Journal: Antioxidants

Article Title: Cell-Based Antioxidant Properties and Synergistic Effects of Natural Plant and Algal Extracts Pre and Post Intestinal Barrier Transport

doi: 10.3390/antiox11030565

Figure Lengend Snippet: Comparison of AOP1 EC 50 s evaluated in Caco-2 and HepG2 cells for the 12 natural extracts selected for the intestinal barrier transport study. Extracts were selected according to the best EC 50 s obtained with AOP1 assay. In order to increase the diversity of tested extracts, some Vitis vinifera extracts were removed from the list because of redundancy with each other. ND = not determined (no full effect observed).

Article Snippet: The HepG2/Nrf2 cells were treated with conditions for 17 h; then, they were treated with a mix (BPS Bioscience, USA) comprising cell lysis solution and luciferin (substrate of luciferase) for 40 min. Luminescence was read on a Varioskan Flash Spectral Scanning Multimode Reader.

Techniques:

Journal: Antioxidants

Article Title: Cell-Based Antioxidant Properties and Synergistic Effects of Natural Plant and Algal Extracts Pre and Post Intestinal Barrier Transport

doi: 10.3390/antiox11030565

Figure Lengend Snippet: Indirect antioxidant ARE/Nrf2 assay data obtained for Syzygium aromaticum extract (given as an example) in HepG2/Nrf2 cells. All other extracts were subjected to the same protocol and data analyses. ( A ) Assay of increasing concentrations showing the typical bell-shaped profile (see text for explanation). Sulforaphane 25 µM was used as positive control; ( B ) dose–response and sigmoid fit curve used for Efficacy Concentration (EC) estimations.

Article Snippet: The HepG2/Nrf2 cells were treated with conditions for 17 h; then, they were treated with a mix (BPS Bioscience, USA) comprising cell lysis solution and luciferin (substrate of luciferase) for 40 min. Luminescence was read on a Varioskan Flash Spectral Scanning Multimode Reader.

Techniques: Positive Control, Concentration Assay

Journal: Antioxidants

Article Title: Cell-Based Antioxidant Properties and Synergistic Effects of Natural Plant and Algal Extracts Pre and Post Intestinal Barrier Transport

doi: 10.3390/antiox11030565

Figure Lengend Snippet: ARE/Nrf2 EC 50 s established in HepG2 cells for the 12 natural extracts selected for the Caco-2 intestinal barrier transport study. ND = not determined; R 2 = determination coefficient.

Article Snippet: The HepG2/Nrf2 cells were treated with conditions for 17 h; then, they were treated with a mix (BPS Bioscience, USA) comprising cell lysis solution and luciferin (substrate of luciferase) for 40 min. Luminescence was read on a Varioskan Flash Spectral Scanning Multimode Reader.

Techniques:

Journal: Antioxidants

Article Title: Cell-Based Antioxidant Properties and Synergistic Effects of Natural Plant and Algal Extracts Pre and Post Intestinal Barrier Transport

doi: 10.3390/antiox11030565

Figure Lengend Snippet: Effect of post-intestinal barrier transport compartments on ARE/NRF2 pathway activation in HepG2 cells. Results are expressed as fold increase compared to constitutive medium ARE/Nrf2 gene expression. Values represent means of quadruplicates obtained from two independent intestinal barrier transport experiments. Sulforaphane 25 µM data is provided as positive control; *** p < 0.005.

Article Snippet: The HepG2/Nrf2 cells were treated with conditions for 17 h; then, they were treated with a mix (BPS Bioscience, USA) comprising cell lysis solution and luciferin (substrate of luciferase) for 40 min. Luminescence was read on a Varioskan Flash Spectral Scanning Multimode Reader.

Techniques: Activation Assay, Expressing, Positive Control

Journal: Antioxidants

Article Title: Cell-Based Antioxidant Properties and Synergistic Effects of Natural Plant and Algal Extracts Pre and Post Intestinal Barrier Transport

doi: 10.3390/antiox11030565

Figure Lengend Snippet: Effect of combinations of post-intestinal barrier compartments on ARE/NRF2 pathway in HepG2 cells. Results are expressed as ARE/Nrf2 gene expression fold increase compared to constitutive medium. Values represent means of pentaplicates for all 50/50 combinations and of triplicates for all others. All data were normalized to constitutive medium values; *** p < 0.001; ** p < 0.01.

Article Snippet: The HepG2/Nrf2 cells were treated with conditions for 17 h; then, they were treated with a mix (BPS Bioscience, USA) comprising cell lysis solution and luciferin (substrate of luciferase) for 40 min. Luminescence was read on a Varioskan Flash Spectral Scanning Multimode Reader.

Techniques: Expressing

Journal: Antioxidants

Article Title: Cell-Based Antioxidant Properties and Synergistic Effects of Natural Plant and Algal Extracts Pre and Post Intestinal Barrier Transport

doi: 10.3390/antiox11030565

Figure Lengend Snippet: Synergistic effects on ARE/Nrf2 gene expression of post-intestinal barrier compartment combinations. Combin. FI = gene expression fold increase (FI) measure for the combination; S indep. FI = sum of the increase in gene expression fold increase (FI) measures of each of the two extracts taken independently as depicted in Figure 6 ; Var. (%) = variation in percentage of the two values. A positive percentage value indicates a synergistic effect.

Article Snippet: The HepG2/Nrf2 cells were treated with conditions for 17 h; then, they were treated with a mix (BPS Bioscience, USA) comprising cell lysis solution and luciferin (substrate of luciferase) for 40 min. Luminescence was read on a Varioskan Flash Spectral Scanning Multimode Reader.

Techniques: Expressing

Journal: Cancers

Article Title: Silibinin Overcomes EMT-Driven Lung Cancer Resistance to New-Generation ALK Inhibitors

doi: 10.3390/cancers14246101

Figure Lengend Snippet: Targeted effects of silibinin against the TGFβ/TGFβR/SMAD signaling pathway. ( A ) Expression levels of E-cadherin, SNAIL, vimentin, phospho-SMAD2/3, total SMAD2/3 were detected by immunoblotting in H2228/H2228CR and H3122/H3122CR cells using specific antibodies. The intensity of the bands was measured using the ImageJ software. Fold-change of each protein relative to parental cells was calculated using GAPDH as a loading control. The figure shows representative immunoblots of multiple ( n ≥ 5) independent experiments. E: Epithelial; M: Mesenchymal. ( B ) Top: Relative luciferase activity using SBE Reporter–HEK293 cells pre-incubated during 4–5 h with graded concentrations of SB525334 and silibinin before stimulation with TGFβ1. Bottom: Expression levels of phospho-SMAD2/3 and total SMAD2/3 were detected by immunoblotting in HEK293 cells stimulated with TGFβ1 (0, 6, and 24 h) in the absence/presence of either silibinin or SB431542 using specific antibodies. The intensity of the bands was measured using the ImageJ software. Fold-change of each protein relative to untreated samples was calculated using GAPDH as a loading control. The figure shows representative immunoblots of multiple ( n ≥ 3) independent experiments. ( C ) Volcano plots of the results from analyses of the Applied Biosystems TM TaqMan TM Array Human TGFβ Pathway in H2228/H2228CR cells cultured in the absence/presence of silibinin (100 μmol/L) for 48 h. Each dot represents a transcript with its corresponding mean Log2 fold-change (FC) ( x axis) and Benjamini–Hochberg corrected p -value (−log10, y axis). Colored dots illustrate differential lipid species, using a cutoff of p < 0.05 and log2FC > 1 or < 1. ( D ) The figure depicts the backbone of the overall crystal structure of TGFβR1 (5E8S) and TGFβR2 (5E8Y) with rainbow colors showing the best docked poses of silibinin A and silibinin B at the catalytic site. The uncropped western blot figures were presented in .

Article Snippet: The SBE Reporter–HEK293 cell line (Cat. #60653; BPS Bioscience, San Diego, CA, USA) was employed for monitoring the impact of silibinin on the activity of the TGFβ/SMAD signaling pathway.

Techniques: Expressing, Western Blot, Software, Luciferase, Activity Assay, Incubation, Cell Culture

Journal: Function

Article Title: Acidic Cannabinoids Suppress Proinflammatory Cytokine Release by Blocking Store-operated Calcium Entry

doi: 10.1093/function/zqac033

Figure Lengend Snippet: Effect of cannabinoids on NFAT activity in standard culture medium. Pure cannabinoids were tested at 5 µ m against NFAT activity. Jurkat T cells expressing NFAT reporter gene were pretreated with the cannabinoids before stimulation using 1 µ m ionomycin and 20 ng/mL PMA. Bioluminescence emitted by NFAT-mediated luciferase expression was measured in arbitrary units 5 h poststimulation. All data points represent mean ± SEM of 3 independent runs ( N = 3), with each run including n = 3 replicates per condition. * P < .05, ** P < .01. Key: NT = nontreated cells, NS = nonstimulated, MeOH = methanol, MeCN = acetonitrile, DMSO = dimethylsulfoxide.

Article Snippet: Jurkat NFAT reporter cell line (BPS Bioscience, San Diego, CA, USA) was maintained in RPMI 1640 medium supplemented with 10% FBS.

Techniques: Activity Assay, Expressing, Luciferase

Journal: Function

Article Title: Acidic Cannabinoids Suppress Proinflammatory Cytokine Release by Blocking Store-operated Calcium Entry

doi: 10.1093/function/zqac033

Figure Lengend Snippet: CBGA inhibitory potency is decreased by increasing serum concentrations. Serum-dependent effects of CBGA on I CRAC and NFAT activity in the presence of 0%, 1%, and 10% FBS. (A) Time course of CRAC currents recorded in Jurkat T cell in whole-cell patch-clamp experiments. Bath solution was supplemented with 0%, 1%, or 10% FBS (0% serum data is the same as in ). To elicit the active calcium depletion from the ER, 50 µ m IP 3 in the recording pipette was utilized. The resulting CRAC currents were allowed to reach maximal activation prior to extracellular application of CBGA at various concentrations. Current amplitudes were measured at −120 mV for each voltage ramp and were normalized to the control at 120 s. n represents the number of cells patched for each condition. (B) Concentration–response effects of CBGA on NFAT activity in the presence of 0%, 1%, and 10% FBS. A 10- and 12-point concentration-response curves, ranging from 100 n m to 100 µ m and 100 n m to 300 µ m , respectively, were established. The 12-point d/r was used for 10% FBS condition due to the weak activity of CBGA in the ten-point concentration range under this condition. Values are average ± SEM of 12–15 replicates, normalized to the control (cells treated with PMA + Ionomycin only).

Article Snippet: Jurkat NFAT reporter cell line (BPS Bioscience, San Diego, CA, USA) was maintained in RPMI 1640 medium supplemented with 10% FBS.

Techniques: Activity Assay, Patch Clamp, Transferring, Activation Assay, Concentration Assay

Journal: Function

Article Title: Acidic Cannabinoids Suppress Proinflammatory Cytokine Release by Blocking Store-operated Calcium Entry

doi: 10.1093/function/zqac033

Figure Lengend Snippet: Isobolographic analysis of CBGA and other cannabinoids against SOCE. (A) An isobole showing dose pairs of CBGA and CBD that are expected to inhibit SOCE by 50%. The y- and the x-intercepts represent the IC 50 values of CBGA and CBD, respectively. (B) Inhibition of SOCE observed when Jurkat NFAT cells were treated with concentration pairs of CBGA and CBD. The dashed line depicts the expected 50% inhibition of SOCE at each dose pair. The bold line connects the actual inhibition observed when treated separately with CBGA and the other cannabinoid (CBD) at individual IC 50 . The bars represent the observed inhibition when treated with various concentration pairs of the two compounds. Here, the bars fall on the bold line, indicating that the two compounds follow a simple additivity behavior. (C) Dose pairs of CBGA and THCVA show deviation from expected inhibition. The green bars above the bold line suggest supra-additive (synergistic) effects between the two compounds. (D) Concentration pairs of CBGA and Δ8-THC show deviation in the opposite direction, suggesting subadditive (inhibitory) effects.

Article Snippet: Jurkat NFAT reporter cell line (BPS Bioscience, San Diego, CA, USA) was maintained in RPMI 1640 medium supplemented with 10% FBS.

Techniques: Inhibition, Concentration Assay