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Figure 3. MAVS signaling mediates sensing of inactivated rotavirus dsRNA in intestinal epithelial cells (A–D) Human ileal enteroids cultured in transwells were stimulated with infectious RRV strain (MOI = 100), equivalent particle numbers of inactivated RRV, or recombinant human IFN-l3 protein (1 mg/mL) for 24 h. The mRNA levels of IFNL3 (A), ISG15 (C), and rotavirus NSP5 (D) were assessed by RT-qPCR, and levels of secreted IFN-l (B) were quantified by <t>ELISA.</t> (E) WT, RIG-I KO, MDA5 KO, RIG-I/MDA5 DKO, and MAVS KO HT-29 cells were inoculated with infectious RRV (MOI = 3) or equivalent particle numbers of inactivated rotavirus. IFNL3 transcript levels were assessed by RT-qPCR. (F) 5-day-old Mavsfl/fland Mavsfl/flAlpi-CreERT2 C57BL/6J mice were orally inoculated with inactivated RRV (107 PFU equivalent per mouse), and diarrhea occurrence was monitored from 0 to 9 dpi.
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Figure 3. MAVS signaling mediates sensing of inactivated rotavirus dsRNA in intestinal epithelial cells (A–D) Human ileal enteroids cultured in transwells were stimulated with infectious RRV strain (MOI = 100), equivalent particle numbers of inactivated RRV, or recombinant human IFN-l3 protein (1 mg/mL) for 24 h. The mRNA levels of IFNL3 (A), ISG15 (C), and rotavirus NSP5 (D) were assessed by RT-qPCR, and levels of secreted IFN-l (B) were quantified by <t>ELISA.</t> (E) WT, RIG-I KO, MDA5 KO, RIG-I/MDA5 DKO, and MAVS KO HT-29 cells were inoculated with infectious RRV (MOI = 3) or equivalent particle numbers of inactivated rotavirus. IFNL3 transcript levels were assessed by RT-qPCR. (F) 5-day-old Mavsfl/fland Mavsfl/flAlpi-CreERT2 C57BL/6J mice were orally inoculated with inactivated RRV (107 PFU equivalent per mouse), and diarrhea occurrence was monitored from 0 to 9 dpi.
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Figure 3. MAVS signaling mediates sensing of inactivated rotavirus dsRNA in intestinal epithelial cells (A–D) Human ileal enteroids cultured in transwells were stimulated with infectious RRV strain (MOI = 100), equivalent particle numbers of inactivated RRV, or recombinant human IFN-l3 protein (1 mg/mL) for 24 h. The mRNA levels of IFNL3 (A), ISG15 (C), and rotavirus NSP5 (D) were assessed by RT-qPCR, and levels of secreted IFN-l (B) were quantified by <t>ELISA.</t> (E) WT, RIG-I KO, MDA5 KO, RIG-I/MDA5 DKO, and MAVS KO HT-29 cells were inoculated with infectious RRV (MOI = 3) or equivalent particle numbers of inactivated rotavirus. IFNL3 transcript levels were assessed by RT-qPCR. (F) 5-day-old Mavsfl/fland Mavsfl/flAlpi-CreERT2 C57BL/6J mice were orally inoculated with inactivated RRV (107 PFU equivalent per mouse), and diarrhea occurrence was monitored from 0 to 9 dpi.
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Figure 3. MAVS signaling mediates sensing of inactivated rotavirus dsRNA in intestinal epithelial cells (A–D) Human ileal enteroids cultured in transwells were stimulated with infectious RRV strain (MOI = 100), equivalent particle numbers of inactivated RRV, or recombinant human IFN-l3 protein (1 mg/mL) for 24 h. The mRNA levels of IFNL3 (A), ISG15 (C), and rotavirus NSP5 (D) were assessed by RT-qPCR, and levels of secreted IFN-l (B) were quantified by ELISA. (E) WT, RIG-I KO, MDA5 KO, RIG-I/MDA5 DKO, and MAVS KO HT-29 cells were inoculated with infectious RRV (MOI = 3) or equivalent particle numbers of inactivated rotavirus. IFNL3 transcript levels were assessed by RT-qPCR. (F) 5-day-old Mavsfl/fland Mavsfl/flAlpi-CreERT2 C57BL/6J mice were orally inoculated with inactivated RRV (107 PFU equivalent per mouse), and diarrhea occurrence was monitored from 0 to 9 dpi.

Journal: Cell host & microbe

Article Title: Innate immune sensing of rotavirus by intestinal epithelial cells leads to diarrhea.

doi: 10.1016/j.chom.2025.02.005

Figure Lengend Snippet: Figure 3. MAVS signaling mediates sensing of inactivated rotavirus dsRNA in intestinal epithelial cells (A–D) Human ileal enteroids cultured in transwells were stimulated with infectious RRV strain (MOI = 100), equivalent particle numbers of inactivated RRV, or recombinant human IFN-l3 protein (1 mg/mL) for 24 h. The mRNA levels of IFNL3 (A), ISG15 (C), and rotavirus NSP5 (D) were assessed by RT-qPCR, and levels of secreted IFN-l (B) were quantified by ELISA. (E) WT, RIG-I KO, MDA5 KO, RIG-I/MDA5 DKO, and MAVS KO HT-29 cells were inoculated with infectious RRV (MOI = 3) or equivalent particle numbers of inactivated rotavirus. IFNL3 transcript levels were assessed by RT-qPCR. (F) 5-day-old Mavsfl/fland Mavsfl/flAlpi-CreERT2 C57BL/6J mice were orally inoculated with inactivated RRV (107 PFU equivalent per mouse), and diarrhea occurrence was monitored from 0 to 9 dpi.

Article Snippet: Compound SAKURA Cat# 4583 Albumin, Bovine Fraction V [BSA] Research Products International Cat# A30075 Triton X-100 Sigma-Aldrich Cat# T8787 DL-dithiothreitol Sigma-Aldrich Cat# 43815 UltraPure 0.5M EDTA Invitrogen Cat# 15575020 Matrigel Corning Cat# 356234 Y-27632 dihydrochloride R&D Cat# 1254 SB 431542 R&D Cat# 1614 Gastrin Sigma-Aldrich Cat# G9145 A83-01 Sigma-Aldrich Cat# SML0788 DAPT Selleckchem Cat# S2215 Mouse EGF R&D Cat# 2028-EG Paraformaldehyde (PFA) Electron Microscopy Sciences Cat# 15710 Collagen Sigma-Aldrich Cat# C5533 TrypLE Express Enzyme Gibco Cat# 12604013 Human EGF Invitrogen Cat# A42556 Critical commercial assays The human IL-29/IL-28B (IFN-lambda 1/3) DuoSet ELISA kit R&D Systems Cat# DY1598B05 Serotonin ELISA kit IBL International Cat# RE59121 RNeasy Plus Mini Kit QIAGEN Cat# 74136 High-Capacity cDNA Reverse Transcription Kit Thermo Fisher Scientific Cat# 4368814 SYBR Green qPCR ReadyMix Sigma-Aldrich Cat# KCQS00 TaqMan assays Applied Biosystems Cat# 4444557 Deposited data scRNA-seq datasets This study GSE288639 Experimental models: Cell lines African green monkey: MA104 cells ATCC Cat# CRL-2378.1 Mouse: L929 cells ATCC Cat# CCL-1 (Continued on next page) Cell Host & Microbe 33, 408–419.e1–e8, March 12, 2025 e2

Techniques: Cell Culture, Recombinant, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay