Journal: Cell Death Discovery

Article Title: Abrogation of store-operated Ca 2+ entry protects against crystal-induced ER stress in human proximal tubular cells

doi: 10.1038/s41420-019-0203-5

Figure Lengend Snippet: Crystal internalization induces upregulation of STIM and ORAI channel expression. Control (noncrystal) CaP, CaOx, and CaP + CaOx (mixed) crystals were introduced into HK2 cells for 24 h. a mRNA expression levels of STIM1, 2, and ORAI channels were analyzed by PCR. ORAI and STIM expression is mediated by SOCE in HK2 cells. HK2 cells were incubated and representative blots were obtained, relative mRNA expression was quantified and represented as bar diagram. b To confirm knockdown following siRNA (10 nM), b STIM1; c STIM2; and d ORAI3 transcript were determined by PCR, representative blots were obtained and quantified as bar diagrams. e To ascertain the role of STIM12 and ORAI3 channel in crystal internalization, STIM1, 2, and ORAI3 expression was knocked down using siRNA, cells were then introduced with or without CaP, CaOx, and mixed (CaP + CaOx) crystals for 24 h. Following crystal internalization, cells were loaded with fura-2AM and Ca 2+ influx was determined and represented as d [Ca 2+ ] rise and e Ca 2+ influx was determined and represented as reduction in [Ca 2+ ] response. Two-tailed t -test was used for statistical comparison. Statistically significant differences are indicated (mean ± SEM) from three different experiments. Levels of significance are indicated as * p < 0.05; ** p < 0.01 as shown in the bar diagrams

Article Snippet: HK2 cells were transfected with (10 nM) siRNA against STIM1 (Santa Cruz Biotechnology, Santa Cruz CA; sc-76589), STIM2 (Santa Cruz sc-76591), ORAI3 (Santa Cruz sc-76005), and scrambled siRNA-A (Santa Cruz sc-37007) as negative control.

Techniques: Expressing, Control, Incubation, Knockdown, Two Tailed Test, Comparison

Journal: Cell Death Discovery

Article Title: Abrogation of store-operated Ca 2+ entry protects against crystal-induced ER stress in human proximal tubular cells

doi: 10.1038/s41420-019-0203-5

Figure Lengend Snippet: Control (noncrystal) CaP, CaOx, and CaP + CaOx (mixed) crystals were introduced into HK2 cells for 24 h. a mRNA levels of ER stress genes ERN (ERN1), CLDN (CLDN1), and GRP78 were analyzed by PCR and representative blots were obtained and quantified. STIM1, STIM2, and ORAI3 expression was knocked down using siRNA transfection (10 nM). Following transfection, b CaP; c CaOx, and d Mixed crystals were introduced into HK2 cells and mRNA levels of ER stress genes ERN1, CLDN1, and GRP78 were analyzed by PCR and representative blots were obtained and quantified. Two-tailed t -test was used for statistical comparison. Statistically significant differences are indicated (mean ± SEM) from three different experiments. Levels of significance are indicated as * p < 0.05; ** p < 0.01 as shown in the bar diagrams

Article Snippet: HK2 cells were transfected with (10 nM) siRNA against STIM1 (Santa Cruz Biotechnology, Santa Cruz CA; sc-76589), STIM2 (Santa Cruz sc-76591), ORAI3 (Santa Cruz sc-76005), and scrambled siRNA-A (Santa Cruz sc-37007) as negative control.

Techniques: Control, Expressing, Transfection, Two Tailed Test, Comparison

Journal: Cell Death Discovery

Article Title: Bcl-2 regulates store-operated Ca 2+ entry to modulate ER stress-induced apoptosis

doi: 10.1038/s41420-018-0039-4

Figure Lengend Snippet: a Control vector (C)-, wild-type Bcl-2 (WT)-, and Bcl-2 mutant (mt)-overexpressing MDCK cells were treated with DMSO or TG for 5 min. Subsequently, immunofluorescence staining was obtained to label STIM1 and nucleus. Representative fluorescence images were obtained using confocal microscopy (scale bar, 20 μm). Arrows indicate the translocation of STIM1 to the juxta-plasma membrane region. b , c Pre-incubation of cells with 2 μM fura-2/AM at 37 °C for 30 min for cytosolic Ca 2+ measurement using a single cell fluorimeter. Depletion of ER lumen-resident Ca 2+ was induced by treating cells in Ca 2+ -free buffer with 2 μM TG for 10 min. The subsequent elevation of Ca 2+ indicated that SOCE occurred during the exchange of Ca 2+ -free buffer to 2 mM-Ca 2+ buffer. b The data in representative curves for the measurement of SOCE are represented as mean ± SEM (where, n ≥ 60 cells) from three independent experiments. c Quantitative analysis of the changes in the peak Ca 2+ levels. All values are represented as mean ± SEM, and the data were found to be statistically significant at p < 0.001 (indicated by ***) compared to control vector (C)-overexpressing cells (Student’s t -test). d Western blotting of SOCE related molecules, such as ER Ca 2+ sensors (STIM1 and STIM2), plasma membrane Ca 2+ channels (Orai1, Orai2, Orai3, and TRPC1), and the internal control β-actin. e Cells were pre-incubated with 2 mM EGTA for 30 min and treated with DMSO or 2 μM TG for 24 h. Representative images of cells were obtained under a bright-field microscope (scale bar, 100 μm). f Bcl-2 mutant (mt)-overexpressing cells were pre-incubated with 20 μM BAPTA/AM, 2 mM EGTA, or 2 μM 2-APB for 30 min and treated with DMSO or 2 μM TG for 24 h. Quantitative analysis of the apoptosis ratio was assessed from the hypodiploid DNA peak of propidium iodide (PI)-stained cells from three independent experiments by flow cytometry. The data were found to be statistically significant at p < 0.01 (indicated by **) compared to the DMSO control (Student’s t -test)

Article Snippet: Cell lysates were harvested in RIPA buffer (150 mM NaCl, 1 mM EGTA, 50 mM Tris at pH 7.4, 10% glycerol, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, and Complete TM ), and the lysates were analyzed by Western blotting using antibodies against Bcl-2 (DAKO, Grostrup, Denmark), Bax, Bak, calnexin, SERCA2, β-actin (Santa Cruz Biotechnology, Santa Cruz, CA), porin (Molecular Probes, Eugene, OR), caspase-8, caspase-9, caspase-12, SERCA3, pSer70-Bcl-2, pThr167-Bax (Cell Signaling Technology, Beverly, MA), Grp78, STIM1, STIM2, IP3R3 (BD, Franklin Lakes, NJ), Orai1, Orai2, Orai3, and TRPC1 (ProSci, Poway, CA).

Techniques: Control, Plasmid Preparation, Mutagenesis, Immunofluorescence, Staining, Fluorescence, Confocal Microscopy, Translocation Assay, Clinical Proteomics, Membrane, Incubation, Western Blot, Microscopy, Flow Cytometry

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: RHBDL2 prevents inappropriate CRAC channel activation in resting cells (A) Scheme of the hOrai1-K-GECO and Cyto-G-GECO reporter assay for spontaneous CRAC channel activity. K-GECO fluorescence is a measure of CRAC channel activity; G-GECO fluorescence is a measure of global cytoplasmic Ca 2+ fluctuations. (B) Four representative line traces of K-GECO fluorescence in single HaCaT cells treated with control siRNA or RHBDL2 siRNA. Spontaneous CRAC channel activation is defined as a >20% increase in the peak amplitude (ΔF/F0). (C) Quantification of spontaneous CRAC channel activity, defined as a >20% increase above the baseline K-GECO fluorescence (see Figure S4 C). Quantification of the percentage of cells displaying spontaneous CRAC channel activity (38 individual cells analyzed in control, 45 individual cells analyzed in RHBDL2 siRNA cells), and the K-GECO fluorescence spike frequency (number of spontaneous CRAC channel activity events over 10 min, relative to the number of cells). (D) TaqMan assay for TNF-alpha in HaCaT cells treated with control or RHBDL2 siRNAs for 48 h. Cyclosporin A (1 μm) was added for the final 24 h. Error bars represent RQ standard error. Each bar represents one of at least four biological replicates. (E) A scheme of the Stim1-BirA experiment in (F) and (G), illustrating the biotinylation of V5-hOrai1 (orange) by Stim1-BirA ∗ (green) at PM-ER contact sites, and the consequence of RHBDL2 (blue) activity. Biotin is indicated by green dots. For simplicity, Stim1 and Orai1 are illustrated as monomeric. (F and G) Western blots of neutravidin agarose-based biotin preparations (in F and G, upper) and total cell lysates (in G, lower) from wild-type (WT) or RHBDL2 knockout (R2 KO) HaCaT cells. The expression of V5-hOrai1 and Stim1-BirA ∗ was induced with doxycycline (DOX; 250 μg/mL final) for 96 h in the presence of 50 μm biotin. Six hours prior to lysis, bafilomycin A1 (BAF; 100 nm final) was added to block lysosomal degradation. In (G), cells were treated twice with Stim2 siRNAs for 96 h prior to lysis. Blots were probed for the N-terminal or C-terminal epitopes recognized by V5 and O1 antibodies, respectively. Stim1 and Stim1-BirA ∗ were probed for using an anti-Stim1 antibody. Different full-length forms of Orai1 and their cleavage products are indicated by the blue arrowheads. (H) Western blots of HaCaT lysates after treatment with control and RHBDL2 siRNA for 72 h, and when used, Stim2 siRNA for 96 h, labeled for endogenous Orai1, Stim1, and beta-actin. Full-length Orai1 (FL) and Orai1β (FLβ) are indicated by arrowheads. (I) Quantification of the fold change in Orai1 protein abundance, from three independent experiments performed as in (H). Error bars represent SEM.

Article Snippet: The Applied Biosystems TaqMan probes, all purchased through Life Technologies, were as follows: RHBDL2 (Hs00983274_m1), TNF alpha (Hs00174128_m1), IL-6 (Hs00985639_m1), Stim2 (Hs00957788_m1) and GAPDH (Hs02786624_g1).

Techniques: Activation Assay, Reporter Assay, Activity Assay, Fluorescence, Control, TaqMan Assay, Western Blot, Knock-Out, Expressing, Lysis, Blocking Assay, Labeling, Quantitative Proteomics

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 Applied Biosystems TaqMan probes, all purchased through Life Technologies, were as follows: RHBDL2 (Hs00983274_m1), TNF alpha (Hs00174128_m1), IL-6 (Hs00985639_m1), Stim2 (Hs00957788_m1) and GAPDH (Hs02786624_g1).

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

Journal: Biochimica et biophysica acta

Article Title: Presenilin-dependent expression of STIM proteins and dysregulation of capacitative Ca2+ entry in familial Alzheimer's disease.

doi: 10.1016/j.bbamcr.2008.11.008

Figure Lengend Snippet: Fig. 2. Cellular level of STIM1 protein is elevated and that of STIM2 is decreased in MEF DKO cells. (A,D) Representative immunoblots of protein extracts from B cell lines developed with anti-STIM1 and anti-STIM2 antibodies and results of densitometric analysis of five independent experiments. (B,C) Results of RT-PCR quantification of STIM1 and STIM2 mRNA levels in MEF cell lines derived from wt, PS1−/−, PS2−/−or DKO (lacking both PS1 and PS2) animals.

Article Snippet: As a double band was seen on immunoblots on human B lymphocytes and HEK293 cells developed with anti-STIM2 antibody (ProSci Incorporated), we analyzed the lower band which, according to antibody manufacturer information, represents STIM2 protein.

Techniques: Western Blot, Reverse Transcription Polymerase Chain Reaction, Derivative Assay

Journal: Biochimica et biophysica acta

Article Title: Presenilin-dependent expression of STIM proteins and dysregulation of capacitative Ca2+ entry in familial Alzheimer's disease.

doi: 10.1016/j.bbamcr.2008.11.008

Figure Lengend Snippet: Fig. 3. CCE is attenuated but STIM proteins' levels are not affected by overexpression of PS1 FAD mutants in HEK293 cells. (A) Averaged traces obtained by ratiometric Fura-2 analysis of HEK293 cell lines overexpressing wt PS1 and PS1 P117R and PS1 E318G mutants. (B) Results of quantification of CCE in analyzed HEK293 cell lines (⁎⁎⁎Pb0,001). (C) Representative immunoblot of protein extracts from HEK293 cell lines developed with anti-STIM1 and anti-STIM2 (D) antibodies and results of densitometric analysis of three independent experiments.

Article Snippet: As a double band was seen on immunoblots on human B lymphocytes and HEK293 cells developed with anti-STIM2 antibody (ProSci Incorporated), we analyzed the lower band which, according to antibody manufacturer information, represents STIM2 protein.

Techniques: Over Expression, Western Blot

Journal: Biochimica et biophysica acta

Article Title: Presenilin-dependent expression of STIM proteins and dysregulation of capacitative Ca2+ entry in familial Alzheimer's disease.

doi: 10.1016/j.bbamcr.2008.11.008

Figure Lengend Snippet: Fig. 5. STIM2 mRNA and protein levels are decreased in B lymphocytes isolated from FAD patients. (A,C) Results of RT-PCR quantification of STIM1 and STIM2 mRNA levels in cell populations from healthy controls and FAD patients. (B,D) Representative immunoblot of protein extracts from B lymphocytes lines developed with anti-STIM1 and anti-STIM2 (D) antibodies and results of densitometric analysis of three independent experiments.

Article Snippet: As a double band was seen on immunoblots on human B lymphocytes and HEK293 cells developed with anti-STIM2 antibody (ProSci Incorporated), we analyzed the lower band which, according to antibody manufacturer information, represents STIM2 protein.

Techniques: Isolation, Reverse Transcription Polymerase Chain Reaction, Western Blot

Journal: Annals of Clinical and Translational Neurology

Article Title: A familial t(4;8) translocation segregates with epilepsy and migraine with aura

doi: 10.1002/acn3.51040

Figure Lengend Snippet: (A) Array‐CGH ratio profiles of proband III‐2. Chromosomes 4 and 8 ideograms showing the 27.3 Mb 4p terminal duplication, the 3.8 Mb 8p terminal deletion, and magnifications of the breakpoint regions showing gene content and genomic positions, according to the genome build CGRh37/hg19. (B) Array‐CGH profiles of two rare CNVs identified in III‐1 genome. 1q43 deletion, identified in III‐1 and her mother (II‐1), located about 112 kb from the 3’UTR of RGS7 gene, and 5q12.3 deletion, partially involving the RGS7BP gene, identified in III‐1, III‐2, and their father (II‐2). Gene content and genomic positions are shown, according to the genome build CGRh37/hg19. (C) FISH analysis of translocation breakpoints. FISH with BAC clone CTD‐322006 (I,II), which spans the translocation bkp at 8p23.3, yields hybridization signal on chromosome 8 and signals of diminished intensity on both derivative chromosomes in II‐2 (I), while in III‐2 (II) the probe produces a signal on chromosome 8 and a signal of diminished intensity on der(8). FISH with BAC clone CTD‐2182P13 (III,IV), partially covering the STIM2 gene sequence at 4p15.2, yields hybridization signals of equal intensity on chromosomes 4 and der(8) in II‐2 (III), but produces three signals of the same intensity on both chromosomes 4 and on der(8) in the proband III‐2 (IV). (D) Physical map of the genomic regions involved in the bkps. 4p15.2‐15.1 region showing the mapped genes, the position of the 4p translocation bkp and its distance from the 3’UTR of STIM2 gene, and the probe CTD‐2182P13used for FISH analysis. 8p23.2 region showing the position of the 8p translocation bkp disrupting the CSMD1 gene at IVS3 and the probe CTD‐322006 used for FISH analysis. (E) STIM2 gene expression analysis . Relative expression of STIM2 blood mRNA in the proband (III‐2), his father (II‐2), and his sister (III‐1), compared to 10 healthy controls. STIM2 showed a significant overexpression in III‐2, while a normal expression level was found in II‐2 and III‐1 compared to controls. Data were normalized against TBP as housekeeping gene; similar results were obtained using GAPDH as normalizer (data not shown). [Corrections added on 21 May 2020 after first publication: Figure 2 has been corrected.]

Article Snippet: All assays were provided by Thermo Fisher Scientific (TaqMan Gene Expression Assays: ID# Hs00372705_m1 STIM2; Hs99999905_m1 GAPDH; Hs99999910_m1 TBP).

Techniques: Translocation Assay, Hybridization, Sequencing, Gene Expression, Expressing, Over Expression