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: Frontiers in Physiology

Article Title: Thiamet-G facilitates reparative dentin formation via modulating O-GlcNAcylation and inflammation

doi: 10.3389/fphys.2025.1739168

Figure Lengend Snippet: Effect of Thiamet-G on human dental pulp stem cells (hDPSCs). (A) Experimental design for MTS assay to examine cell viability and proliferation. (B) MTS assay shows that Thiamet-G treatment facilitates dose-dependent hDPSC proliferation. (C) Experimental design for ALP activity assay. (D) Thiamet-G treatment significantly induces ALP activity in hDPSCs compared to the control, particularly after 14 days (E) Thiamet-G treatment increases the expression of reparative dentin-related markers, including Alp, Bmp2, Bsp, Dspp, Opn, Gsk3β, and RUNX2, compared to the control. Ns non-significant, * p < 0.03, ** p < 0.002, *** p < 0.0002, and **** p < 0.0001. NC negative control, GM growth medium, OM osteogenic medium.

Article Snippet: After 24 h, the media was changed with osteogenic media (Alfa-MEM with 1% penicillin-streptomycin, 5% fetal bovine serum, 50 μM L-ascorbic acid, 10 mM β-glycerophosphate, and 100 nM dexamethasone) in presence or absence of Thiamet-G (S7213, Selleckchem.com ) with various concentrations.

Techniques: MTS Assay, ALP Activity Assay, Activity Assay, Control, Expressing, Negative Control

Journal: Carbohydrate polymers

Article Title: Chitosan - Rosmarinic acid conjugates with antioxidant, anti-inflammatory and photoprotective properties.

doi: 10.1016/j.carbpol.2021.118619

Figure Lengend Snippet: Fig. 5. Cell viability human epidermal fibroblasts (HFB), human epidermal keratinocytes (HEK) and murine macrophages (RAW 264.7) exposed to CSRA lixiviates (A–C) or and free RA (D–F). The diagrams include the mean, SD (n = 16), and the ANOVA results (*p < 0.05 statistically significant difference between the cells in DMEM (control) and treated cells, and #p < 0.05 between the cells treated with different CSRA conjugates or RA concentrations (brackets)).

Article Snippet: The HEK cell line was cultured in the Keratinocytes Medium Kit from Innoprot (Derio, Bizkaia, Spain) and maintained over permissive conditions in a humidified incubator with 5% CO2.

Techniques: Control

Journal: Respiratory Research

Article Title: Cigarette smoke destabilizes NLRP3 protein by promoting its ubiquitination

doi: 10.1186/s12931-016-0485-6

Figure Lengend Snippet: Cigarette Smoke Extract (CSE) decreases NLRP3 abundance. a THP1 cells (total 3 × 10 6 cells) were treated with the indicated concentrations of CSE for 16 h. Cell lysates (Lys) and culture medium (supernatants, Sup) were collected to measure protein levels of NLRP3, NLRP6 (negative control), ASC, Caspase-1, IL-1β, IL-18 and β-actin/GAPDH (loading control) by immunoblotting. Membranes were stripped to detect multiple different proteins. Immunoblot shown is representative of four independent experiments. The lysate blots are from two blots that are loaded at the same time from identical cell lysates. NLRP3, NLRP6 and β-actin are obtained from one, and p45, ASC, and GAPDH are from another blot. For supernatant blots, NLRP3, NLRP6, and p20 are from the same blot, and IL-1β and IL-18 are from a different blot. b THP1 cells were treated with the indicated concentrations of CSE for 16 h before RNA isolation. Shown is the NLRP3 mRNA expression fold changes determined by qRT-PCR in a box plot. Data are representative of four independent experiments. P value was determined by a Kruskal-Wallis test. c Whole lung lysates from non-smoked or smoked C57BL/6 mice were immunoblotted for NLRP3, NLRP6, and β-actin protein. The relative densitometries of NLRP3 protein for the immunoblots are shown in the right panel. P value was determined by a Mann-Whitney test

Article Snippet: Human peripheral blood macrophages and human macrophage cell culture medium were purchased from Celprogen (Torrance, CA).

Techniques: Negative Control, Western Blot, Isolation, Expressing, Quantitative RT-PCR, MANN-WHITNEY

Journal: Respiratory Research

Article Title: Cigarette smoke destabilizes NLRP3 protein by promoting its ubiquitination

doi: 10.1186/s12931-016-0485-6

Figure Lengend Snippet: CSE decreases the half-life of the NLRP3 protein. a THP1 cells were incubated with or without CSE at 80 μg/mL for 18 h prior to CHX exposure at 40 μg/mL for the indicated time periods. Cells were collected and assayed for NLRP3 and β-actin (loading control) by immunoblotting for a half-life study. Representative images are shown. b Densitometric plots of NLRP3 protein decay versus time of CHX exposure with best fit lines, depicting the pooled data mean ± S.D. of three independent experiments. c Primary human macrophages from peripheral blood were transfected with 4 μg of either WT NLRP3 -V5 or the point mutant K689R NLRP3 -V5 plasmid. Following transfection, the cells were incubated with or without CSE at 120 μg/mL for 21 h, prior to CHX exposure at 40 μg/mL at different time points for a half-life study. Cells were collected and assayed for NLRP3-V5 and β-actin by immunoblotting. d Densitometric plots of adjusted NLRP3 protein decay over time under different conditions were best fitted. The half-life of WT NLRP3 protein was reduced with CSE exposure, while a K689R NLRP3 mutant was not. Data are mean ± S.D. of two independent experiments

Article Snippet: Human peripheral blood macrophages and human macrophage cell culture medium were purchased from Celprogen (Torrance, CA).

Techniques: Incubation, Western Blot, Transfection, Mutagenesis, Plasmid Preparation

Journal: Respiratory Research

Article Title: Cigarette smoke destabilizes NLRP3 protein by promoting its ubiquitination

doi: 10.1186/s12931-016-0485-6

Figure Lengend Snippet: CSE reduces LPS-induced NLRP3 abundance and release of cytokines. a THP1 cells were incubated with or without 80 μg/mL of CSE, with or without 400 ng/mL of LPS, for 18 h (four groups; none, CSE, LPS, and LPS + CSE). Human primary macrophages derived from monocytes were incubated with or without 120 μg/mL of CSE, with or without 200 ng/mL of LPS, for 20 h. Protein levels of NLRP3, NLRP6, β-actin or GPADH were determined by immunoblotting. Culture medium (supernatants) were also collected for immunoblotting of NLRP3 and NLRP6. The relative densitometries of NLRP3 protein adjusted for loading control (β-actin or GPADH) are shown in the bottom panels. Data are mean ± S.D. of two independent experiments. b Equal numbers of THP1 (3 × 10 6 cells) were plated in equivalent amounts of culture medium and incubated with or without 80 μg/mL of CSE, with or without 400 ng/mL of LPS, for 18 h. Cells were then exposed to 5 mM of ATP for 15 min. Culture medium was precipitated with TCA for immunoblotting of IL-1β, IL-18, and caspase-1. Also, equal numbers of human monocyte-derived macrophages in equal amounts of culture medium were incubated with or without 120 μg/mL of CSE, with or without LPS at 400 ng/mL, for 40 h. Cells were then exposed to 5 mM of ATP for 30 min. Cell lysates and culture medium were collected for immunoblotting. p45 and p20 are probed in the same blot, but presented in different exposure. IL-1β and IL-18 are from the same blot

Article Snippet: Human peripheral blood macrophages and human macrophage cell culture medium were purchased from Celprogen (Torrance, CA).

Techniques: Incubation, Derivative Assay, Western Blot

Journal: BMC Cancer

Article Title: Neuropilin-1 promotes the oncogenic Tenascin-C/ integrin β3 pathway and modulates chemoresistance in breast cancer cells

doi: 10.1186/s12885-018-4446-y

Figure Lengend Snippet: Tenascin C contributes to NRP-1 associated migration. a. Dual immunofluorescence staining (40× magnification scale bar 10 μm) of NRP-1 and TNC on BT-474 and BT-474 NRP-1 cells indicates their colocalization in the cytoplasm. Treatment of BT-474 NRP-1 cells with TNC targeted siRNA molecules, b , reduced TNC gene expression, c , reduced migratory capacity and d , downregulated NRP-1 and vimentin expression. (TNC protein was not detected on western blot due to the lack of specific antibody for this application.) The gene expression fold change was measured by comparing the basal levels detected in the empty plasmid transfected BT-474 or in the case of the siRNA experiment, to the control siRNA treated BT-474 and normalized to β-Actin and GUSB reference gene expression. Wound healing assay images (panel d, 5× magnification, scale bar 500 μm) taken on day 0 and day 2 after siRNA transfection. Graphs represent the mean ± SEM of three independent experiments. Statistical analysis using independent samples t-test, p-value < 0.05 considered as statistically significant. * p < 0.05, ** p < 0.01, *** p < 0.001

Article Snippet: Cells were treated with 80 pmol of a pool of three human TNC-targeted siRNA (Santa Cruz) or control siRNA in transfection reagent and serum-free siRNA transfection medium (Santa Cruz) according to the manufacturer’s instructions.

Techniques: Migration, Immunofluorescence, Staining, Gene Expression, Expressing, Western Blot, Plasmid Preparation, Transfection, Control, Wound Healing Assay

Journal: BMC Cancer

Article Title: Neuropilin-1 promotes the oncogenic Tenascin-C/ integrin β3 pathway and modulates chemoresistance in breast cancer cells

doi: 10.1186/s12885-018-4446-y

Figure Lengend Snippet: NRP-1 overexpression activates integrin β3 and TNFR2 pathways. Representative western blot images of protein lysates from untransfected BT-474, BT-474 NRP-1 and empty vector control cells blotted with indicated antibodies involved in a. Integrin signaling, and downstream signaling targets FAK, Akt, GSK3-β and NF-kB b. siRNA-mediated TNC downregulation decreased phosphorylation of FAK and Akt-473. c. Blots show levels of tumor necrosis factor receptors (TNFRs). GAPDH protein expression is indicated as a loading control. (The prefix P beside the antibody names indicates the phosphorylated form)

Article Snippet: Cells were treated with 80 pmol of a pool of three human TNC-targeted siRNA (Santa Cruz) or control siRNA in transfection reagent and serum-free siRNA transfection medium (Santa Cruz) according to the manufacturer’s instructions.

Techniques: Over Expression, Western Blot, Plasmid Preparation, Control, Phospho-proteomics, Expressing