Arwen A. Tisdale

Supplementary Table S1. Compound requisition and reconstitution information

Ligands of known ALP receptors only partially phenocopy ALP signature changes. A and B, Fold change in RNA expression of respective cytokines as determined by RT-qPCR following 24 hours of treatment of C7-TA-PSC cells with A, 10 or 100 μmol/L of the indicated compound, or B, 0.05, 0.5, 5, or 50 μmol/L ALP relative to its vehicle control (0 μmol/L). C, Ratio of Ct values for each gene relative to that of GAPDH in C7-TA-PSC cells at baseline as determined by RT-qPCR. D, Heatmap of relative RNA levels in normal and tumor fibroblasts profiled in a human scRNA-seq dataset (26). E, Fold change in RNA expression for TSPO and ALP signature cytokines in siNTC and siTSPO transfected C7-TA-PSC cells as determined by RT-qPCR. Data in A were collected in technical duplicate and biological triplicate (n = 3). Data in B, C, and E were collected in technical and biological triplicate (n = 3). Data are represented as mean ± SD. N.D. indicates that Ct values for at least two technical replicates of each biological replicate were beyond the limit of detection. ns, not significant; *, P < 0.001; ****, P < 0.0001.

ALP suppresses TLR4 signaling in PDAC CAFs. A, Heatmap of relative RNA expression levels for TLRs in a scRNA-seq profile of human fibroblasts from normal pancreas and PDAC tumor tissues analyzed in ref. (26). B, RT-qPCR validation of TLR4 RNA expression in C7-TA-PSC cells at baseline relative to GAPDH. C, Fold change in cytokine RNA expression levels determined by RT-qPCR following 24 hours of treatment with DMSO, 20 μmol/L ALP, and/or 2.5 μg/mL LPS. Experiments in B and C were conducted in technical and biological triplicate (n = 3). Data are represented as mean ± SD. Statistical analyses in C were conducted using one-way ANOVA with a Tukey correction for multiple comparisons. ns, not significant; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

Unbiased phosphoproteomics reveals ALP-dependent changes in kinase activity. A, Fold change in cytokine RNA expression levels determined by RT-qPCR following treatment of C7-TA-PSC cells with 20 μmol/L ALP or DMSO for the indicated durations relative to the 0-hour vehicle control. Samples were analyzed in technical duplicate and biological triplicate (n = 3). B, Graphical representation of the phosphoproteomics experimental design, conducted in biological triplicate (n = 3). C, Venn diagrams representing the number of significantly upregulated or downregulated phosphopeptides upon 20 μmol/L ALP treatment at each designated time point relative to the 0-minute control. D, Heatmap depicting z-scores of changing phosphopeptides over time. E, Volcano plots of differentially enriched phosphopeptides upregulated (magenta) and downregulated (cyan) following 10, 30, and 60 minutes of 20 μmol/L ALP treatment relative to the 0-minute time point. F, Volcano plots of kinases in which activity is predicted to be enriched (magenta) or diminished (cyan) based on the differentially expressed phosphopeptide substrates at each time point.

Supplementary Table S2. Primer requisition information