rat lncrna/mrna microarray  (Arraystar inc)

Journal: Acta Biochimica et Biophysica Sinica

Article Title: Endogenous RBM4 prevents Ang II-induced cardiomyocyte hypertrophy via downregulating the expression of PTBP1

doi: 10.3724/abbs.2024103

Figure Lengend Snippet: The expression of RBM4 in cardiomyocyte is mediated by m6A-YTHDF1 (A) The potential m6A sites in RBM4 mRNA predicted by SRAMP. (B) The m6A methylation of RBM4 mRNA in cardiomyocyte is increased by Ang II stimulation. The N6-methylated RNAs captured from cardiomyocytes treated with Ang II or saline (con) by m6A antibody or control IgG, were purified with TRIzol. The RBM4 mRNA level was analyzed by qPCR. ****P < 0.0001. n = 3. (C) The levels of m6A writer or eraser proteins in cardiomyocytes were analyzed by immunoblotting. n = 3. (D) The interaction between RBM4 mRNA and YTHDF1 was increased by Ang II stimulation. The RNA captured from the lysates of cardiomyocytes treated with Ang II or saline (con) by YTHDF1 antibody or control IgG was purified with TRIzol. The RBM4 mRNA level was analyzed by qPCR. *P < 0.05, **P < 0.01, ***P < 0.001. n = 3. (E,F) YTHDF1 enhances RBM4 translation during cardiomyocyte hypertrophy. Cardiomyocytes were transfected with control siRNA or YTHDF1 siRNA and then treated with Ang II. (E) The protein levels of RBM4 and YTHDF1 were analyzed by immunoblotting. (F) The RBM4 mRNA level was analyzed by qPCR. ns, P ≥ 0.05. n = 3.

Article Snippet: Double-strand small interfering RNA (siRNA) oligonucleotides specific for rat RBM4 mRNA, PTBP1 mRNA and YTHDF1 mRNA were designed and purchased from GenePharma Co., Ltd. (Shanghai, China).

Techniques: Expressing, Methylation, Saline, Control, Purification, Western Blot, Transfection

Journal: Biological Research

Article Title: Control of astrocytic Ca 2+ signaling by nitric oxide-dependent S-nitrosylation of Ca 2+ homeostasis modulator 1 channels

doi: 10.1186/s40659-024-00503-3

Figure Lengend Snippet: The Cx hemichannel- and Panx-1 channel-mediated Ca 2+ signaling depends on the activation of purinergic receptors. A Time course of the increase in [Ca 2+ ] i induced in primary cultures of astrocytes by 10 µM glutamate in control conditions and after the blockade of purinergic receptors with 100 µM PPADS or the inhibition of CALHM1 channels with 20 µM ruthenium red (RuR). B Analysis of the increase in ethidium uptake rate induced by glutamate in control conditions and in the presence of PPADS or RuR. The rate of ethidium uptake was assessed by calculating the slope of the increase in fluorescence intensity (expressed as arbitrary units, AU) along the time in basal conditions and during the stimulation with glutamate. C ATP release evoked by glutamate in control conditions and after the treatment with 100 µM N ω -nitro-L-arginine (L-NA) to inhibit NO production, the mimetic peptide 37,43 Gap27 (Gap27, 100 µM) to block hemichannels formed by Cx37 or Cx43, 10 Panx (100 µM) to block Panx1 channels or RuR. ATP was measured after 3 min of stimulation with glutamate. Numbers inside the bars indicate the n value. D Time course of the increase in [Ca 2+ ] i elicited by 100 nM ATP in primary cultures of astrocytes in control conditions and in the presence of 37,43 Gap27 or 10 Panx. Values are means ± SEM. *P < 0.05 vs Control by one-way ANOVA plus Bonferroni post hoc test. † P < 0.05 vs Baseline by paired Student’s t-test

Article Snippet: The expression of CALHM1 was downregulated using a siRNA designed to target rat Calhm1 mRNA that was obtained from QIAGEN (Düsseldorf, Germany, Cat. #SI02901241).

Techniques: Activation Assay, Inhibition, Fluorescence, Blocking Assay

Journal: Biological Research

Article Title: Control of astrocytic Ca 2+ signaling by nitric oxide-dependent S-nitrosylation of Ca 2+ homeostasis modulator 1 channels

doi: 10.1186/s40659-024-00503-3

Figure Lengend Snippet: The increase in [Ca 2+ ] i , activation of Cx hemichannels and Panx-1 channels and ATP release induced by glutamate depends on the expression of CALHM1 channels. A Detection of the expression of CALMH1 (red) by co-immunofluorescence analysis with glial fibrillary acidic protein (GFAP, green) in primary cultures of astrocytes treated with a control siRNA (siControl) or a siRNA directed against Calhm1 (siCALHM1). The cell nuclei are highlighted by the staining with DAPI (blue). B – D Representative Western blot and densitometric analysis of CALHM1 ( B ), Cx43 ( C ) and Panx-1 ( D ) expression in the primary cultures of astrocytes shown in A . Numbers inside the bars indicate the n value. The whole images of the representative Western blots are shown in Additional file : Fig. S7. E , Maximal increase in [Ca 2+ ] i observed in response to 10 µM glutamate in astrocytes cultures treated with siControl or siCALHM1. F Time course of the increase in ethidium uptake induced by 10 µM glutamate in primary cultures of astrocytes treated with siControl or siCALHM1. The horizontal bar indicates the stimulation period. G ATP release attained 3 min after the stimulation with glutamate in the same conditions than those shown in E and F . Values are means ± SEM. *P < 0.05 vs siControl by unpaired Student’s t-test. † P < 0.05 vs siControl by two-way ANOVA

Article Snippet: The expression of CALHM1 was downregulated using a siRNA designed to target rat Calhm1 mRNA that was obtained from QIAGEN (Düsseldorf, Germany, Cat. #SI02901241).

Techniques: Activation Assay, Expressing, Immunofluorescence, Staining, Western Blot

Journal: Biological Research

Article Title: Control of astrocytic Ca 2+ signaling by nitric oxide-dependent S-nitrosylation of Ca 2+ homeostasis modulator 1 channels

doi: 10.1186/s40659-024-00503-3

Figure Lengend Snippet: Stimulation with glutamate is associated with the S-nitrosylation of CALHM1 in primary cultures of astrocytes. A Analysis performed through Proximity Ligation Assay (PLA) of the spatial association of eNOS or nNOS with CALHM1. Note that CALHM1 is associated with eNOS, but not with nNOS. A control in which primary antibodies were omitted (negative control) is also shown. B Representative Western blots (left) and densitometric analysis (right) of the changes in the levels of CALHM1 S-nitrosylation (CALHM1 S-NO) detected by biotin switch in primary cultures of astrocytes 3 min after the stimulation with 10 µM glutamate (Glut), 3 µM SNAP or the vehicle of glutamate (Vh). In addition, the effect of the inhibition of NO production with 100 µM N ω -nitro-L-arginine (L-NA) on the glutamate-elicited increase in CALHM1 S-NO is also shown. Variations in the level of CALHM1 S-NO are expressed in arbitrary units (A.U.). C Representative Western blot (WB) of CALHM1 S-nitrosylation using an anti-S-Nitroso-Cysteine antibody in astrocytes samples previously submitted to CALHM1 immunoprecipitation (IP). *P < 0.05 vs Vehicle by unpaired Student’s t-test

Article Snippet: The expression of CALHM1 was downregulated using a siRNA designed to target rat Calhm1 mRNA that was obtained from QIAGEN (Düsseldorf, Germany, Cat. #SI02901241).

Techniques: Proximity Ligation Assay, Negative Control, Western Blot, Inhibition, Immunoprecipitation

Journal: Biological Research

Article Title: Control of astrocytic Ca 2+ signaling by nitric oxide-dependent S-nitrosylation of Ca 2+ homeostasis modulator 1 channels

doi: 10.1186/s40659-024-00503-3

Figure Lengend Snippet: NO activates CALHM1 channels in a heterologous expression system. A Representative current traces before and after application of 10 μM SNAP in a non-injected oocytes (NI) or oocytes expressing CALHM1 obtained at 1.8 mM extracellular Ca 2+ . Three CALHM1 expressing oocytes were additionally treated with 50 µM ascorbic acid, 10 µM ODQ or 20 µM ruthenium red (RuR). Oocytes were clamped to − 80 mV, and square pulses from − 80 mV to + 40 mV (in 10 mV steps) were then applied for 2 s. At the end of each pulse, the membrane potential was returned to − 80 mV. Note that ODQ did not affect NO-induced CALHM1 currents. B Normalized currents were obtained from the ratio between recorded current after and before 10 µM SNAP treatment. C Normalized currents at 0 mV oocytes resting membrane potential before and after 10 µM SNAP stimulation. Comparisons between groups were made using two-way ANOVA plus Tukey post-hoc test, *P < 0.05 vs Non-Injected (NI). D Time course of tail current peaks after reaching current saturation during a depolarizing pulses from − 80 to 20 mV (yellow box). Tail current peaks were obtained in the absence and presence of SNAP, and during RuR application. E Representative Western blot showing the expression and NO-mediated S-nitrosylation of CALHM1 in oocytes. From left to right, the first two lanes correspond to Western blots in non-injected oocytes (NI) and oocytes expressing CALHM1, and the next two lanes show the Biotin Switch analysis of CALHM1 S-nitrosylation observed in oocytes expressing CALHM1 in control conditions (lane 3) and after the stimulation with SNAP (lane 4)

Article Snippet: The expression of CALHM1 was downregulated using a siRNA designed to target rat Calhm1 mRNA that was obtained from QIAGEN (Düsseldorf, Germany, Cat. #SI02901241).

Techniques: Expressing, Injection, Membrane, Western Blot

Journal: Biological Research

Article Title: Control of astrocytic Ca 2+ signaling by nitric oxide-dependent S-nitrosylation of Ca 2+ homeostasis modulator 1 channels

doi: 10.1186/s40659-024-00503-3

Figure Lengend Snippet: Schematic model of the signaling events that mediate the increase in [Ca 2+ ] i initiated by metabotropic glutamate receptor (mGluR) activation in astrocytes. Glutamate released during an increase in neuronal activity can exit the synaptic cleft and activate receptors on astrocyte processes. The activation of astrocyte mGluRs leads to an initial increase in [Ca 2+ ] i by the release of Ca 2+ from the intracellular Ca 2+ stores through activation of an inositol (1,4,5)-triphosphate (IP 3 )-mediated pathway. This astrocytic Ca 2+ signal triggers an increment in nitric oxide (NO) production by the endothelial NO synthase isoform (eNOS), which, in turn, evokes the opening of CALHM1 channels by the S-nitrosylation of this protein. The activation of CALHM1 channels play a pivotal role in the response by providing a pathway for ATP release and the sequential activation of P2 receptors (P2R), leading to the opening of Cx43 hemichannels and Panx-1 channels. Finally, the Ca 2+ influx through these membrane channels contributes to amplify the intracellular Ca 2+ store-initiated Ca 2+ signaling. In addition, the increase in [Ca 2+ ] i can be coordinated through the propagation of an inter-astrocyte Ca 2+ signal via ATP release or directly by gap junction communication (GJ)

Article Snippet: The expression of CALHM1 was downregulated using a siRNA designed to target rat Calhm1 mRNA that was obtained from QIAGEN (Düsseldorf, Germany, Cat. #SI02901241).

Techniques: Activation Assay, Activity Assay, Membrane