trpm8 sirna  (Genechem)

Journal: bioRxiv

Article Title: Orai1-mediated Ca 2+ Entry Regulates Lipolysis and Mitochondrial Activation in Brown Adipose Thermogenesis

doi: 10.64898/2026.04.24.718619

Figure Lengend Snippet: a Intracellular Ca 2+ levels in primary brown adipocytes with Orai1, Trpv2 , and Trpm8 knockdown upon temperature shift from 37 °C to 15 °C. Black lines are average of each traces. b AUC analysis of a. c Quantification of Orai1 mRNA expression in brown adipose tissue of Orai1 control and BKO mice exposed to acute cold exposure (4 °C for 6 hrs). d Expressional profiles of SOCE components under chronic cold exposure (3 days), obtained from the GSE70437 dataset. Data are presented as mean ± SEM. Statistical significance: * p < 0.05, ** p < 0.01, **** p < 0.0001.

Article Snippet: Cells were transfected with siRNAs targeting Orai1, Stim1, Trpv2, and Trpm8 (Bioneer) using Lipofectamine RNAiMAX (Thermo Fisher).

Techniques: Knockdown, Expressing, Control

Journal: Cell Death & Disease

Article Title: Rewiring melanoma cell fate: TRPM8 modulators trigger apoptosis and boost NK cell cytotoxicity

doi: 10.1038/s41419-026-08469-8

Figure Lengend Snippet: A – C WM266-4 cells were transfected with control siRNA (siRNA ctrl) or TRPM8-targeting siRNA (siRNA TRPM8). A Western blot analysis was performed on total cell lysates using the indicated antibodies. B Cells were then left untreated or treated with compound 4 or 9 (1 μM, 24 h), and cell death was assessed by PI staining. Total cells were stained in green with acridin orange. C Quantification of PI-positive cells corresponding to ( B ). Representative Western blots showing transient TRPM8 overexpression (TRPM8 OE) in WM266-4 ( D ) and AMM16 ( F ) melanoma cells. E , G Densitometric analysis of TRPM8 and GAPDH protein levels, represented as TRPM8/GAPDH ratios (from three independent experiments). WM266-4 ( H ) and AMM16 ( I ) cells transfected with control plasmid (ctrl plasmid) or TRPM8 plasmid (TRPM8 OE) were left untreated or treated with compounds 4 and 9 (1 or 10 μM) for 6 h. Representative images (contrast phase, PI staining, and overlays) and corresponding quantitative graphs (below panels) are shown. Scale bar, 100 μm. In C , E , G , H , I Data are presented as mean ± SD from n independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001. In H , I Red asterisks indicate statistical significance between ctrl and OE groups at the corresponding treatment conditions.

Article Snippet: Small interfering RNAs (siRNAs) targeting human TRPM8 (sc-95009) and TP53 (sc-29435) were purchased from Santa Cruz Biotechnology (Dallas, TX, USA) and transfected into melanoma cells using Lipofectamine 2000 (Thermo Fisher Scientific, Invitrogen), following the manufacturer’s instructions.

Techniques: Transfection, Control, Western Blot, Staining, Over Expression, Plasmid Preparation

Journal: Cell Death & Disease

Article Title: Rewiring melanoma cell fate: TRPM8 modulators trigger apoptosis and boost NK cell cytotoxicity

doi: 10.1038/s41419-026-08469-8

Figure Lengend Snippet: A Representative images of crystal violet-stained colonies, derived from WM266-4 cells, after 21-day treatment with TRPM8 modulators. B Western blot analysis showing ULBP1 expression in WM266-4 cells after treatment with TRPM8 modulators. The α-tubulin was used as a loading control. C WM266-4 derived spheroids treated for 21 days as indicated, in absence (upper panel; -NK cells) or presence (lower panel; + NK cells) of NK cells. Images are representative of three different experiments. Bar, 100 µm. D The graph represents the dead cells/total cells. Values of dead (red stained cells) and total cells (green stained cells) were analyzed using NIH Image J. They derive from red fluorescence mean/green fluorescence mean intensity and are expressed as mean ± SD of 3 different experiments ( n = 3); ** p < 0.01; *** p < 0.001. E NK cell cytotoxicity assay. WM266-4 cells pre-treated with TRPM8 modulators were co-cultured with primary NK cells at the indicated effector:target (E:T) ratios. Where indicated, neutralizing antibodies against ULBP1 or NKG2D were added 1 h before co-culture to melanoma cells or NK, respectively. Data are presented as percentage of lysis. * p < 0.05; ** p < 0.01. F WM266-4 cells were transfected with control siRNA (siRNA ctrl) or TP53-targeting siRNA (siRNA p53) at two different concentrations (300 pmol and 400 pmol, respectively). After 4 days, cells were collected, lysed, and Western blot analysis was performed on cell lysates using the indicated antibodies. α-Tubulin was used as a loading control. G WM2664 cells transfected with control siRNA (siRNA ctrl) or TP53-targeting siRNA (siRNA p53; 300 pmol) were unstimulated or stimulated with compounds 4 and 9 (at 1 μM) for 4 days and then collected and lysed. Western blot analysis was performed using the indicated antibodies. α-Tubulin was used as a loading control. H Phosphorylated AKT (Ser473) levels in melanoma cells treated with the PI3K agonist, 740 Y-P, in absence or presence of TRPM8 modulators (used at 1 μM) for 4 days, analyzed by Western blot. GAPDH was used as a loading control.

Article Snippet: Small interfering RNAs (siRNAs) targeting human TRPM8 (sc-95009) and TP53 (sc-29435) were purchased from Santa Cruz Biotechnology (Dallas, TX, USA) and transfected into melanoma cells using Lipofectamine 2000 (Thermo Fisher Scientific, Invitrogen), following the manufacturer’s instructions.

Techniques: Staining, Derivative Assay, Western Blot, Expressing, Control, Fluorescence, Cytotoxicity Assay, Cell Culture, Co-Culture Assay, Lysis, Transfection

Journal: Cancers

Article Title: Inorganic Polyphosphate Promotes Colorectal Cancer Growth via TRPM8 Receptor Signaling Pathway

doi: 10.3390/cancers16193326

Figure Lengend Snippet: CRC displays a high level of TRPM8 receptor. ( A ). Cellular extracts from 50 human biopsies, where the tumoral sample (T) was plotted against the peritumoral (P) counterpart of the same patient (Pt), were analyzed by immunoblotting for the TRPM8 expression level. Actin was used as a loading control for the normalization. The uncropped bolts are shown in . ( B ). The Spearman correlation between iPolyP and TRPM8 in the total cohort, denoting a strong positive correlation (**** p < 0.0001). Fold changes versus peritumoral (P), normalized to 1.

Article Snippet: For the TRPM8-knockout experiments using siRNA, HCEC-1CT, Caco-2 and SW620 were electrophoresed with two TRPM8 Silencer siRNAs (Thermo Fisher Scientific, Cat. No. 4392420, ID: S35489 and ID: S35490, respectively) or with Silencer siRNA Negative Control (Thermo Fisher Scientific, Cat. No. 4390843).

Techniques: Western Blot, Expressing, Control

Journal: Cancers

Article Title: Inorganic Polyphosphate Promotes Colorectal Cancer Growth via TRPM8 Receptor Signaling Pathway

doi: 10.3390/cancers16193326

Figure Lengend Snippet: iPolyP enhances PCNA expression and promotes Caco-2 colorectal cancer proliferation. ( A ). Cellular extracts from WT and siRNA-mediated TRPM8 knockdown Caco-2 cell lines were analyzed by immunoblotting for the PCNA expression level. GAPDH was used as a loading control. The uncropped bolts are shown in . ( B ). Representative micrographs of the crystal violet assay performed on WT and siRNA-mediated TRPM8 knockdown Caco-2 cell lines upon treatment for 96 h with iPolyP, a TRPM8 inhibitor, or both. Scale bar, 10 µm. Images are representative of three independent experiments. ( C ). Statistical analysis of the crystal violet assay by Student’s t -test, respectively, for panel ( C ) (*** p < 0.001 and **** p < 0.0001). Fold changes versus control, untreated (UT), normalized to 1. Data are presented as the mean ± SD for triplicate wells from three independent experiments.

Article Snippet: For the TRPM8-knockout experiments using siRNA, HCEC-1CT, Caco-2 and SW620 were electrophoresed with two TRPM8 Silencer siRNAs (Thermo Fisher Scientific, Cat. No. 4392420, ID: S35489 and ID: S35490, respectively) or with Silencer siRNA Negative Control (Thermo Fisher Scientific, Cat. No. 4390843).

Techniques: Expressing, Knockdown, Western Blot, Control, Crystal Violet Assay

Journal: Cancers

Article Title: Inorganic Polyphosphate Promotes Colorectal Cancer Growth via TRPM8 Receptor Signaling Pathway

doi: 10.3390/cancers16193326

Figure Lengend Snippet: iPolyP promotes colorectal cancer patient-derived organoids and Caco-2- and SW620-derived 3D spheroids. ( A , B ). Two independent experiments of a CRC patient’s derived organoids, assessed by light microscopy, in the absence of iPolyP (UT) or incubated for 10 days in the presence of iPolyP. Scale bar, 100 µm. Fold changes versus control, untreated (UT), normalized to 1. Statistical analysis was performed by Student’s t -test (*** p < 0.001). ( C ). Representative bright-field images of 24 h- and 96 h-induced spheroid formation derived from the HCEC-1CT, Caco-2, and SW620 cell line, respectively, upon treatment with iPolyP, a TRPM8 inhibitor, or both for 96 h. Scale bar, 100 µm. Images are representative of three independent experiments. ( D ). Quantification relative to panel ( C ). Fold changes versus control, untreated (UT). Statistical analysis was performed by Student’s t -test (**** p < 0.0001). Data are presented as the mean ± SD for triplicate wells from three independent experiments. The uncropped bolts are shown in .

Article Snippet: For the TRPM8-knockout experiments using siRNA, HCEC-1CT, Caco-2 and SW620 were electrophoresed with two TRPM8 Silencer siRNAs (Thermo Fisher Scientific, Cat. No. 4392420, ID: S35489 and ID: S35490, respectively) or with Silencer siRNA Negative Control (Thermo Fisher Scientific, Cat. No. 4390843).

Techniques: Derivative Assay, Light Microscopy, Incubation, Control

Journal: Cancers

Article Title: Inorganic Polyphosphate Promotes Colorectal Cancer Growth via TRPM8 Receptor Signaling Pathway

doi: 10.3390/cancers16193326

Figure Lengend Snippet: iPolyP drives cells into G2/M phase. ( A ) Real-time PCR on the iPolyP-treated Caco-2 cell line for 72 h on cyclins implicated in different phases of the cell cycle; untreated (UT) samples were normalized to 1. * p < 0.05; ** p < 0.01; **** p < 0.0001. ( B ) Sketch representing cyclins-dependent cell-cycle regulation consisting of Gap 1 (G1), synthesis (S), Gap 2 (G2), and mitosis (M). Figure was created with BioRender. ( C ) Representative micrographs of the cell-cycle assay on Caco-2 ( upper panel ) and SW620 ( lower panel ) cell line treated for 72 h with iPolyP, a TRPM8 inhibitor, or both. Scale bar = 10 µm. Images are representative of three independent experiments. ( D ) Percentage of cells in G2/M phase. Statistical analysis was performed by Student’s t -test (*** p < 0.001). Fold changes versus control, untreated (UT). Data are presented as the mean ± SD for triplicate wells from three independent experiments.

Article Snippet: For the TRPM8-knockout experiments using siRNA, HCEC-1CT, Caco-2 and SW620 were electrophoresed with two TRPM8 Silencer siRNAs (Thermo Fisher Scientific, Cat. No. 4392420, ID: S35489 and ID: S35490, respectively) or with Silencer siRNA Negative Control (Thermo Fisher Scientific, Cat. No. 4390843).

Techniques: Real-time Polymerase Chain Reaction, Cell Cycle Assay, Control

Journal: Cancers

Article Title: Inorganic Polyphosphate Promotes Colorectal Cancer Growth via TRPM8 Receptor Signaling Pathway

doi: 10.3390/cancers16193326

Figure Lengend Snippet: Schematic of the role of iPolyP derived from platelets and microbiota on macrophages and CRC cancer cells. The iPolyP–TRPM8 axis stimulates colorectal cancer expansion by enhancing the level of the ccnb1 gene, which coordinates the M phase of the cell cycle, alongside the expression of two bona fide proliferative markers, PCNA and Ki-67.

Article Snippet: For the TRPM8-knockout experiments using siRNA, HCEC-1CT, Caco-2 and SW620 were electrophoresed with two TRPM8 Silencer siRNAs (Thermo Fisher Scientific, Cat. No. 4392420, ID: S35489 and ID: S35490, respectively) or with Silencer siRNA Negative Control (Thermo Fisher Scientific, Cat. No. 4390843).

Techniques: Derivative Assay, Expressing