Journal: Journal of Lipid Research

Article Title: Crosstalk between ORMDL3, serine palmitoyltransferase, and 5-lipoxygenase in the sphingolipid and eicosanoid metabolic pathways

doi: 10.1016/j.jlr.2021.100121

Figure Lengend Snippet: Identification of the ORMDL3/5-LO/SPTLC1/SPTLC2 complex. A: ORMDL3-MYC immunoprecipitated from lysates of BMMCL expressing ORMDL3-MYC or empty vectors (control) and activated or not with thapsigargin. The immunoprecipitates were size-fractionated by SDS-PAGE and stained by Coomassie blue. ←5-LO indicates position of 5-LO as identified by mass spectrometry. B: 5-LO interacts with ORMDL3, SPTLC1, and SPTLC2. Twin-Strep-tag (TS) affinity purification from lysates of HEK293FT cells stably transduced with FLAG-ORMDL3 alone or in combination with TS-5-LO and activated for 0, 3, 15, or 25 min with thapsigargin. The proteins were analyzed by immunoblotting using the corresponding antibodies. C: Colocalization of endogenous 5-LO with ORMDLs (left panel) or STIM1 (right panel) in nonactivated (0 min) or activated HMC-1.1 cells for 10 or 30 min with ionomycin. Scale bars represent 5 μm. D: Physical interaction of 5-LO with ORMDL3. FLAP, ORMDL3, LTC4S, and 5-LO were tagged with GST and expressed in bacteria. GST-tagged proteins and GST alone were purified on glutathione-coated beads in the presence of in vitro translate expressing 35 S-5-LO. SDS-PAGE followed by autoradiography was used to detect 35 S-5-LO. The gel was stained with Coomassie brilliant blue (CB staining) to determine the loading levels of GST and GST-tagged proteins. E: SDS-PAGE of lysates from HEK293 cells stably transduced with empty vector (control), murine 5-LO, or murine ORMDL3 developed with the indicated protein-specific antibodies. F and G: LC-ESI-MS/MS analysis of sphingolipids in resting HEK293 cells stably transduced with murine 5-LO (n = 13), murine ORMDL3 (n = 7), or empty vector (control, n = 12). F: Total sphingosines, the sum of C18:1 and C18:0 is calculated. G: The sum of total ceramide fatty acid chain molecular species (including 2-hydroxy ceramide molecular species), derived from d18:1 sphingosine, was calculated. Data in B and D are representative of three independent experiments. Quantitative data in F and G are mean ± SEM, calculated from n, which show numbers of biological replicates of independently transduced cells. P values were determined by one-way ANOVA with Bonferoni post hoc test.

Article Snippet: Antibodies against HA tag (NB600-363) and stromal interaction molecule 1 (STIM1) (NB110-60547S) were obtained from Novus Biologicals.

Techniques: Immunoprecipitation, Expressing, Control, SDS Page, Staining, Mass Spectrometry, Strep-tag, Affinity Purification, Stable Transfection, Transduction, Western Blot, Bacteria, Purification, In Vitro, Autoradiography, Plasmid Preparation, Tandem Mass Spectroscopy, Derivative Assay

Journal: Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology

Article Title: Inhibition of Lithium Sensitive Orai1/ STIM1 Expression and Store Operated Ca2+ Entry in Chorea-Acanthocytosis Neurons by NF-κB Inhibitor Wogonin.

doi: 10.1159/000495229

Figure Lengend Snippet: Fig. 2. Effect of lithium treatment without and with NFκB inhibitor wogonin on Orai1, STIM1 and STIM2 protein abundance in iPSC-derived neurons from ChAc patients. A,B,C. Original Western blot of (A) Orai1, (B) STIM1 and (C) STIM2 protein abundance in neurons differentiated from iPSCs derived from ChAc patients (ChAc) without or with lithium (24 h, 2 mM) treatment without and with presence of NFκB inhibitor wogonin (50 µM). D,E,F. Arithmetic means (± SEM, n = 5) of (D) Orai1, (E) STIM1 and (F) STIM2 protein abundance in neurons generated from iPSCs derived from ChAc patients without (blue bars) or with (red and grey bars) lithium (24 h, 2 mM) treatment without (red bars) and with (grey bars) presence of NFκB inhibitor wogonin (50 µM). #(p<0.05), ##(p<0.01) indicates statistically significant difference to respective value in the absence of treatment, §(p<0.05), §§§(p<0.001) indicates statistically significant difference to respective value in the absence of wogonin.

Article Snippet: The proteins were separated by 10% SDS-PAGE in Glycine-Tris buffer and electro-transferred onto nitrocellulose membranes for 90 min. After blocking with 5% milk in TBST at room temperature for 1 h, the membranes were incubated with primary anti-ORAI1 antibody (1:1000, Proteintech), anti-STIM1 antibody (1:1000, Cell Signaling), anti-STIM2 antibody (1:1000, Cell Signaling) and anti-GAPDH antibody (1:1000, Cell Signaling) at 4°C overnight.

Techniques: Quantitative Proteomics, Derivative Assay, Western Blot, Generated

Journal: Molecular Cell

Article Title: Conformational surveillance of Orai1 by a rhomboid intramembrane protease prevents inappropriate CRAC channel activation

doi: 10.1016/j.molcel.2021.10.025

Figure Lengend Snippet:

Article Snippet: The following antibodies were used for western blotting (WB) and immunofluorescence (IF): mouse anti-beta-actin (Santa Cruz, catalog number sc-47778; WB 1:2000), mouse anti-HA (ENZO, catalog number ENZ-ABS120-0200; WB 1:1000), mouse anti-transferrin receptor (Invitrogen, catalog number 13–6800; WB 1:1000), rabbit anti-Stim1 (Cell Signaling Technology, catalog number 5668S (D88E10); WB 1:2000), rabbit anti-Orai1 (Sigma Aldrich, catalog number O8264; WB 1:2500), goat anti-myc tag (Abcam, catalog number ab9132; IF 1:2000), rabbit anti-RHBDL2 (Proteintech, catalog number 12467-1-AP; WB 1:250 – only detected RHBDL2 in HaCaT lysates), rabbit anti-V5 tag (Cell Signaling Technology, catalog number 13202S; WB and IF 1:2000).

Techniques: Virus, Recombinant, Gene Expression, Control, Negative Control, shRNA, Mutagenesis, Plasmid Preparation, Software

Journal: Frontiers in Pharmacology

Article Title: Phemindole, a Synthetic Di-indole Derivative Maneuvers the Store Operated Calcium Entry (SOCE) to Induce Potent Anti-Carcinogenic Activity in Human Triple Negative Breast Cancer Cells

doi: 10.3389/fphar.2016.00114

Figure Lengend Snippet: Phemindole downregulates STIM1 Expression. (A) Immunofluorescence images showing decreased expression of STIM1 in the MDAMB-231 cells in response to Phemindole treatment. Anti STIM1 antibody was FITC tagged (green) and nuclei were stained with DAPI (blue) (left panel). Corrected Fluoroscence intensity of STIM1 protein was quantified and represented as bar diagram (Right panel). Pictures showed representative cells of each population and were representative of three independent experiments. (B) Western blot analysis depicting the changes in the expression pattern of STIM1 and Orai1. MDAMB-231 cells were treated with Phemindole and Western blotted with respective antibodies as labeled in the figure (upper-left panel). Quantification of WB were depicted as bar diagram (upper-right panel). MDAMB-231 cells were treated with 10 μM Phemindole for 24 h. Lysates were subjected to SDS–PAGE followed by Western blotting (with anti-Orai1 antibodies. β–actin was used as a loading control (middle-left panel). Band intensity was quantified and represented as a bar histogram (middle-right panel). Cells were treated with 10 μM Phemindole and were harvested at 0, 6, 12, or 24 h after Phemindole treatment. Western blotting was done. Antibodies used are labeled and β-actin was used as loading control (lower-left panel). Lower-right panel represent the normalized intensity of the bands. Blots are representative of three independent experiments and quantifications are indicated by Bar diagram. Values were normalized by intensity of loading control. (C) MDAMB-231 cells were treated with Phemindole or Tg under different conditions, lysed and proteins were subjected to immune precipitation by anti STIM1 antibody and followed by Western blotting with respective antibody (upper panel). (D) Representative Fluorescence images showed the association between STIM1 (red) and ORAI1 (green) at plasma membrane. Nuclei were stained with DAPI (blue). Enlargements of the areas indicated by the color rectangles showed overlap formation of STIM1 and Orai1 in plasma membrane due to Tg treatment (Left-middle) which is invisible in Phemindole treated set (left-lower) and not prominent in un-stimulated control set (left-upper panel). Pictures showed representative cells of each population and were representative of three independent experiments. Right panel, the Pearson’s and Manders coefficients determining the level of overlap of RITC and FITC on treatment with Control, thapsigargin ( TG ) and Phemindole has been represented graphically. ∗ , ∗∗ , ∗∗∗ , # indicate p < 0.05, p < 0.01, p < 0.0005, p > 0.05 respectively versus untreated or control group.)

Article Snippet: MDAMB-231 cells were transfected with 300 pmol of STIM1 siRNA (Santa Cruz Biotechnology) and Lipofectamine 2000 (Invitrogen) separately for 12 h. Levels of STIM1 proteins were estimated by Western blotting.

Techniques: Expressing, Immunofluorescence, Staining, Western Blot, Labeling, SDS Page, Control, Fluorescence, Clinical Proteomics, Membrane

Journal: Frontiers in Pharmacology

Article Title: Phemindole, a Synthetic Di-indole Derivative Maneuvers the Store Operated Calcium Entry (SOCE) to Induce Potent Anti-Carcinogenic Activity in Human Triple Negative Breast Cancer Cells

doi: 10.3389/fphar.2016.00114

Figure Lengend Snippet: Inhibitions of STIM1 mimics the effects of Phemindole and over expression of STIM1 showed opposite effect. (A) Ca 2+ imaging was performed in control, in the presence of SKF 96365 (SOCE inhibitor) and STIM1 si-RNA treated MDAMB-231 cells. Analog plots of the fluorescence ratio (340/380) of the cells were shown. Knockdown of STIM1 protein was confirmed by western blot analysis (Inset) (B) Quantification (mean ± SD) of fluorescence ratio (340/380). ∗ P < 0.05 versus control. (C) Bar diagram showed MTT assay under SKF 96365, STIM1 si-RNA, treated conditions for 24 h in MDAMB231 cells. (D) STIM1 protein levels were restored by Transfection of STIM1 clones and also other protein levels were measured in presence and absence of Phemindole in STIM1 transfected cells by western blot (left panel). Quantification of band intensity in respect to loading control was shown in bar diagram (left panel). Blots are representative of three independent experiments and quantifications are indicated by bar diagram. Values were normalized by intensity of loading control. (E) Fura-2 Ca 2+ imaging showing the results of a quantitative analysis of Ca 2+ content (or Ca 2+ release in after Tg treatment) and Ca 2+ influx during SOCE as mentioned previously. Values are the mean ± SEM for all cells in plates ∗ p < 0.05 versus control group. (F) Bar diagram represented the cell proliferation pattern of STIM1 over expressed MDAMB-231 cells in presence of Phemindole treatment. Each bar gives the mean ± SEM of three separate experiments. ∗ , ∗∗ , ∗∗∗ , # indicate p < 0.05, p < 0.01, p < 0.0005, p > 0.05 respectively versus untreated or control group.

Article Snippet: MDAMB-231 cells were transfected with 300 pmol of STIM1 siRNA (Santa Cruz Biotechnology) and Lipofectamine 2000 (Invitrogen) separately for 12 h. Levels of STIM1 proteins were estimated by Western blotting.

Techniques: Over Expression, Imaging, Control, Fluorescence, Knockdown, Western Blot, MTT Assay, Transfection, Clone Assay

Journal: Frontiers in Pharmacology

Article Title: Phemindole, a Synthetic Di-indole Derivative Maneuvers the Store Operated Calcium Entry (SOCE) to Induce Potent Anti-Carcinogenic Activity in Human Triple Negative Breast Cancer Cells

doi: 10.3389/fphar.2016.00114

Figure Lengend Snippet: Assessment of toxicity of Phemindole. (A) 4T1 induced tumors excised from the aforementioned sets were analyzed histopathologically using eosin haematoxylin staining and phase contrast images were captured. (B) Immuno-histochemical analysis of the excised tumors from the untreated and Phemindole treated sets using anti-STIM1 antibody (left panel). Corrected Fluorescence intensity of STIM1 protein was quantified and represented as bar diagram (right panel). (C) Histological sections of liver and kidney from the animals were stained with haematoxylin and counter-stained with eosin and microscopically analyzed for histopathologically examinations of tissue toxicity like cellular damage and vacuolization. (D) Blood was collected after sacrifice the mice from control, untreated tumor and Phemindole treated sets and subsequent Liver function test and Kidney Function Test were performed. All measurements of three groups were repeated at least three times, Values are mean ± SEM of three independent experiments in each case or representative of typical experiment. ∗∗∗ P < 0.001, (E) Schematic diagram depicting the detailed molecular mechanisms of antitumorogenic activity of Phemindole against triple negative breast cancer cells.

Article Snippet: MDAMB-231 cells were transfected with 300 pmol of STIM1 siRNA (Santa Cruz Biotechnology) and Lipofectamine 2000 (Invitrogen) separately for 12 h. Levels of STIM1 proteins were estimated by Western blotting.

Techniques: Staining, Fluorescence, Control, Activity Assay

Journal: Pediatric diabetes

Article Title: Expansion of CD4+CD25+FOXP3+ regulatory T cells in infants of mothers with type 1 diabetes

doi: 10.1111/j.1399-5448.2012.00852.x

Figure Lengend Snippet: Expression of FOXP3, IL-10, NFATc2, STIM1, and transforming growth factor (TGF)-β specific mRNA in cord blood mononuclear cells in infants of mothers with type 1 diabetes (A) and of unaffected mothers (B) after 72 h stimulation with human insulin (hi) or medium alone (neg). Results of FOXP3, IL-10, and NFATc2 are presented as 1000× relative transcription and STIM1 and TGF-β are presented as 100× relative transcription. Median and interquartile range are shown. The asterisk indicates significant differences (*p < 0.05, **p < 0.01; Wilcoxon test).

Article Snippet: Determined targets were FOXP3 (cat. no Hs00203958_m1), NFATc2 (Hs00905451_m1), STIM1 (Hs00162394_m1), IL-10 (Hs00174086_m1), and TGF-β (Hs00171257_m1).

Techniques: Expressing

Journal: Pediatric diabetes

Article Title: Expansion of CD4+CD25+FOXP3+ regulatory T cells in infants of mothers with type 1 diabetes

doi: 10.1111/j.1399-5448.2012.00852.x

Figure Lengend Snippet: Upregulation of STIM1 and NFATc2-specific mRNA expressed as fold change in relation to the PTPN22 genotype in infants of mothers with type 1 diabetes (n = 19). Fold change in STIM1 (A) and NFATc2 (B) was calculated by dividing the relative mRNA level of insulin-stimulated (72 h) cord blood mononuclear cells (CBMCs) by the relative mRNA level of non-stimulated CBMCs. Horizontal lines represent median values. p Values comparing the groups (Mann–Whitney U test) are shown.

Article Snippet: Determined targets were FOXP3 (cat. no Hs00203958_m1), NFATc2 (Hs00905451_m1), STIM1 (Hs00162394_m1), IL-10 (Hs00174086_m1), and TGF-β (Hs00171257_m1).

Techniques: MANN-WHITNEY