primary antibody against ip3r  (Servicebio Inc)

Journal: bioRxiv

Article Title: MYC shapes ER-mitochondria calcium transfer by directly targeting ITPR1 : implications for MYC-induced safeguard mechanisms and cancer

doi: 10.1101/2024.08.28.610025

Figure Lengend Snippet: A. RT-qPCR of ITPR1 , ITPR2 and ITPR3 genes at day 3 after treatment of MRC5-MYC:ER cells with 4OHT. Mean +/- SEM of n = 5 independent experiments. Two-Way ANOVA. P-values are indicated. B. RT-qPCR of ITPR1 , BIM and PUMA genes at the indicated times after treatment of MRC5-MYC:ER cells with 4OHT. Mean +/- SEM of n = 3 independent experiments. Two-Way ANOVA. P-values are indicated. C. ChIP-seq analysis of MYC occupancy at the ITPR1 promoter region in MYC-overexpressing cells (U2OS, HeLa) (GSE44672).

Article Snippet: Membranes were then blocked with 5% milk in TBS with 0.01% Tween-20 (Sigma-Aldrich) TBST for 1 h at room temperature and incubated with mouse primary antibodies against ITPR1 (sc-271197, Santa Cruz Biotechnology) or α-Tubulin (T6199, Sigma-Aldrich) at 4°C overnight with gentle shaking.

Techniques: Quantitative RT-PCR, ChIP-sequencing

Journal: bioRxiv

Article Title: MYC shapes ER-mitochondria calcium transfer by directly targeting ITPR1 : implications for MYC-induced safeguard mechanisms and cancer

doi: 10.1101/2024.08.28.610025

Figure Lengend Snippet: MRC5-MYC:ER cells were transfected with a control siRNA pool (siControl) or with a siRNA pool targeting ITPR1 (siITPR1) and then treated or not with 4OHT. A. RT-qPCR of ITPR1 gene at day 3 after 4OHT treatment. Mean +/- SEM of n = 5 independent experiments. Two-Way ANOVA. P-values are indicated. B. Western blot showing ITPR1 protein levels at day 3 after 4OHT treatment and α-Tubulin protein levels as loading control. Representative images of n = 3 independent experiments. C . Crystal violet staining at day 10 after 4OHT treatment. Representative image of n = 3 independent experiments. D . Percentage of trypan blue-positive cells counted at day 3 after 4OHT treatment. Mean +/- SEM of n = 3 independent experiments. Two-Way ANOVA. P-values are indicated. E . Cell death monitored using SYTOX Green in the first 40h after 4OHT treatment. Representative experiment from n=3 independent experiments.

Article Snippet: Membranes were then blocked with 5% milk in TBS with 0.01% Tween-20 (Sigma-Aldrich) TBST for 1 h at room temperature and incubated with mouse primary antibodies against ITPR1 (sc-271197, Santa Cruz Biotechnology) or α-Tubulin (T6199, Sigma-Aldrich) at 4°C overnight with gentle shaking.

Techniques: Transfection, Control, Quantitative RT-PCR, Western Blot, Staining

Journal: bioRxiv

Article Title: MYC shapes ER-mitochondria calcium transfer by directly targeting ITPR1 : implications for MYC-induced safeguard mechanisms and cancer

doi: 10.1101/2024.08.28.610025

Figure Lengend Snippet: A. MRC5-MYC:ER cells were transfected with a control siRNA pool (siControl) or with a siRNA pool targeting ITPR1 (siITPR1) or VDAC3 (siVDAC3) and then treated or not with 4OHT. RT-qPCR of ITPR1 gene (left) or VDAC3 gene (right) at day 3 after 4OHT treatment. Mean +/- SEM of n = 3 independent experiments. Two-Way ANOVA. P-values are indicated. B . MRC5-MYC:ER cells overexpressing mito-GEM-GECO1 ratiometric mitochondrial Ca 2+ gene reporter were transfected with a control siRNA pool (siControl) or with a siRNA pool targeting ITPR1 (siITPR1) or VDAC3 (siVDAC3) and then treated or not with 4OHT for 2 days. Resting mitochondrial Ca 2+ levels according to the ratio F(437-499)/F(520-755) were measured. Representative images are shown as well as quantitative results from n = 3 independent experiments. siControl -4OHT: n = 148 cells, siITPR1 -4OHT: n = 129 cells, siVDAC3 - 4OHT: n = 198 cells, siControl +4OHT: n = 122 cells, siITPR1 +4OHT: n = 152 cells, siVDAC3 +4OHT: n = 159 cells. Mean ± SEM are shown. Two-way ANOVA test. P-values are indicated. C . Percentage of trypan blue-positive cells counted in MRC5-MYC:ER cells transfected with siControl, siITPR1 or siVDAC3 and then treated or not with 4OHT for 3 days. Mean +/- SEM of n = 3 independent experiments. Two-Way ANOVA. P-values are indicated.

Article Snippet: Membranes were then blocked with 5% milk in TBS with 0.01% Tween-20 (Sigma-Aldrich) TBST for 1 h at room temperature and incubated with mouse primary antibodies against ITPR1 (sc-271197, Santa Cruz Biotechnology) or α-Tubulin (T6199, Sigma-Aldrich) at 4°C overnight with gentle shaking.

Techniques: Transfection, Control, Quantitative RT-PCR

Journal: bioRxiv

Article Title: MYC shapes ER-mitochondria calcium transfer by directly targeting ITPR1 : implications for MYC-induced safeguard mechanisms and cancer

doi: 10.1101/2024.08.28.610025

Figure Lengend Snippet: A. Violin plots depicting the distribution of ITPR1 expression levels in normal tissue samples compared to tumor tissue samples across various cancer types from The Cancer Genome Atlas (TCGA) dataset. Wilcox test. P-values are indicated. B-E . U2OS cancer cells were infected with a MYC:ER encoding retroviral vector and puromycin selected. B. RT-qPCR of ITPR1 gene at the indicated times after 4OHT treatment. Mean +/- SEM of n = 3 independent experiments. Two-Way ANOVA. P-values are indicated. C-E . U2OS-MYC:ER cells were transfected with a control siRNA pool (siControl) or with a siRNA pool targeting ITPR1 (siITPR1) and then treated or not with 4OHT. C . RT-qPCR of ITPR1 gene 3 days after 4OHT treatment. Mean +/- SEM of n = 4 independent experiments. Two-Way ANOVA. P-values are indicated. D . Cell density assessed by crystal violet staining 6 days after 4OHT treatment. Representative of n=3 experiments. E . Percentage of trypan blue-positive cells 6 days after 4OHT treatment. Mean +/- SEM of n = 3 independent experiments. Two-Way ANOVA. P-values are indicated.

Article Snippet: Membranes were then blocked with 5% milk in TBS with 0.01% Tween-20 (Sigma-Aldrich) TBST for 1 h at room temperature and incubated with mouse primary antibodies against ITPR1 (sc-271197, Santa Cruz Biotechnology) or α-Tubulin (T6199, Sigma-Aldrich) at 4°C overnight with gentle shaking.

Techniques: Expressing, Infection, Retroviral, Plasmid Preparation, Quantitative RT-PCR, Transfection, Control, Staining

Journal: bioRxiv

Article Title: MYC shapes ER-mitochondria calcium transfer by directly targeting ITPR1 : implications for MYC-induced safeguard mechanisms and cancer

doi: 10.1101/2024.08.28.610025

Figure Lengend Snippet: Expression of ITPR1 , BCL2 and BCL2L1 (BCL-xL) genes in 1,019 cancer cell lines extracted from the Cancer Cell Line Encyclopedia. A . Correlation between ITPR1 and BCL2 or BCL1L1 levels of expression. The cancer cell lines displaying the highest expression levels of ITPR1 (>3) and of BCL2 (>3) were selected from the left-hand graph (upper right square: 54 cancer cell lines). B. A circular diagram showing the distribution of the cancer cell lines according to their tissue of origin in the 54 cancer cell lines displaying high ITPR1 and BCL2 expression levels (left) and in the other 965 cancer cell lines (right). C. Expression level of MYCN and MYC in these cancer cell lines. The 54 cancer cell lines were split into 2 categories: MYCN high, and MYCN intermediate or MYCN low. One-Way ANOVA. D. Expression data of BCL2 and ITPR1 extracted from a human neuroblastoma dataset (Tumor Neuroblastoma – SEQC – 498 – custom – ad44kcwolf) using the R2 Genomics Analysis and Visualization Platform. Correlation of expression between BCL2 and ITPR1 mRNA levels is shown in 92 MYCN -amplified neuroblastomas.

Article Snippet: Membranes were then blocked with 5% milk in TBS with 0.01% Tween-20 (Sigma-Aldrich) TBST for 1 h at room temperature and incubated with mouse primary antibodies against ITPR1 (sc-271197, Santa Cruz Biotechnology) or α-Tubulin (T6199, Sigma-Aldrich) at 4°C overnight with gentle shaking.

Techniques: Expressing, Amplification

Journal: bioRxiv

Article Title: MYC shapes ER-mitochondria calcium transfer by directly targeting ITPR1 : implications for MYC-induced safeguard mechanisms and cancer

doi: 10.1101/2024.08.28.610025

Figure Lengend Snippet: A. Representation of NMYC ChIP-seq peaks on the ITPR1 promoter in the Kelly MYCN -amplified neuroblastoma cell line (GSE80151). The cCRE track summarizes the ENCODE Candidate Cis-Regulatory Elements (cCREs) combined from all cell types with red indicating promoter and orange and yellow indicating proximal and distal enhancer-like signatures, respectively. B. RT-qPCR of ITPR1 gene in Kelly cells treated with DMSO (negative control) or NMYC inhibitor VPC-70619. Mean +/- SEM of n = 3 independent experiments. T-test. P-values are indicated. C. ChIP-seq analysis of NMYC occupancy at the ITPR1 promoter region in neuroblastoma cells expressing or not NMYC (SHEP21 TET-OFF system). D. RNA-seq analysis of ITPR1 expression level after 24 h of NMYC depletion in SHEP21 TET-OFF system. Wilcoxon test. P-value is shown. E. Expression data of MYCN and ITPR1 extracted from a human neuroblastoma dataset (Tumor Neuroblastoma – SEQC – 498 – custom – ad44kcwolf) using the R2 Genomics Analysis and Visualization Platform. Correlation of expression between MYCN and ITPR1 is shown in 92 MYCN -amplified neuroblastomas (left panel) and in 401 MYCN non-amplified neuroblastomas (right panel).

Article Snippet: Membranes were then blocked with 5% milk in TBS with 0.01% Tween-20 (Sigma-Aldrich) TBST for 1 h at room temperature and incubated with mouse primary antibodies against ITPR1 (sc-271197, Santa Cruz Biotechnology) or α-Tubulin (T6199, Sigma-Aldrich) at 4°C overnight with gentle shaking.

Techniques: ChIP-sequencing, Amplification, Quantitative RT-PCR, Negative Control, Expressing, RNA Sequencing

Journal: bioRxiv

Article Title: MYC shapes ER-mitochondria calcium transfer by directly targeting ITPR1 : implications for MYC-induced safeguard mechanisms and cancer

doi: 10.1101/2024.08.28.610025

Figure Lengend Snippet: A. Kaplan-Meier survival curves drawn from GSE49710 dataset from patients with MYCN -amplified neuroblastoma. The 2 groups were formed according to the lowest quartile of ITPR1 expression. B-C . Cell death in Kelly and SKNAS neuroblastoma cells monitored using SYTOX Green in the first 24 h after treatment with Scrambled or Bird2 peptides. B . Representative curve over a 24 h course. Mean +/- SD of n=3 experiments. C. Analysis after 24 h treatment with the peptides. Mean +/- SEM of n = 3 independent experiments. Two-Way ANOVA. P-values are indicated. D . Quantification of mitochondrial calcium levels in Kelly cells overexpressing Mitycam, a mitochondrial calcium genetic sensor, in response to treatment with Scrambled or Bird2 peptides. The ratio (F1-F0)/F0 (F1: measurement 3 seconds after injection and F0: measurement 3 seconds before injection) was calculated. n = 3 independent experiments. Scrambled: n = 1,480 cells, Bird2: n = 913 cells. Mean ± SEM are shown. T-test. P-values are indicated. E. Schematic representation of the protocol used to assess Bird2 efficacy on neuroblastoma using a model of graft in the chick embryo. F. Size of the tumor in the embryo measured through GFP fluorescence. A representative image of neuroblastoma for each condition is shown (left) and quantification of the normalized neuroblastoma volume is displayed for each embryo (right). Scrambled peptide n=21, Bird2 peptide n=12. Mean +/- SEM. T-test. P-values are indicated.

Article Snippet: Membranes were then blocked with 5% milk in TBS with 0.01% Tween-20 (Sigma-Aldrich) TBST for 1 h at room temperature and incubated with mouse primary antibodies against ITPR1 (sc-271197, Santa Cruz Biotechnology) or α-Tubulin (T6199, Sigma-Aldrich) at 4°C overnight with gentle shaking.

Techniques: Amplification, Expressing, Injection, Fluorescence

Journal: Redox Biology

Article Title: LncRNA H19 inhibition impairs endoplasmic reticulum-mitochondria contact in hepatic cells and augments gluconeogenesis by increasing VDAC1 levels

doi: 10.1016/j.redox.2023.102989

Figure Lengend Snippet: Inhibition of H19 induces mitochondrial dysfunction in Hepa 1 – 6 cells. (A) Hepa 1–6 cells transfected with either the scramble or H19 siRNA (5 nM, 48h) were subjected to in-situ proximity ligation assay using anti-VDAC1 and anti-IP3R antibodies. Representative confocal images of the cells are shown and the interaction between IP3R and VDAC1 is indicated by red dots. Nuclei are indicated by blue staining using DAPI. Quantification of IP3R-VDAC1 interactions is represented by red blobs per nucleus. Scale: 20 μm; Magnification: 60X. At least five images were captured per incubation. (B) Hepa 1–6 cells transfected with either the scramble or H19 siRNA (5 nM, 48h) were evaluated for total mitochondrial (B) and cytosolic (C) Ca 2+ levels using Rhod-2 AM (1 μM) and Fluo-3 AM (1 μM), respectively, by FACS analysis. Mitochondrial ROS levels as detected by Mitosox (5 μM) (D) and total ATP (normalized to total protein) content (E) in Hepa 1–6 cells transfected with either the scramble or H19 siRNA (5 nM, 48h) are presented. (F) OCR of Hepa 1–6 cells transfected with either the scramble or H19 siRNA (5 nM) for 48 h in the presence of 2 μM oligomycin (ATP synthase inhibitor), 1.25 μM FCCP (uncoupling agent) and 2 μM rotenone and 2 μM antimycin (complex I and III inhibitors, respectively). Basal respiration, maximal respiration, proton leak, spare respiratory capacity and ATP production were determined as described in the “Methods” section. (G) Mitochondria was isolated from scramble and H19 siRNA transfected cells as described in the “Methods” section. OCR in isolated mitochondria (5 μg) from scramble and H19 siRNA transfected cells was performed in the presence of ADP (4 mM), oligomycin (2.5 μg/ml), FCCP (4 μM) and antimycin A (4 μM). (H) Total RNA was isolated from Hepa 1–6 cells transfected with either the scramble or H19 siRNA as in “A”, reverse transcribed and the transcript levels of genes specific to Complexes I, III, IV and V of the mitochondrial electron transport chain were evaluated by RT-PCR. 18S rRNA was taken as the normalization control. Scramble and H19 siRNA (5 nM, 48h) transfected cells were evaluated for mitochondrial membrane potential and integrity using JCI (I) and for mitochondrial number using mitochondrial short d-loop specific primers by qRT-PCR. β-2 microglobulin was used as normalization control (J). (K) Glycolytic rates in scramble and H19 siRNA transfected cells were assessed in the presence of glucose (10 mM), oligomycin (2 μM) and 2-Deoxy- d -glucose (100 mM). Glycolysis, glycolytic capacity and glycolytic reserve as determined are presented. All experiments were done thrice and data are presented are means ± SEM.**p < 0.01, *p < 0.05, ns: non-significant. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: On termination of incubation, cells were fixed, permeablized, blocked and incubated overnight with primary antibodies against VDAC1 (1:200, Cat no. 14734, Abcam, Cambridge, UK) and IP3R (1:200, Cat no. 05–1210, Sigma, St. Louis, USA) at 4 °C followed by incubation with secondary antibodies linked to complementary oligonucleotides (anti-rabbit PLUS and anti-mouse MINUS), ligation and amplification.

Techniques: Inhibition, Transfection, In Situ, Proximity Ligation Assay, Staining, Incubation, Isolation, Reverse Transcription Polymerase Chain Reaction, Membrane, Quantitative RT-PCR

Journal: Redox Biology

Article Title: LncRNA H19 inhibition impairs endoplasmic reticulum-mitochondria contact in hepatic cells and augments gluconeogenesis by increasing VDAC1 levels

doi: 10.1016/j.redox.2023.102989

Figure Lengend Snippet: VDAC1 inhibition rescues H19 siRNA mediated effects in Hepa1-6 cells. (A) Hepa 1–6 cells were transfected with either the scramble or VDAC1 siRNA (5–20 nM) for 48h, lysed and probed for the levels of VDAC1 by Western Blot analysis. Hepa 1–6 cells were co-transfected with H19 siRNA (5 nM) and VDAC1 siRNA (5 nM) and after 48h, the levels of PCK1 (B), G6Pase (C), FBP1 (D), p -JNK1/2/JNK1/2 (E) and p-IRS1(Ser307)/IRS1 (F) were evaluated by Western Blot analyses using specific antibodies. Vinculin or HSC70 was used as the loading control. Representative blots are shown and densitometric analyses of three blots are given in the panels below. (G) Hepa 1–6 cells transfected as in “A” were subjected to in-situ proximity ligation assay using anti-VDAC1 and anti-IP3R antibodies. Representative confocal images of the cells are shown and the interaction between IP3R and VDAC1 is indicated by red dots. Nuclei are indicated by blue staining using DAPI. Quantification of IP3R-VDAC1 interactions is depicted by red blobs per nucleus. Scale: 20 μm; Magnification: 60X. At least five images were captured per incubation. (H) OCR in the presence of 2 μM oligomycin (ATP synthase inhibitor), 1.25 μM FCCP (uncoupling agent) and 2 μM rotenone and 2 μM antimycin (complex I and III inhibitors, respectively) of Hepa 1–6 cells transfected as in “A”. Basal respiration, maximal respiration, proton leak, spare respiratory capacity and ATP production were determined as described in the “Materials and Methods” section. Experiments were done three times and data presented as means ± SEM. ***p < 0.001, **p < 0.01, *p < 0.05. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: On termination of incubation, cells were fixed, permeablized, blocked and incubated overnight with primary antibodies against VDAC1 (1:200, Cat no. 14734, Abcam, Cambridge, UK) and IP3R (1:200, Cat no. 05–1210, Sigma, St. Louis, USA) at 4 °C followed by incubation with secondary antibodies linked to complementary oligonucleotides (anti-rabbit PLUS and anti-mouse MINUS), ligation and amplification.

Techniques: Inhibition, Transfection, Western Blot, In Situ, Proximity Ligation Assay, Staining, Incubation

Journal: Redox Biology

Article Title: LncRNA H19 inhibition impairs endoplasmic reticulum-mitochondria contact in hepatic cells and augments gluconeogenesis by increasing VDAC1 levels

doi: 10.1016/j.redox.2023.102989

Figure Lengend Snippet: Metformin rescues H19 siRNA mediated effects in Hepa1-6 cells. (A) Hepa 1–6 cells were incubated either without (Control) or with metformin (20–1000 μM) for 42h; after competition of incubation, total RNA was isolated and subjected to qRT-PCR using VDAC1 (A) or H19 (B) specific primers. 18S rRNA was used as the normalization control. Hepa 1–6 cells were transfected with either scramble or H19 siRNA (5 nM) in the presence or absence of metformin (1000 μM) for 42h; after competition of incubation, total RNA was isolated and subjected to qRT-PCR to determine the transcript levels of Fbp1 (C), G6Pase (D), Pcx (E) and Pck1(F). 18S rRNA was used as the normalization control. (G) Hepa 1–6 cells transfected as in “C” were subjected to in-situ proximity ligation assay using anti-VDAC1 and anti-IP3R antibodies. Representative confocal images of the cells are shown and the interaction between IP3R and VDAC1 is indicated by red dots. Nuclei are indicated by blue staining using DAPI. Quantification of IP3R-VDAC1 interactions is represented by red blobs per nucleus. Scale: 20 μm; Magnification: 60X. At least five images were captured per incubation. Each data point is the mean of three independent experiments and is presented as means ± SEM. ***p < 0.001, **p < 0.01, *p < 0.05. ns: non-significant (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: On termination of incubation, cells were fixed, permeablized, blocked and incubated overnight with primary antibodies against VDAC1 (1:200, Cat no. 14734, Abcam, Cambridge, UK) and IP3R (1:200, Cat no. 05–1210, Sigma, St. Louis, USA) at 4 °C followed by incubation with secondary antibodies linked to complementary oligonucleotides (anti-rabbit PLUS and anti-mouse MINUS), ligation and amplification.

Techniques: Incubation, Isolation, Quantitative RT-PCR, Transfection, In Situ, Proximity Ligation Assay, Staining