Journal: Scientific Reports

Article Title: TRPM8 channel inhibitor-encapsulated hydrogel as a tunable surface for bone tissue engineering

doi: 10.1038/s41598-021-81041-w

Figure Lengend Snippet: Functional TRPM8 is endogenously expressed in osteoblasts. (A) Confocal images of Rat Calvarial Osteoblasts (RCOs) cultured for 7 days in differentiation medium and probed for endogenous TRPM8 (green) using anti-TRPM8 antibody in absence (upper panel) or in presence of its antigenic peptide (lower panel). ( B) Confocal images of mice pre-osteoblasts MC3T3-E1 cultured in differentiation media for 7 days and probed for endogenous TRPM8 (green) using anti-TRPM8 antibody in absence (upper panel) or in presence of its antigenic peptide (lower panel). ( C) Confocal images of bone marrow derived Mesenchymal Stem Cells (MSCs) undifferentiated (upper panel) and differentiated to osteoblasts (lower panel) are shown. Higher TRPM8 signal is observed in differentiated MSCs. ( D) Confocal images of human osteosarcoma cell line Saos2 depicting TRPM8 expression (green) are shown. ( E) Western blot of RCOs cultured for 7 days in differentiation medium are shown. Blot was probed for endogenous TRPM8 using anti-TRPM8 antibody. ( F,G) Time series images of Ca 2+ -intensity in live Saos2 cells. Cells were loaded with Fluo-4-AM and fluorescence intensity is represented in pseudo color (red and blue indicating highest and lowest intensity respectively). Calcium imaging was performed at the speed of 2 Frames per second. At 100th frame, cells were treated with TRPM8 activator WS12 (1 µM) (F) or inhibitor AMTB Hydrate (1 µM) (G) . ( H,I) Quantification of Fluo-4-AM intensity (in AU) over 500 frames, with the initial value normalized at 100%. Increased fluorescence intensity is observed in most cells treated with WS12, which declines gradually over time. TRPM8 inhibitor (AMTB) doesnot alter Fluo-4-AM intensity over time (G) .

Article Snippet: Rabbit polyclonal antibody raised against extracellular loop region of TRPM8 and a specific blocking peptide (SDVDGTTYDFAHC) were was purchased from ALOMONE Labs (Israel).

Techniques: Functional Assay, Cell Culture, Derivative Assay, Expressing, Western Blot, Fluorescence, Imaging

Journal: Scientific Reports

Article Title: TRPM8 channel inhibitor-encapsulated hydrogel as a tunable surface for bone tissue engineering

doi: 10.1038/s41598-021-81041-w

Figure Lengend Snippet: TRPM8 inhibition enhances osteoblast differentiation and mineralization. (A,B) Bar graphs represent 7-day Alkaline Phosphatase (ALP) activity of mice pre-osteoblasts MC3T3-E1 treated with increasing concentrations of TRPM8 activator WS12 ( A , n = 6) or inhibitor AMTB Hydrate ( B , n = 6). ( C,D) . Bar graphs represent ALP activity of Rat Calvarial Osteoblasts (RCOs) grown for 2 days ( C , n = 6) and 7 days ( D , n = 4) in presence of TRPM8 inhibitor AMTB Hydrate in increasing concentrations. ( E) Photographs represents mineralization nodules of Rat Bone Marrow cells grown for 21 days in presence of DMSO control or AMTB Hydrate (1 nM) as detected by Alizarin Red staining. ( F) Corresponding quantification of Alizarin Red intensity is shown as a measure of bone mineralization due to AMTB Hydrate (1 nM) for 21 days (n = 3). ( G,I ) Graphs depict the RT-PCR fold change values of osteogenic genes upon TRPM8 inhibition by AMTB Hydrate for 48 h. Transcript levels (n = 3) of RunX2 increases by ~ 4.2 fold, Bmp2 increases by ~ 1.8 fold and Col1 increases by ~ 1.5 fold. ANOVA test, p values: * < 0.05, ** < 0.01, *** < 0.001.

Article Snippet: Rabbit polyclonal antibody raised against extracellular loop region of TRPM8 and a specific blocking peptide (SDVDGTTYDFAHC) were was purchased from ALOMONE Labs (Israel).

Techniques: Inhibition, Activity Assay, Control, Staining, Reverse Transcription Polymerase Chain Reaction

Journal: Scientific Reports

Article Title: TRPM8 channel inhibitor-encapsulated hydrogel as a tunable surface for bone tissue engineering

doi: 10.1038/s41598-021-81041-w

Figure Lengend Snippet: Hydrogel (CMT:HEMA) as a suitable surface for drug coating and growing of Mesenchymal Stem Cells. (a) Shown are the Atomic Force Microscopic images of CMT only and CMT:HEMA. CMT:HEMA provides a surface with optimum network and stiffness suitable for growth of Mesenchymal Stem Cells. C-0 corresponds to pure carboxymethyl tamarind while C-10 corresponds to Hydroxyethylmethacrylate incorporated carboxymethyl tamarind hydrogel (i.e. CMT-HEMA hydrogel). ( b) Release pattern of AMTB drug from hydrogel (CMT:HEMA) are shown. ( c) Scanning Electron Microscopic images of Mesenchymal Stem Cells grown on hydrogel (CMT:HEMA) only and coated with different drugs modulating TRPM8 ion channel are shown in left side. Magnified images of same or different view field are shown in right side.

Article Snippet: Rabbit polyclonal antibody raised against extracellular loop region of TRPM8 and a specific blocking peptide (SDVDGTTYDFAHC) were was purchased from ALOMONE Labs (Israel).

Techniques:

Journal: Scientific Reports

Article Title: TRPM8 channel inhibitor-encapsulated hydrogel as a tunable surface for bone tissue engineering

doi: 10.1038/s41598-021-81041-w

Figure Lengend Snippet: Hydrogel-mediated drug release alters the morphology of bone marrow derived mesenchymal stem cell population. BM-MSCPs were isolated and grown on only glass, only hydrogel or on hydrogel-coated with different drugs affecting TRPM8 ion channel. (A) Morphology of cells grown on different surface are shown. Cells were stained with Phalloidin (green) and DAPI (blue). (B) Different morphology parameters of the cells grown on different surface are shown. The statistical values are: ANOVA test, p values: * < 0.05, ** < 0.01, *** < 0.001, **** < 0.0001.

Article Snippet: Rabbit polyclonal antibody raised against extracellular loop region of TRPM8 and a specific blocking peptide (SDVDGTTYDFAHC) were was purchased from ALOMONE Labs (Israel).

Techniques: Derivative Assay, Isolation, Staining

Journal: Scientific Reports

Article Title: TRPM8 channel inhibitor-encapsulated hydrogel as a tunable surface for bone tissue engineering

doi: 10.1038/s41598-021-81041-w

Figure Lengend Snippet: Hydrogel coated with TRPM8 activators but not with inhibitor specifically stimulates ROS signaling by decreasing mitochondrial oxidative potential. (A,B) Fluorescence microscopic images from cells grown on only glass, only hydrogel or the same hydrogel coated with different activators and inhibitor are shown. ROS signal (green in left panel) and DIC images of the cells are shown in left side (A) . Similarly, mitochondrial oxidative potential analyzed by JC1 dye is shown in right side (B) . JC1 fluorescence emission at 590 nm (red panel, indicative of high oxidative potential) is reduced in cells when grown on hydrogel coated with TRPM8 activators. ( C,D) Quantified values representing the fluorescence values from individual cells grown on different surface as shown in figure (A,B) . The statistical values are as follows: ANOVA test, p values: ** < 0.01, *** < 0.001, ns non-significant.

Article Snippet: Rabbit polyclonal antibody raised against extracellular loop region of TRPM8 and a specific blocking peptide (SDVDGTTYDFAHC) were was purchased from ALOMONE Labs (Israel).

Techniques: Fluorescence

Journal: Scientific Reports

Article Title: TRPM8 channel inhibitor-encapsulated hydrogel as a tunable surface for bone tissue engineering

doi: 10.1038/s41598-021-81041-w

Figure Lengend Snippet: Hydrogel (CMT:HEMA)-mediated release of TRPM8 agonist and antagonist modulates osteoblasts functions. ( A) Photographs (upper panel) represents mineralization nodules of Mesenchymal Stem Cells (MSCs) grown for 15 days in absence or presence of hydrogel (CMT:HEMA) as detected by Alizarin Red staining (n = 3). The lower panel photographs represent the extent of mineralization nodules formed when cells were grown on WS12 or AMTB encapsulated CMT:HEMA hydrogel. Scale bar 40 µm. ( B,C) Bone marrow-derived Mesenchymal Stem Cell population (BM-MSCPs) grown on these hydrogels or hydrogels-encapsulated with AMTB or WS12 reveals dose-dependent differentiation (B) and mineralization (C) (n = 3). The significance values are as follows: ANOVA test, p values: * < 0.05, ** < 0.01, *** < 0.001.

Article Snippet: Rabbit polyclonal antibody raised against extracellular loop region of TRPM8 and a specific blocking peptide (SDVDGTTYDFAHC) were was purchased from ALOMONE Labs (Israel).

Techniques: Staining, Derivative Assay

Journal: Poultry Science

Article Title: Differential expressions of hypothalamic thermosensitive TRP ion channels may underlie the posthatching ontogeny of brain cooling capacity in broiler chickens

doi: 10.1016/j.psj.2023.102782

Figure Lengend Snippet: PCR primers of relevant genes used in this study.

Article Snippet: The membrane was then incubated with specific primary antibodies (1:1,000 dilution) overnight at 4°C: TRPV1 (#AF8250, rabbit polyclonal antibody from Beyotime), TRPV2 (#bs-10297R, rabbit polyclonal antibody from Beijing Biosynthesis Biotechnology Co., Ltd., China), TRPV3 (#BA2875-2, rabbit polyclonal antibody from Boster Biological Technology Co., Ltd., Wuhan, Hubei, China), TRPV4 (#AF8253, rabbit polyclonal antibody from Beyotime), TRPA1 (#AF8241, rabbit polyclonal antibody from Beyotime), TRPM8 (#PB0882, rabbit polyclonal antibody from Boster), and GAPDH (#AF0006, mouse monoclonal antibody from Beyotime).

Techniques: Sequencing