Journal: eLife

Article Title: Circadian regulation of endoplasmic reticulum calcium response in cultured mouse astrocytes

doi: 10.7554/eLife.96357

Figure Lengend Snippet:

Article Snippet: Antibody , Rabbit polyclonal anti-ITPR2 , Alomone Labs , Cat# ACC-116; Lot: ACC116AN015; RRID: AB_2340910 , 1:1000.

Techniques: Transfection, Construct, Plasmid Preparation, Sequencing, Negative Control, Software, Microscopy

Journal: Cell Death Discovery

Article Title: IP3R2-mediated Ca 2+ release promotes LPS-induced cardiomyocyte pyroptosis via the activation of NLRP3/Caspase-1/GSDMD pathway

doi: 10.1038/s41420-024-01840-8

Figure Lengend Snippet: A , B Representative traces of Fluo4/AM changes over time by adding of 1 μM ATP or 10 μM ATP, respectively (left). Scatter plot showing the ATP-evoked calcium peak amplitude after adding 1 μM ATP or 10 μM ATP (right) ( n = 20 cells). C IP3R1, IP3R2 and IP3R3 mRNA levels in NRCMs detected via RT-qPCR analysis ( n = 4). D Western blotting images showing the expression of IP3R2 protein in NRCMs increased with an increase in LPS concentration. E Western blotting images and summarized data showing the IP3R2 protein expression level significantly increased in the myocardium of LPS group rats ( n = 6). Data are shown as mean ± SEM. ** P < 0.01. ns: no significant statistical difference.

Article Snippet: Briefly, siRNA targeting IP3R2 (si-IP3R2) and the negative scrambled control (si-NC) were obtained from RuiBo Biology Co., Ltd. (Guangzhou, China).

Techniques: Quantitative RT-PCR, Western Blot, Expressing, Concentration Assay

Journal: Cell Death Discovery

Article Title: IP3R2-mediated Ca 2+ release promotes LPS-induced cardiomyocyte pyroptosis via the activation of NLRP3/Caspase-1/GSDMD pathway

doi: 10.1038/s41420-024-01840-8

Figure Lengend Snippet: A NRCMs were pre-treated with IP3R inhibitor XeC (0.1 μM) and monitored for changes in intracellular Ca 2+ levels stimulated by ATP (10 μM). B Detection of IP3R2 mRNA levels in NRCMs with si-IP3R2 intervention using RT-qPCR analysis ( n = 4). C Western blotting images and the summarized data showing the IP3R2 protein expression level significantly decreased in NRCMs with si-IP3R2 intervention ( n = 4). D Representative traces of Fluo4/AM changes over time by adding of 10 μM ATP (left). Scatter plot showing the ATP-evoked calcium peak amplitude after adding ATP (right) ( n = 20 cells). E , F Flow cytometry analysis was conducted to examine the effects of XeC or si-IP3R2 on apoptosis in NRCMs induced by LPS ( n = 4). G , H IL-18 and IL-1β mRNA levels were detected in NRCMs with XeC or si-IP3R2 intervention using RT-qPCR analysis ( n = 4). I Western blotting images and summarized data showing the protein expression level of NLRP3, GSDMD-NT, Caspase-1 p10, IL-18 and IL-1β ( n = 4) significantly decreased in NRCMs with si-IP3R2 intervention. Data are shown as mean ± SEM. * P < 0.05, ** P < 0.01.

Article Snippet: Briefly, siRNA targeting IP3R2 (si-IP3R2) and the negative scrambled control (si-NC) were obtained from RuiBo Biology Co., Ltd. (Guangzhou, China).

Techniques: Quantitative RT-PCR, Western Blot, Expressing, Flow Cytometry

Journal: Cell Death Discovery

Article Title: IP3R2-mediated Ca 2+ release promotes LPS-induced cardiomyocyte pyroptosis via the activation of NLRP3/Caspase-1/GSDMD pathway

doi: 10.1038/s41420-024-01840-8

Figure Lengend Snippet: A Western blotting images and the summarized data showing IP3R2 protein expression level significantly decreased in NRCMs following 4-PBA intervention ( n = 4). B Representative traces of Fluo-4/AM changes over time after adding 10 μM ATP (left). Scatter plot showing the ATP-evoked calcium peak amplitude after adding ATP (right) ( n = 20 cells). C , D Detection of ATF4 and CHOP mRNA levels in NRCMs with XeC or si-IP3R2 intervention via RT-qPCR analysis ( n = 4). E Western blotting images and the summarized data showing the protein expression level of p-eIF2α, CHOP and ATF4 ( n = 4) significantly decreased in NRCMs following si-IP3R2 intervention. Data are shown as mean ± SEM. * P < 0.05, ** P < 0.01. ns: no significant statistical difference.

Article Snippet: Briefly, siRNA targeting IP3R2 (si-IP3R2) and the negative scrambled control (si-NC) were obtained from RuiBo Biology Co., Ltd. (Guangzhou, China).

Techniques: Western Blot, Expressing, Quantitative RT-PCR

Journal: Cell Death Discovery

Article Title: IP3R2-mediated Ca 2+ release promotes LPS-induced cardiomyocyte pyroptosis via the activation of NLRP3/Caspase-1/GSDMD pathway

doi: 10.1038/s41420-024-01840-8

Figure Lengend Snippet: LPS stimulation induces IP3R2 upregulation in NRCMs. Then, IP3R2-mediated Ca2+ release participates in LPS induced-cardiomyocytes pyroptosis. NLRP3 inflammasome is activated by IP3R-mediated Ca2+ release, characterized by upregulated NLRP3 expression. The activation of NLRP3 inflammasome governs the cleavage and activation of pro-Caspase-1. Activated Caspase-1 cleaves the full-length gasdermin D (GSDMD-FL) protein to release the N-terminal of GSDMD (GSDMD-NT), which results in GSDMD pore formation. Meanwhile, mature IL-18 and IL-1β are cleaved by the activated NLRP3/Caspase-1 pathway, and the cleaved IL-18 and IL-1β are released through the GSDMD pore in the cell membrane and induces pyroptosis. It is worth mentioning that the mutual regulation of IP3R2 and ER stress further exacerbates the pyroptosis process. Thus, inhibiting IP3R2 could be a promising strategy for the prevention of SIC.

Article Snippet: Briefly, siRNA targeting IP3R2 (si-IP3R2) and the negative scrambled control (si-NC) were obtained from RuiBo Biology Co., Ltd. (Guangzhou, China).

Techniques: Expressing, Activation Assay, Membrane

Journal: Cell Death Discovery

Article Title: IP3R2-mediated Ca 2+ release promotes LPS-induced cardiomyocyte pyroptosis via the activation of NLRP3/Caspase-1/GSDMD pathway

doi: 10.1038/s41420-024-01840-8

Figure Lengend Snippet: Information about the primers we have used in this study.

Article Snippet: Briefly, siRNA targeting IP3R2 (si-IP3R2) and the negative scrambled control (si-NC) were obtained from RuiBo Biology Co., Ltd. (Guangzhou, China).

Techniques: Sequencing

Journal: bioRxiv

Article Title: Neuron-astrocyte Coupling in Lateral Habenula Mediates Depressive-like Behaviors

doi: 10.1101/2024.11.03.621722

Figure Lengend Snippet: Neuron-to-Astrocyte Crosstalk in LHb during FS (A) Schematic illustrating optogenetic activation of ChrimsonR-expressing neurons and fiber photometry recording of nearby GCaMP6f-expressing astrocytes in LHb in vivo. (D) Schematic illustrating optogenetic activation of ChrimsonR-expressing neurons and two-photon imaging of nearby GCaMP6f-expressing astrocytes in LHb slices in vitro. (B, E) Astrocytic calcium responses to LHb neuronal ChrimsonR activation (1s, 40 Hz) in vivo (B) and in LHb slices (E). (C, F) Mean astrocytic calcium responses to varying stimulation frequencies in vivo (C) and in LHb slices (F). (G) Schematic of in vivo fiber photometry recording of LHb astrocytes in response to FS after i.p. injection of various receptor blockers. (H) Bar graph showing effects of MPEP (mGluR5 antagonist), LY341495 (mGluR2/3 antagonist), Scopolamine (mAChR antagonist), Mecamylamine (nAChR antagonist), Prazosin (α1-AR antagonist), Propranolol (β-AR antagonist) and Atipamezole (α2-AR antagonist) on FS-evoked calcium signals in LHb astrocytes. Each circle represents one mouse. (I) Bar graph showing effects of a1-AR subtype-selective antagonists, including Silodosin (α 1A -AR antagonist), L-765314 (α 1B -AR antagonist), BMY 7378 (α 1D -AR antagonist) on FS-evoked calcium signals in LHb astrocytes. Each circle represents one mouse. (J-L) In vivo fiber photometry recording of NE sensor signals in LHb during FS. Viral expression (J, stained with antibodies against GFP and Hoechst) after injection of AAV2/9-hSyn-NE2h and optic fiber placement (indicated by the yellow dotted line) in LHb, the boundary of which is outlined by the white dotted line. Scale bar, 100 μm. Example trace (K) of LHb NE sensor signals during FS (black) and homecage (HC, grey). Scale bars, 5% delta F/F, 50s. Plots (L, left) of delta F/F ratio of NE sensor signals aligned to the onset of FS. Solid lines indicate mean and shaded areas indicate SEM. Heatmap (L, right) of delta F/F ratio of NE sensor signals in the LHb aligned to the onset of FS (n = 46 trials from 5 mice). Color bars on the left indicate different mice. (M) Plots of delta F/F ratio of calcium signals from LHb neurons, LHb astrocytes, and LHb-NE-sensor signals aligned to onset of FS stress. Solid lines indicate mean and shaded areas indicate SEM. Data from 2E-H and 3L. (N) Average time to peak of LHb neuronal calcium signals, LHb-NE-sensor signals and LHb astrocytic calcium signals from FS onset (left) and average time to inflection of LHb neuronal calcium signals and LHb-NE-sensor signals (right). Data from 2E-H and 3L. (O) Schematic illustrating optogenetic activation of LHb-LC terminals and fiber photometry recording of LC NE neurons in TH-Cre mice. (P) Calcium response of LC NE neurons to optogenetic activation of LHb-LC terminals (1s, 40 Hz). Plots of delta F/F ratio of calcium signal aligned to laser onset. Red line represents light-on. Solid lines indicate mean and shaded areas indicate SEM. (Q) Schematic illustrating optogenetic activation of LC TH -LHb terminals and fiber photometry recording of LHb astrocytes in TH-Cre mice. (R) Calcium response of LHb astrocytes to optogenetic activation of LC TH -LHb terminals (1s, 40Hz). Plots of averaged delta F/F ratio of astrocytic calcium signal induced by optogenetic activation of LC TH -LHb terminals after i.p. injection of saline (black) or Prazosin (green, α1-AR antagonist) aligned to laser onset. Red line represents light-on. Solid lines indicate mean and shaded areas indicate SEM. (S) Bar graph showing effects of Prazosin (α1-AR antagonist) on astrocytic calcium signals induced by optogenetic activation of LC TH -LHb terminals. Each circle represents one mouse. (T) Working model summarizing the neuron-to-astrocyte crosstalk in the LHb during FS. Stress activates LHb neurons, which recruit LC NE neurons to elevate LHb astrocytic calcium via NE-α 1A signaling. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; n.s., not significant. Data are represented as mean ± SEM.

Article Snippet: For manipulation experiments of astrocytes, the following viruses were used: AAV2/5- GfaABC 1 D-hM3Dq-mCherry (titre: 1.45 × 10 13 vector genome (v. g.)/ml, dilution: 1: 5, 0.2μl per side bilateral into LHb, plasmid from Addgene); AAV2/5- GfaABC 1 D-mCherry (titre: 2.08 × 10 13 vector genome (v. g.)/ml, dilution: 1: 5, 0.2μl per side bilateral into LHb, Sunbio Medical Biotechnology); AAV2/8-GfaABC 1 D-cOpn5-T2A-mCherry-WPRE-pA (0.2μl per side bilateral into LHb, provided by M. Luo); AAV2/5-GfaABC 1 D-cOpn5-T2A-mCherry-WPRE-pA (0.2μl per side bilateral into LHb, Taitool Bioscience); AAV2/5-GfaABC 1 D-iβARK-p2A-mCherry-WPRE-pA (titre: 1.9 × 10 13 vector genome (v. g.)/ml, 0.2μl per side bilateral into LHb, plasmid from Addgene); AAV2/5-GfaABC 1 D-IP3R2-shRNA-mCherry-WPRE-pA (titre: 2.61 × 10 13 vector genome (v. g.)/ml, 0.2μl per side bilateral into LHb, Sunbio Medical Biotechnology).

Techniques: Activation Assay, Expressing, In Vivo, Imaging, In Vitro, Injection, Staining, Saline