ip3r1  (Santa Cruz Biotechnology)

Journal: Science Advances

Article Title: Carbonic anhydrase VIII regulates IP3R1-dependent Ca 2+ signaling to coordinate insulin secretion and hepatic glucose production

doi: 10.1126/sciadv.aeh4948

Figure Lengend Snippet: CA8 negatively regulates GIIS and HGP by inhibiting IP3R1-mediated Ca 2+ release from ER. This characteristic implies that CA8 is involved in orchestration of glucose homeostasis. Altered CA8 expression in islets and hepatocytes of ob/ob mice is accompanied with lower GIIS and elevated HGP, respectively. Furthermore, chronic exposure of glucagon and exendin-4 elevated CA8 expression, consequently limited HGP in vivo. Created in BioRender. Murakami, T. (2026) https://BioRender.com/qpiyzwk .

Article Snippet: s63273, s63274, s63275, Ambion, TX, USA) or IP3R1 (25 μM, catalog no. sc-42476, Santa Cruz Biotechnology, TX, USA) was used.

Techniques: Expressing, In Vivo

Journal: Cellular & molecular biology letters

Article Title: IP 3 R1-mediated MAMs formation contributes to mechanical trauma-induced hepatic injury and the protective effect of melatonin.

doi: 10.1186/s11658-023-00509-x

Figure Lengend Snippet: Fig. 3 Melatonin inhibited IP3R1 elevation and suppressed MAMs formation in the liver following MT. A, B Representative blots and quantitative analysis of representative functional proteins (VDAC1, IP3R1, GRP75), structural proteins (Mfn1, Mfn2) and apoptotic related protein (Bap31, Fis1) of MAMs (n = 4). C Representative TEM images at × 15,000 magnification. Scale bars: 1 μm. D Quantification of the minimum distance between ER and mitochondria. E Quantification of ER length close to mitochondria normalized by total ER length. All of the values are shown as the means ± SEM. n = 8 in each group. *P < 0.05, **P < 0.01 vs Sham; #P < 0.05, ##P < 0.01 vs MT + V

Article Snippet: ERK1/2 siRNA (sc-29308, sc-35336, Santa Cruz, USA), IP3R1 siRNA (sc42476, Santa Cruz, USA) or negative control siRNA (sc-37007, Santa Cruz, USA) were transfected to hepatocytes using OPTI-MEM (Invitrogen, USA) and Lipofectamine RNAi MAX reagent (Invitrogen, USA) respectively.

Techniques: Functional Assay

Journal: Cellular & molecular biology letters

Article Title: IP 3 R1-mediated MAMs formation contributes to mechanical trauma-induced hepatic injury and the protective effect of melatonin.

doi: 10.1186/s11658-023-00509-x

Figure Lengend Snippet: Fig. 4 Melatonin inhibited cell death and MAMs formation in the hepatocytes cultured in TS. A Cell viability (percentage of SS). B, C Representative blots and quantitative analysis of IP3R1, cleaved-caspase 3 (n = 4). D Relative activity of caspase 3. E Representative confocal images of primary hepatocytes double-stained by mitotracker (red) and ER-localized virus (green) at × 600 magnification. F, G The representative figures of scatter plot were analyzed by colocalization finder in Image J software. Statistical quantification of the colocalization area between mitochondria and ER. H Representative images and quantitative analysis of MitoSOX-stained mitochondria-derived superoxide production. All of the values are shown as the means ± SEM. n = 6 in each group. **P < 0.01 vs SS; ##P < 0.01 vs TS

Article Snippet: ERK1/2 siRNA (sc-29308, sc-35336, Santa Cruz, USA), IP3R1 siRNA (sc42476, Santa Cruz, USA) or negative control siRNA (sc-37007, Santa Cruz, USA) were transfected to hepatocytes using OPTI-MEM (Invitrogen, USA) and Lipofectamine RNAi MAX reagent (Invitrogen, USA) respectively.

Techniques: Cell Culture, Activity Assay, Staining, Virus, Software, Derivative Assay

Journal: Cellular & molecular biology letters

Article Title: IP 3 R1-mediated MAMs formation contributes to mechanical trauma-induced hepatic injury and the protective effect of melatonin.

doi: 10.1186/s11658-023-00509-x

Figure Lengend Snippet: Fig. 6 Melatonin inhibited MAMs formation and mitochondrial calcium overload in TS-treated hepatocytes by suppressing IP3R1 expression. A, B Representative blots and quantitative analysis of IP3R1, cleaved-caspase 3 (n = 4). C Relative activity of caspase 3. D The levels of ALT release (fold difference compared with TS + Ad-EV). E Representative confocal images of primary hepatocytes double-stained by mitotracker (red) and ER-localized virus (green) at × 600 magnification. F, G Statistical quantification of the colocalization area between mitochondria and ER. H Representative images and quantitative analysis of MitoSOX-stained mitochondria-derived superoxide production. I Quantification of fluorescence intensity normalized by TS + Ad-EV. J Representative traces of ionomycin-induced changes of mitochondrial Ca2+ concentration (n = 3). K Quantification of the relative increment of fluorescence intensity in mitochondrial Ca2+ concentration after ionomycin stimulation normalized by TS + Ad-EV. L Cell viability (percentage of TS + Ad-EV). M, N Oxygen consumption rate (OCR) measured by Seahorse and quantitative statistical analysis of OCR (n = 3). All of the values are shown as the means ± SEM. n = 6 in each group. *P < 0.05, **P < 0.01 vs TS + Ad-EV; ##P < 0.01 vs TS + Mel + Ad-EV

Article Snippet: ERK1/2 siRNA (sc-29308, sc-35336, Santa Cruz, USA), IP3R1 siRNA (sc42476, Santa Cruz, USA) or negative control siRNA (sc-37007, Santa Cruz, USA) were transfected to hepatocytes using OPTI-MEM (Invitrogen, USA) and Lipofectamine RNAi MAX reagent (Invitrogen, USA) respectively.

Techniques: Expressing, Activity Assay, Staining, Virus, Derivative Assay, Fluorescence, Concentration Assay

Journal: Cellular & molecular biology letters

Article Title: IP 3 R1-mediated MAMs formation contributes to mechanical trauma-induced hepatic injury and the protective effect of melatonin.

doi: 10.1186/s11658-023-00509-x

Figure Lengend Snippet: Fig. 7 Melatonin inhibited IP3R1-mediated MAMs via ERK1/2 signaling pathway. A, B Several specific pharmacological inhibitors, including Bisindolylmaleimide XI hydrochloride (Bis XI, a PKC inhibitor), Dorsomorphin (DSM, an AMPK inhibitor), PD98059 (a MEK/ERK inhibitor), Luzindole (a melatonin receptor inhibitor), Ruxolitinib (Ruxo, a JAK inhibitor), EX-527 (a SIRT1 inhibitor) and Wortmannin (a PI3K/Akt inhibitor) were pre-administered to the hepatocytes in the TS + Mel group and then the expression of IP3R1 was quantified (n = 4). C, E Representative blots and quantitative analysis of phosphorylated ERK1/2 (p-ERK1/2) and total ERK1/2 (n = 4). F Cell viability (percentage of SS). G The levels of ALT release (fold difference compared with SS). H Quantitative analysis of IP3R1 mRNA expression determined by real-time PCR. I–L ERK1/2 was knocked down by siRNA, after which the cells were subjected to TS with or without melatonin. Representative blots and quantitative analysis of phosphorylated ERK1/2 (p-ERK1/2), total ERK1/2 and IP3R1 (n = 4). M Quantitative analysis of IP3R1 mRNA expression determined by real-time PCR. N Cell viability (percentage of TS + Scramble RNAi). O The levels of ALT release (fold difference compared with TS + Scramble RNAi). P Representative confocal images of primary hepatocytes double-stained by mitotracker (red) and ER-tracker (green) at × 600 magnification. Q, R Statistical quantification of the colocalization area between mitochondria and ER. S Representative images and quantitative analysis of MitoSOX-stained mitochondria-derived superoxide production. All of the values are shown as the means ± SEM. n = 6 in each group. *P < 0.05, **P < 0.01 vs SS or TS + Scramble RNAi; ##P < 0.01 vs TS or TS + Mel + Scramble RNAi; &P < 0.05 &&P < 0.01 vs TS + Mel

Article Snippet: ERK1/2 siRNA (sc-29308, sc-35336, Santa Cruz, USA), IP3R1 siRNA (sc42476, Santa Cruz, USA) or negative control siRNA (sc-37007, Santa Cruz, USA) were transfected to hepatocytes using OPTI-MEM (Invitrogen, USA) and Lipofectamine RNAi MAX reagent (Invitrogen, USA) respectively.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Staining, Derivative Assay

Journal: Cellular & molecular biology letters

Article Title: IP 3 R1-mediated MAMs formation contributes to mechanical trauma-induced hepatic injury and the protective effect of melatonin.

doi: 10.1186/s11658-023-00509-x

Figure Lengend Snippet: Fig. 8 Melatonin-ERK1/2 suppressed IP3R1-mediated MAMs via inhibiting FoxO1-mediated transcription. A Quantitative analysis of IP3R1 mRNA expression determined by real-time PCR (AS1842856, a JUNB inhibitor) (n = 6). B–D FoxO1 was overexpressed by adenovirus, after which the cells were subjected to TS or TS + Mel. Representative blots and quantitative analysis of FoxO1 and IP3R1 (n = 4). E Quantitative analysis of IP3R1 mRNA expression determined by real-time PCR. F, G Representative blots and quantitative analysis of nuclear FoxO1 (n = 4). H Cell viability (percentage of SS + Ad-EV). I The levels of ALT release (fold difference compared with SS + Ad-EV). J Representative confocal images of primary hepatocytes double-stained by mitotracker (red) and ER-localized virus (green) at × 600 magnification. K, L Statistical quantification of the colocalization area between mitochondria and ER. M Representative images and quantitative analysis of MitoSOX-stained mitochondria-derived superoxide production. N Chromatin immunoprecipitation (ChIP) and real-time PCR analysis for the binding of FoxO1 to IP3R1 promoter (n = 3). All of the values are shown as the means ± SEM. n = 6 in each group. **P < 0.01 vs SS or SS + Ad-EV; #P < 0.05, ##P < 0.01 vs TS + Ad-EV; &&P < 0.01 vs TS + Mel + Ad-EV

Article Snippet: ERK1/2 siRNA (sc-29308, sc-35336, Santa Cruz, USA), IP3R1 siRNA (sc42476, Santa Cruz, USA) or negative control siRNA (sc-37007, Santa Cruz, USA) were transfected to hepatocytes using OPTI-MEM (Invitrogen, USA) and Lipofectamine RNAi MAX reagent (Invitrogen, USA) respectively.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Staining, Virus, Derivative Assay, Chromatin Immunoprecipitation, Binding Assay

Journal: Cellular & molecular biology letters

Article Title: IP 3 R1-mediated MAMs formation contributes to mechanical trauma-induced hepatic injury and the protective effect of melatonin.

doi: 10.1186/s11658-023-00509-x

Figure Lengend Snippet: Fig. 9 Schematic figure showing that melatonin attenuates mechanical trauma-induced hepatic injury by inhibiting IP3R1-mediated MAMs formation via activating the melatonin receptor-ERK1/2-FoxO1 pathway. Melatonin binds to melatonin receptor and induces the phosphorylation of ERK1/2 that subsequently interacts with FoxO1 and induces the phosphorylation and inactivation of the FoxO1. Inactivated FoxO1 reduced FoxO1 binding to the IP3R1 promoter to inhibit IP3R1-mediated MAMs. Subsequently, melatonin-induced downregulation of IP3R1 inhibits MAMs formation and Ca2+ overload, and mitigates mitochondrial dysfunction and mechanical trauma-induced hepatic injury

Article Snippet: ERK1/2 siRNA (sc-29308, sc-35336, Santa Cruz, USA), IP3R1 siRNA (sc42476, Santa Cruz, USA) or negative control siRNA (sc-37007, Santa Cruz, USA) were transfected to hepatocytes using OPTI-MEM (Invitrogen, USA) and Lipofectamine RNAi MAX reagent (Invitrogen, USA) respectively.

Techniques: Phospho-proteomics, Binding Assay

Journal: Frontiers in Immunology

Article Title: IP3R-dependent mitochondrial dysfunction mediates C5b-9-induced ferroptosis in trichloroethylene-caused immune kidney injury

doi: 10.3389/fimmu.2023.1106693

Figure Lengend Snippet: Deposition of C5b-9 activated IP3R. (A) IP3R protein in the mouse renal cortex was detected using immunoblotting. (B) Quantitative analysis of IP3R protein levels. (C) Ip3r mRNA levels in the mouse renal cortex were measured using RT-PCR. (D) IP3R protein expression in HK-2 cells was detected using immunoblotting. (E) Quantitative analysis of IP3R protein levels. (F) Ip3r mRNA levels in HK-2 cells were measured using RT-PCR. Quantitative data are shown as the mean ± SEM. (n = 4-6 per group). a P < 0.05 compared with the Vehicle group, b P < 0.05 compared with the TCE+ group, * P < 0.05.

Article Snippet: To explore the underlying mechanism whereby C5b-9 induces ferroptosis, HK-2 cells were treated with IP3R siRNA, 2 μM BAPTA-AM (Selleck, Cat. S7534) or 10 nM cyclosporin A (CsA, Selleck, Cat. S2286).

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

Journal: Frontiers in Immunology

Article Title: IP3R-dependent mitochondrial dysfunction mediates C5b-9-induced ferroptosis in trichloroethylene-caused immune kidney injury

doi: 10.3389/fimmu.2023.1106693

Figure Lengend Snippet: IP3R activation induced cytosolic Ca 2+ overload and mitochondrial dysfunction. The HK-2 cells were cultured with C5b6 and NHS to assemble C5b-9 on the cytomembrane after IP3R siR was transfected. (A) Relative cytosolic Ca 2+ concentration of TCE-sensitized mouse primary tubular epithelial cells was detected by monitoring the fluorescence intensity at Ex/Em = 490/525 nm using a multimode plate reader (PerkinElmer). (B) The relative cytosolic Ca 2+ concentration of HK-2 cells was detected. (C) MCU protein in the mouse renal cortex was detected using immunoblotting. (D) Quantitative analysis of MCU protein levels. (E) Mcu mRNA levels in the mouse renal cortex were measured using RT-PCR. (F) The mitochondrial membrane potential of TCE-sensitized mouse primary tubular epithelial cells was detected using JC-1 dye. The scale bar represents 50 μm. (G) The essential components of OXPHOS in the mouse renal cortex were detected using immunoblotting. (H–L) Quantitative analysis of the essential components of OXPHOS. (M) IP3R and MCU proteins of HK-2 cells were detected using immunoblotting. (N, O) Quantitative analysis of IP3R and MCU protein levels. (P, Q) Ip3r and Mcu mRNA levels in HK-2 cells were measured using RT-PCR. (R) The mitochondrial membrane potential of HK-2 cells was detected using JC-1 dye. The scale bar represents 50 μm. (S) Flow cytometry results showing the mitochondrial membrane potential of HK-2 cells. (T) The essential components of OXPHOS in HK-2 cells were detected using immunoblotting. (U–Y) Quantitative analysis of the essential components of OXPHOS. Quantitative data are shown as the mean ± SEM. (n = 4-6 per group). a P < 0.05 compared with the Vehicle group, b P < 0.05 compared with the TCE+ group, * P < 0.05.

Article Snippet: To explore the underlying mechanism whereby C5b-9 induces ferroptosis, HK-2 cells were treated with IP3R siRNA, 2 μM BAPTA-AM (Selleck, Cat. S7534) or 10 nM cyclosporin A (CsA, Selleck, Cat. S2286).

Techniques: Activation Assay, Cell Culture, Transfection, Concentration Assay, Fluorescence, Western Blot, Reverse Transcription Polymerase Chain Reaction, Membrane, Flow Cytometry

Journal: Frontiers in Immunology

Article Title: IP3R-dependent mitochondrial dysfunction mediates C5b-9-induced ferroptosis in trichloroethylene-caused immune kidney injury

doi: 10.3389/fimmu.2023.1106693

Figure Lengend Snippet: IP3R siR protected HK-2 cells from ferroptosis induced by C5b-9. The HK-2 cells were cultured with C5b6 and NHS to assemble C5b-9 on the cytomembrane after IP3R siR was transfected. (A) Cell viability of HK-2 cells after 24 hr of C5b-9 assembly. (B) ACSL4, COX2 and GPX4 proteins in HK-2 cells were detected using immunoblotting after treatment with IP3R siR. (C–E) Quantitative analysis of ACSL4, COX2 and GPX4 proteins. (F–H) Acsl4 , Ptgs2 and Gpx4 mRNA levels in HK-2 cells were measured using RT-PCR after treatment with IP3R siR. (I) GSH levels in HK-2 cells after treatment with siR. (J) MDA level of HK-2 cells after treatment with IP3R siR. (K) Lipid ROS were detected by BODIPY™ 581/591 C11 after treatment with IP3R siR. The green fluorescence represents oxidized lipids, and the red fluorescence represents non-oxidized lipids. The scale bar represents 50 μm. (L) Fe 2+ was detected by FerroOrange dye. The scale bar represents 50 μm. Quantitative data are shown as the mean ± SEM. (n = 4-6 per group). * P < 0.05.

Article Snippet: To explore the underlying mechanism whereby C5b-9 induces ferroptosis, HK-2 cells were treated with IP3R siRNA, 2 μM BAPTA-AM (Selleck, Cat. S7534) or 10 nM cyclosporin A (CsA, Selleck, Cat. S2286).

Techniques: Cell Culture, Transfection, Western Blot, Reverse Transcription Polymerase Chain Reaction, Fluorescence

Journal: Frontiers in Immunology

Article Title: IP3R-dependent mitochondrial dysfunction mediates C5b-9-induced ferroptosis in trichloroethylene-caused immune kidney injury

doi: 10.3389/fimmu.2023.1106693

Figure Lengend Snippet: mPTP inhibition alleviated IP3R-induced mitochondrial dysfunction in HK-2 cells. (A) The mPTP of HK-2 cells was detected by mPTP staining dye. (B) The mitochondrial membrane potential of HK-2 cells was detected using JC-1 dye. The scale bar represents 50 μm. (C) Flow cytometry results showing the mitochondrial membrane potential of HK-2 cells. (D) The essential components of OXPHOS in HK-2 cells were detected using immunoblotting. (E–I) Quantitative analysis of the essential components of OXPHOS. Quantitative data are shown as the mean ± SEM. (n = 4-6 per group). * P < 0.05.

Article Snippet: To explore the underlying mechanism whereby C5b-9 induces ferroptosis, HK-2 cells were treated with IP3R siRNA, 2 μM BAPTA-AM (Selleck, Cat. S7534) or 10 nM cyclosporin A (CsA, Selleck, Cat. S2286).

Techniques: Inhibition, Staining, Membrane, Flow Cytometry, Western Blot