Journal: Cell reports

Article Title: Inducing ferroptosis to impede metastasis by inhibiting the calcium channel TRPC6

doi: 10.1016/j.celrep.2025.116543

Figure Lengend Snippet: (A) Relative viability of p27 + and p27 − sorted p27-mVenus CAL-51 cells after 24 h treatment with either DMSO or IKE in the presence or absence of ferrostatin-1, measured using the CellTiter-Glo (CTG) assay. (B) Expression of TRPC6 in p27 + and p27 − CAL-51 cells, assessed by immunoblotting. (C) p27 + CAL-51 cells were treated for 24 h with either DMSO or increasing concentrations of IKE in the presence of either BI-749327 (10 μM) or ferrostatin-1 (2 μM), or a combination of BI-749327 and ferrostatin-1. Viability was measured using the CTG assay. (D) Mice injected with either CAL-51 p27 − or CAL-51 p27 + cells were sacrificed 6 weeks post tail-vein injection, and metastases in the lung were assessed by H&E staining. The percentage of metastasis was quantified and is presented. (E) Expression of TRPC6 mRNA in primary tumor and circulating tumor cells (CTCs) in breast cancer patients obtained from ctcRbase. (F) Relative viability of LM2 cells after treatment with DMSO, IKE (1 μM), ferrostatin-1 (2 μM), or their combination for 24 h. (G) Relative viability of parental LM2 cells (Par) and an LM2 IKE-resistant (IKE-Res) sub-population after 48 h, measured using the CTG assay. (H) Quantification of p27 mRNA by qPCR in LM2 parental and IKE-Res cells. (I) Quantification of TRPC6 mRNA by qPCR in LM2 parental and IKE-Res cells. (J) Time course of Ca 2+ flux measured by mean fluorescence intensity following CaCl 2 exposure in LM2 parental and LM2 IKE-Res cells pretreated with Fluo4-AM. (K) Relative viability of LM2 parental cells and LM2 cells that had been transfected with a TRPC6 expression plasmid (TRPC6-WT) and then treated with increasing IKE concentrations for 24 h, measured using the CTG assay. (L) Quantification of p27 mRNA by qPCR in LM2 parental and LM2 TRPC6-WT-overexpressing cells. The bars in graphs represent mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: Rabbit polyclonal Anti-TRPC6 Antibody , Alomone Labs , Cat#ACC-017; RRID: AB_2040243.

Techniques: CTG Assay, Expressing, Western Blot, Injection, Staining, Fluorescence, Transfection, Plasmid Preparation

Journal: Cell reports

Article Title: Inducing ferroptosis to impede metastasis by inhibiting the calcium channel TRPC6

doi: 10.1016/j.celrep.2025.116543

Figure Lengend Snippet: (A) The level of GSH was quantified in p27 + and p27 − sorted p27-mVenus CAL-51 cells. (B) The level of GSH levels was quantified in p27 + CAL-51 cells that have been pretreated with either BI-749327 (10 μM) or DMSO for 48 h and subsequently treated with IKE (10 μM) or DMSO for 24 h. (C) Expression of glutamate-cysteine ligase catalytic (GCLC) subunit mRNA in LM2 (parental) and LM2 IKE-resistant (IKE-Res) cells was quantified by qPCR. (D) GSEA of transcriptomic data from p27 + and p27 − CAL-51 cells shows enrichment of c-Myc targets in p27 − cells. (E) Expression of c-Myc in p27 + and p27 − CAL-51 cells and in LM2 parental and LM2 IKE-Res cells shows enrichment of c-Myc targets in p27 − CAL-51 and LM2 cells, assessed by immunoblotting. (F) Expression of c-Myc mRNA in primary tumor and circulating tumor cells (CTCs) in breast cancer patients obtained from ctcRbase. (G) mRNA expression of TRPC6 , c-Myc , CDC25a , and GCLC was quantified in p27 + CAL-51 cells that had been treated with siTRPC6 or siCTRL for 24 h. (H) The level of GSH was quantified after 30 min in suspension in CAL-51 p27 + cells treated with siTRPC6 or siCTRL for 24 h. (I) mRNA expression of TRPC6 , c-Myc , and GCLC was quantified in CAL-51 p27 − parental or CAL-51 p27 − TRPC6-overexpressing (TRPC6-WT) cells. (J) The concentration of GSH was quantified after 30 min in suspension in CAL-51 p27 − parental and TRPC6-WT cells. (K) Relative viability of p27 − CAL-51 cells that had been treated with increasing concentrations of IKE in the presence of either DMSO or 10074-G5 (50 μM) for 24 h. (L) Relative viability of LM2 cells that had been treated with increasing concentrations of IKE in the presence of either DMSO or 10074-G5 (50 μM) for 24 h. (M) The concentration of GSH was quantified in p27 − CAL-51 cells after 24-h treatment with either 10074-G5 (50 μM) or DMSO. (N) The level of GSH was quantified in LM2 cells after 24-h treatment with either 10074-G5 (50 μM) or DMSO. (O) GCLC mRNA expression was quantified by qPCR in LM2 cells that had been treated with 10074-G5 (50 μM) or DMSO for 24 h. The bars in graphs represent mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: Rabbit polyclonal Anti-TRPC6 Antibody , Alomone Labs , Cat#ACC-017; RRID: AB_2040243.

Techniques: Expressing, Western Blot, Suspension, Concentration Assay

Journal: Cell reports

Article Title: Inducing ferroptosis to impede metastasis by inhibiting the calcium channel TRPC6

doi: 10.1016/j.celrep.2025.116543

Figure Lengend Snippet: (A) Total ROS measured by DCF fluorescence of p27 + and p27 − CAL-51 cells 30 min after matrix detachment. (B) Relative viability of p27 + and p27 − CAL-51 cells 48 h after matrix detachment as measured by trypan blue exclusion. (C) Total ROS measured by DCF fluorescence in p27 − CAL-51 cells after pretreatment with 10074-G5 (50 μM) or DMSO for 24 h and matrix detachment for 30 min (D) The level of GSH was quantified in p27 − CAL-51 cells after pretreatment with 10074-G5 (50 μM) or DMSO for 24 h and matrix detachment for 30 min (E) Relative viability of p27 + CAL-51 cells assessed by trypan blue exclusion after pretreatment with BI-749327 (10 μM) or DMSO for 24 h and matrix detachment for 48 h in the presence of either DMSO or ferrostatin-1 (2 μM). (F) Relative viability of p27 − CAL-51 cells that were engineered to express TRPC6 (TRPC6-WT) after pretreatment with BI-749327 (10 μM) or DMSO for 24 h and matrix detachment for 48 h in the presence of either DMSO or ferrostatin-1 (2 μM). (G) Relative viability of IKE-Res, TRPC6-expressing (TRPC6-WT), and parental LM2 cells pretreated with either BI-749327 (10 μM) or DMSO for 24 h before matrix detachment for 48 h. (H) Relative viability of LM2 IKE-Res and LM2 IKE-Res cells that were engineered to express c-Myc after matrix detachment for 48 h. (I) GSH was quantified in LM2 cells pretreated with 10074-G5 (50 μM) or DMSO for 24 h before matrix detachment for 30 min (J) Total ROS measured by DCF fluorescence of LM2 cells pretreated with either 10074-G5 (50 μM) or DMSO for 24 h before matrix detachment for 30 min (K) Relative viability assay of LM2 cells pretreated with 10074-G5 (50 μM) or DMSO for 24 h before matrix detachment for 48 h as assessed by trypan blue exclusion. The bars in graphs represent mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: Rabbit polyclonal Anti-TRPC6 Antibody , Alomone Labs , Cat#ACC-017; RRID: AB_2040243.

Techniques: Fluorescence, Expressing, Viability Assay

Journal: Nucleic Acids Research

Article Title: A novel transient receptor potential C3/C6 selective activator induces the cellular uptake of antisense oligonucleotides

doi: 10.1093/nar/gkae245

Figure Lengend Snippet: Effects of the TRPC3/C6/C7 inhibitor, knockdown of TRPC3/C6, and Ca 2+ chelator on ASO uptake, and examination of L687-mediated uptake pathways. ( A ) ASO uptake was analysed by incubating cells with or without a TRPC inhibitor (SKF96365). Alexa647-AmNA#26 (10 nM) was added to either 10 μM L687, 20 μM GSK1702934A, or 30 μM CBD with or without 20 μM SKF96365 in the medium. After 24 h, the intracellular fluorescence intensities were analysed by flow cytometry. Data are shown as the relative MFI of ASO in DMSO. All data are presented as mean ± standard error of the mean (SEM) of three independent experiments ( n = 3). Statistical significance was determined by comparing with values of DMSO using Tukey's test. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. ( B ) siRNA-mediated knockdown of the TRPC3/C6 channels. TRPC3 siRNA (30 nM) or TRPC6 siRNA (10 nM) was transfected into A549 cells using Lipofectamine3000, and cells were collected after 48 h. The relative expression of TRPC3/C6 was analysed and compared with that in the untreated cells. ( C ) siRNA-mediated knockdown of TRPC3/C6 channel. TRPC3 siRNA (30 nM) or TRPC6 siRNA (10 nM) was transfected into A549 cells using Lipofectamine3000, and cells were collected after 48 h. The relative expression of TRPC3/C6 was analysed by western blot and compared with that in untreated cells. ( D ) The effects of siRNA-mediated TRPC3/C6 channel knockdown on ASO uptake. TRPC3 siRNA (30 nM), TRPC6 (10 nM), or a combination of both were transfected into A549 cells for 48 h. The medium was replaced with Alexa647-AmNA#26 containing L687, and intracellular fluorescence intensities were analysed after 24 h. Data are shown as the relative MFI of ASO in DMSO. ( E ) Analysis of ASO uptake after incubating cells with a Ca 2+ chelator (BAPTA-AM). ASO and L687 were added to the medium, with or without 10 μM BAPTA-AM. After 24 h, intracellular fluorescence intensities were analysed by flow cytometry. ( F ) Analysis of dextran uptake mediated by L687. Alexa647-labelled dextran (1 and 3 μM) with 10 and 30 μM L687 was added to the medium, and A549 cells were cultured for 24 h. Intracellular fluorescence was analysed by flow cytometry, and the relative MFI was compared with 1 μM of Alexa647-dextran with DMSO. ( G ) Analysis of ASO uptake by incubating the cells with a macropinocytosis inhibitor (Cytochalasin D). L687 (30 μM) was then added to the medium. The following day, cells were then washed twice with PBS and incubated with cytochalasin D in the medium for 1 h. Then, cells were washed twice with PBS and incubated with Alexa647-AmNA#26 (10 nM) and L687 (30 μM). After 4 h, intracellular fluorescence intensities were analysed by flow cytometry. ( H ) Analysis of ASO uptake by incubating cells with a macropinocytosis inhibitor (EIPA). ASO and L687 were added to the medium with or without 100 μM EIPA. After 24 h, intracellular fluorescence intensities were analysed by flow cytometry. ( I ) Fluorescence imaging analysis of ASO incorporated into cells. Alexa647-AmNA#26 (100 nM) and L687 (30 μM) were added to the medium, and staining with Lysotracker-green and Hoechst, fluorescence microscopy imaging, and image analysis were performed after 48 h. ASO, antisense oligonucleotide; CBD, cannabidiol; DMSO, dimethyl sulfoxide; MFI, mean fluorescence intensity; TRPC, transient receptor potential canonical.

Article Snippet: Incubation with primary polyclonal rabbit anti-TRPC3 antibody (#ACC-016; Alomone Labs, Jerusalem, Israel), primary polyclonal rabbit anti-TRPC6 antibody (#ACC-120; Alomone Labs, Jerusalem, Israel) at 1:2000 dilution and mouse monoclonal anti-GAPDH (#AM4300; Thermo Fisher Scientific, MA, USA) at 1:2000 dilution was performed at 4°C overnight.

Techniques: Knockdown, Fluorescence, Flow Cytometry, Transfection, Expressing, Western Blot, Cell Culture, Incubation, Imaging, Staining, Microscopy

Journal: ACS Nano

Article Title: A Dual Role of Mesenchymal Stem Cell Derived Small Extracellular Vesicles on TRPC6 Protein and Mitochondria to Promote Diabetic Wound Healing

doi: 10.1021/acsnano.3c09814

Figure Lengend Snippet: TRPC6 is impaired in the diabetic wound. (A, B) Fura-2 AM (5 μ M) detected cytosolic Ca 2+ levels in DFs when treated with AGE-BSA. (C) Schematic diagram of calcium channels expressed in the skin. (D) Quantitative real-time PCR from three independent experiments showing the expression levels of TRPC6 in DFs treated with AGE-BSA for 48 h. (E) Western blotting analysis from three independent tests showed the levels of TRPC6 in DFs treated with AGE-BSA for 48 h. (F) Dox diagram of TRPC6 expression. (G) Heat map of correlation between TRPC6 and other genes (Bubble size represents correlation r value; “*” indicates a significant correlation; red indicates positive correlation). (H) Immunohistochemical staining for TRPC6 in skin tissues from surgical specimens of diabetic ulcer patients. Scale bar: 100 μ m. Data are presented as mean ± standard error of the mean (SEM). ns, not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Article Snippet: After blocking with a 5% BSA solution, the skin sections were incubated overnight with a specific rabbit polyclonal anti-TRPC6 antibody (Novus, #NBP1-77260, 1:500) at 4 °C.

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

Journal: ACS Nano

Article Title: A Dual Role of Mesenchymal Stem Cell Derived Small Extracellular Vesicles on TRPC6 Protein and Mitochondria to Promote Diabetic Wound Healing

doi: 10.1021/acsnano.3c09814

Figure Lengend Snippet: TRPC6 knockdown decreases Ca 2+ influx and impairs the biological function of DFs in wound healing. (A–C) Fura-2 AM (5 μ M) and Fluo-4 AM (5 μ M) detected cytosolic Ca 2+ levels in DFs when treated with small interfering RNA of TRPC6 (Si-TRPC6) for 48 h. (D) Immunofluorescence analysis showed the level of α -SMA and collagen I of DFs when transfected with Si-TRPC6 for 48 h. Scale bar: 100 μ m. (E) Western blotting analysis of α -SMA, collagen I expression in DFs treated with Si-TRPC6 for 48 h. (F) Quantitative real-time PCR analysis of the expression levels of IL-1β, IL-6, and TNF-α in DFs when treated with Si-TRPC6 for 48 h. (G) Quantitative real-time PCR analysis of the expression levels of TRPC6 in skin tissues when infected in shTRPC6 for 4 weeks. (H) Western blotting analysis of TRPC6 in skin tissues when infected in shTRPC6 for 4 weeks. (I, J) HE and Masson staining evaluated wound and collagen distribution in skin tissues when infected in shTRPC6 for 4 weeks. Scale bar: 100 μ m. (K) Immunohistochemical staining evaluated the expression of CD31. Scale bar: 100 μ m. Data are presented as mean ± standard error of the mean (SEM). ns, not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Article Snippet: After blocking with a 5% BSA solution, the skin sections were incubated overnight with a specific rabbit polyclonal anti-TRPC6 antibody (Novus, #NBP1-77260, 1:500) at 4 °C.

Techniques: Knockdown, Small Interfering RNA, Immunofluorescence, Transfection, Western Blot, Expressing, Real-time Polymerase Chain Reaction, Infection, Staining, Immunohistochemical staining

Journal: ACS Nano

Article Title: A Dual Role of Mesenchymal Stem Cell Derived Small Extracellular Vesicles on TRPC6 Protein and Mitochondria to Promote Diabetic Wound Healing

doi: 10.1021/acsnano.3c09814

Figure Lengend Snippet: HucMSC-sEVs restore TRPC6 expression in vitro and in vivo . (A) Quantitative real-time PCR from three independent experiments showing the expression levels of TRPC6 in DFs treated with hucMSC-sEVs (1.0 × 10 10 particles/mL) for 48 h. (B) Western blotting analysis of TRPC6 expression in DFs treated with hucMSC-sEVs (1.0 × 10 10 particles/mL) for 48 h. (C) Immunofluorescence analysis showed the location and level of TRPC6 (red) in DFs when treated with DIO-labeled hucMSC-sEVs (1.0 × 10 10 particles/mL) for 48 h. Scale bar: 10 μ m. (D) Immunohistochemical staining evaluated the expression of TRPC6 in diabetic rats’ skin. Scale bar: 50 μ m. (E) Immunofluorescent staining for the expression of TRPC6 in diabetic rats’ skin. Scale bar: 100 μ m. (F–H) Fura-2 AM (5 μ M) and Fluo-4 AM (5 μ M) detected cytosolic Ca 2+ levels in DFs when treated with hucMSC-sEVs (1.0 × 10 10 particles/mL) for 48 h. Data are presented as mean ± standard error of the mean (SEM). ns, not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Article Snippet: After blocking with a 5% BSA solution, the skin sections were incubated overnight with a specific rabbit polyclonal anti-TRPC6 antibody (Novus, #NBP1-77260, 1:500) at 4 °C.

Techniques: Expressing, In Vitro, In Vivo, Real-time Polymerase Chain Reaction, Western Blot, Immunofluorescence, Labeling, Immunohistochemical staining, Staining

Journal: ACS Nano

Article Title: A Dual Role of Mesenchymal Stem Cell Derived Small Extracellular Vesicles on TRPC6 Protein and Mitochondria to Promote Diabetic Wound Healing

doi: 10.1021/acsnano.3c09814

Figure Lengend Snippet: HucMSC-sEVs transfer transcription factor SP2 to DFs and activate TRPC6 gene expression. (A) Diagram of a database cross to confirm transcription factor SP2. (B) Western blotting analysis of SP2 expression in hucMSC-sEVs. (C) Western blotting analysis of SP2 expression in the nucleus and cytoplasm of DFs when treated with hucMSC-sEVs (1.0 × 10 10 particles/mL) for 48 h. (D) Double luciferase reporter gene assay showed SP2 binding to the TRPC6 promoter. (E) ChIP analysis of SP2 binding to the TRPC6 promoter. (F) Schematic structure of the full-length TRPC6 promoter-reporter and its deletion mutant constructs. (G, H) Double luciferase reporter gene assay showed the SP2 binding sites. (I) Quantitative real-time PCR analysis of the expression levels of TRPC6 in DFs when treated with SiSP2-hucMSC-sEVs (1.0 × 10 10 particles/mL) for 48 h. (J) Western blotting analysis of TRPC6 and collagen I expression in DFs when treated with SiSP2-hucMSC-sEVs (1.0 × 10 10 particles/mL) for 48 h. (K) CCK-8 assay showed proliferation of DFs treated with SiSP2-hucMSC-sEVs (1.0 × 10 10 particles/mL) for 48 h. (L) Immunofluorescence analysis showed the level of collagen I of DFs when treated with SiSP2-hucMSC-sEVs (1.0 × 10 10 particles/mL) for 48 h. Scale bar: 100 μ m. Data are presented as mean ± standard error of the mean (SEM). ns, not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Article Snippet: After blocking with a 5% BSA solution, the skin sections were incubated overnight with a specific rabbit polyclonal anti-TRPC6 antibody (Novus, #NBP1-77260, 1:500) at 4 °C.

Techniques: Gene Expression, Western Blot, Expressing, Luciferase, Reporter Gene Assay, Binding Assay, Mutagenesis, Construct, Real-time Polymerase Chain Reaction, CCK-8 Assay, Immunofluorescence

Journal: ACS Nano

Article Title: A Dual Role of Mesenchymal Stem Cell Derived Small Extracellular Vesicles on TRPC6 Protein and Mitochondria to Promote Diabetic Wound Healing

doi: 10.1021/acsnano.3c09814

Figure Lengend Snippet: HucMSC-sEVs protect the function of mitochondria to maintain calcium balance. (A) Quantitative real-time PCR analysis of the expression levels of TRPC6 in DFs transfected with overexpression plasmid of TRPC6 for 48 and 96 h. (B) Calcium ion assay of DFs treated with AGE-BSA, overexpression plasmid of TRPC6 (OE-TRPC6), overexpression of controlled plasmid (OE-Vector), CaCl 2 (2 mM), and hucMSC-sEVs (1.0 × 10 10 particles/mL) for 24, 48, 72, and 96 h. (C) Annexin V/PI staining of DFs treated with AGE-BSA, overexpression plasmid of TRPC6 (OE-TRPC6), overexpression of controlled plasmid (OE-Vector), CaCl 2 (2 mM), and hucMSC-sEVs (1.0 × 10 10 particles/mL) for 24, 48, 72, and 96 h. (D) The wound healing assay showed migration of DFs when treated with AGE-BSA, overexpression plasmid of TRPC6 (OETRPC6), overexpression of controlled plasmid (OE-Vector), CaCl 2 (2 mM), and hucMSC-sEVs (1.0 × 10 10 particles/mL) for 24, 48, 72, and 96 h. (E) Mitochondrial membrane potential assay with JC-1 of DFs treated with AGE-BSA and hucMSC-sEVs (1.0 × 10 10 particles/mL) for 48 h. Scale bar: 100 μ m. (F) Western blotting analysis of MCU, MICU1, and NCLX expression in DFs treated with AGE-BSA and hucMSC-sEVs (1.0 × 10 10 particles/mL) for 48 h. (G) Fluorescent Ca 2+ indicator of Rhod-2 AM (5 μ M) showed mitochondrial calcium level in DFs treated with AGE-BSA and hucMSC-sEVs (1.0 × 10 10 particles/mL) for 48 h. Scale bar: 200 μ m. (H) Relative mitochondrial Ca 2+ uptake in DFs treated with AGE-BSA and hucMSC-sEVs (1.0 × 10 10 particles/mL) for 48 h. Data are presented as mean ± standard error of the mean (SEM). ns, not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Article Snippet: After blocking with a 5% BSA solution, the skin sections were incubated overnight with a specific rabbit polyclonal anti-TRPC6 antibody (Novus, #NBP1-77260, 1:500) at 4 °C.

Techniques: Real-time Polymerase Chain Reaction, Expressing, Transfection, Over Expression, Plasmid Preparation, Staining, Wound Healing Assay, Migration, Membrane, Western Blot

Journal: Cell reports

Article Title: The calcium channel TRPC6 promotes chemotherapy-induced persistence by regulating integrin α6 mRNA splicing

doi: 10.1016/j.celrep.2023.113347

Figure Lengend Snippet: (A) Heatmap of differentially expressed genes in the CSC and non-CSC populations from a published model system. TRPC6 is highlighted in the heatmap. False discovery rate (FDR) < 0.05 and |log2FC| (fold change) > 1. (B) Expression of TRPC6 and other calcium channels was compared in the same CSC vs. non-CSC populations by qPCR. (C) TRPC6 expression was compared in MDA-MB-231 cells and their TE3 variants. (D) HMLER cells were sorted into CD104 − /CD24 − (CSC; HMLER −/− ) and CD104 + /CD24 + (non-CSC; HMLER +/+ ) populations, and TRPC6 expression was quantified by qPCR. (E) The CSC (CD44 high /CD24 low ) and non-CSC (CD44 low /CD24 high ) populations was sorted from a TNBC PDX (left), and TRPC6 expression levels measured were quantified by qPCR. (F and G) TRPC6 expression was knocked down in either TE3 (F) or CAL-51 (G) cells using shRNA, and expression was rescued with either a wild-type TRPC6 construct (WT) or a pore mutant (G757D) that is deficient in calcium uptake (MUT). These populations were assessed for self-renewal by serial passage of mammospheres (P1, passage 1; P2, passage 2). (H) CAL-51 control shRNA (shCTRL) or TRPC6 knockdown (shTRPC6) cells were injected into the mammary fat pads of NSG mice in limiting dilution (10 6 , 10 5 , and 10 4 cells), and the frequency of tumor incidence was determined (right). Tumor incidence was plotted utilizing ELDA in a log plot to estimate the frequency of tumor-initiating cells in each group. (I) CAL-51 cells (shCTRL, shTRPC6–1, sh+ WT TRPC6, and sh+ mut TRPC6) were injected into the mammary fat pads of NSG mice, and tumor onset in terms of days post-injection was compared among the groups. The TRPC6 expression data shown in (C) and (D) represent the mean ± SD of a representative experiment performed three times independently. The mammosphere data shown represent the mean ± SD of three independent experiments. For (H), data are presented as log-log plot, and the frequency of tumor-initiating cells is calculated by extreme limiting-dilution analysis. The tumor onset data represent the median days post-injection between the groups. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.

Article Snippet: Rabbit polyclonal anti-TRPC6 , Proteintech , Cat# 18236–1-AP; RRID: AB_10859822.

Techniques: Expressing, shRNA, Construct, Mutagenesis, Injection

Journal: Cell reports

Article Title: The calcium channel TRPC6 promotes chemotherapy-induced persistence by regulating integrin α6 mRNA splicing

doi: 10.1016/j.celrep.2023.113347

Figure Lengend Snippet: (A) Characterization of CAL-51 chemotherapy-resistant cells. CAL-51 cells were made resistant to paclitaxel as described in the and then compared to parental cells (termed sensitive) for viability in response to increasing concentrations of paclitaxel. (B) TRPC6 mRNA expression was quantified in CAL-51-sensitive and -resistant cells. (C) CAL-51-sensitive and -resistant cells were assayed for the frequency of CSCs using a limiting-dilution mammosphere assay, and the data were analyzed by ELDA. (D) CAL-51-resistant cells were treated with vehicle control or BI-749327 (10 μM) for 24 h, and the frequency of CSCs was determined using a limiting-dilution mammosphere assay and ELDA. (E) CAL-51-resistant (CAL-51-R) cells were treated with increasing concentrations of paclitaxel in combination with either DMSO or BI-749327 (10 μM) for 24 h, and cell viability was assessed. (F) TRPC6 mRNA expression was quantified in chemotherapy-sensitive and -resistant models of a patient-derived organoid (9883T) as described in the . (G) The organoid models described in (F) were treated with either DMSO, paclitaxel (PTX; 20 nM), BI-749437 (10 μM), or PTX+BI-749327 (BI) for 96 h, and cell viability was measured. (H) Tumors volumes (in mm 3 ) in mice that had been implanted orthotopically with a human TNBC PDX (PDX HCI028). The mice were divided into 4 groups of 5 mice each. When tumors reached an approximate volume of 100 m 3 , the mice were treated with either vehicle, PTX (15 mg/kg), BI (15mg/kg), or a combination of PTX and BI. PTX was injected intraperitoneally (i.p.) twice a week, and BI was administered by oral gavage 4 days a week. Day 0 on the x axis indicates the start of the treatments. Tumor volume was measured every 5 days. For (C) and (D), data are presented as a log-log plot, and the frequency of stem cells is calculated by extreme limiting-dilution analysis. The viability data shown (E and G) represent the mean ± SD of a representative experiment performed three times independently. The tumor volume data shown in (H) are represented as mean ± SEM of the number of mice in the respective groups. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.

Article Snippet: Rabbit polyclonal anti-TRPC6 , Proteintech , Cat# 18236–1-AP; RRID: AB_10859822.

Techniques: Expressing, Derivative Assay, Injection

Journal: Cell reports

Article Title: The calcium channel TRPC6 promotes chemotherapy-induced persistence by regulating integrin α6 mRNA splicing

doi: 10.1016/j.celrep.2023.113347

Figure Lengend Snippet: (A) Heatmap showing genes that are differentially expressed in vehicle- and BI (10 μM)-treated TE3 cells (12 h). FDR < 0.05 and |log2FC| > 1. (B and C) ESRP1 mRNA (B) and protein (C) expression was assessed in TE3 cells treated with either vehicle control or BI (10 μM) for 24 h. (D) TE3 and CAL-51 cells were treated with either vehicle control or BI (10 μM) for 24 h, and the expression of the α6A and α6B integrin splice variants was assessed by immunoblotting. (E) The expression of TRPC6, ESRP1, α6A, and α6B was assessed in the CAL-51-sensitive and CAL-51-R cells by immunoblotting. (F) The expression of ESRP1, α6A, and α6B in CAL-51-R cells that had been treated with either DMSO or BI (10 μM) for 24 h was assessed by immunoblotting. (G) CAL-51-R cells were stably transfected with either a control plasmid (vector) or an ESRP1 expression plasmid (ESRP1-HA), and the expression of ESRP1, HA, and α6B was assessed by immunoblotting. (H) The same cells as in (G) were assayed for their sensitivity to increasing concentrations of PTX. (I) Parental CAL-51 cells that had been depleted of α6 integrin using CRISPR were stably transfected with either α6A or α6B plasmids. Cell viability in response to increasing concentrations of PTX was measured. The TRPC6 expression data shown in (B) represent the mean ± SD of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.

Article Snippet: Rabbit polyclonal anti-TRPC6 , Proteintech , Cat# 18236–1-AP; RRID: AB_10859822.

Techniques: Expressing, Western Blot, Stable Transfection, Transfection, Plasmid Preparation, CRISPR

Journal: Cell reports

Article Title: The calcium channel TRPC6 promotes chemotherapy-induced persistence by regulating integrin α6 mRNA splicing

doi: 10.1016/j.celrep.2023.113347

Figure Lengend Snippet: (A) Myc mRNA expression was quantified by qPCR in CAL-51-sensitive (CAL-51-S) and CAL-51-R cells. (B) TRPC6 expression (log2fold) was analyzed from a published dataset of chemoresistant (doxorubicin) organoid models that were derived from patient tumors or cell lines (see Dupont et al. ). (C and D) Myc mRNA expression was quantified in TE3 (C) or CAL-51 (D) cells that had been stably transfected with either a control (shCTRL) or TRPC6 shRNAs (shTRPC6–1, shTRPC6–2). (E) TE3 cells were treated with either DMSO or BI (10 μM) for 24 h. Myc mRNA expression was quantified by qPCR in TE3 cells. (F) Myc protein expression by immunoblotting was detected in TE3 cells treated with either vehicle or 10 μM BI for 24 h. Freshly harvested PDX tumors that had been treated with either vehicle or BI (see ) were used to extract protein and RNA. Protein lysates were immunoblotted with a Myc antibody. Densitometric values are shown below the immunoblot. (G) CAL-51-R cells were transfected with either vector alone or a Myc expression vector, and their sensitivity to increasing concentrations of PTX was assessed by quantifying cell viability (left). Immunoblot showing Myc protein levels in CAL-51-R cells transfected with either an empty vector or Myc overexpression plasmid (right). (H) CAL-51 parental cells that has been pretreated with either DMSO or 10074-G5 (50 μM) for 24 h were assessed for their sensitivity to increasing concentrations of PTX, either alone or in combination with 1007-G5 (50 μM) for an additional 24 h. (I) Expression of the Myc target gene CDC25A was quantified by qPCR in CAL-51 cells treated with either DMSO or 10074-G5 (50 μM). The Myc expression levels in (A), (C), (D), and (E) represent the mean ± SD of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.

Article Snippet: Rabbit polyclonal anti-TRPC6 , Proteintech , Cat# 18236–1-AP; RRID: AB_10859822.

Techniques: Expressing, Derivative Assay, Stable Transfection, Transfection, Western Blot, Plasmid Preparation, Over Expression

Journal: Cell reports

Article Title: The calcium channel TRPC6 promotes chemotherapy-induced persistence by regulating integrin α6 mRNA splicing

doi: 10.1016/j.celrep.2023.113347

Figure Lengend Snippet: (A) SETD4 mRNA expression was quantified in CAL-51 cells that had been stably transfected with either a control (shCTRL) or TRPC6 shRNAs (shTRPC6–1,shTRPC6–2). (B) SETD4 mRNA expression was quantified in a TNBC PDX that had been treated with either vehicle or BI. (C) CAL-51 cells expressing a p27-mVenus reporter were treated with either vehicle or BI for 24 h. Cells were detached and processed for flow cytometry (mean fluorescence indicated by the number on the top left). Bar graph plotted using mean fluorescence intensity is shown on the right. (D) Comparison of TRPC6, SETD4, and ESRP1 mRNA expression between the CAL-51 p27 high and p27 low populations. (E) The p27 high population of CAL-51 cells was treated with either vehicle or BI for 24 h, and their ability to form mammospheres was assessed. (F) HCC-1806 cells expressing the p27-mVenus reporter were treated with either vehicle or BI for 24 h and processed for flow cytometry. Bar graph of mean fluorescence intensity is shown on the right. (G) HCC-1806 cells were transfected with a p27-mVenus reporter and sorted into p27 high and p27 low populations. The expression of TRPC6, SETD4, and ESRP1 mRNAs was compared between these populations. (H) Mammosphere formation was assayed in the p27 high population of HCC-1806 cells that had been treated with either DMSO or BI (10 μM). (I) The sensitivity of the p27 high and p27 low populations of CAL-51 to increasing concentrations of PTX was assessed. The SETD4 expression data shown represent the mean ± SD of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.

Article Snippet: Rabbit polyclonal anti-TRPC6 , Proteintech , Cat# 18236–1-AP; RRID: AB_10859822.

Techniques: Expressing, Stable Transfection, Transfection, Flow Cytometry, Fluorescence, Comparison

Journal: Cell reports

Article Title: The calcium channel TRPC6 promotes chemotherapy-induced persistence by regulating integrin α6 mRNA splicing

doi: 10.1016/j.celrep.2023.113347

Figure Lengend Snippet: (A and B) Either TE3 (A) or CAL-51 (B) TRPC6 knockdown cells (shTRPC6–1, shTRPC6–2) were retransfected with an α6B expression plasmid (α6B-GFP) followed by knockdown of the endogenous α6 integrin (shITGA6). Myc mRNA expression was quantified by qPCR. (C) Myc expression in the non-CSC and CSC populations described in . (D) TE3 cells that had been stably transfected with either a control (shCTRL) or TRPC6 shRNAs (shTRPC6–1, shTRPC6–2) were retransfected with an α6B expression plasmid (α6B-GFP) followed by knockdown of the endogenous α6 integrin (shITGA6). Expression of ESRP1, CTGF, and ANKRD1 mRNAs was quantified by qPCR. (E and F) Myc mRNA expression was quantified in TE3 (E) and CAL-51 (F) cells that had been transfected with siCTRL or siRNA targeting TAZ (siTAZ). (G) Myc mRNA expression was quantified in CAL-51 cells transfected with either shCTRL or TRPC6 shRNAs (shTRPC6–1, shTRPC6–2) that were re-transfected with a constitutively active TAZ-4SA plasmid (4SA). The Myc expression data shown in (A), (B), (E), and (F) represent the mean ± SD of two independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.

Article Snippet: Rabbit polyclonal anti-TRPC6 , Proteintech , Cat# 18236–1-AP; RRID: AB_10859822.

Techniques: Expressing, Plasmid Preparation, Stable Transfection, Transfection

Journal: Cell reports

Article Title: The calcium channel TRPC6 promotes chemotherapy-induced persistence by regulating integrin α6 mRNA splicing

doi: 10.1016/j.celrep.2023.113347

Figure Lengend Snippet: This schematic depicts the key findings in this study. Treatment of a heterogeneous tumor with chemotherapy results in the survival of persister cells that are resistant to chemotherapy. These cells have high TRPC6 expression levels and stem cell properties. TRPC6 sustains the expression of α6B in this population through suppression of the splicing factor ESRP1. Enrichment of α6B in this population drives chemoresistance through a TAZ-mediated MYC suppression. This suppression of Myc maintains these cells in a quiescent state to survive chemotherapeutic stress.

Article Snippet: Rabbit polyclonal anti-TRPC6 , Proteintech , Cat# 18236–1-AP; RRID: AB_10859822.

Techniques: Expressing

Journal: Cell reports

Article Title: The calcium channel TRPC6 promotes chemotherapy-induced persistence by regulating integrin α6 mRNA splicing

doi: 10.1016/j.celrep.2023.113347

Figure Lengend Snippet:

Article Snippet: Rabbit polyclonal anti-TRPC6 , Proteintech , Cat# 18236–1-AP; RRID: AB_10859822.

Techniques: Virus, Derivative Assay, Recombinant, Mutagenesis, Bacteria, Sequencing, CRISPR, Plasmid Preparation, Software