Fig 2: Knockdown of MAGT1 expression inhibits the proliferation, migration and invasion of pancreatic carcinoma cells. (A) Protein and (B) mRNA expression level of MAGT1 in BxPC-3 cells transfected with shMAGT1-1, sh-MAGT1-2 or sh-NC was analyzed using western blot analysis and reverse transcription-quantitative PCR, respectively. Untransfected cells were used as the control. (C) Cell Counting kit-8 assay was used to analyze cell viability in cells transfected with sh-NC and sh-MAGT1-2 at different time points (24, 48 and 72 h). (D) Representative western blots and quantification showing the protein expression levels of Ki67. and PCNA in BxPC-3 cells were decreased in cells transfected with sh-MAGT1-2 compared with that in cells transfected with sh-NC. (E) The BxPC-3 cells transfected with sh-MAGT1-2 had decreased cell migration and invasion compared with that in cells transfected with sh-NC. (F) Representative western blots and quantification showing the protein expression level of MMP2, and MMP9 was significantly increased in the BxPC-3 cells transfected with sh-MAGT1-2 compared with that in cells transfected with sh-NC. *P<0.05, **P<0.01, ***P<0.001 vs. sh-NC group. MAGT1, magnesium transporter 1; NC, negative control; sh, short hairpin.

Fig 3: Overexpression of MAGT1 promotes the proliferation, migration and invasion of pancreatic carcinoma cells. (A) Protein and (B) mRNA expression levels of MAGT1 in the BxPC-3 cell line transfected with pcDNA3.1-MAGT1, and pcDNA3.1-NC plasmids using western blot analysis and reverse transcription-quantitative PCR, respectively. Untransfected cells were used as a control. (C) Cell Counting Kit-8 assay was used to analyze cell viability in cells transfected with pcDNA3.1-NC and pcDNA3.1-MAGT1 at different time points (24, 48 and 72 h). (D) Representative western blots and quantification showing the protein expression levels of Ki67, and PCNA in the BxPC-3 cells were increased in cells transfected with pcDNA3.1-MAGT1 compared with that in cells transfected with pcDNA3.1-NC. (E) The BxPC-3 cells transfected with pcDNA3.1-MAGT1 had increased cell migration and invasion compared with that in cells transfected with pcDNA3.1-NC. (F) Representative western blots and quantification showing the protein expression level of MMP2, and MMP9 was significantly increased in the BxPC-3 cells transfected with pcDNA3.1-MAGT1 compared with that in cells transfected with pcDNA3.1-NC. *P<0.05, **P<0.01, ***P<0.001 vs. pcDNA3.1-NC group. MAGT1, magnesium transporter 1; NC, negative control.

Fig 4: Transcriptional profiling of CDH18-silenced EPI cells.a PCA of CDH18-silenced d12 and d24 EPI cells as well as induced EPDCs compared to unspecific siRNA-treated control (scr) [d24•si18, n = 2; d12•si18, n = 2; iSMC, n = 1; CF; n = 1]. b Agglomerative hierarchical sample cluster: The Agnes dendrogram was plotted by applying Manhattan distances between samples. c Chord diagram representing the flow and the detailed relationship between d12•si18 DEGs (left semicircle perimeter) and their enriched GO biological processes (right semicircle perimeter). d Heatmap showing the expression analysis of the gene set enriched for epicardial, SMC and CF genes in iSMCs, CDH18-downregulated and control (scr) cells. e Heatmap showing the expression analysis of the gene set enriched for epicardial, SMC and CF genes in CDH18-downregulated and control (scr) cells under different culture conditions. f, g Expression levels for f EMT specific transcription factors (EMT-TFs) ZEB1, ZEB2, SNAI2 and TWIST1 and g CDKN1A, CDKN1B, CDKN2A, BUB1, MKI67, MCM6, MCM4 and MCM2 in CDH18-downregulated cells. h IPA analysis of CDH18-silenced d12 EPI cells (si18) versus control (scr) cells showing the activation pattern of signature genes involved in major important developmental pathways. i Pathway enrichment analysis showing the proportional correlation of genes affected in important developmental pathways contributing to SMC differentiation. [analysis of Fig. 4 was based on RNA-Seq dataset GSE165450].

Fig 5: Loss of CDH18 triggers cell-fate specific EMT towards smooth muscle cells.a Illustration of the experimental workflow. b and c Downregulation of b CDH18 [n = 3; unpaired Students t-test, ***p = 0.0003] and c CDH18 protein expression after 8 days of CDH18 silencing in d24 EPI cells (d24•si18). d Phase contrast microscopy displaying d24•si18 cells with lost cobblestone morphology and elongated shape (yellow arrowheads). e Growth curve of d24 cells silenced for CDH18 (green) and control (scr) (gray) for 2-8 days [n = 3; **p = 0.0038, ****p < 0.0001]. f SNAI1 western blot of d24•si18 cells. g CDH1 and CDH2 expression in d24•si18 cells [control (scr); n = 3; *p = 0.0141, ****p < 0.0001]. h Western blot of CDH18 and SNAI1 after 8 days of CDH18 silencing in d24 EPI cells (d12•si18). i Microscopy analysis of d12•si18 showing changed morphology (yellow arrowheads) and loss of Ki67 (pink) expression [scale bar 50 µm]. j Expression of CDH1 and CDH2 in d12•si18 EPI cells compared to their control cells [n = 3; *p = 0.0231, ***p = 0.0001]. k Boyden chamber assay evaluating cell migration capacity in d12•si18 cells [n = 3; **p = 0.002, ***p = 0.0004]. Right panel shows invaded cells stained by crystal violet. l Western blot analysis showing reduction of CDH18 and TCF21, increased LEF1 and ß-catenin with decreased phosphorylated (p)-ß-catenin. m Expression analysis of SMC markers ACTA2 and CNN1 in d12•si18 cells and iSMC [n = 3; unpaired Students t-test: ns = not significant]. n Immunocytochemistry analysis of aSMA and ZO1 expression in d12•si18 cells [scale bar 50 µm]. [statistical analysis performed by two-way ANOVA with Sidak’s multiple comparison test, unless otherwise indicated; all error bars represent SEM; scale bars 100 µm, unless otherwise indicated].