Journal: Molecular neurobiology

Article Title: CD38 Coordinates with NF-κB to Promote Cochlear Inflammation in Noise-Induced Hearing Loss: the Protective Effect of Apigenin.

doi: 10.1007/s12035-024-04675-7

Figure Lengend Snippet: Fig. 9 CD38 coordinates with NF-κB to promote cochlear inflammation in NIHL. As the closest immune cell to organ of Corti, activated macrophages can express CD38 and consume NAD in organ of Corti, causing NAD + metabolism dysfunction and ultimately leads to cochlear inflammation. In this process, inflammatory factors such as IL-1, IL-6, and TNF-α pro- moted the activation of NF-κB signaling pathway, forming a positive feedback cycle

Article Snippet: Immunofluorescence The cochlear basilar membrane was prepared with the surface preparation method, antibodies used in this experiment were rabbit anti-NF-κB p65 polyclonal antibody (Abcam, ab32536), rat anti-F4/80 polyclonal antibody (Abcam, ab6640), and mouse anti-CD38 monoclonal antibody (Proteintech, 60,006–1-lg).

Techniques: Activation Assay

Journal: Experimental & Molecular Medicine

Article Title: CD38/ADP-ribose/TRPM2-mediated nuclear Ca 2+ signaling is essential for hepatic gluconeogenesis in fasting and diabetes

doi: 10.1038/s12276-023-01034-9

Figure Lengend Snippet: a After isolating nuclei with sucrose ultracentrifugation, the outer membrane of the nuclei was separated by treatment with sodium citrate. Nucleoplasts were fixed with 3.8% paraformaldehyde for 20 min, permeabilized, and then stained with anti-mouse CD38 antibody (green). Lamin B1 was used as a marker for the inner membrane of the nucleus. The nucleus was stained with DAPI (blue). Scale bar, 5 μm. b After nuclei were isolated from Cd38 +/+ or Cd38 − / − mouse primary hepatocytes, the outer membrane of the nucleus was removed by sodium citrate treatment. The nucleoplasts (outer membrane-deprived nuclei) were stained with an anti-mouse CD38 antibody (C90). Scale bar, 5 μm. c Adenoviral vector constructs of Control (Ad-NLS), catalytic active-nCD38 (wtCd38), and catalytic inactive-nCD38 (Cd38E230D). d Hepatocytes were transduced with adenoviral vectors expressing Ad-NLS, wtCd38-NLS, or Cd38E230D-NLS at an MOI of 50 for 24 h, and then cytoplasmic and nuclear extracts were isolated. Immunoblotting was performed to detect Myc, LAMP-1 (a marker of lysosomes), GAPDH (a cytoplasmic marker), and Lamin B1 (a nuclear marker). e NAD glycohydrolase activity. f ADPR levels in hepatocytes treated with glucagon (100 nM) for 30 sec. g , h qRT-PCR of G6pc ( g ) and Pck1 ( h ) after incubation with glucagon (100 nM) for 4 h. i Glucose production after incubation with glucagon (100 nM) for 5 h. e – i n = 6 independent experiments, mean values ± SEMs are shown, and P values were calculated by one-way ANOVA followed by Tukey’s multiple comparison test. ** P < 0.01, *** P < 0.001, and not significant (NS). For a , b , d , representative images of three independent experiments are shown.

Article Snippet: Generation of NLS-CD38 and NLS-Flag Cx43 Adenovirus: CD38 or Cx43 was amplified via PCR from pCMV-CD38 (Sino Biological Inc., MG50191-UT) or pCMV3- Flag Cx43 (Sino Biological Inc., G52427-NF) by using primers to add NLS, which was inserted into the pCMV/myc/nuc vector (Thermo Fisher Scientific, V82120).

Techniques: Staining, Marker, Isolation, Plasmid Preparation, Construct, Transduction, Expressing, Western Blot, Activity Assay, Quantitative RT-PCR, Incubation

Journal: Experimental & Molecular Medicine

Article Title: CD38/ADP-ribose/TRPM2-mediated nuclear Ca 2+ signaling is essential for hepatic gluconeogenesis in fasting and diabetes

doi: 10.1038/s12276-023-01034-9

Figure Lengend Snippet: a , b Hepatocytes were infected with GCaMP6m and NLS-R-GECO adenoviruses, and Ca 2+ signals were recorded. The time point where 100 nM glucagon (Gcg) is added is indicated by the arrow. Scale bar, 10 μm. c Cytosolic and nuclear Ca 2+ responses to glucagon after preincubation with BAPTA-am (10 μM). d – f Cytosolic and nuclear Ca 2+ responses to glucagon after incubation with various inhibitors of Ca 2+ second messengers [8-Br-cADPR (100 μM, d ), 8-Br-ADPR (100 μM, e ), or Ned19 (10 μM, f )]. g , h Cytosolic and nuclear Ca 2+ responses to glucagon after overexpression of nCD38 using wtCd38-NLS adenovirus in Cd38 +/+ hepatocytes ( g ) or Cd38 − / − hepatocytes ( h ). n = 20 cells for each condition in b – h .

Article Snippet: Generation of NLS-CD38 and NLS-Flag Cx43 Adenovirus: CD38 or Cx43 was amplified via PCR from pCMV-CD38 (Sino Biological Inc., MG50191-UT) or pCMV3- Flag Cx43 (Sino Biological Inc., G52427-NF) by using primers to add NLS, which was inserted into the pCMV/myc/nuc vector (Thermo Fisher Scientific, V82120).

Techniques: Infection, Incubation, Over Expression

Journal: Experimental & Molecular Medicine

Article Title: CD38/ADP-ribose/TRPM2-mediated nuclear Ca 2+ signaling is essential for hepatic gluconeogenesis in fasting and diabetes

doi: 10.1038/s12276-023-01034-9

Figure Lengend Snippet: a Glucagon-induced ADPR production in Cd38 +/+ or Cd38 − / − primary hepatocytes. n = 6 independent experiments. ** P < 0.01; Cd38 +/+ vs. Cd38 +/+ . b ADPR levels in hepatocytes treated with glucagon (100 nM) for 30 sec after preincubation with vehicle or SKF 96365 (SKF, 50 μM), or under Ca 2+ -deprived extracellular conditions. n = 6 independent experiments. ** P < 0.01. c , d Effects of SOCE on glucagon-induced mRNA levels of G6pc ( c ) and Pck1 ( d ). n = 8 independent experiments. *** P < 0.001. e , f Effects of 8-Br-ADPR on glucagon-induced mRNA levels of G6pc ( e , left) and Pck1 ( e , right) and glucose production ( f ). 8-Br-ADPR (100 μM) was preincubated for 30 min. n = 6 independent experiments. ** P < 0.01, *** P < 0.001. g Effects of exogenous ADPR on G6pc and Pck1 gene expression. n = 6 independent experiments. *** P < 0.001. h Immunoblotting for pCREB, pCaMKII, pCaMKIV, and β-actin in total cell lysates. i Immunoblotting cytoplasmic and nuclear extracts after treating hepatocytes with glucagon for 30 min. Cells were preincubated with 8-Br-ADPR (100 μM) for 30 min. The PARP-1 antibody was used as a marker of nuclear extracts. j , k Effects of 8-Br-ADPR on CRE luciferase activity in fasting mice ( j , n = 3 mice per group) and the pyruvate tolerance test ( k , n = 5 mice per group). 8-Br-ADPR (32 mg/kg) was intravenously admi n istered to mice. ** P < 0.01 and *** P < 0.001; vehicle vs. 8-Br-ADPR. l Effects of 8-Br-ADPR on G6pc and Pck1 mRNA levels in fasting mice. n = 8 mice per group. *** P < 0.001. Data are represented as the mean ± SEM. Statistics were determined by unpaired t test ( a , g , k , l ) or one-way ANOVA followed by Tukey’s multiple comparison test ( b – f ). h , i Representative images of three independent experiments are shown.

Article Snippet: Generation of NLS-CD38 and NLS-Flag Cx43 Adenovirus: CD38 or Cx43 was amplified via PCR from pCMV-CD38 (Sino Biological Inc., MG50191-UT) or pCMV3- Flag Cx43 (Sino Biological Inc., G52427-NF) by using primers to add NLS, which was inserted into the pCMV/myc/nuc vector (Thermo Fisher Scientific, V82120).

Techniques: Expressing, Western Blot, Marker, Luciferase, Activity Assay

Journal: Experimental & Molecular Medicine

Article Title: CD38/ADP-ribose/TRPM2-mediated nuclear Ca 2+ signaling is essential for hepatic gluconeogenesis in fasting and diabetes

doi: 10.1038/s12276-023-01034-9

Figure Lengend Snippet: Glucagon regulates glucose homeostasis by binding to two G protein-coupled receptors (Gq and Gs) to produce IP 3 and cADPR, respectively. IP 3− and cADPR-mediated Ca 2+ mobilization from ER Ca 2+ stores induces SOCE, resulting in Ca 2+ influx. This activates the nuclear translocation of PLCδ1/3. Nuclear PLCδ1/3 produces IP 3 and DAG, activating PKCδ, which phosphorylates Cx43. Phospho-Cx43 gates NAD from the nucleoplasm into the perinuclear space. Constitutively active CD38, with its catalytic site facing the perinuclear space, produces ADPR, which then activates TRPM2 to trigger nucleoplasmic Ca 2+ release. Nuclear Ca 2+ increases the expression of G6pc and Pck1 , which promote gluconeogenesis through the CaMKII/CaMKIV/CREB pathway. Note that CD38, Cx43, TRPM2, and PKCδ are all located on the inner nuclear membrane.

Article Snippet: Generation of NLS-CD38 and NLS-Flag Cx43 Adenovirus: CD38 or Cx43 was amplified via PCR from pCMV-CD38 (Sino Biological Inc., MG50191-UT) or pCMV3- Flag Cx43 (Sino Biological Inc., G52427-NF) by using primers to add NLS, which was inserted into the pCMV/myc/nuc vector (Thermo Fisher Scientific, V82120).

Techniques: Binding Assay, Translocation Assay, Expressing