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

Journal: Molecular Neurobiology

Article Title: Transcription Factor 2I Regulates Neuronal Development via TRPC3 in 7q11.23 Disorder Models

doi: 10.1007/s12035-018-1290-7

Figure Lengend Snippet: Expression level of TRPC3 in Gtf2i +/Del tissue and neurons. a Representative confocal images from Div8 neuronal culture of Gtf2i mutant mice. TRPC3, green and actin-phalloidin, cyan. Fluorescence intensity was measured using TRPC3/actin ratio and measured in the same area of the cytosol for every cell. b Fluorescence intensity summary showed that TRPC3/actin phalliodin intensity did not significantly change between the 3 genotypes. n = 40 cells per group from 12 slices from 3 different mice. c , d Western blot analysis using cortical brain tissue from 7-day-old Gtf2i mutant mice; n = 6 samples per group.TRPC3 expression level is not significantly different between the WT and samples from their mutant littermates ( P < 0.005)

Article Snippet: Cultured cortical neurons were transfected 4 h after plating with Trpc3-siRNA (20 nM, sc-42667, Santa Cruz) or no siRNA as negative control.

Techniques: Expressing, Mutagenesis, Fluorescence, Western Blot

Journal: Molecular Neurobiology

Article Title: Transcription Factor 2I Regulates Neuronal Development via TRPC3 in 7q11.23 Disorder Models

doi: 10.1007/s12035-018-1290-7

Figure Lengend Snippet: Increased TRPC3 membrane expression in Gtf2i +/ Del neurons. The cellular localization of TRPC3 in Gtf2i +/ Dup and Gtf2i +/ Del cultured cortical neurons was examined using immunofluorescence and confocal imaging. a , b , c Representative confocal images of DIV8 cortical neurons from WT, Gtf2i +/ Dup and Gtf2i +/ Del . TRPC3, green; MAP2, red. d , e , f Quantitative summary of TRPC3/MAP2 ratio of fluorescence intensity ( n = 20 for each group). g Membrane fraction with cadherin as positive control. h Cytosol fraction with GAPDH as control. i TRPC3 expression in Gtf2i +/ Del group is not significantly increased in the membrane fraction comparing with WT and Gtf2i +/ Dup group, respectively (WT, 0.41 ± 0.038; Gtf2i +/ Del , 0.51 ± 0.076, Gtf2i +/ Dup , 0.41 ± 0.07), n = 4. j TRPC3 expression in the cytosol fraction is significantly increased in Gtf2i +/ Dup cortical tissue compared to control (WT, 0.45 ± 0.035; Gtf2i +/ Del , 0.46 ± 0.04, Gtf2i +/ Dup , 0.83 ± 0.054, P < 0.0005; n = 4). * P < 0.05, ** P < 0.005, *** P < 0.0005, indicating the difference to the other groups in the same plot. Scale bar 10 μm

Article Snippet: Cultured cortical neurons were transfected 4 h after plating with Trpc3-siRNA (20 nM, sc-42667, Santa Cruz) or no siRNA as negative control.

Techniques: Expressing, Cell Culture, Immunofluorescence, Imaging, Fluorescence, Positive Control

Journal: Molecular Neurobiology

Article Title: Transcription Factor 2I Regulates Neuronal Development via TRPC3 in 7q11.23 Disorder Models

doi: 10.1007/s12035-018-1290-7

Figure Lengend Snippet: TRPC3 knockdown suppresses Ca 2+ entry. Cortical neurons were transfected with eGFP-sRNA and Trpc3 -siRNA-eGFP 4 h after plating. a Representatives of confocal images of cortical neurons labeled with TRPC3 in red, GFP in green and NeuN in blue, at DIV4. b Quantification of fluorescence intensity of TRPC3 in confocal images shows 40% knockdown of TRPC3 using TRPC3-siRNA in control neurons was confirmed by measuring fluorescence intensity of TRPC3 in confocal images ( P < 0.0005). c Western Blot shows reduction of TRPC3 protein level, confirming TRPC3-knockdown. TRPC3 protein, 97 KDa; β-actin, 42 KDa. (* P < 0.0005). Whole-cell patch-clamp recordings and fura-2 imaging were carried out to study the current density and calcium signals, respectively, in Gtf2i mutants and WT neurons with Trpc3 knockdown. Cortical neurons were transfected with a scrambled-eGFP-siRNA and Trpc3 -siRNA-eGFP 4 h after plating. Representatives of I-V curves of neurons from Gtf2i +/Del mice and their WT littermates designated WT Del ( d ), Gtf2i +/Dup and WT Dup littermates ( e ) and WT neurons treated with TRPC3-siRNA-eGFP (Trpc3-KD) or the control vector ( f ). g Summary of the whole-cell current density showing a significant increase in Gtf2i +/Del neurons (WT De n = 10; Gtf2i +/Del n = 7, P < 0.0005), and smaller in Gtf2i +/Dup neurons ( n = 11) compared to their WT littermates (WT Dup n = 9, P < 0.005). The current density from the neurons treated with TRPC3-siRNA-eGFP was significantly reduced compared to control neurons (eGFP-sRNA n = 9; Trpc3 -siRNA-eGFP n = 7, P < 0.0005). This decrease in current density of TRPC3-siRNA-eGFP treated cells is comparable and not significantly different to the current density seen in Gtf2i +/Dup neurons. h , i , j Ratiometric Fura-2 Ca 2+ signals measured in the soma. Representative Ca 2+ traces show carbachol (CCh)-induced Ca 2+ signal in the absence (showing Ca 2+ release from internal store) or presence (showing extracellular Ca 2+ entry) of Ca 2+ in bath solution. k Quantification of [Ca 2+ ] i release. WT Del = 86; Gtf2i +/Del n = 102; WT Dup n = 88; Gtf2i +/Dup n = 88 ( P < 0.0005); eGFP-sRNA n = 80; Trpc3 -siRNA n = 88 ( P < 0.0005). l Quantification of Ca 2+ entry: WT Del n = 86; Gtf2i +/Del n = 102 ( P < 0.0005); WT Dup n = 88; Gtf2i +/Dup n = 88 ( P < 0.005); eGFP-sRNA n = 80; Trpc3 -siRNA n = 88 ( P < 0.0005). Gtf2i +/Dup , and Trpc3 -KD are both significantly decreased compared to control. * P < 0.05, ** P < 0.005, *** P < 0.0005

Article Snippet: Cultured cortical neurons were transfected 4 h after plating with Trpc3-siRNA (20 nM, sc-42667, Santa Cruz) or no siRNA as negative control.

Techniques: Transfection, Labeling, Fluorescence, Western Blot, Patch Clamp, Imaging, Plasmid Preparation

Journal: Molecular Neurobiology

Article Title: Transcription Factor 2I Regulates Neuronal Development via TRPC3 in 7q11.23 Disorder Models

doi: 10.1007/s12035-018-1290-7

Figure Lengend Snippet: TRPC3 knockdown rescues the phenotype of Gtf2i +/Del neurons. a Representative total neurite traces of neurons at DIV4, 6, 8, and 10. b Total neurite length of Trpc3 -siRNA treated neurons (DIV4 n = 125; DIV6 n = 95; DIV8 n = 80; DIV10 n = 86) was significantly shorter than eGFP-sRNA controls (DIV4 n = 125; DIV6 n = 90; DIV8 n = 81; DIV10 n = 85, P < 0.05). c Representative confocal images of neurons immunostained with MAP2 and Tau-1. d Quantitative summary of dendritic length in both eGFP-sRNA control group and Trpc3 KD group. eGFP-sRNA n = 75; Trpc3 -targeted siRNA n = 70. Unpaired Student’s t test. e Quantitative summary showed that the axonal length of Trpc3 -siRNA-treated neurons ( n = 70) was significantly decreased compared to eGFP-sRNA treated neurons ( n = 75, P < 0.05), unpaired Student’s t test. f Quantitative summary showed that number of branches in axons was significantly decreased in neurons treated with Trpc3 -targeted siRNA ( n = 80) compared to eGFP-sRNA ( n = 80) treated neurons. ( P < 0.0005, unpaired Student’s t test). g Sholl analysis showing Trpc3-KD reduced the number of branches at all the distance measured, as seen in Gtf2i +/Dup . h Representative images of WT and Gtf2i +/ Del neurons immunostained with Tubulin. Total neurite length of GFP positive neurons was traced using the labeling for Tubulin. i Summary shows that total neurite length of Gtf2i +/ Del neurons treated with Trpc3 -siRNA-eGFP was significantly shorter than control siRNA-eGFP treated Gtf2i +/ Del neurons ( n = 40, P < 0.005), and similar to control siRNA-eGFP-treated WT neurons. Again, treated with control siRNA-eGFP, the total length of the Gtf2i +/ Del neurons ( n = 40) was significantly greater than WT ( n = 40; P < 0.0005). j Representative Ca 2+ traces show carbachol (CCh)-induced Ca 2+ signal in the absence (showing Ca 2+ release from internal store) or presence (showing extracellular Ca 2+ entry) of Ca 2+ in bath solution. Additional caffeine and thapsigargin, as indicated by open bar. k Ca 2+ release was not significantly different between the 3 groups ( n = 20). Gtf2i +/ Del neurons treated with Trpc3 -siRNA-eGFP calcium entry were rescued to WT levels ( n = 20; P < 0.0005). * P < 0.05, ** P < 0.005, *** P < 0.0005

Article Snippet: Cultured cortical neurons were transfected 4 h after plating with Trpc3-siRNA (20 nM, sc-42667, Santa Cruz) or no siRNA as negative control.

Techniques: Labeling