Journal: Cells

Article Title: TRPM7 Modulates Human Pancreatic Stellate Cell Activation

doi: 10.3390/cells11142255

Figure Lengend Snippet: Antibodies used in Western blotting.

Article Snippet: TRPM7 , Abcam (ab109438) , 1:1000 , Rabbit.

Techniques: Western Blot

Journal: Cells

Article Title: TRPM7 Modulates Human Pancreatic Stellate Cell Activation

doi: 10.3390/cells11142255

Figure Lengend Snippet: Correlation between TRPM7 expression and PS-1 activation status. ( A ) Lipid droplets were increased after ATRA treatment (as assessed by Nile Red staining). ( B ) Western blot analyses and associated histograms showed a reduced vimentin expression ( n = 3). * indicates p < 0.05 (Mann–Whitney rank sum test). ( C ) Viability of PS-1 stellate cells was decreased upon ATRA treatment ( n = 4). * indicates p < 0.05 (2-ways ANOVA). ( D ) TRPM7 protein expression was decreased in ATRA treated-cells ( n = 3). * indicates p < 0.05 (Mann–Whitney rank sum test). ( E ) mRNA expression levels analyses showed a reduction in TRPM7 transcription following ATRA treatment ( n = 7). *** indicates p < 0.001 (Mann–Whitney rank sum test). ( F ) Viability of PS-1 stellate cells was increased when cells were activated by incubation with Mia PaCa-2 conditioned medium ( n = 3). * indicates p < 0.05 (2-ways ANOVA). ( G ) TRPM7 protein expression was increased when PS-1 cells were cultured in Mia PaCa-2 conditioned medium ( n = 4). * indicates p < 0.05 (Mann–Whitney rank sum test). ( H ) mRNA expression levels analyses revealed an increase in TRPM7 transcription after incubation with Mia PaCa-2 conditioned medium ( n = 3). *** indicates p < 0.001 (Mann–Whitney rank sum test). ( I ) Comparison between PS-1 and RLT-PSC stellate cells showed that RLT-PSC had higher viability than PS-1 cells ( n = 4). *** indicates p < 0.001 (2-ways ANOVA). ( J ) Western blot showing TRPM7 expression in RLT-PSC cells and associated quantification ( n = 6). ** indicates p < 0.01 (Mann–Whitney rank sum test).

Article Snippet: TRPM7 , Abcam (ab109438) , 1:1000 , Rabbit.

Techniques: Expressing, Activation Assay, Staining, Western Blot, MANN-WHITNEY, Incubation, Cell Culture

Journal: Cells

Article Title: TRPM7 Modulates Human Pancreatic Stellate Cell Activation

doi: 10.3390/cells11142255

Figure Lengend Snippet: Proliferation was influenced by TRPM7 expression. ( A ) Immunoblotting of lysates of PS-1 cells transfected with a scramble siRNA (siControl) or a siRNA targeting TRPM7 (siTRPM7), and incubated with anti-TRPM7 antibody ( n = 4). * indicates p < 0.05 (Mann–Whitney Rank Sum Test). ( B ) PSC viability was decreased at 72 h and 96 h following TRPM7 silencing ( n = 4). *** indicates p < 0.001 (2-ways ANOVA). ( C ) TRPM7 silencing led to enrichment of cells in G0/G1 while decreasing the percentages of cells in S and in G2/M phases of the cell cycle ( n = 3). * indicates p < 0.05 and *** indicates p < 0.001 (2-ways ANOVA). ( D ) The pharmacological blocker of TRPM7 channels, NS8593 (25 µM), decreased PS-1 cell viability at 72 and 96 h ( n = 3). ** indicates p < 0.01 and *** indicates p < 0.001 (2-ways ANOVA). ( E ) Following NS8593 treatment, PS-1 cells accumulated in G0/G1 phase, whereas the percentage of cells decreased in S and in G2/M phases ( n = 3). *** indicates p < 0.001 (2-ways ANOVA).

Article Snippet: TRPM7 , Abcam (ab109438) , 1:1000 , Rabbit.

Techniques: Expressing, Western Blot, Transfection, Incubation, MANN-WHITNEY

Journal: Cells

Article Title: TRPM7 Modulates Human Pancreatic Stellate Cell Activation

doi: 10.3390/cells11142255

Figure Lengend Snippet: Influence of TRPM7 on cell cycle regulators (cyclin E, p53, CDK2, and PCNA expressions). ( A ) Immunoblotting of PS-1 protein lysates. The cells were transfected with a scramble siRNA (siControl) or a siRNA targeting TRPM7 (siTRPM7) and incubated with the following antibodies: anti-Cyclin-E, anti-p53, anti-CDK2, anti-PCNA, and anti-GADPH. ( B ) Quantification of Cyclin-E, p53, CDK2, and PCNA protein expression in PS-1 cells transfected with a scramble siRNA (siControl) or a TRPM7 targeting siRNA (siTRPM7) ( n = 4–8). * indicates p < 0.05 and *** indicates p < 0.001 (Mann-Withney Rank Sum Test). ( C ) Cell viability without (Control) or with Nutlin-3, an inhibitor of the main p53 regulator, Mdm-2 ( n = 4). ** indicates p < 0.01 and *** indicates p < 0.001 (2-ways ANOVA). ( D ) Following nutlin-3 treatment, the percentage of PS-1 cells was decreased in S phase ( n = 3). *** indicates p < 0.001 (2-ways ANOVA). ( E ) Western blots showing TRPM7, p53, CDK2, PCNA, GAPDH expression following treatment with Nutlin-3a. ( F ) Relative protein expressions of TRPM7, p53, CDK2 and PCNA following treatment with Nutlin-3a ( n = 3–4). * indicates p < 0.05 and *** indicates p < 0.001 (2-ways ANOVA).

Article Snippet: TRPM7 , Abcam (ab109438) , 1:1000 , Rabbit.

Techniques: Western Blot, Transfection, Incubation, Expressing

Journal: Cells

Article Title: TRPM7 Modulates Human Pancreatic Stellate Cell Activation

doi: 10.3390/cells11142255

Figure Lengend Snippet: TRPM7 is able to regulate PS-1 proliferation through the PI3K/Akt pathway. ( A ) Cell viability assessed without (Control) or with wortmannin (an inhibitor of the PI3K). * indicates p < 0.05 and *** indicates p < 0.001 (2-ways ANOVA). ( B ) Following wortmannin treatment, PS-1 cells accumulated in G0/G1 phase, whereas the percentage of cells decreased in S and in G2/M phases ( n = 3). * indicates p < 0.05 and ** indicates p < 0.01 (2-ways ANOVA). ( C ) Immunoblotting of PS-1 lysates. Cells were either transfected with a scramble siRNA (siControl) or siTRPM7, and incubated with antibodies against phosphorylated and total forms of MDM2, Akt, and ERK. GAPDH expression is used as normalization control. ( D ) Quantifications of relative protein expressions in PS-1 cells of pMDM2/MDM2, pAkt/Akt, and pERK/ERK ratios ( n = 4–8). * indicates p < 0.05 (Mann–Whitney Rank Sum Test).

Article Snippet: TRPM7 , Abcam (ab109438) , 1:1000 , Rabbit.

Techniques: Western Blot, Transfection, Incubation, Expressing, MANN-WHITNEY

Journal: Cells

Article Title: TRPM7 Modulates Human Pancreatic Stellate Cell Activation

doi: 10.3390/cells11142255

Figure Lengend Snippet: TRPM7 is active and regulates cytosolic Mg 2+ homeostasis in PSCs. ( A ) Development of an outward Magnesium-Inhibited Cation (MIC) current recorded at +100 mV during the dialysis of intracellular media by a 0-Mg intrapipette solution in PSCs transfected by a control siRNA ( n = 6) and in PSCs transfected by a siRNA targeting TRPM7 ( n = 4). ( B ) MIC currents are decreased in PSCs treated by a TRPM7 siRNA ( n = 4) compared to cells treated by a control siRNA ( n = 6). * indicates p < 0.05 (Mann–Whitney Rank Sum Test for the current-densities recorded at +100 mV). ( C ) Estimation of constitutive divalent cation entry by Mn 2+ -quenching in PSCs treated by a TRPM7 siRNA ( n = 19) compared to control cells ( n = 23). ( D ) Quantification of constitutive divalent cation entry after TRPM7 silencing. *** indicates p < 0.001 ( t -test). ( E ) Estimation of constitutive divalent cation entry by Mn 2+ -quenching in PSCs treated by NS8593 ( n = 34) compared to control cells ( n = 30). ( F ) Quantification of constitutive divalent cation entry after TRPM7 block. *** indicates p < 0.001 ( t -test). ( G ) Quantification of MagFura-2 fluorescence ratio after treatment with siTRPM7 ( n = 87 for siTRPM7 and n = 92 for siControl), after treatment with NS8593 ( n = 32 for treated cells and n = 39 for control), and after treatment with ATRA ( n = 40 for treated cells and n = 32 for control). *** indicates p < 0.001 ( t -tests). ( H ) Quantification of Fura-2 fluorescence ratio after treatment with siTRPM7 ( n = 47 for siTRPM7 and n = 36 for siControl), after treatment with NS8593 ( n = 23 for treated cells and n = 34 for control), and after treatment with ATRA ( n = 52 for treated cells and n = 27 for control). *** indicates p < 0.001 ( t -tests).

Article Snippet: TRPM7 , Abcam (ab109438) , 1:1000 , Rabbit.

Techniques: Transfection, MANN-WHITNEY, Blocking Assay, Fluorescence

Journal: Cells

Article Title: TRPM7 Modulates Human Pancreatic Stellate Cell Activation

doi: 10.3390/cells11142255

Figure Lengend Snippet: Expression of p53 is magnesium-dependent. ( A ) Cell viability was assessed with usual magnesium concentration (Control) or with reduced magnesium concentration (0.1 mM, n = 3). * indicates p < 0.05 (2-ways ANOVA). ( B ) When magnesium concentration was reduced (0.1 mM), PS-1 cells accumulated in G0/G1 phase, with a decreased percentage of cells in S and in G2/M phases ( n = 3). *** indicates p < 0.001 (2-ways ANOVA). ( C ) Effect of Mg 2+ supplementation, with or without TRPM7 silencing, on cell viability ( n = 3). ( D ) Mg 2+ supplementation reversed the effect of TRPM7 silencing on cell cycle regulation ( n = 3). * indicates p < 0.05; ** indicates p < 0.01; *** indicates p < 0.001 (2-ways ANOVA) ( E ) Western blot showing protein expression of p53 in PS-1 cells. Expressions are shown in different conditions: either transfected with siControl or siTRPM7, or in combination with increased magnesium concentrations (72 h post transfection). ( F ) Quantifications of relative p53 expression in PS-1 cells: magnesium supplementation was able to restore the effect of TRPM7 silencing on p53 expression ( n = 4). *** indicates p < 0.001.

Article Snippet: TRPM7 , Abcam (ab109438) , 1:1000 , Rabbit.

Techniques: Expressing, Concentration Assay, Western Blot, Transfection

Journal: Cells

Article Title: TRPM7 Modulates Human Pancreatic Stellate Cell Activation

doi: 10.3390/cells11142255

Figure Lengend Snippet: TRPM7 and POSTN expressions are increased in Cancer-Associated Fibroblasts (CAFs) in pancreatic cancer. ( A ) Relative mRNA levels of TRPM7, TRPM6 and POSTN in PDAC and a normal pancreas. Boxplots were generated with GEPIA from TCGA and GTEX datasets. Relative levels are expressed as log2 transcripts per million bases (TPM). * indicates p < 0.05 ( t -test). ( B ) Correlation analysis of TRPM7 and POSTN in PAAD TCGA dataset. ** indicates p < 0.01 ( t -test) ( C ) Correlation analysis of TRPM7 and POSTN in GSE28735 dataset. ** indicates p < 0.01 (paired t -test). ( D ) Relative mRNA levels of TRPM7, TRPM6 and POSTN in CD4+, CD8+, endothelial cells, macrophages and cancer associated fibroblasts (CAFs) from TCGA tumors and normal pancreas samples. EPIC deconvolution of the dataset was performed using GEPIA2021 tool. Quantitative comparisons of TRPM7, TRPM6 and POSTN expression were analyzed using ANOVA test.

Article Snippet: TRPM7 , Abcam (ab109438) , 1:1000 , Rabbit.

Techniques: Generated, Expressing

Journal: Cells

Article Title: TRPM7 Modulates Human Pancreatic Stellate Cell Activation

doi: 10.3390/cells11142255

Figure Lengend Snippet: Overview of the pathway activated by TRPM7 and magnesium in PSCs.

Article Snippet: TRPM7 , Abcam (ab109438) , 1:1000 , Rabbit.

Techniques: