Fig 2: Ceramide synthase 1 knockdown causes reduction of GRP78 in Panc1 cells. (a) and (b) qPCR for CerS1 mRNA following transfection with 5 nM si-CERS1 for 24 and 48 h. (c) Bar blot of protein concentration (ng/ml) of CerS1 using ELISA following transfection with 5 nM si-CERS1 for 48, 72 and 96 h. (d) Bar blot of protein concentration (ng/ml) of CerS1 using ELISA following 48 h treatment with cannabidiol at 10, 20 and 40 µM. (e) Western blot (s) of housekeeping gene, alpha-tubulin, CerS1, GRP78 and CHOP following siRNA treatment. (f) Western blot (s) of GRP78, CHOP and alpha-tubulin following 48 h treatment with cannabidiol at 10, 20 and 40 µM

Fig 3: CerS1 upregulation may be needed to induce tumour reduction by cannabidiol (CBD) and increase survival in an in vivo model of PDAC. (a) Dose-response curve for gemcitabine treatment over 48 and 72 h. (b) Dose-response curve for cannabidiol treatment over 48 and 72 h. (c) Kaplan-meier curve for cannabidiol, gemcitabine and abraxane (GA) and gemcitabine, abraxane and cannabidiol (GACBD) groups. (d) Tumour volume change (mm3) for vehicle control group and cannabidiol treatment. (e) Tumour volume change (mm3) for vehicle control group and GA treatment. (f) Tumour volume change (mm3) for vehicle control group and GACBD treatment. (g) H&E and IHC staining for GRP78 and CerS1 in vehicle (control) and CBD treated tumours

Fig 4: Ceramide synthase 1 knockdown reduces protein expression of GRP78 in Panc03.27 cells. (a) and (b) qPCR for CerS1 mRNA following transfection with 5 nM si-CERS1 for 24 and 48 h. (c) Bar blot of protein concentration (ng/ml) of CerS1 using ELISA following transfection with 5 nM si-CERS1 for 48, 72 and 96 h. (d) Bar blot of protein concentration (ng/ml) of CerS1 using ELISA following 48 h treatment with cannabidiol at 10, 20 and 40 µM. (e) Western blot (s) of housekeeping gene, alpha-tubulin, CerS1, GRP78 and CHOP following siRNA treatment. (f) Western blot (s) of GRP78, CHOP and alpha-tubulin following 48 h treatment with cannabidiol at 10, 20 and 40 µM

Fig 5: Ceramide Synthase isoform 1 is upregulated by cannabidiol in pancreatic cancer. (a) Summary plot of mean and error with SD of CerS1 fold changes across all treatment groups of concentraions reflected in table 1’s ic50 values at 48 h, comparing human Panc03.27, Panc1 and murine-3275 cells. The subsequent statements indicate greatest to lowest significance of CerS1 upregulation with gemcitabine, abraxane and CBD (GAC) treatment in Panc03.27 cells followed by CBD treatment, then combination of gemcitabine and CBD (GC) and finally gemcitabine single treatment. In Panc1 cells, GAC treatment followed CBD single treatment and finally gemcitabine and abraxane (GA). Murine 3275 cells showed signficance in CerS1 upregulation in GC combination treatment only in comparison to control. (b) Using publicly available RNA sequencing data (GSEA, gene set enrichment analysis) of PDAC tumours versus normal, a Kaplan-Meier curve indicates higher CERS1 correlates with better overall survival (OS) (HR: hazard ratio = 0.61, Logrank p = 0.018, p = 0.019). Gem = gemcitabine, Abx = abraxane, CBD = cannabidiol