trpm8 silencer sirna s35489  (Thermo Fisher)

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