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

Journal: Cell death & disease

Article Title: Transcription factor activating enhancer-binding protein 2ε (AP2ε) modulates phenotypic plasticity and progression of malignant melanoma.

doi: 10.1038/s41419-024-06733-3

Figure Lengend Snippet: Fig. 5 AP2ε regulates phenotypic plasticity of melanoma cells. A Representative Western Blot images depicting murine MITF and BRN2 protein levels in primary AP2ε-/-/Tg(GRM1) and Tg(GRM1) cells (left panels) and human MITF and BRN2 in Mel Juso cells transfected with the AP2ε-overexpression (hAP2ε) plasmid and control vector pCMX (right panels). β-Actin served as loading control. B Immunohistochemical staining of MITF and BRN2 protein expression in Mel Im melanoma cells bioprinted in CIB (Magnification: 20x). Quantification of percentage of cells with “no”, “weak”, or “strong” MITF and BRN2 expression. Data are represented as mean ± SEM; *p < 0.05; ns: not significant (Two-way- ANOVA with Fisher’s LSD test). C mRNA- and protein expression analysis for Snail1 in AP2ε-/-/Tg(GRM1) and Tg(GRM1) cells (n = 3). D mRNA- and protein expression analysis for Snail1 in Mel Juso cells transfected with the AP2ε-overexpression (hAP2ε) plasmid and control vector pCMX (n = 3). E mRNA- and protein expression analysis for E-cadherin in AP2ε-/-/Tg(GRM1) and Tg(GRM1) cells (n = 3). F mRNA- and protein expression analysis for E-cadherin in Mel Juso cells transfected with the AP2ε-overexpression (hAP2ε) plasmid and control vector pCMX (n = 3). Data information: All data from at least three independent experiments are represented as mean ± SEM; *p < 0.05 (Two-tailed Student’s t-test; ns: not significant).

Article Snippet: Primary antibodies against MITF (1:500 in 5% BSA, Santa Cruz, sc-515925, RRID:AB_2828036), BRN2 (1:500 in 5% BSA/1x TBS-T, Santa Cruz, sc393324, RRID:AB_2737347), Snail1 (1:1,000 in 5% BSA/1x TBS-T, Cell Signaling Technology Cat# 3879, RRID:AB_2255011) and E-cadherin (1:1,000 in 5% BSA/1x TBS-T, Cell Signaling Technology Cat# 3195, RRID:AB_2291471) were incubated overnight at 4 °C.

Techniques: Western Blot, Transfection, Over Expression, Plasmid Preparation, Control, Immunohistochemical staining, Staining, Expressing, Two Tailed Test