bx430  (Cayman Chemical)

Journal: Science (New York, N.Y.)

Article Title: Rotavirus induces intercellular calcium waves through ADP signaling

doi: 10.1126/science.abc3621

Figure Lengend Snippet: (A-B) Number of Ca2+ spikes in MA104-GCaMP cells in RV-infected (SA11cl3-mRuby3) at MOI 0.01 and neighboring (NB)+3 cell, treated with: (A) DMSO, 30μM 18β-glycyrrhetinic acid (18β-gly), or 50μM TAT-Gap19 (Gap19) and imaged ~8–24 hpi or (B) anti-VP7 M60 MAb, anti-NSP4 MAb 622, or rabbit anti-NSP4 antisera 120–147 (Rb Ab) and imaged ~8–24 hpi. (data combined from N=3 independent experiments) (C) Representative images of intercellular calcium waves in MA104-GCaMP cells infected with RV (SA11cl3-mRuby3) at MOI 0.1 and mock- or 10U/mL apyrase-treated (5 U/mL apyrase VI and 5U/mL apyrase VII) and imaged ~10 hpi with (D) representative Ca2+ traces from ~8–25 hpi. (E) Ca2+ spikes in RV-infected and NB cells. (F) Average magnitude of Ca2+ spikes/cell in RV-infected and NB cells, (data combined from N=3 independent experiments). (G) qPCR of purinergic receptor mRNA normalized to 18S mRNA and fold change relative to P2X3 mRNA transcript levels in MA104 cells, (data combined from N=3 independent experiments) (H-I) Ca2+ spikes in MA104-GCaMP cells RV (SA114F)-infected (H) or neighboring (NB) cells (I) and treated with DMSO, 10μM BPTU, 10μM AR-C 118925XX (ARC), 10μM Bx430, or 10μM 5-BDBD (data combined from N=3 independent experiments). (A-B, E-F, H-I) Kruskal-Wallis with Dunn’s multiple comparisons test used. Scale bar = 100 μm. Data represented as mean ± SD, (*p<0.05, ****p<0.0001).

Article Snippet: BPTU, AR-C 118925XX, Bx430, 5-BDBD, MRS2179, MRS2279, MRS2500, 10-Panx, suramin, TAT-Gap19, PPADS (Pyridoxalphosphate-6-azophenyl-2’,4’-disulfonic acid), NOC-7, prostaglandin E2, ARL 67156, and ω-agatoxin were purchased from Tocris Bioscience.

Techniques: Infection

Journal: Science (New York, N.Y.)

Article Title: Rotavirus induces intercellular calcium waves through ADP signaling

doi: 10.1126/science.abc3621

Figure Lengend Snippet: (A) J3 jHIE-GCaMP6s monolayers mock- or RV (Ito)-infected produce intercellular calcium waves originating at a cell (arrowheads), imaged at ~4 hpi. (B) IF images of (J3)HIE-GCaMP6s monolayers mock- or RV-infected, fixed at 24 hpi, and immunostained for RV antigen (pink) and counterstained with DAPI (gray). (C) Ca2+ spikes per field-of-view (FOV), in (J3)HIEs mock- or RV-infected and treated with vehicle, 100 μM carbenoxolone (CBX), or 10 μM 10Panx at 8–22 hpi (data combined from N=3 independent experiments). (D) qPCR of purinergic receptor mRNA normalized to 18S mRNA and fold change relative to P2X3 mRNA transcript levels in (J3)HIE monolayers (data combined from N=3 independent experiments). (E) Representative Ca2+ traces/FOV of (J3)HIE-GCaMP6s monolayers either mock- or RV-infected and treated with DMSO, 100 U/mL apyrase, or 10 μM BPTU between 8.5–22 hpi. Mock- or RV-infected (J3)HIEs, treated with DMSO, apyrase, or purinergic receptor blockers. (F) Ca2+ spikes/FOV in (J3)HIE-GCaMP6s monolayers treated with DMSO, 100U/mL apyrase (50 U/mL apyrase VI and 50U/mL apyrase VII), 10 μM BPTU, 10 μM AR-C 118925XX, 10 μM Bx430, or 10 μM 5-BDBD and (G) average magnitude of Ca2+ spikes/FOV for 8.5–22 hpi, (data combined from N=3 independent experiments) (H) Ca2+ spikes/FOV of patient J2 jHIE-GCaMP6s monolayers mock- or RV-infected and treated with DMSO, 10 μM BPTU, 10 μM MRS2179, 10 μM MRS2279, or 10 μM MRS2500 and (I) average magnitude of Ca2+ spikes/FOV for 8–22 hpi, (data combined from N=3 independent experiments). (C,H,I) One-way ANOVA with Bonferroni’s and (F,G) Kruskal-Wallis with Dunn’s multiple corrections test used. Scale bar = 50 μm. Data represented as mean ± SD, (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001)

Article Snippet: BPTU, AR-C 118925XX, Bx430, 5-BDBD, MRS2179, MRS2279, MRS2500, 10-Panx, suramin, TAT-Gap19, PPADS (Pyridoxalphosphate-6-azophenyl-2’,4’-disulfonic acid), NOC-7, prostaglandin E2, ARL 67156, and ω-agatoxin were purchased from Tocris Bioscience.

Techniques: Infection

Journal: Science (New York, N.Y.)

Article Title: Rotavirus induces intercellular calcium waves through ADP signaling

doi: 10.1126/science.abc3621

Figure Lengend Snippet: (A) Rotavirus yield from SA114F-infected MA104 cells treated with DMSO vehicle, 10U/mL apyrase, 10μM BPTU, 10μM AR-C 118925XX, 20μM Bx430, 20μM 5-BDBD, 300μM suramin, or 10μM PPADS by fluorescent focus assay (data combined from N=3 independent experiments). (B) Plaque assay yield of RV (SA114F)-infected MA104-GCaMP (Par) or MA104-GCaMP P2Y1 knockout (KO) cells (data combined from N=3 independent experiments). (C-E) qPCR of mRNA transcripts normalized to 18S mRNA transcripts and fold change relative to the Mock-DMSO transcript levels in (J3)HIE monolayers mock- or RV (Ito)-infected and treated with DMSO vehicle, 100U/mL apyrase, or 10 μM BPTU (data combined from N=3 independent experiments). (F-G) Serotonin secretion from RV (Ito)-infected jHIE (F) monolayers and (G) transwells treated with DMSO, 100U/mL apyrase, 10μM BPTU, or 300nM ω-agatoxin (data combined from N=3 independent experiments). (H) Enteroid swelling assay: 3D jHIE-GCaMP6s enteroids mock- or RV (Ito)-infected and treated with DMSO or 10μM BPTU. Cross-sectional area of the internal lumen (pink outline) used for percent increase between basal and max swelling (left panels). Scale bar = 100μm. (n ≥ 68 HIEs per condition, data combined from N=3 independent experiments) (I-K) C57Bl/6J mouse pups with diarrhea infected with Rhesus RV and vehicle- or BPTU-treated and the (J) mean diarrhea score. (data combined from 4 cages of each condition, 26–30 pups total per condition, mean ± SEM) (K) Summary model of RV-induced ICWs mediated by extracellular ADP. (A-B, F-G) One-way ANOVA with Bonferroni multiple comparisons test, (C-J) Kruskal-Wallis with Dunn’s multiple comparisons, or (I-J) Mann-Whitney tests used. (A-H) Data represented as mean ± SD, (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001).

Article Snippet: BPTU, AR-C 118925XX, Bx430, 5-BDBD, MRS2179, MRS2279, MRS2500, 10-Panx, suramin, TAT-Gap19, PPADS (Pyridoxalphosphate-6-azophenyl-2’,4’-disulfonic acid), NOC-7, prostaglandin E2, ARL 67156, and ω-agatoxin were purchased from Tocris Bioscience.

Techniques: Infection, Plaque Assay, Knock-Out, MANN-WHITNEY