Journal: Molecular oncology
Article Title: Postbiotics of Lacticaseibacillus paracasei CECT 9610 and Lactiplantibacillus plantarum CECT 9608 attenuates store-operated calcium entry and FAK phosphorylation in colorectal cancer cells.
doi: 10.1002/1878-0261.13629
Figure Lengend Snippet: Fig. 1. Expression of Orai and stromal interaction molecule (STIM) proteins and Store-operated Ca2+ entry (SOCE) is enhanced in the colorectal adenocarcinoma cell lines HT-29 and Caco-2. (A–E) NCM460, HT-29 and Caco-2 cells were lysed and the whole cell lysates were analyzed by western blotting using anti-Orai1 (A), anti-Orai2 (B), anti-Orai3 (C), anti-STIM1 (D) or anti-STIM2 (E) antibody. Molecular masses indicated on the right were determined using molecular-mass markers run in the same gel. Membranes were probed with anti-b-actin antibody for protein loading control. These results are representative of four separate experiments. Quantification of protein expression in NCM460 (n = 4), HT-29 (n = 4) and Caco-2 (n = 4) cells normalized to the b-actin expression is depicted in the bar graph. Data are represented as mean standard error of the mean (SEM) and were statistically analyzed using Kruskal–Wallis test combined with Dunn’s post hoc test. **P < 0.01 and ****P < 0.0001. (F) Representative Ca2+ mobilization in response to 2 lM thapsigargin (TG) measured using fura-2 in NCM460 (n = 3 [62 cells]), HT-29 (n = 3 [69 cells]) and Caco-2 (n = 3 [36 cells]) cells. Cells were superfused with a Ca2+-free Hepes Buffer Saline (HBS) (100 lM ethylene glycol-bis(2-aminoethylether)-N,N,N0,N0-tetraacetic acid (EGTA) added) and stimulated with 2 lM TG, followed by re-addition of CaCl2 (1.8 mM) to estimate Ca2+ influx. (G, H) Quantification of TG-evoked Ca2+ release from the intracellular stores and entry in NCM460 (n = 3 [62 cells]), HT-29 (n = 3 [69 cells]) and Caco-2 (n = 3 [36 cells]) cells is shown in the scatter plots. Data in bar graphs are represented as mean SEM and were statistically analyzed using Kruskal–Wallis test combined with Dunn’s post hoc test. ****P < 0.0001. a.u., arbitrary units; AUC, area under the curve.
Article Snippet: Rabbit polyclonal anti-STIM2 antibody (catalog number 4917S) was from Cell Signaling Technology (Leiden, the Netherlands).
Techniques: Expressing, Western Blot, Control, Saline
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![Fig. 1. Expression of Orai and stromal interaction molecule (STIM) proteins and Store-operated Ca2+ entry (SOCE) is enhanced in the colorectal adenocarcinoma cell lines HT-29 and Caco-2. (A–E) NCM460, HT-29 and Caco-2 cells were lysed and the whole cell lysates were analyzed by western blotting using anti-Orai1 (A), anti-Orai2 (B), anti-Orai3 (C), anti-STIM1 (D) or <t>anti-STIM2</t> (E) antibody. Molecular masses indicated on the right were determined using molecular-mass markers run in the same gel. Membranes were probed with anti-b-actin antibody for protein loading control. These results are representative of four separate experiments. Quantification of protein expression in NCM460 (n = 4), HT-29 (n = 4) and Caco-2 (n = 4) cells normalized to the b-actin expression is depicted in the bar graph. Data are represented as mean standard error of the mean (SEM) and were statistically analyzed using Kruskal–Wallis test combined with Dunn’s post hoc test. **P < 0.01 and ****P < 0.0001. (F) Representative Ca2+ mobilization in response to 2 lM thapsigargin (TG) measured using fura-2 in NCM460 (n = 3 [62 cells]), HT-29 (n = 3 [69 cells]) and Caco-2 (n = 3 [36 cells]) cells. Cells were superfused with a Ca2+-free Hepes Buffer Saline (HBS) (100 lM ethylene glycol-bis(2-aminoethylether)-N,N,N0,N0-tetraacetic acid (EGTA) added) and stimulated with 2 lM TG, followed by re-addition of CaCl2 (1.8 mM) to estimate Ca2+ influx. (G, H) Quantification of TG-evoked Ca2+ release from the intracellular stores and entry in NCM460 (n = 3 [62 cells]), HT-29 (n = 3 [69 cells]) and Caco-2 (n = 3 [36 cells]) cells is shown in the scatter plots. Data in bar graphs are represented as mean SEM and were statistically analyzed using Kruskal–Wallis test combined with Dunn’s post hoc test. ****P < 0.0001. a.u., arbitrary units; AUC, area under the curve.](https://pub-med-unpaywalled-images-cdn.bioz.com/pub_med_ids_ending_with_4909/pm38514909/pm38514909__page2_image1.jpg)