Journal: Renal Failure

Article Title: Piezo1 facilitates the initiation and progression of renal fibrosis by mediating cell apoptosis and mitochondrial dysfunction

doi: 10.1080/0886022X.2024.2415519

Figure Lengend Snippet: Yoda1 facilitated renal injury and fibrosis by Bax mediated apoptosis and mitochondrial dysfunction. (A) The WB of Bax, Bcl2, Cytc, Caspase3 and Cleaved-caspase3 ( n = 3 per group). (B) Bar plot represents the proteins quantification results of Bax, Bcl2, Cytc, Caspase3 and Cleaved-caspase3. (C) The mRNA expression level of Bax, Bcl2, Bax/Bcl2 and Caspase3 after Yoda1 treatment. (D) Flow cytometry scatter plot of AnnexinV-PI/7ADD staining in TCMK-1 cells after Yoda1 intervention ( n = 3 per group). (E) the immunostaining of mitochondrial membrane potential after Yoda1 treatment ( n = 3 per group, 3 random sights per sample), Scale bar 100 μm. (F) Detection of intracellular ATP content in TCMK-1 cells after Yoda1 intervention. (G) the intracellular calcium concentration was detected with fluorescence microscopy after Yoda1 intervention, Scale bar 100 μm. Statistical analyses were calculated using 2-tailed student’s t test for 2 groups. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001

Article Snippet: The following antibodies were used Piezo1(Proteintech,15939-1-AP, 1:1000), Bcl-2 (Proteintech, 68103-1-Ig, 1:1000), Bax (Proteintech, 50599-2-Ig,1:1000), alpha-smooth muscle actin (α-SMA) (Abcam, ab32575, 1:1000), Fn(Servicebio,GB114491,1:1000), CollagenI (Servicebio, GB11022, 1:1000), Caspase3 (CST, 9662S, 1:1000), Cleaved-caspase3 (CST, 9661S, 1:1000), Cytc (Proteintech, 12245-1-AP, 1:1000), KIM1 (Boster, ba3537, 1:1000), NGAL (Boster, ba9609, 1:1000), Tubulin (ZENBIO, 380628, 1:5000), GAPDH(ZENBIO, 380626, 1:5000), HRP-Anti-Rabbit lgG (ZENBIO, 511203, 1:5000), HRP-Anti-mouse lgG (ZENBIO, 511103, 1:5000).

Techniques: Expressing, Flow Cytometry, Staining, Immunostaining, Membrane, Concentration Assay, Fluorescence, Microscopy

Journal: Renal Failure

Article Title: Piezo1 facilitates the initiation and progression of renal fibrosis by mediating cell apoptosis and mitochondrial dysfunction

doi: 10.1080/0886022X.2024.2415519

Figure Lengend Snippet: The rescue experiments that activated Piezo1 and inhibited Bax. (A) The WB expression levels of Bax, Bcl2, Cytc, Caspase3, Cleaved-caspase3, KIM1, Collagen I and Fn ( n = 3 per group). (B) Bar plot represents the proteins quantification results of Bax, Bcl2, Cytc, Caspase3, Cleaved-caspase3, KIM1, Collagen1 and Fn. (C) The mRNA expression levels of Bax, Bcl2, Bax/Bcl2, α-SMA, Collagen I and Fn. (D) Flow cytometry scatter plot of JC1 staining after Yoda1 or/and BAI1 co-treatment, the FITC+ cells present the decreased of mitochondrial membrane potential ( n = 3 per group). (E) Detection of intracellular ATP content in TCMK-1 cells after Yoda1 and/or BAI1 intervention. Statistical analysis was calculated using one-way ANOVA, * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001.

Article Snippet: The following antibodies were used Piezo1(Proteintech,15939-1-AP, 1:1000), Bcl-2 (Proteintech, 68103-1-Ig, 1:1000), Bax (Proteintech, 50599-2-Ig,1:1000), alpha-smooth muscle actin (α-SMA) (Abcam, ab32575, 1:1000), Fn(Servicebio,GB114491,1:1000), CollagenI (Servicebio, GB11022, 1:1000), Caspase3 (CST, 9662S, 1:1000), Cleaved-caspase3 (CST, 9661S, 1:1000), Cytc (Proteintech, 12245-1-AP, 1:1000), KIM1 (Boster, ba3537, 1:1000), NGAL (Boster, ba9609, 1:1000), Tubulin (ZENBIO, 380628, 1:5000), GAPDH(ZENBIO, 380626, 1:5000), HRP-Anti-Rabbit lgG (ZENBIO, 511203, 1:5000), HRP-Anti-mouse lgG (ZENBIO, 511103, 1:5000).

Techniques: Expressing, Flow Cytometry, Staining, Membrane

Journal: Renal Failure

Article Title: Piezo1 facilitates the initiation and progression of renal fibrosis by mediating cell apoptosis and mitochondrial dysfunction

doi: 10.1080/0886022X.2024.2415519

Figure Lengend Snippet: Inhibition Piezo1 could relief renal injury and fibrosis. (A) The WB expression levels of Bax, Bcl2, Caspase3, Cleaved-caspase3, KIM1 and Fn ( n = 3 per group). (B) The WB quantitative results of Bax, Bcl2, Caspase3, Cleaved-caspase3, Kim1 and Fn. (C) The mRNA level of Bax, Caspase3, KIM1 and Fn. (D) Flow cytometry scatter plot of AnnexinV-PI/7ADD staining in TCMK-1 cells after TGFβ1 and/or GsMTx4 intervention ( n = 3 per group). (E) The immunostaining of mitochondrial membrane potential after TGFβ1 and/or GsMTx4 in intervention ( n = 3 per group, 3 random sights per sample), JC1 aggregates (red), JC1 monomers (green), Scale bar 100 μm. (F) The WB expression levels of Kim1, α-SMA, Bax, Bcl2, Cytc, Caspase3 and Cleaved-caspase3 in UUO mice models intervened by GsMTx4. (G) The WB quantitative results of KIM1,α-SMA, Bax, Bcl2, Cytc, Caspase3 and Cleaved-caspase3 in UUO mice models intervened by GsMTx4. Statistical analyses were calculated using one-way ANOVA. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001.

Article Snippet: The following antibodies were used Piezo1(Proteintech,15939-1-AP, 1:1000), Bcl-2 (Proteintech, 68103-1-Ig, 1:1000), Bax (Proteintech, 50599-2-Ig,1:1000), alpha-smooth muscle actin (α-SMA) (Abcam, ab32575, 1:1000), Fn(Servicebio,GB114491,1:1000), CollagenI (Servicebio, GB11022, 1:1000), Caspase3 (CST, 9662S, 1:1000), Cleaved-caspase3 (CST, 9661S, 1:1000), Cytc (Proteintech, 12245-1-AP, 1:1000), KIM1 (Boster, ba3537, 1:1000), NGAL (Boster, ba9609, 1:1000), Tubulin (ZENBIO, 380628, 1:5000), GAPDH(ZENBIO, 380626, 1:5000), HRP-Anti-Rabbit lgG (ZENBIO, 511203, 1:5000), HRP-Anti-mouse lgG (ZENBIO, 511103, 1:5000).

Techniques: Inhibition, Expressing, Flow Cytometry, Staining, Immunostaining, Membrane

Journal: Cell Death Discovery

Article Title: The dark side of the light (UVA): melanoma microenvironment and cell survival strategies

doi: 10.1038/s41420-025-02751-y

Figure Lengend Snippet: A Immunostaining staining using mutated phospho anti-p53N239Y antibody and anti-GDF-15 (upper panel) shows that nuclear expression of the mutated p53 is most of the time present in the melanoma cells, the GDF-15 localization in the nucleus during post-UVA irradiation makes promoter hypomethylation via p53 N239Y /GDF-15 axis better as compared to p53 Ser392 /GDF-15 axis where the p53 Ser392 expression in the nucleus is enhanced only after post-UVA irradiation. This effect was also reconfirmed with WT p53 (lower panel). B Colony Formation Units (CFU) assay was carried out on post-irradiated 10 5 A375 melanoma cells, which are either GDF-15 KDN /blue or GDF-15 WT /Red, or GDF-15 TG /Green. The cells are sorted and post-irradiated and mixed further in equal proportions in Human methylcellulose with complete cytokine media (IL-6 20 ng/mL, HSC005, R&D system) shows that GDF-15 KDN shows poor survival as compared to GDF-15WT or GDF-15TG. IL-6 in the methylcellulose could equally hypomethylate GDF-15 WT and GDF-15 TG , but high endogenous GDF-15 levels in GDF-15TG might have given early protection against UVA-induced apoptosis. Taken together, we observe that increased activated p53 along with loss of phosho-MDM2 and epigenetic unlocking of GDF-15 with reduced DNMT1 and enhanced APOBEC as shown in Suppl figure 2E. Together fathoms pro-survival in melanoma under genotoxic stress. *P < 0.01, ***P < 0.001, ****P < 0.0001 by ANOVA multiple comparison with Tukey`s post hoc test. −as represented by the bar denotes 200 times magnification.

Article Snippet: The cells are sorted and post-irradiated and mixed further in equal proportions in Human methylcellulose with complete cytokine media (IL-6 20 ng/mL, HSC005, R&D system) shows that GDF-15 KDN shows poor survival as compared to GDF-15WT or GDF-15TG.

Techniques: Immunostaining, Staining, Expressing, Irradiation, Colony-forming Unit Assay, Comparison

Journal: Human Reproduction (Oxford, England)

Article Title: Complete spermatogenesis in intratesticular testis tissue xenotransplants from immature non-human primate

doi: 10.1093/humrep/dey373

Figure Lengend Snippet: Germ cell differentiation in intratesticular marmoset testis tissue xenotransplants . Morphological identification of post-meiotic germ cells by H/PAS, and on VIMENTIN and MAGE-A4 stained sections and immunohistochemical confirmation by CREM-1 expression and acrosome visualisation by immunostaining for ACROSIN. Arrowheads indicate round spermatids; arrows indicate elongating/elongated spermatids.

Article Snippet: Following another washing step, non-specific adhesion sites were blocked with 4% normal goat serum (Tebu-bio, Boechout, Belgium) diluted in PBS or with Cas-block (Life Technologies) (only for CREM-1 antibody) for 30 min. Next, primary antibodies against VIMENTIN (marmoset specific), MAGE-A4 (mouse monoclonal anti-melanoma-associated antigen 4; dilution 1:200; gift from Dr Spagnoli; marker for spermatogonia and primary spermatocytes, marmoset specific), BOLL (mouse monoclonal anti-BOLL; dilution 1:400; H00066037-M03; R&D system, Abingdon, UK; marker for spermatocytes (meiotic cells)), CREM-1 (rabbit polyclonal anti-CREM-1; dilution 1:400; sc-440; Santa-Cruz Biotechnology, Heidelberg, Germany; marker for round spermatids (post-meiotic cells)) and ACROSIN (rabbit polyclonal anti-ACROSIN; dilution 1:500; sc-67151; Santa-Cruz; marker for acrosome visualisation in round, elongating and elongated spermatids (post-meiotic cells), marmoset specific), were applied to the sections and incubated in a humidified chamber overnight at 4°C.

Techniques: Cell Differentiation, Staining, Immunohistochemical staining, Expressing, Immunostaining

Journal: Stem Cells International

Article Title: Periostin Is a Candidate Regulator of the Host Microenvironment in Regeneration of Pulp and Dentin Complex and Periodontal Ligament in Transplantation with Stem Cell-Conditioned Medium

doi: 10.1155/2024/7685280

Figure Lengend Snippet: Human primers for reverse transcription-polymerase chain reaction.

Article Snippet: Immunostaining was performed using mouse anti-DSPP antibody (Santa Cruz, sc-73632), mouse anti-nestin antibody (GeneTex, GT623), rabbit anti-DMP1 antibody (Affinity Biosciences, DF8825), anti-Runx2 antibody (Proteintech, 20700-11AP), and anti-periostin antibody (abcam, ab14041), 7, 14, and 21 days after dentin induction.

Techniques: Sequencing, Amplification

Journal: Stem Cells International

Article Title: Periostin Is a Candidate Regulator of the Host Microenvironment in Regeneration of Pulp and Dentin Complex and Periodontal Ligament in Transplantation with Stem Cell-Conditioned Medium

doi: 10.1155/2024/7685280

Figure Lengend Snippet: Murine primers for reverse transcription-polymerase chain reaction.

Article Snippet: Immunostaining was performed using mouse anti-DSPP antibody (Santa Cruz, sc-73632), mouse anti-nestin antibody (GeneTex, GT623), rabbit anti-DMP1 antibody (Affinity Biosciences, DF8825), anti-Runx2 antibody (Proteintech, 20700-11AP), and anti-periostin antibody (abcam, ab14041), 7, 14, and 21 days after dentin induction.

Techniques: Sequencing, Amplification

Journal: Stem Cells International

Article Title: Periostin Is a Candidate Regulator of the Host Microenvironment in Regeneration of Pulp and Dentin Complex and Periodontal Ligament in Transplantation with Stem Cell-Conditioned Medium

doi: 10.1155/2024/7685280

Figure Lengend Snippet: Expression of DSPP , nestin , DMP1 , Runx2 , and periostin by dentin induction of dental pulp stem cells. Immunohistochemical staining images after the start of dentin induction 21 days. (Bule: Hoechst 33342 and Green: (a) DSPP, (b) nestin, (c) DMP1, (d) Runx2, and (e) periostin).

Article Snippet: Immunostaining was performed using mouse anti-DSPP antibody (Santa Cruz, sc-73632), mouse anti-nestin antibody (GeneTex, GT623), rabbit anti-DMP1 antibody (Affinity Biosciences, DF8825), anti-Runx2 antibody (Proteintech, 20700-11AP), and anti-periostin antibody (abcam, ab14041), 7, 14, and 21 days after dentin induction.

Techniques: Expressing, Immunohistochemical staining, Staining

Journal: Stem Cells International

Article Title: Periostin Is a Candidate Regulator of the Host Microenvironment in Regeneration of Pulp and Dentin Complex and Periodontal Ligament in Transplantation with Stem Cell-Conditioned Medium

doi: 10.1155/2024/7685280

Figure Lengend Snippet: Gene expression of DSPP , nestin , DMP1 , Runx2 , and periostin by dentin induction of dental pulp stem cells. Relative gene expression levels 7, 14, and 21 days after dentin induction was initiated. (Relative quantification by ΔΔCt method after qPCR analysis; (a) DSPP , (b) nestin , (c) DMP1 , (d) Runx2 , and (e) periostin ). Mean ± standard error with n = 4, ∗ P < 0.05 and ∗∗ P < 0.01.

Article Snippet: Immunostaining was performed using mouse anti-DSPP antibody (Santa Cruz, sc-73632), mouse anti-nestin antibody (GeneTex, GT623), rabbit anti-DMP1 antibody (Affinity Biosciences, DF8825), anti-Runx2 antibody (Proteintech, 20700-11AP), and anti-periostin antibody (abcam, ab14041), 7, 14, and 21 days after dentin induction.

Techniques: Gene Expression, Quantitative Proteomics

Journal: Stem Cells International

Article Title: Periostin Is a Candidate Regulator of the Host Microenvironment in Regeneration of Pulp and Dentin Complex and Periodontal Ligament in Transplantation with Stem Cell-Conditioned Medium

doi: 10.1155/2024/7685280

Figure Lengend Snippet: PCR evaluation of DSPP , nestin , DMP1 , Runx2 , and periostin expression in regenerated dental pulp tissues Relative gene expression levels 7, 14, and 21 days in regenerated dental pulp tissue. (Relative quantification by ΔΔCt method after qPCR analysis; (a) DSPP , (b) nestin , (c) DMP1 , (d) Runx2 , and (e) periostin ). Mean ± standard error with n = 4, ∗ P < 0.05 and ∗∗ P < 0.01.

Article Snippet: Immunostaining was performed using mouse anti-DSPP antibody (Santa Cruz, sc-73632), mouse anti-nestin antibody (GeneTex, GT623), rabbit anti-DMP1 antibody (Affinity Biosciences, DF8825), anti-Runx2 antibody (Proteintech, 20700-11AP), and anti-periostin antibody (abcam, ab14041), 7, 14, and 21 days after dentin induction.

Techniques: Expressing, Gene Expression, Quantitative Proteomics

Journal: Journal of neurochemistry

Article Title: p120-catenin is necessary for neuroprotection induced by CDK5 silencing in models of Alzheimer's disease

doi: 10.1111/jnc.13697

Figure Lengend Snippet: A) Blots of p120 ctn, N-cadherin, β-catenin, α-N-catenin, GluN2B and PSD95 from Alzheimer’s disease brains. B) Dot plot representing the quantification of each patient band shown in A. Tubulin was used as a loading control. C) Representative blots of PSD95, GluN2B and p120 ctn from the PSD95 immunoprecipitation assay. D; E) Quantitative representation of the results shown in C. The data were normalized to IgG load for evaluating changes in the complex (D) and relativized to PSD95 expression in order to detect variation in the association of the proteins (E). Densitometric quantification was performed. The data are presented as the means ± SEM (relative units). n = 6-7, *p < 0.05 (Student’s t-test). MW: Molecular weight.

Article Snippet: Isolated primary neurons were plated at a low density (52 cells/mm 2 ) for immunofluorescence analysis or at a high density (1500 cells/mm 2 ) for Western blot. p120 ctn RNAi Mouse p120 ctn shRNA (m) and control shRNA lentiviral particles were synthesized by Santa Cruz Biotechnology, Inc. To knock down gene expression, p120 ctn shRNA (m) viral particles containing target-specific constructs encoding a 19-25 nt (short hairpin) shRNA were designed.

Techniques: Control, Immunoprecipitation, Expressing, Molecular Weight

Journal: Journal of neurochemistry

Article Title: p120-catenin is necessary for neuroprotection induced by CDK5 silencing in models of Alzheimer's disease

doi: 10.1111/jnc.13697

Figure Lengend Snippet: A) Representative GFP fluorescence from mice injected with AAV2-shSCRmiR or AAV2-shCDK5miR. Confocal images were captured at 60X magnification. Scale bar, 20 μm. The images shown are representative of 4 independent experiments. B) Representative blots of the CDK5, p35 and p25 protein levels in 3xTgAD and control mice treated with shCDK5miR or shSCRmiR. C) Quantitative representation of the results shown in B. D) The 3xTgAD mice showed an increased p25/p35 expression ratio, and this effect was reversed by CDK5 silencing. The synaptic and adhesion protein levels were decreased in the 3xTgAD mouse model, and CDK5 silencing ameliorated these effects. E) Representative blots of N-cadherin, p120 catenin, β-catenin, α-N-catenin, GluN2B and PSD95 in 3xTgAD and control mice treated with shCDK5miR or shSCRmiR. F) Quantitative representation of the results shown in A. Tubulin was used as a loading control. Densitometric quantification was performed. G) Representative blots of N-cadherin, p120 catenin, β-catenin, GluN2B and PSD95 in the PSD95 immunoprecipitation assays. H) and I) Quantitative representation of the results shown in G. Densitometric quantification was performed. The data were normalized to IgG load for analyzing changes in the complex expression (H) and relativized to PSD95 in order to evaluate the association between proteins (I) The data are presented as the mean relative units (RUs) ± SEM. n = 3-4. *p < 0.05, **p < 0.01, ***p < 0.001 (ANOVA followed by Tukey’s post hoc test or the Kruskal-Wallis test followed by Dunnett’s post hoc test, Student’s t-test between experimental groups). MW: Molecular weight .

Article Snippet: Isolated primary neurons were plated at a low density (52 cells/mm 2 ) for immunofluorescence analysis or at a high density (1500 cells/mm 2 ) for Western blot. p120 ctn RNAi Mouse p120 ctn shRNA (m) and control shRNA lentiviral particles were synthesized by Santa Cruz Biotechnology, Inc. To knock down gene expression, p120 ctn shRNA (m) viral particles containing target-specific constructs encoding a 19-25 nt (short hairpin) shRNA were designed.

Techniques: Fluorescence, Injection, Control, Expressing, Immunoprecipitation, Molecular Weight

Journal: Journal of neurochemistry

Article Title: p120-catenin is necessary for neuroprotection induced by CDK5 silencing in models of Alzheimer's disease

doi: 10.1111/jnc.13697

Figure Lengend Snippet: The p120 ctn protein levels were decreased in the neurites of a glutamate-induced excitotoxicity model but were recovered by CDK5 silencing or roscovitine treatment. A) Representative blots and quantitative representation of the CDK5 and p120 ctn levels in a glutamate-induced excitotoxicity model after CDK5 silencing. Tubulin was used as a loading control. Densitometric quantification was performed. The data are presented as the means ± SEM. n = 3, performed in duplicate. *p < 0.05, **p < 0.01, ***p < 0.001. B) Relative units of fluorescence intensity from soma and dendrites were individually measured. p120 ctn intensity was determined in primary, secondary and tertiary neurites. Data are presented as the means ± SEM; n = 5, performed in duplicate. *, #, + represent comparisons of p120 ctn intensity for primary, secondary and tertiary neurites, respectively. *,#,+ p<0.05, **,##,++ p<0.01. ; ANOVA with Tukey’s test. C) Neurons transduced with AAV carrying eGFP-tagged SCR or CDK5 shRNA were immunostained for p120 ctn. Arrows: neurites formation; White squares: Merge crop. Confocal images were captured at 60X magnification. Scale bar, 20 μm. The images shown are representative of 4 independent experiments. D) p120 ctn immunostaining rainbow pseudo-color representation and scaling intensity profile in a surface Plot. Signal Intensity is showed at the scale in bits. E) Representative blots and quantitative graphs of the CDK5 and p120 ctn levels in a glutamate-induced excitotoxicity model after roscovitine treatment. Tubulin was used as a loading control. Densitometric quantification was performed. The data are presented as the means ± SEM. n = 3, performed in duplicate. *p < 0.05, **p < 0.01. The p120 ctn levels were increased in CDK5 KO mice. F) Representative blots and quantitative representation of the CDK5 and p120 ctn protein levels in heterozygous and homozygous CDK5 KO mice. Tubulin was used as a loading control. Densitometric quantification was performed. The data are presented as the means ± SEM. n = 3, performed in duplicate. *p < 0.05, **p < 0.01 (ANOVA followed by Tukey’s post hoc test or the Kruskal-Wallis test followed by Dunnett’s post hoc test).

Article Snippet: Isolated primary neurons were plated at a low density (52 cells/mm 2 ) for immunofluorescence analysis or at a high density (1500 cells/mm 2 ) for Western blot. p120 ctn RNAi Mouse p120 ctn shRNA (m) and control shRNA lentiviral particles were synthesized by Santa Cruz Biotechnology, Inc. To knock down gene expression, p120 ctn shRNA (m) viral particles containing target-specific constructs encoding a 19-25 nt (short hairpin) shRNA were designed.

Techniques: Control, Fluorescence, Transduction, shRNA, Immunostaining

Journal: Journal of neurochemistry

Article Title: p120-catenin is necessary for neuroprotection induced by CDK5 silencing in models of Alzheimer's disease

doi: 10.1111/jnc.13697

Figure Lengend Snippet: A) CDK5 silencing-induced neuroprotection was reversed by p120 ctn knockdown. Neuronal cytotoxicity is presented as the percentage of LDH release from the cells. The data are presented as the means ± SEM of 3 experiments performed in triplicate. p120 ctn over-expression protects against glutamate-induced cytotoxicity. Percentage of condensed nuclei relative to total nuclei in transfected neurons. B) Rac and RhoA activity were quantified after 30 mins of glutamate treatment as the amount of Rac-GTP and RhoA- GTP by ELISA (k = 490 nm). The data are presented as the means ± SEM from n = 3 per duplicate. Control shRNA: gray black bars and sh p120: gray light bars. * represents the comparison between each treatment of the control shRNA neurons or # sh p120ctn neurons. * or # p < 0.05, *** or ### p < 0.001; ANOVA with Tukey’s test. C) Representative blots and quantitative bars of the CDK5 and p120 ctn levels in a glutamate-induced excitotoxicity model and the respective controls after silencing both CDK5 and p120 ctn. Tubulin was used as a loading control. Densitometric quantification was performed. The data are presented as the means ± SEM. n = 3; each experiment was performed in duplicate. *p < 0.05, ***p < 0.001. D) Morphological characteristics are shown for neurons transfected with a plasmid expressing GFP-tagged (green) LZRS-eGFP or LZRS-p120ctn. The nuclei were stained with Hoechst (blue). The arrowhead shows the condensed nucleus for comparison with a normal nucleus (arrow). Magnification, 60X. Scale bar, 20 μm. E) Graphic representation of the numerical results shown in C. The data are presented as the means ± SEM of 3 experiments performed in duplicate. *p < 0.05. F) Representative blots and quantitation of the p120 ctn protein levels in HEK203T cells after LZRS-eGFP or LZRS-p120ctn transfection. Actin was used as a loading control. (ANOVA followed by Tukey’s post hoc test or the Kruskal-Wallis test followed by Dunnett’s post hoc test.)

Article Snippet: Isolated primary neurons were plated at a low density (52 cells/mm 2 ) for immunofluorescence analysis or at a high density (1500 cells/mm 2 ) for Western blot. p120 ctn RNAi Mouse p120 ctn shRNA (m) and control shRNA lentiviral particles were synthesized by Santa Cruz Biotechnology, Inc. To knock down gene expression, p120 ctn shRNA (m) viral particles containing target-specific constructs encoding a 19-25 nt (short hairpin) shRNA were designed.

Techniques: Knockdown, Over Expression, Transfection, Activity Assay, Enzyme-linked Immunosorbent Assay, Control, shRNA, Comparison, Plasmid Preparation, Expressing, Staining, Quantitation Assay

Journal: Journal of cell science

Article Title: Regulation of transient receptor potential canonical channel 1 (TRPC1) by sphingosine 1-phosphate in C2C12 myoblasts and its relevance for a role of mechanotransduction in skeletal muscle differentiation.

doi: 10.1242/jcs.035402

Figure Lengend Snippet: Fig. 1. Expression and subcellular localization of TRPC1 in C2C12 myoblasts. (A) Transient receptor potential canonical (TRPC) channel mRNA isoforms in C2C12 myoblasts. Lanes show amplified products of RT-PCR reactions. Total RNA (1 μg) obtained from C2C12 myoblasts was retro-transcripted and cDNA amplified as described in Materials and Methods. The PCR products were visualized on an ethidium-bromide- stained agarose gel. Data are representative of three independent experiments. β-actin amplification, used as an internal control, is shown. (B) TRPC1 expression in subcellular fractions of C2C12 myoblasts. Aliquots of proteins (25 μg) from cell lysates (Lys), cytosol (cyt), Triton-soluble (Ms) or Triton-insoluble (Mi) membrane were processed for western blotting analysis. A blot representative of three is shown. (C) Effect of TRPC1 silencing on channel expression. C2C12 myoblasts were transfected with SCR-siRNA (–) and TRPC1- siRNA (+) as described in Materials and Methods. Aliquots of proteins (30 μg) from cell lysate were subjected to western blotting analysis. A blot representative of three independent experiments with similar results is shown. Band intensity is reported as relative percentage with s.e.m. less than 15%. (D) Confocal immunofluorescence analysis of TRPC1 cell localization. C2C12 cells were grown on glass coverslips, fixed and stained with the primary antibody against TRPC1 (green). The cells were counterstained with TRITC-phalloidin (red) to reveal actin filaments. In the inset, a superimposed fluorescence and DIC image shows co-localization of TRPC1 with TRITC-conjugated WGA at the cell surface. Note that the immunostaining is markedly reduced in TRPC1-silenced cells compared with native and SCR-siRNA treated ones. The images are representative of at least three independent experiments with similar results.

Article Snippet: To inhibit the expression of TRPC1, short interference RNA duplexes (siRNA) (Santa Cruz Biotechnology, Santa Cruz, CA) corresponding to three distinct regions of the DNA sequence of mouse TRPC1 gene (NM_011643): GAGAAAUGCUGUUACC - AUA, CCAGAAUAUUCAACAACGA and GAACUUAAGUCGUCUGAAA) were used in combination.

Techniques: Expressing, Amplification, Reverse Transcription Polymerase Chain Reaction, Staining, Agarose Gel Electrophoresis, Control, Membrane, Western Blot, Transfection, Immunofluorescence, Fluorescence, Immunostaining

Journal: Journal of cell science

Article Title: Regulation of transient receptor potential canonical channel 1 (TRPC1) by sphingosine 1-phosphate in C2C12 myoblasts and its relevance for a role of mechanotransduction in skeletal muscle differentiation.

doi: 10.1242/jcs.035402

Figure Lengend Snippet: Fig. 2. Effect of TRPC1 silencing on stretch-activated Ca2+-transients and SAC currents in C2C12 myoblasts. (A) C2C12 myoblasts were pre-loaded with Fluo3-AM and mechanically stretched with the tip of an AFM probe (thick grey line). Fluorescence images were acquired soon after mechanical stimulation at a rate of 1 image/second using a digital camera. The pseudo- colouring represents the global Ca2+ increase as indicated by the colour bar. Note the marked reduction and the absence of the Ca2+ transient in TRPC1 silenced cells. The images are representative of at least five to six independent experiments with similar results (number of stretched cells for each group=5). (B) Two patch pipettes were attached to the cells at (a) resting length and (b) after 20% cell stretching induced by the movement of the upper pipette in the longitudinal direction. (C) Representative total current traces, (a) Im* recorded in bath solution by whole cell path-clamp; (b) leak current, Im,leak, recorded in the presence of GdCl3 added in the bath solution and (c-j) SAC-mediated current; Im, evaluated by detracting Im,leak from Im*; (c-f) SAC-mediated currents (Im) in SCR- and TRPC1-siRNA cells unstimulated and (g-j) S1P- stimulated evaluated before (c,e,g,i) and after (d,f,h,j) the application of the mechanical stretch; (k,l), currents normalized for Cm elicited by ramp voltage pulses from the same cell reported in panels a-j. siTRPC1, TRPC1-siRNA transfected cells; str, stretched cells.

Article Snippet: To inhibit the expression of TRPC1, short interference RNA duplexes (siRNA) (Santa Cruz Biotechnology, Santa Cruz, CA) corresponding to three distinct regions of the DNA sequence of mouse TRPC1 gene (NM_011643): GAGAAAUGCUGUUACC - AUA, CCAGAAUAUUCAACAACGA and GAACUUAAGUCGUCUGAAA) were used in combination.

Techniques: Fluorescence, Transferring, Transfection

Journal: Journal of cell science

Article Title: Regulation of transient receptor potential canonical channel 1 (TRPC1) by sphingosine 1-phosphate in C2C12 myoblasts and its relevance for a role of mechanotransduction in skeletal muscle differentiation.

doi: 10.1242/jcs.035402

Figure Lengend Snippet: Fig. 5. Effect of TRPC1 silencing on skeletal myogenic differentiation of C2C12 myoblasts. (A) Western analysis of TRPC1 silencing on the expression of myogenic markers. Confluent C2C12 myoblasts were transfected with SCR-siRNA (–) or TRPC1-siRNA (+), stimulated with (+) or without (–) 1 μM S1P and differentiation started as described in the Materials and Methods. The content of TRPC1, myogenin and α-sarcomeric actin were analysed by western blotting. A blot representative of at least three independent experiments with similar results and the relative percentage is shown (mean ± s.e.m.). (B) Confocal immunofluorescence and phase-contrast analysis of differentiating myoblasts. SCR-siRNA and TRPC1-siRNA cells were cultured on glass coverslips in DM, fixed and stained with the primary antibody against myogenin (green), and counterstained with TRITC-phalloidin to detect actin filaments (red). Parallel experiments were performed to reveal myotube formation by phase contrast. Note that silenced cells reveal reduced nuclear myogenin staining and polyhedral morphology typical of the undifferentiated cells and are unable to form multinucleate myotubes compared to SCR-siRNA cells. The images are representative of at least three separate experiments with similar results.

Article Snippet: To inhibit the expression of TRPC1, short interference RNA duplexes (siRNA) (Santa Cruz Biotechnology, Santa Cruz, CA) corresponding to three distinct regions of the DNA sequence of mouse TRPC1 gene (NM_011643): GAGAAAUGCUGUUACC - AUA, CCAGAAUAUUCAACAACGA and GAACUUAAGUCGUCUGAAA) were used in combination.

Techniques: Western Blot, Expressing, Transfection, Immunofluorescence, Cell Culture, Staining

Journal: Journal of cell science

Article Title: Regulation of transient receptor potential canonical channel 1 (TRPC1) by sphingosine 1-phosphate in C2C12 myoblasts and its relevance for a role of mechanotransduction in skeletal muscle differentiation.

doi: 10.1242/jcs.035402

Figure Lengend Snippet: Fig. 8. Effects of TRPC1-siRNA and 2-APB on Gm/Cm in differentiating unstimulated and S1P-stimulated C2C12 myoblasts. C2C12 differentiating cells were transfected with scrambled-siRNA (SCR) and TRPC1-siRNA (siTRPC1) or treated with 2-APB in the presence or in the absence of S1P and incubated in DM for 24 hours. Significance of differences: *P<0.05, **P<0.01 with respect to relative controls; §§P<0.01 of TRPC1-siRNA (siTRPC1) with respect to SCR-siRNA (one-way ANOVA). Data are mean ± s.e.m. of 14-18 independent cell recordings.

Article Snippet: To inhibit the expression of TRPC1, short interference RNA duplexes (siRNA) (Santa Cruz Biotechnology, Santa Cruz, CA) corresponding to three distinct regions of the DNA sequence of mouse TRPC1 gene (NM_011643): GAGAAAUGCUGUUACC - AUA, CCAGAAUAUUCAACAACGA and GAACUUAAGUCGUCUGAAA) were used in combination.

Techniques: Transfection, Incubation