Journal: PLoS ONE

Article Title: Calcium Sensing Receptor Modulates Extracellular Calcium Entry and Proliferation via TRPC3/6 Channels in Cultured Human Mesangial Cells

doi: 10.1371/journal.pone.0098777

Figure Lengend Snippet: Human MCs were starved for 24-free medium prior to stimulation with 1 mM (Ctl) or 5 mM [Ca 2+ ] o for 24 h. (A) Real-time PCR experiments showed that the administration of 5 mM [Ca 2+ ] o for 24 h significantly increased the TRPC3 and TRPC6 mRNA levels but did not affect the mRNA levels of TRPC1 or TRPC4 (**p<0.01 vs. Ctl, n = 3). (B) Representative Western blot and corresponding quantitative analysis showing that treatment with 5 mM [Ca 2+ ] o for 24 h increased the TRPC3 and TRPC6 protein expression but did not affect the protein expression of TRPC1 or TRPC4 (**p<0.01 vs. Ctl, n = 3). Asterisks indicate the statistical significance (**p<0.01), with respect to 1 mM [Ca 2+ ] o conditions (Ctl). Data are shown as the means ± SEMs.

Article Snippet: Human MCs were transiently transfected with human TRPC3 siRNA, TRPC6 siRNA or scrambled siRNA (Santa Cruz, USA) using the Xtreme GENE siRNA transfection reagent (Roche, Germany) according to the manufacturer's instructions.

Techniques: Real-time Polymerase Chain Reaction, Western Blot, Expressing

Journal: PLoS ONE

Article Title: Calcium Sensing Receptor Modulates Extracellular Calcium Entry and Proliferation via TRPC3/6 Channels in Cultured Human Mesangial Cells

doi: 10.1371/journal.pone.0098777

Figure Lengend Snippet: (A, B) Real-time PCR experiments showed that the TRPC3 siRNA (A) and TRPC6 siRNA (B) decreased the mRNA expression of TRPC3 and TRPC6, respectively (*p<0.05 vs. Ctl, n = 3), without affecting other TRPC channels (p>0.05, n = 3). (C, D) Western blot experiments showed that transfection with TRPC3 siRNA (C) and TRPC6 siRNA (D) reduced TRPC3 and TRPC6 protein expression (**p<0.01 vs. Scr, n = 3), respectively, without affecting other TRPC channels (p>0.05, n = 3) compared with transfection with scramble siRNA. Asterisks indicate the statistical significance (**p<0.01). Data are shown as the means ± SEMs.

Article Snippet: Human MCs were transiently transfected with human TRPC3 siRNA, TRPC6 siRNA or scrambled siRNA (Santa Cruz, USA) using the Xtreme GENE siRNA transfection reagent (Roche, Germany) according to the manufacturer's instructions.

Techniques: Real-time Polymerase Chain Reaction, Expressing, Western Blot, Transfection

Journal: PLoS ONE

Article Title: Calcium Sensing Receptor Modulates Extracellular Calcium Entry and Proliferation via TRPC3/6 Channels in Cultured Human Mesangial Cells

doi: 10.1371/journal.pone.0098777

Figure Lengend Snippet: [Ca 2+ ] i dynamics were monitored by Fura-3 fluorescence methods. (A, B) Representative traces showing that transfection with TRPC3 siRNA (A) or TRPC6 siRNA (B) significantly inhibit the 3 mM spermine-induced [Ca 2+ ] i increase (p<0.05 vs. Scr, n = 5), respectively, in contrast with cells transfected with scrambled siRNA. (C) Summary of data showing that transfection with TRPC3 siRNA or TRPC6 siRNA significantly inhibits the average value of the plateau of the 3 mM spermine-induced [Ca 2+ ] i increase (**p<0.01 vs. Scr, n = 5), respectively. (D, E) Representative traces showing that transfection with TRPC3 siRNA (D) or TRPC6 siRNA (E) significantly inhibits the 5 mM [Ca 2+ ] o -induced [Ca 2+ ] i increase (p<0.05 vs. Scr, n = 5), respectively, in contrast with cells transfected with scrambled siRNA. (F) Summary of data showing that transfection with TRPC3 siRNA or TRPC6 siRNA significantly inhibits the average value of the plateau of the 5 mM [Ca 2+ ] o -induced [Ca 2+ ] i increase (**p<0.01 vs. Scr, n = 5), respectively. Data are shown as the means ± SEMs. The results were from at least three independent experiments, and each experiment measured 20 to 40 cells.

Article Snippet: Human MCs were transiently transfected with human TRPC3 siRNA, TRPC6 siRNA or scrambled siRNA (Santa Cruz, USA) using the Xtreme GENE siRNA transfection reagent (Roche, Germany) according to the manufacturer's instructions.

Techniques: Fluorescence, Transfection

Journal: PLoS ONE

Article Title: Calcium Sensing Receptor Modulates Extracellular Calcium Entry and Proliferation via TRPC3/6 Channels in Cultured Human Mesangial Cells

doi: 10.1371/journal.pone.0098777

Figure Lengend Snippet: [Ca 2+ ] i dynamics were monitored by Fura-3 fluorescence methods. Representative traces are shown in A-D. (A, B) Representative traces (A–B) and summary of data (C) showing that transfection with TRPC3 siRNA (A) or TRPC6 siRNA (B) significantly reduces the 100 µM OAG-induced Ca 2+ influx (p<0.01 vs. Scr, n = 4), respectively, compared with transfection with scrambled siRNA. Data are shown as the means ± SEMs. (D, E) The 5 mM [Ca 2+ ] o (D)- and spermine(E)-induced [Ca 2+ ] i increase is significantly enhanced by pretreatment with 100 µM OAG for 20 min (p<0.05 vs. Ctl, n = 4). The results were from at least three independent experiments, and each experiment measured 20 to 40 cells.

Article Snippet: Human MCs were transiently transfected with human TRPC3 siRNA, TRPC6 siRNA or scrambled siRNA (Santa Cruz, USA) using the Xtreme GENE siRNA transfection reagent (Roche, Germany) according to the manufacturer's instructions.

Techniques: Fluorescence, Transfection

Journal: PLoS ONE

Article Title: Calcium Sensing Receptor Modulates Extracellular Calcium Entry and Proliferation via TRPC3/6 Channels in Cultured Human Mesangial Cells

doi: 10.1371/journal.pone.0098777

Figure Lengend Snippet: Cell proliferation was measured by a Cell Proliferation ELISA BrdU kit. After starvation for 24-free medium, cells were incubated in the same medium supplemented with different [Ca 2+ ] o (1–5 mM) in the presence or absence of various inhibitors for 24 h. Untreated cells cultured in medium containing 1 mM Ca 2+ were used as a control. (A) Incubation of cells for 24 h with 3 mM and 5 mM [Ca 2+ ] o increase proliferation of human MCs, respectively, compared with 1 mM [Ca 2+ ] o (Ctl) (*p<0.05, **p<0.01 vs. Ctl, n = 3). (B) Pretreatment with 10 µM NPS2390 significantly reduces the 5 mM [Ca 2+ ] o -induced cell proliferation (**p<0.01 vs. Ctl, # p<0.05 vs. 5 mM [Ca 2+ ] o without NPS2390, n = 3). Incubation with NPS2390 alone do not affect cell proliferation at 1 mM [Ca 2+ ] o (p>0.05, n = 3). (C) The [Ca 2+ ] o -mediated cell proliferation is significantly inhibited by pretreatment with 50 µM SKF96365 or 100 µM 2-APB (**p<0.01 vs. Ctl, #p<0.05 vs. 5 mM [Ca 2+ ] o without inhibitors, n = 3). (D) Transfection of TRPC3 siRNA and TRPC6 siRNA significantly attenuate the promotion of proliferation by 5 mM [Ca 2+ ] o (**p<0.01 vs. Scr+1 mM [Ca 2+ ] o , #p<0.05 vs. Scr+5 mM [Ca 2+ ] o ), respectively, compared with scramble siRNA treated with 5 mM [Ca 2+ ] o . Cells were transfected with TRPC3 siRNA, TRPC6 siRNA or scrambled siRNA followed by treatment with 5 mM [Ca 2+ ] o for 24 h. Untransfected cells cultured in medium containing 1 mM Ca 2+ were used as a control. Data are shown as the means ± SEMs.

Article Snippet: Human MCs were transiently transfected with human TRPC3 siRNA, TRPC6 siRNA or scrambled siRNA (Santa Cruz, USA) using the Xtreme GENE siRNA transfection reagent (Roche, Germany) according to the manufacturer's instructions.

Techniques: Enzyme-linked Immunosorbent Assay, Incubation, Cell Culture, Transfection