Journal: eLife

Article Title: Piezo1/2 mediate mechanotransduction essential for bone formation through concerted activation of NFAT-YAP1-ß-catenin

doi: 10.7554/eLife.52779

Figure Lengend Snippet: ( a ) Luciferase reporter assays of NFATc1 (pGL3-NFAT), YAP1 (8XGTIIC) and CTNNB1 (SUPERTOPFLASH) transcription activities in HEK293T cells. N = 3, data are shown as means ± SD. *p<0.05, **p<0.01, ***p<0.001, two-tailed unpaired Student’s t -test and one-way ANOVA followed by Tukey’s multiple comparisons tests when ANOVA was significant . Expression one of NFATc1, YAP1 and CTNNB1 led to activation of the other two. The only exception is that YAP1 overexpression reducd NFATc1 activties. ( b, d ) Immunoprecipitation (IP) assays of HEK293T cell lysates with the indicated expression constructs. ( b ) Yoda1 treatment (40 μM, 4 hr) promoted complex formation of NFATc1 and YAP1, which was inhibited by CsA or Gd 3+ treatment. ( c ) Western blotting assay of HEK 293 T cells transfected with HA-tagged YAP1 and S-tagged PPP3CA as indicated. DMSO (vehicle) or Yoda1 (40 μM) treatment was performed 24 hr after transfection for 4 hr. Arrow indicates endogenous PPP3CA. ( d ) Expression of NFATc1, YAP1 and CTNNB1 together promoted their complex formation. ( e ) Western blotting analyses of Piezo1/2 deficient BMSCs treated with Yoda1. Yoda1 treatment failed to reduce phosphorylation of Ctnnb1 and Yap1. Ppp3ca: Catalytic subunit of CaN. ( f ) Western blotting analyses of wild-type primary BMSCs treated by Gd 3+ . Increased Nfatc1 phosphorylation (blue arrow), as well as increased phosphorylation of Yap1 and Ctnnb1 was observed . Figure 9—figure supplement 1—source data 1. Original Western blots.

Article Snippet: Cell line ( Homo sapiens ) , HEK 293T , ATCC , Cat# ACS-4500, RRID: CVCL_4V93 , .

Techniques: Luciferase, Two Tailed Test, Expressing, Activation Assay, Over Expression, Immunoprecipitation, Construct, Western Blot, Transfection, Phospho-proteomics

Journal: eLife

Article Title: Piezo1/2 mediate mechanotransduction essential for bone formation through concerted activation of NFAT-YAP1-ß-catenin

doi: 10.7554/eLife.52779

Figure Lengend Snippet:

Article Snippet: Cell line ( Homo sapiens ) , HEK 293T , ATCC , Cat# ACS-4500, RRID: CVCL_4V93 , .

Techniques: Transfection, Construct, Negative Control, Isolation, Recombinant, Plasmid Preparation, Luciferase, Sequencing, Gel Extraction, RNAscope, Gene Expression, Reporter Assay, Enzyme-linked Immunosorbent Assay, Staining, TUNEL Assay, SYBR Green Assay, Reverse Transcription, Labeling, Blocking Assay, Software

Journal: EMBO Molecular Medicine

Article Title: A clinical and mechanistic study of topical borneol‐induced analgesia

doi: 10.15252/emmm.201607300

Figure Lengend Snippet: A–D Representative whole‐cell currents in HEK 293 cells expressing TRPV1 (A), ASIC3 (B), P2X2 (C), or P2X4 (D) in response to capsaicin (A), acidic pH (B), or ATP (C, D) in the absence of presence of borneol ( n = 5 for each channel). E The effect of different concentrations of borneol on the enzymatic activity of human COX‐2. The number of independent measurements is marked on top of each bar. F Averaged intracellular Ca 2+ increases in cultured mouse DRG neurons in response to consecutive applications of 200 μM borneol, 200 μM menthol, and 67 mM KCl. A total of 81 in 1,689 neurons from four mice were found to be borneol‐sensitive and were included in the analysis. G Representative intracellular Ca 2+ signals in HEK 293 cells expressing human TRPM8 (hTRPM8) in response to different concentrations of borneol. After each application of borneol, Ca 2+ ionophore ionomycin was applied to calibrate Ca 2+ response. RFU: relative fluorescence unit. H Dose–response curves of the borneol‐ or menthol‐induced increase in intracellular Ca 2+ in hTRPM8‐expressing HEK 293 cells. Smooth curves are fit to the Hill equation with an EC 50 of 65 μM and a Hill coefficient of 2.0 for borneol ( n = 15) and an EC 50 of 13 μM and a Hill coefficient of 2.0 for menthol ( n = 6 at concentrations of 0.1, 0.3, and 1 μM; n = 9 at concentration of 3 μM; n = 13 at concentrations of 10, 30, 100, and 300 μM). The data were normalized to ionomycin‐induced intracellular Ca 2+ increases. I Time course of menthol‐ and subsequently applied borneol‐induced whole‐cell currents in hTRPM8‐expressing HEK 293 cells ( n = 6). Data information: All the data are presented as the mean ± standard error of the mean (SEM).

Article Snippet: Intracellular calcium imaging of HEK 293 cells was performed using the FlexStation 3 microplate reader (Molecular Devices).

Techniques: Expressing, Activity Assay, Cell Culture, Fluorescence, Concentration Assay

Journal: EMBO Molecular Medicine

Article Title: A clinical and mechanistic study of topical borneol‐induced analgesia

doi: 10.15252/emmm.201607300

Figure Lengend Snippet: Representative intracellular Ca 2+ signals in HEK 293 cells transfected with empty vector in response to borneol and subsequent applied Ca 2+ ionophore ionomycin ( n = 6). Representative intracellular Ca 2+ signals in HEK 293 cells expressing hTRPM8 in response to different concentrations of menthol and the subsequently applied Ca 2+ ionophore ionomycin.

Article Snippet: Intracellular calcium imaging of HEK 293 cells was performed using the FlexStation 3 microplate reader (Molecular Devices).

Techniques: Transfection, Plasmid Preparation, Expressing

Journal: EMBO Molecular Medicine

Article Title: A clinical and mechanistic study of topical borneol‐induced analgesia

doi: 10.15252/emmm.201607300

Figure Lengend Snippet: Quantification of consecutively applied 100 μM menthol‐ and 600 μM borneol‐induced hTRPM8 currents. Currents were normalized to 100 μM menthol‐induced currents at +80 mV ( n = 6). Representative intracellular Ca 2+ signals in HEK 293 cells expressing mouse TRPM8 in response to different concentrations of borneol. Dose–response curves of borneol‐induced increase in intracellular Ca 2+ in mouse TRPM8‐expressing HEK 293 cells. The smooth curve is a fit to the Hill equation with an EC 50 of 116 μM ( n = 12). The data were normalized to ionomycin‐induced intracellular Ca 2+ increases. Time course of menthol‐ and subsequently applied borneol‐induced whole‐cell currents in mouse TRPM8‐expressing HEK 293 cells ( n = 5). Data information: All the data are presented as the mean ± standard error of the mean (SEM).

Article Snippet: Intracellular calcium imaging of HEK 293 cells was performed using the FlexStation 3 microplate reader (Molecular Devices).

Techniques: Expressing

Journal: Scientific reports

Article Title: Altered functional properties of the codling moth Orco mutagenized in the intracellular loop-3.

doi: 10.1038/s41598-021-83024-3

Figure Lengend Snippet: Figure 3. Comparison of HEK cell responses expressing wild-type and mutagenized form of Orco. (a)— Universal Orco agonist, VUAA1, elicits dose-dependent Ca++ i increase in HEK293A cells expressing either CpomOrco (blue) or CpomOrcoQ417H (red). (b)—VUAA1 concentration dependencies. Data points represent the mean response amplitudes (± SE). Data were fit to a Hill equation for CpomOrco (blue smooth line) or CpomOrcoQ417H (red smooth line) respectively, providing maximum responses of ~ 6.3 ± 0.1 and ~ 3.9 ± 1.3 ∆F, EC50s of ~ 157.1 ± 3.58 and ~ 261.8 ± 165.6 µM and Hill coefficients of ~ 2.4 ± 0.1 and ~ 2.1 ± 1.9; total number of cells analysed: N = 123 and 94. (c)—Effects of Pear ester on the activity of CpomOrco+OR3 (blue) and CpomOrcoQ417H+OR3 (red) heteromers. (d)—Pear ester concentration dependencies. Data points represent the mean response amplitudes (± SE) of cells from at least three experiments. Data were fit to a Hill equation providing the following parameters: maximum responses of ~ 6.6 ± 0.3 and 3.7 ± 0.1 ∆F; EC50s of ~ 210 ± 14.2 and ~ 733.5 ± 26.9 µM; Hill coefficients of ~ 4.6 ± 2.4 and ~ 4.6 ± 0.4, for CpomOR3/CpomOrco (blue smooth line) or CpomOR3/CpomOrcoQ417H (red smooth line) respectively. Total number of cells analysed: N = 160 and 262. Traces in A and C represent the mean responses of cells from one experiment. Data in B and D were not normalized. Scales in B and D are different.

Article Snippet: HEK293 cells lines (HEK293A/HEK293T) were grown in HEK cell media [Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum (MP Biomedicals, Solon, OH, USA), 2.0 mM L-glutamine, and 100 μg/mL penicillin/streptomycin (Invitrogen)] at 37 °C and 5% CO2.

Techniques: Comparison, Expressing, Concentration Assay, Activity Assay

Journal: Scientific reports

Article Title: Altered functional properties of the codling moth Orco mutagenized in the intracellular loop-3.

doi: 10.1038/s41598-021-83024-3

Figure Lengend Snippet: Figure 4. Testing pH sensitivity of HEK cell expressing wild-type and mutagenized form of Orco. (a)— Decrease in fluorescence intensity of the pH sensitive probe, BCECF, possibly reflects acidification of cytoplasm in response to low pH extracellular conditions. (b)—Comparison of VUAA1 concentration dependencies obtained after 30 min incubation at low pHe for CpomOrco or CpomOrcoQ417H. Left panels—VUAA1 activated calcium responses. Right panels—VUAA1 concentration dependencies. Data points represent the mean response amplitudes (± SE). Data were fit to a Hill equation with the following parameters: maximum responses of ~ 1.3 ± 0.7 and ~ 1.8 ± 0.2 ∆F; EC50s of ~ 260 ± 187 and ~ 263.3 ± 45.3 µM; Hill coefficients of ~ 2.5 ± 4.3 and ~ 2.2 ± 0.5, for CpomOrco (blue smooth line, n = 84) or CpomOrcoQ417H (red smooth line, n = 63) respectively. Constraints were applied to fit greatly scattered data in B. Traces in B (left panels) represent the mean responses of cells from one experiment. Data in B (right panels) were not normalized. Concentration dependencies obtained in physiologically relevant control conditions were taken from Fig. 3.

Article Snippet: HEK293 cells lines (HEK293A/HEK293T) were grown in HEK cell media [Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum (MP Biomedicals, Solon, OH, USA), 2.0 mM L-glutamine, and 100 μg/mL penicillin/streptomycin (Invitrogen)] at 37 °C and 5% CO2.

Techniques: Expressing, Fluorescence, Comparison, Concentration Assay, Incubation, Control