Journal: Cancer medicine

Article Title: TRPM8 overexpression suppresses hepatocellular carcinoma progression and improves survival by modulating the RTP3/STAT3 pathway.

doi: 10.1002/cam4.70109

Figure Lengend Snippet: FIGURE 4 TRPM8 level determination and TRPM8-silencing cell function experiments. (A–D) TRPM8 inhibition in PLC/PRF/5 and HUH-7 cells transfected with shTRPM8 lentivirus was verified by Western blot and qRT-PCR. (E–I) Effect of TRPM8 loss-of-function on PLC/RPF/5 and HUH-7 cell proliferation was determined by colony formation assay (E), CCK-8 (F-G) and EdU assay (H, I). (J, K) The effects of TRPM8 inhibition on the cell migration and invasion of PLC/PRF/5 and HUH-7 cells were detected using Transwell assays. Scale bars: 100 μm. *p < 0.05.

Article Snippet: Specific shRNAs targeting TRPM8 were transfected with TRPM8- RNAi lentiviruses (Genechem Co., LTD, Shanghai, China) to generate TRPM8 silencing HUH7 and PLC/PRF/5 cells.

Techniques: Cell Function Assay, Inhibition, Transfection, Western Blot, Quantitative RT-PCR, Colony Assay, CCK-8 Assay, EdU Assay, Migration

Journal: Cancer medicine

Article Title: TRPM8 overexpression suppresses hepatocellular carcinoma progression and improves survival by modulating the RTP3/STAT3 pathway.

doi: 10.1002/cam4.70109

Figure Lengend Snippet: FIGURE 6 AD80 promoted the expression of TRPM8 and inhibited the proliferation, migration and invasion of HCC cells in vitro. (A, B) Calcium imaging was used to detected the changes of the fluorescence intensity of WS12 (A) and AD80 (B) groups on time in SNU-449 cells. (C, D) Effects of WS12 (C) and AD80 (D) on the expression of TRPM8 in SNU-449 cells were detected by Western blot. (E-G) EdU (E) and CCK-8 assays (F) were used to detect the effect of WS12 on the proliferation of SNU-449. (G) The effects of WS12 on the cell migration and invasion of SSNU-449 cells were detected using Transwell assays. (H–J) EdU (H) and CCK-8 assays (I) were used to detect the effect of WS12 on the proliferation of SSNU-449. (J) The effects of AD80 on the cell migration and invasion of SNU-449 cells were detected using Transwell assays. Scale bars: 100 μm. *p < 0.05.

Article Snippet: Specific shRNAs targeting TRPM8 were transfected with TRPM8- RNAi lentiviruses (Genechem Co., LTD, Shanghai, China) to generate TRPM8 silencing HUH7 and PLC/PRF/5 cells.

Techniques: Expressing, Migration, In Vitro, Imaging, Fluorescence, Western Blot, CCK-8 Assay

Journal: Cancer medicine

Article Title: TRPM8 overexpression suppresses hepatocellular carcinoma progression and improves survival by modulating the RTP3/STAT3 pathway.

doi: 10.1002/cam4.70109

Figure Lengend Snippet: FIGURE 7 Schematic depicting the possible mechanism of the TRPM8- mediated RTP3/STAT3 pathway in HCC.

Article Snippet: Specific shRNAs targeting TRPM8 were transfected with TRPM8- RNAi lentiviruses (Genechem Co., LTD, Shanghai, China) to generate TRPM8 silencing HUH7 and PLC/PRF/5 cells.

Techniques:

Journal: Frontiers in Physiology

Article Title: Transient Receptor Potential Melastatin 8 (TRPM8)-Based Mechanisms Underlie Both the Cold Temperature-Induced Inflammatory Reactions and the Synergistic Effect of Cigarette Smoke in Human Bronchial Epithelial (16HBE) Cells

doi: 10.3389/fphys.2019.00285

Figure Lengend Snippet: Roles of cold temperature or/and CSE in TRPM8 mediated increase in intracellular Ca 2+ level in 16HBE cells. Intracellular Ca 2+ levels were measured by Fluo3-AM fluorescent probe assay. (A) Cells were exposed to 18°C or/and CSE for 30 min. (B) Representative images of fluorescence-positive cells were exposed to18°C for 6 min, to CSE for 3 min, to both cold temperature and CSE for 6 min and with or without transfection of TRPM8 shRNA or scramble shRNA. (C) Fluorescence intensity for intracellular calcium concentration was analyzed with the quantification tools. Data in each group are mean ± SD; n = 4. ∗ p < 0.05 vs. control, # p < 0.05 vs. 18°C alone, Δ p < 0.05 vs. CSE alone.

Article Snippet: Exon 18-specific TRPM8 shRNA and scramble shRNA were purchased from Santa Cruz Biotechnology.

Techniques: Fluorescence, Transfection, shRNA, Concentration Assay, Control

Journal: Frontiers in Physiology

Article Title: Transient Receptor Potential Melastatin 8 (TRPM8)-Based Mechanisms Underlie Both the Cold Temperature-Induced Inflammatory Reactions and the Synergistic Effect of Cigarette Smoke in Human Bronchial Epithelial (16HBE) Cells

doi: 10.3389/fphys.2019.00285

Figure Lengend Snippet: Roles of MAPKs/NF-κB signaling pathway in the cold temperature-induced production of inflammatory cytokines in 16HBE cells. (A) Protein expression of ERK, P38, JNK, and IκBα was analyzed by western blotting in 16HBE cells exposed to medium, 18°C, and 18°C with TRPM8 shRNA transfection or scramble shRNA transefection, respectively. (B) NF-KB activity was measured with luciferase assay in 16HBE cells exposed to medium, 18°C, and 18°C with TRPM8 shRNA transfection or scramble shRNA transefection, respectively. (C) mRNA and protein expression of IL-6, IL-8, and TNF-α were separately measured with RT-RCR and ELISA in 16HBE cells exposed to 18°C with pretreatment with an ERK inhibitor (U0126), a P38 inhibitor (SB203580), or a NF- κ B inhibitor (BAY 11-7085; BAY), p- and t- represent phospho- and total-, respectively. Data in each group are mean ± SD n = 3. ∗ p < 0.05 vs. control.

Article Snippet: Exon 18-specific TRPM8 shRNA and scramble shRNA were purchased from Santa Cruz Biotechnology.

Techniques: Expressing, Western Blot, shRNA, Transfection, Activity Assay, Luciferase, Enzyme-linked Immunosorbent Assay, Control

Journal: Frontiers in Physiology

Article Title: Transient Receptor Potential Melastatin 8 (TRPM8)-Based Mechanisms Underlie Both the Cold Temperature-Induced Inflammatory Reactions and the Synergistic Effect of Cigarette Smoke in Human Bronchial Epithelial (16HBE) Cells

doi: 10.3389/fphys.2019.00285

Figure Lengend Snippet: Roles of MAPKs/NF-κB signaling pathway in the synergistic effect of CSE on the cold temperature-induced production of inflammatory cytokines in 16HBE cells. (A) Protein expression of ERK, P38, JNK, and IκBα was analyzed by western blotting in 16HBE cells exposed to medium, CSE, both CSE and 18°C with or without transfection of TRPM8 shRNA or scramble shRNA, respectively. (B) NF-KB activity was measured with luciferase assay in 16HBE cells exposed to medium, CSE, both CSE and 18°C with or without transfection of TRPM8 shRNA or scramble shRNA, respectively. (C) mRNA and protein expression of IL-6, IL-8, and TNF-α were separately measured with RT-RCR and ELISA in 16HBE cells exposed to both CSE and 18°C with pretreatment with an ERK inhibitor (U0126), a P38 inhibitor (SB203580), or a NF-κB inhibitor (BAY 11-7085). p- and t- represent phospho- and total-, respectively. Data in each group are mean ± SD; n = 3. ∗ p < 0.05 vs. control, # p < 0.05 vs. cold alone, Δ p < 0.05 vs. CSE alone.

Article Snippet: Exon 18-specific TRPM8 shRNA and scramble shRNA were purchased from Santa Cruz Biotechnology.

Techniques: Expressing, Western Blot, Transfection, shRNA, Activity Assay, Luciferase, Enzyme-linked Immunosorbent Assay, Control

Journal: Frontiers in Physiology

Article Title: Transient Receptor Potential Melastatin 8 (TRPM8)-Based Mechanisms Underlie Both the Cold Temperature-Induced Inflammatory Reactions and the Synergistic Effect of Cigarette Smoke in Human Bronchial Epithelial (16HBE) Cells

doi: 10.3389/fphys.2019.00285

Figure Lengend Snippet: Primer sequences used for RT-PCR analysis of selected cytokine genes.

Article Snippet: Exon 18-specific TRPM8 shRNA and scramble shRNA were purchased from Santa Cruz Biotechnology.

Techniques:

Journal: Frontiers in Physiology

Article Title: Transient Receptor Potential Melastatin 8 (TRPM8)-Based Mechanisms Underlie Both the Cold Temperature-Induced Inflammatory Reactions and the Synergistic Effect of Cigarette Smoke in Human Bronchial Epithelial (16HBE) Cells

doi: 10.3389/fphys.2019.00285

Figure Lengend Snippet: Effects of cold temperature or/and CSE on TRPM8 expression in 16HBE cells relative quantification of transient receptor potential melastatin 8 (TRPM8) mRNA and protein in 16HBE cells exposed to18°C or/and CSE. (A) The levels of the TRPM8 protein were determined by western blot analysis. (B) The levels of TRPM8 mRNA were determined by real-time RT-PCR, and a comparative Ct method (2 −ΔΔCt ) was used for the relative mRNA quantification. (C) Densitometry quantification of the bands in A was performed using Quantity One software, and the results are expressed as the ratio of the expression of TRPM8 to β-actin. The values in B and C are shown as the means ± SD; n = 4. ∗ p < 0.05 vs. control.

Article Snippet: Exon 18-specific TRPM8 shRNA and scramble shRNA were purchased from Santa Cruz Biotechnology.

Techniques: Expressing, Quantitative Proteomics, Western Blot, Quantitative RT-PCR, Software, Control

Journal: Frontiers in Physiology

Article Title: Transient Receptor Potential Melastatin 8 (TRPM8)-Based Mechanisms Underlie Both the Cold Temperature-Induced Inflammatory Reactions and the Synergistic Effect of Cigarette Smoke in Human Bronchial Epithelial (16HBE) Cells

doi: 10.3389/fphys.2019.00285

Figure Lengend Snippet: Roles of cold temperature or/and CSE in TRPM8 mediated increase in intracellular Ca 2+ level in 16HBE cells. Intracellular Ca 2+ levels were measured by Fluo3-AM fluorescent probe assay. (A) Cells were exposed to 18°C or/and CSE for 30 min. (B) Representative images of fluorescence-positive cells were exposed to18°C for 6 min, to CSE for 3 min, to both cold temperature and CSE for 6 min and with or without transfection of TRPM8 shRNA or scramble shRNA. (C) Fluorescence intensity for intracellular calcium concentration was analyzed with the quantification tools. Data in each group are mean ± SD; n = 4. ∗ p < 0.05 vs. control, # p < 0.05 vs. 18°C alone, Δ p < 0.05 vs. CSE alone.

Article Snippet: Exon 18-specific TRPM8 shRNA and scramble shRNA were purchased from Santa Cruz Biotechnology.

Techniques: Fluorescence, Transfection, shRNA, Concentration Assay, Control

Journal: Frontiers in Physiology

Article Title: Transient Receptor Potential Melastatin 8 (TRPM8)-Based Mechanisms Underlie Both the Cold Temperature-Induced Inflammatory Reactions and the Synergistic Effect of Cigarette Smoke in Human Bronchial Epithelial (16HBE) Cells

doi: 10.3389/fphys.2019.00285

Figure Lengend Snippet: Roles of MAPKs/NF-κB signaling pathway in the cold temperature-induced production of inflammatory cytokines in 16HBE cells. (A) Protein expression of ERK, P38, JNK, and IκBα was analyzed by western blotting in 16HBE cells exposed to medium, 18°C, and 18°C with TRPM8 shRNA transfection or scramble shRNA transefection, respectively. (B) NF-KB activity was measured with luciferase assay in 16HBE cells exposed to medium, 18°C, and 18°C with TRPM8 shRNA transfection or scramble shRNA transefection, respectively. (C) mRNA and protein expression of IL-6, IL-8, and TNF-α were separately measured with RT-RCR and ELISA in 16HBE cells exposed to 18°C with pretreatment with an ERK inhibitor (U0126), a P38 inhibitor (SB203580), or a NF- κ B inhibitor (BAY 11-7085; BAY), p- and t- represent phospho- and total-, respectively. Data in each group are mean ± SD n = 3. ∗ p < 0.05 vs. control.

Article Snippet: Exon 18-specific TRPM8 shRNA and scramble shRNA were purchased from Santa Cruz Biotechnology.

Techniques: Expressing, Western Blot, shRNA, Transfection, Activity Assay, Luciferase, Enzyme-linked Immunosorbent Assay, Control

Journal: Frontiers in Physiology

Article Title: Transient Receptor Potential Melastatin 8 (TRPM8)-Based Mechanisms Underlie Both the Cold Temperature-Induced Inflammatory Reactions and the Synergistic Effect of Cigarette Smoke in Human Bronchial Epithelial (16HBE) Cells

doi: 10.3389/fphys.2019.00285

Figure Lengend Snippet: Roles of MAPKs/NF-κB signaling pathway in the synergistic effect of CSE on the cold temperature-induced production of inflammatory cytokines in 16HBE cells. (A) Protein expression of ERK, P38, JNK, and IκBα was analyzed by western blotting in 16HBE cells exposed to medium, CSE, both CSE and 18°C with or without transfection of TRPM8 shRNA or scramble shRNA, respectively. (B) NF-KB activity was measured with luciferase assay in 16HBE cells exposed to medium, CSE, both CSE and 18°C with or without transfection of TRPM8 shRNA or scramble shRNA, respectively. (C) mRNA and protein expression of IL-6, IL-8, and TNF-α were separately measured with RT-RCR and ELISA in 16HBE cells exposed to both CSE and 18°C with pretreatment with an ERK inhibitor (U0126), a P38 inhibitor (SB203580), or a NF-κB inhibitor (BAY 11-7085). p- and t- represent phospho- and total-, respectively. Data in each group are mean ± SD; n = 3. ∗ p < 0.05 vs. control, # p < 0.05 vs. cold alone, Δ p < 0.05 vs. CSE alone.

Article Snippet: Exon 18-specific TRPM8 shRNA and scramble shRNA were purchased from Santa Cruz Biotechnology.

Techniques: Expressing, Western Blot, Transfection, shRNA, Activity Assay, Luciferase, Enzyme-linked Immunosorbent Assay, Control