Fig 2: Targeting ABCC3 in KPC primary cell line reduces cell proliferation through STAT3 and HIF1α dysregulation. Primary cell lines were established from the pancreatic tumours of KPC mice as described in the Methods section. KPC cells were treated with indicated doses of MCI-715 and number of cells was assessed after 72 h. For the assessment of anchorage-independent growth, cells were plated on soft agar, treated with indicated concentrations of MCI-715 and number of colonies was counted after 4 weeks. a Data presents the effects of MCI-715 dose response treatment in established KPC primary cell lines on anchorage dependent and independent cell growth. Blue: Ist established cell line, Green: 2nd established cell line. Data is expressed as fold change of cells treated with vehicle (DMSO). The results are presented as mean ± SEM of 5 (blue) and 3 (green) independent experiments, One-was ANOVA was performed for statistical analysis, *p < 0.5, **p < 0.01, ***p < 0.001, ****p < 0.0001; Representative Western blot images showing the effects of knockdown of ABCC3 with two specific siRNAs b and treatment with 10 μM MCI-715 c on the expression of pSTAT3 Y705 and HIF1α in the KPC primary cell line. Cells were collected 48 h post transfection/treatment. Quantitative analysis is presented as mean ± SEM of 3 (pSTAT3 Y705) and 4 (HIF1α) independent experiments and expressed as fold change of expression in control sample in reference to loading control; unpaired Student’s t-test was performed for statistical analysis, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001

Fig 3: TC treatment enhances canalicular and basolateral BA transporters. (A) Fecal BA excretion (n = 4) and urinary BA concentration (n = 3) in TC‐treated (1 week) DKO and BKO. (B) mRNA levels of BA transporters (n = 4). (C) Immunostaining of ABCB11, ABCC3, and ABCC4 (magnification ×200). Arrowhead, positive area. Student t test; *P < 0.05, ***P < 0.005 compared to CO‐treated group. Abbreviation: MDR, multidrug resistance.

Fig 4: MCI-715 is a potent inhibitor of ABCC3 and its application significantly reduces PDAC cell growth. a Western blot probed with anti-ABCC3 antibody (C-18; Santa Cruz) confirming the overexpression of ABCC3 in transfected cells. Lane 1, whole cell lysate prepared from untransfected HEK293T; lane 2, whole cell lysate prepared from HEK293T cells transiently-transfected with pcDNA3.1-ABCC3. b ABCC3 effluxes Calcein-AM to reduce accumulation in HEK293T cells (left histogram). Flow cytometry histogram comparing Calcein accumulation in red-fluorescent dsRed-expressing HEK293T cells co-transfected with pcDNA3-ABCC3 (red trace) compared to non-expressing cells (blue trace). c ABCC3 is fully inhibited by 750 μM MCI-715 (right histogram; colour coding as before). d MCI-715 is a more potent inhibitor of calcein-AM efflux by ABCC3 than sulindac sulphide. IC50 data are presented as mean ± SEM of 3 independent experiments, statistical analysis was performed by unpaired Student’s t-test p = 0.0033; The effect of the treatment of indicated PDAC cell lines with increasing doses of MCI-715 on anchorage-dependent e and anchorage-independent f cell growth. PDAC cells were treated with indicated doses of MCI-715 and number of cells was assessed after 72 h. For the assessment of anchorage-independent growth, cells were plated on soft agar, treated with indicated concentrations of MCI-715 and number of colonies was counted after 4 weeks. Data is expressed as fold change of cells treated with vehicle (DMSO). All experiments are presented as mean ± SEM of 3 independent experiments. One-way ANOVA was used for statistical analysis, **p < 0.01, ***p < 0.001, ****p < 0.0001

Fig 5: ABCC3 pharmacological inhibition reduces PDAC progression in in vivo mouse models. Mice were injected with HPAFII cells and treated with 25 mg/kg MCI-715 for 28 days as described in Methods section. Data presents the comparison of the tumour growth (a) and survival (b) of HPAFII xenograft mouse model treated with vehicle (n = 4) and MCI-715 (n = 6), Multiple-t-test was performed for statistical analysis of tumour growth, p = 0.0343, Logrank (Mantel-Cox) was performed for statistical analysis of the survival, p = 0.0033; (c) PDX mouse model was created and treated with 25 mg/kg MCI-715 as described in Methods section. Tumour growth in PDX mouse model of pancreatic cancer treated with vehicle (n = 5) and MCI-715 (n = 5) shows significant difference between the two treatment groups. Arrows indicate start and the end of the treatment period, Multiple-t-test was performed for statistical analysis of tumour growth *p < 0.05; (d) KPC transgenic mouse model was treated with 25 mg/kg MCI-715 as described in Methods section. Kaplan Meier survival curve of KPC mice treated with vehicle (n = 8) and MIC-715 (n = 6). Logrank (Mantel-Cox) test was performed for statistical analysis, p = 0.0010