAPI (blue). Scale bar=250 μm, n=10 biological replicates.Representative immunohistochemistry staining images of Ly6G (C in aortic roots (scale bar=25 μm, n=10 biological replicates).Representative immunohistochemistry staining images of Ly6C (E in aortic roots (scale bar=25 μm, n=10 biological replicates).Scatter diagram (G) and quantification of monocyte in plasma measured by an automated blood cell analyzer (n=10 biological replicates). SSC, side scatter; SFL, side fluorescence. Data information: Data were shown as mean ± SEM; *, P < 0.05; ns, not significant, two-tailed unpaired Student's t-test.(J-K) Protein (J) and mRNA (K) levels of inflammatory cytokines TNF-α and IL-6 in serum and aortas. The values of mRNA levels were normalized to Rn18s (n=10 biological replicates). Data information: Data were shown as mean ± SEM; *, P < 0.05; ns, not significant, two-tailed unpaired Student's t-test.(L) mRNA levels of proinflammatory chemokines and adhesion molecules in aortas (n=10 biological replicates). The values were normalized to Rn18s. Data information: Data were shown as mean ± SEM; *, P < 0.05; ns, not significant, two-tailed unpaired Student's t-test.
Figure 4(A-B) Mouse primary peritoneal macrophages (MPMs) isolated from DCLK1f/f and DCLK1MCKO mice were challenged with oxLDL (50 μg/mL) for 24 h. Protein levels of TNF-α (A) and IL-6 (B) were analyzed using ELISA (n=3 biological replicates). Data information: Data were shown as mean ± SEM; *, P < 0.05; ns, not significant, two-tailed unpaired Student's t-test.(C-D) MPMs isolated from DCLK1f/f and DCLK1MCKO mice were challenged with oxLDL (50 μg/mL) for 6 h. mRNA levels of Tnf-α (C) and Il-6 (D) were determined via RT-qPCR (n=3 biological replicates). The values were normalized to β-actin. Data information: Data were shown as mean ± SEM; *, P < 0.05; ns, not significant, two-tailed unpaired Student's t-test.(J, K) Western blot analysis (J) and densitometric quantification (K) of p-p65 and IκBα. GAPDH and p65 were used as loading controls (n=3 biological replicates). Data information: Data were shown as mean ± SEM; *, P < 0.05; ns, not significant, two-tailed unpaired Student's t-test.(L-M) MPMs were treated as described in panel (J). Western blot analysis (L) and densitometric quantification (M) of p65 in nucleus and cytoplasm. GAPDH was used as the loading control for cytosolic fractions. Lamin B1 was used as the loading control for nuclear fractions (n=3 biological replicates). Data information: Data were shown as mean ± SEM; *, P < 0.05; ns, not significant, two-tailed unpaired Student's t-test.(N-O) MPMs were treated as described in panel (J). Representative immunofluorescence staining images (N) and quantification (O) of NF-κB p65 (red) translocating into nucleus in MPMs. Cells were counterstained with DAPI (blue). Scale bar=25 μm, n=3 biological replicates. Data information: Data were shown as mean ± SEM; *, P < 0.05; ns, not significant, two-tailed unpaired Student's t-test.(P-Q) Representative immunofluorescence staining images (P) and quantification (Q) of p-p65 (red) in aortic roots. Tissues were counterstained with DAPI (blue). Scale bar=100 μm, n=10 biological replicates. p-p65 area was quantified by the proportion of p-p65 positive area to plaque area. Data information: Data were shown as mean ± SEM; *, P < 0.05; ns, not significant, two-tailed unpaired Student's t-test.
Figure 5(B) 293T cells were transfected with Flag-DCLK1 plasmid for 24 h. Control cells were transfected with empty vector (EV). Levels of Flag were measured by Western blot (n=3 biological replicates). Data information: Data were shown as mean ± SEM; *, P < 0.05; ns, not significant, two-tailed unpaired Student's t-test.(D) Co-immunoprecipitation of DCLK1 and IKKβ in 293T cells transfected with Flag-DCLK1. Flag-DCLK1 was immunoprecipitated by anti-Flag antibody. IgG, immunoglobulin G. (E) Co-immunoprecipitation of DCLK1 and IKKβ in MPMs challenged with oxLDL (50 μg/mL) for 1 h. DCLK1 was immunoprecipitated by anti-DCLK1 antibody.293T cells were co-transfected with Flag-DCLK1 and si-IKKβ for 24 h. Western blot analysis (H) of IκBα, p-IKKβ and p-p65. GAPDH, IKKβ and p65 were used as loading controls (n=3 biological replicates).
Figure 6(B) MPMs were pretreated with DCLK1-IN-1 (5 and 10 μM) or vehicle (DMSO, 1‰) for 1 h, followed by exposure of oxLDL (50 μg/mL) for 24 h. Protein levels of TNF-α and IL-6 were analyzed using ELISA (n=3 biological replicates). Data information: Data were shown as mean ± SEM; *, P < 0.05, two-tailed unpaired Student's t-test.(D-E) MPMs were pretreated with DCLK1-IN-1 (5 and 10 μM) or vehicle (DMSO, 1‰) for 1 h, followed by exposure of oxLDL (50 μg/mL) for 1 h. Western blot analysis (D) and densitometric quantification (E) of IκBα, p-IKKβ and p-p65. GAPDH, IKKβ and p65 were used as loading controls (n=3 biological replicates). Data information: Data were shown as mean ± SEM; *, P < 0.05, two-tailed unpaired Student's t-test.(F-G) MPMs were treated as described in panel (E). Western blot analysis (F) and densitometric quantification (G) of p65 in nucleus and cytoplasm. GAPDH was used as the loading control for cytosolic fractions. Lamin B1 was used as the loading control for nuclear fractions (n=3 biological replicates). Data information: Data were shown as mean ± SEM; *, P < 0.05, two-tailed unpaired Student's t-test.(H-I) MPMs were treated as described in panel (D). Representative immunofluorescence staining images (H) and quantification (I) of NF-κB p65 (red) translocating into nucleus in MPMs. Cells were counterstained with DAPI (blue). Scale bar=25 μm, n=3 biological replicates. Data information: Data were shown as mean ± SEM; *, P < 0.05, two-tailed unpaired Student's t-test.
Figure 7Representative images (C) of plaque lesion area in aortic arches (n=10 biological replicates). The plaque area was quantified by the proportion of plaque area to aortic arches area.(K-N) Protein (K-L) and mRNA (M-N) levels of inflammatory cytokines TNF-α and IL-6 in serum and aortas. The values of mRNA levels were normalized to Rn18s (n=10 biological replicates). Data information: Data were shown as mean ± SEM; *, P < 0.05, two-tailed unpaired Student's t-test.
Figure 1D Microscale thermophoresis (MST) curves for binding of peptides to labelled GyraseA14. The circles indicate the experimental values and the solid lines indicate the fitted fluorescence values obtained upon analysis using the MO.Affinity Analysis software (version 2.2.5, NanoTemper Technologies). Data information: In panels D, data are obtained from n = 3 biological replicates; wherein for each biological replicate, the experiment is performed in duplicates (technical duplicates for each biological replicate). In panel D, the data are presented as mean ± SD values obtained from all the replicates.
Figure 2B, In vitro bactericidal action of the CP1-WT peptide on E. coli Top10 cells. B Growth profile of E. coli Top10 in the presence of increasing concentrations of the CP1-WT peptide. Data information: data are obtained from n = 3 biological replicates; wherein for each biological replicate, the experiment is performed in duplicates (technical duplicates for each biological replicate). In panels B data are presented as mean ± SD values obtained from all the replicates. In panel B, N.T. - No treatment; 4.5% DMSO (solvent control); CP1-WT (test peptide); CP1-Mut (negative control peptide) and Cip - 10µg/mL ciprofloxacin (positive control).C In vitro bactericidal action of the CP1-WT peptide on E. coli Top10 cells. C Estimation of Minimum Inhibitory Concentration (MIC), indicated in terms of OD600 and Minimum Bactericidal Dose (MBD), indicated in terms of CFU/mL, show that the CP1-WT peptide has similar MIC and MBD values of 40µM (~130µg/mL). Data information: data are obtained from n = 3 biological replicates; wherein for each biological replicate, the experiment is performed in duplicates (technical duplicates for each biological replicate). In panels C, data are presented as mean ± SD values obtained from all the replicates.
Figure 3A-D Peptide-induced membrane damage was studied by confocal microscopy and measured by propidium iodide (PI) influx (% PI uptake) in (A) S. Typhimurium, (B) A. baumannii, (C) S. aureus and (D) P. aeruginosa. Data information: In panels A-D, n = 5 biological replicates; scale bars: 5µm. Representative images are shown. In the bar graphs, data are presented as mean ± SD values of percentage of propidium iodide uptake (% PI uptake), averaged across all the replicates. 3%, 7%, 8% and 3% DMSO, and 80µM, 50µM, 50µM and 80µM of the peptides in the respective DMSO concentrations were used to treat S. Typhimurium, A. baumannii, S. aureus and P. aeruginosa respectively.E-H Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Dose (MBD) estimation of CP1-WT on stationary phase cells of (E) S. Typhimurium, (F) A. baumannii, (G) S. aureus and (H) P. aeruginosa. No bactericidal action is observed for P. aeruginosa. In each of the panels and its corresponding bar graph, the black lines and black bars represent the MIC data indicated by their OD600 values, and the red lines and red bars represent the MBD data indicated by their CFU/mL values. The bar graphs in each panel depict control reactions used for each strain where the cells were treated with DMSO, peptides and ciprofloxacin (Cip - postive control; 10µg/mL). The strain of A. baumannii used was reported to be ciprofloxacin-resistant (Nagarajan et al, 2018) as is observed here (inset in panel F). In panels E-H, n = 5 biological replicates. Data are presented as mean ± SD values, averaged across all the replicates. For the bar graphs in these panels, 7%, 13%, 8% and 4% DMSO, and 80µM, 100µM, 100µM and 50µM of the peptides, were used for S. Typhimurium, A. baumannii, S. aureus and P. aeruginosa respectively. N.T- No treatment; WT - CP1-WT peptide; Mut - CP1-Mut peptide (negative control). P values are calculated by Student's unpaired t-test; nsP > 0.05; *** P < 0.001; **** P < 0.0001 Student's unpaired t-test.
Figure 4A-D Transmission electron micrographs of the indicated bacterial strains (left) depicting the loss of membrane integrity upon treatment with the CP1-WT peptide which is time-dependent. Arrows indicate membrane blebs, generalized membrane damage and the exclusion of cytoplasmic contents out of the cell. 1h, 2h and 3h indicate different treatment times of 1, 2 and 3 hours respectively with the CP1-WT and CP1-Mut peptides as indicated in the panels. Untreated cells, used as controls, are also shown. Scale bars: 2μm. Data information: In each panel, across each fluorescent channel, the unstriped and striped bars indicate the data for cells treated with DMSO or peptides for 0 (immediate addition) and 2 hours respectively. nsP > 0.05, * P < 0.05, ** P < 0.01; Student's unpaired t-test. WT and Mut indicate CP1-WT and CP1-Mut treatments respectively.E-H Mean fluorescence intensities across the three fluorescent channels (FM 4-64FX, FAM and DAPI fluorophores) were estimated from confocal microscopy experiments to understand if the peptide was bound to the membrane or internalized in the cytosol of the bacteria indicated in each of the panels (top). Triple staining procedure with FM 4-64FX (membrane-binding dye; red), FAM-conjugated peptides (green) and DAPI (nuclear staining dye; blue) was used and the mean fluorescence intensities across these three fluorescent channels were calculated using ZEN 2.3 software. The bacterial cells were treated with 2% DMSO/ FAM-labelled CP1-WT/ FAM-labelled CP1-Mut for 0 (0hr) or 2 hours (2hr), followed by staining with FM 4-64FX and DAPI consecutively. The 0th hour data represents the data obtained after immediate addition of DMSO or peptide. In each panel, green circles represent autofluorescence (cells emitting light of wavelength corresponding to that observed for FAM fluorophore) of DMSO-treated cells at 0 and 2 hours. Data information: In panels E-H, n = 5 biological replicates are used. In each of these panels, the mean ± SD intensity values (averaged across all the replicates) obtained across each fluorescent channel (FM 4-64FX, FAM and DAPI) are presented for a particular bacterial strain (indicated on top of the panel). In each panel, across each fluorescent channel, the unstriped and striped bars indicate the data for cells treated with DMSO or peptides for 0 (immediate addition) and 2 hours respectively. nsP > 0.05, * P < 0.05, ** P < 0.01; Student's unpaired t-test. WT and Mut indicate CP1-WT and CP1-Mut treatments respectively.
Figure 5In vivo toxicity determination of the CP1-WT peptide using a murine model. BALB/c mice (Five mice per cohort) were intraperitoneally injected with 1.6% DMSO or varying peptide doses as indicated and monitored for 14 days. A Changes in body weights relative to day 0 were noted. The mean percentage changes in body weights for each cohort are plotted across all the days. Data information: n = 2 biological replicates with 5 mice per cohort were used. In panel A, across the entire experimental tenure of 14 days, data are presented as mean ± SD values for each cohort, averaged across both the replicates for all the five mice (studied in the respective cohort). Multiple t test with Bonferroni Dunn correction was used to compare percentage change in body weights across DMSO-treated and the respective peptide treated cohorts (ns P > 0.05).In vivo toxicity determination of the CP1-WT peptide using a murine model. BALB/c mice (Five mice per cohort) were intraperitoneally injected with 1.6% DMSO or varying peptide doses as indicated and monitored for 14 days. B, After 14 days post peptide treatment, blood was collected from the animals by retro-orbital puncture and analysed for (B) alanine transaminase (SGPT), alanine aminotransferase (SGOT), alkaline phosphatase (ALP) Relative to the values obtained for the DMSO cohort, insignificant changes in serum levels of these parameters indicate that the peptide shows no significant hepatotoxic (as seen in panel B) Data information: biological replicates with 5 mice per cohort were used. In the panels B representative data for only one biological replicate across all the cohorts (five mice per cohort) are presented as mean ± SD values. P values were calculated in comparison to the DMSO-treated cohorts using Mann-Whitney rank sum test (ns P > 0.05).In vivo toxicity determination of the CP1-WT peptide using a murine model. BALB/c mice (Five mice per cohort) were intraperitoneally injected with 1.6% DMSO or varying peptide doses as indicated and monitored for 14 days. C After 14 days post peptide treatment, blood was collected from the animals by retro-orbital puncture and analysed for (C) creatinine and blood urea nitrogen (BUN) levels. Relative to the values obtained for the DMSO cohort, insignificant changes of these parameters indicate that the peptide shows no significant nephrotoxic effects (as seen in panel C). Data information: n = 2 biological replicates with 5 mice per cohort were used. In panels C, representative data for only one biological replicate across all the cohorts (five mice per cohort) are presented as mean ± SD values. For panels C, P values were calculated in comparison to the DMSO-treated cohorts using Mann-Whitney rank sum test (ns P > 0.05).
Figure 1(F) UMAP plot showing the expression levels of EMT markers. The magenta dotted lines indicate cluster 6 (partial EMT).
Figure 2(E), (F) Heatmap of DEGs in ROR1high and ROR1low cells in the S2-VP10 xenograft (E) or PDO#1 xenograft (F).
Figure 3(A) Kaplan-Meier disease-specific survival curves based on ROR1 expression levels in PDAC patients from the TCGA database (n = 154) . Data information: Graphs are presented as mean ± s.e.m., *, p < 0.05; log-rank test (A)(C) FACS gating to sort ROR1high and ROR1low cells and the number of organoids or colonies (n = 3 or n = 5, biological replicates). Mouse cells expressing H-2Kd/H-2Dd were eliminated. Data information: Graphs are presented as mean ± s.e.m., *, p < 0.05; two-sided t-test (C)(G) Representative images of tissue stained with H&E and AB/PAS, and for ROR1 in the PDO#1 xenograft and of tumor derived from ROR1high or ROR1low cells in the PDO#1 xenograft. Data information: Scale bars, 100 µm (G).
Figure 4(H) The expression level of AURKB in PANC-1 cells transfected with control or ROR1 siRNA and in ROR1high or ROR1low cells from S2-VP10 and PDO#1 xenografts (n = 3). Data information: Data are presented as mean ± s.e.m., two-sided t-test. *, p < 0.05; n.s., not significant.(I) Western blot analysis of ROR1, AURKB, and β-actin in S2-VP10 and S2-013 cells transfected with control or ROR1 siRNA.(J) Treatment of PDO#1 and S2-VP10 organoids with DMSO (vehicle) or Aurora kinase inhibitors (n = 3, biological replicates). Tozasertib, pan-Aurora kinase inhibitor; Barasertib, selective Aurora B kinase inhibitor. Representative images of organoids are shown. An area of 2,000 μm2 and more was identified as an organoid. The area of organoids is shown in the violin plot. Black or white solid lines indicate the median value for each violin. Data information: Scale bars 1 mm (J) Data are presented as mean ± s.e.m., two-sided t-test. *, p < 0.05; n.s., not significant.(K) Organoid formation assay of ROR1high or ROR1low cells derived from S2-VP10 EGFP (control) or S2-VP10 AURKB-EGFP xenografts (n = 3, biological replicates). Representative images and the number of organoids are shown. Data information: Scale bars, 1 mm (K). Data are presented as mean ± s.e.m., two-sided t-test. *, p < 0.05; n.s., not significant.(L) Western blot analysis of ROR1, AKT, phospho-AKT (Ser473), and β-actin in S2-VP10 and S2-013 cells transfected with control or ROR1 siRNA.(N) Western blot analysis of ROR1, c-Myc, phospho-RB (Ser780), E2F1, and β-actin in S2-VP10 and S2-013 cells transfected with control or ROR1 siRNA.
Figure 5(C) Tumor growth curve of the four groups (control, Dox only, gemcitabine only, and Dox + gemcitabine) (n = 8). The endpoint for each group is shown by a slash. Data information: Data are presented as mean ± s.e.m., two-sided t-test. *, p < 0.05.
Figure 6(A)-(C) Representative images of ROR1, mCherry, and aSMA staining in the primary tumor (pancreas) (A) and metastatic lesions in the lung (B) and mesenteric lymph node (C) after orthotopic transplantation of S2-VP10-mCherry cells into the pancreas of BRJ mice. Data information: Scale bars, 100 µm(D), (E) Representative images of S2-013 orthotopic xenografts stained with H&E and for ROR1 in the pancreas (D) and mesenteric lymph node (E). Data information: Scale bars, 100 µm
Figure 7(A), H3K4me1, K3K4me3, H3K27ac, and open chromatin profiles around the ROR1 gene by CUT&RUN and ATAC-sequencing in cultured S2-VP10 cells (A) The ROR1 promoter (blue-shaded boxes) and enhancer elements (green-shaded box) of ROR1 are indicated.
Figure 8(E) YAP occupancy at the ROR1 enhancer as determined by ChIP-qPCR. The known YAP targets, CTGF and CYR61 promoters, were tested as positive controls, and ROR1 intron 1 and AFM promoter were tested as negative controls (n = 4, biological replicates).(G) Western blot analysis of YAP, TAZ, ROR1, and β-actin in S2-VP10 cells transfected with control or YAP1/WWTR1 siRNA.(I) Relative expression of ROR1 and YAP target genes (CTGF and CYR61) in S2-VP10 cells treated with DMSO or verteporfin (VP) (n = 3, biological replicates). mRNA levels are normalized to that of RPS18.(M) Relative luciferase activity using the ROR1 reporter in S2-VP10 cells treated with DMSO or BET inhibitor (JQ1) (n = 3, biological replicates). Data information: Data are presented as mean ± s.e.m., *, p < 0.05. ; two-sided t-test (M)(O), Treatment of PDO#1 and S2-VP10 organoids with DMSO or JQ1 (n = 3, biological replicates). Relative expression of ROR1 (O), mRNA levels are normalized to that of RPS18. Data information: Data are presented as mean ± s.e.m., *, p < 0.05. ; two-sided t-test (O)(P) Treatment of PDO#1 and S2-VP10 organoids with DMSO or JQ1 (n = 3, biological replicates). representative images of organoids, and organoid area (P) are shown. The area of organoids is shown in the violin plot. Black or white solid lines indicate the median value for each violin. Data information: Scale bars, 1 mm (P). Data are presented as mean ± s.e.m., *, p < 0.05. ; two-sided t-test (P).
Figure 1CD14+ monocytes were cultured with M-CSF, IL-4 and TNFα and analyzed by scRNA-seq at different time points (n=2 biological replicates). (D) Expression patterns of monocytes (top row), macrophages (middle row) and DC (bottom row) markers.