Posted on July 12, 2021
Supplementary MaterialsS1 Fig: A) 2CMC is definitely non-toxic in HIE at operating concentration
Supplementary MaterialsS1 Fig: A) 2CMC is definitely non-toxic in HIE at operating concentration. (716K) GUID:?88428422-7E1E-4550-89DB-6DF1422005FD S2 Fig: Changes in transcript levels of select genes in undifferentiated vs. differentiated HIE. The differentiation status of J2 and D124 HIE was monitored at 0 and 6 days post-differentiation (dpd) (after WNT3A removal) by measuring transcripts of A) olfactomedin 4 (OLFM4), a stem cell marker, and B) alkaline phosphatase, cytochrome P450, SLC15A1, and SLC11A2 (markers of enterocytes). C) differentiation Pomalidomide-PEG4-C-COOH status of J2 HIE monitored at 0 and 3 days post differentiation after WNT3A removal (CMGF-) or VA1 illness without removal of WNT3A (CMGF+). Transcript levels were measured by qPCR. Data are from 3 experiments; error = mean SD. Collapse change is relative to GAPDH, and is statistically significantly different (P 0.05) from your 0 dpd inside a) and B). In C) ***P 0.001. Abbreviations: D = duodenum, J = jejunum. The number associated with each letter shows the patient identifier. NS = not significant.(TIF) ppat.1008057.s002.tif (1.9M) GUID:?C0A9FB46-A06E-4633-8E87-5042F519976D S3 Fig: Representative flow plots of the percent of VA1-infected cells (reddish) vs mock-infected cells (black). Differentiated HIE I124 were infected with VA1 (MOI of 1 1) and a single cell suspension was generated at 3 dpi. Cells were stained Pomalidomide-PEG4-C-COOH with the surface markers lysozyme (paneth cells), CD44 ITGB8 (progenitor cells), chromogranin A (enteroendocrine cells), MUC2 (goblet cells) and sucrase isomaltase (adult enterocytes) and the intracellular dsRNA antibody conjugated with biotin followed by the secondary streptavidin APC-Cy7 antibody. The circulation cytometry data were generated on BD LSRFortessa and analyzed with FlowJo. The gating strategy from Cells to Solitary cells to Alive cells is definitely illustrated for one representative sample in the remaining column, whereas the gating strategy for each cell type and the related infected cells is definitely illustrated for one VA1-infected (reddish) and one Mock-infected (black) sample in the middle and right column, respectively.(TIF) ppat.1008057.s003.tif (8.6M) GUID:?4C4B615D-164E-4106-99AC-1EC03D073ABF S4 Fig: RNAseq analysis of VA1-infected HIE. A) The correlation between genome copies per D124 HIE as determined by RT-qPCR and the proportion of viral transcripts in the pool of sequenced RNA collected from your same HIE cultures. B) IFN , , and are up-regulated upon VA1 illness in the RNAseq dataset. CCD) Top genes positively- (C) and negatively- (D) correlated with viral weight as determined by RNA-seq and RT-qPCR.(TIF) ppat.1008057.s004.tif (3.6M) GUID:?8556BCCC-1E5B-4073-82AF-E3DCFCA56F3E S5 Fig: HIE mounts IFN response to virus infection. A) ISG15 transcript levels at 1 dpi or post poly I:C treatment. Undifferentiated C68 HIE were infected with VA1, Sindbis disease or VSV (all MOI = 1) for 1 day or treated with 50 g/ml poly I:C for 24 hours. Cellular RNA was extracted for ISG15 transcript quantification as collapse increase over 0 dpi by qPCR. GADPH was used as internal control. B) VSV and Sindbis disease titer dedication at 1 dpi by plaque assay with Vero cells. N 3; error = mean SD.(TIF) ppat.1008057.s005.tif (438K) GUID:?E58105D9-6027-4F68-9B42-B214D86A39AD S1 Table: RNA-seq gene summary for VA1 over mock. (DOCX) ppat.1008057.s006.docx (14K) GUID:?B20841ED-032B-4DF3-9789-C7C9909E78D7 S2 Table: RNA-seq list of significantly regulated genes Pomalidomide-PEG4-C-COOH (Adj P 0.05) for VA1 over mock. (DOCX) ppat.1008057.s007.docx (31K) GUID:?8EA7F20A-5E54-424D-B430-87E3C300B0D0 S3 Table: CT ideals for ISG15, IFN-, IFN- and IFN- mRNA expression in VA1 infected I124 HIE as detected by RT-qPCR. (DOCX) ppat.1008057.s008.docx (15K) GUID:?81A0976D-DC59-4CB4-A7D3-0A7438D225ED S4 Table: CT values for ISG15, IFN-, IFN- and IFN- mRNA expression in VA1 infected Caco2 cells as detected by RT-Qpcr. (DOCX) ppat.1008057.s009.docx (16K) GUID:?181E7B1C-37B6-47F3-AA29-A402E6AB7DDF S5 Table: Summary of the HIE lines used in this study. (DOCX) ppat.1008057.s010.docx (15K) GUID:?95751281-9E9A-41CE-B5DE-16F4DFB90E94 S6 Table: List of commercial antibodies. (DOCX) ppat.1008057.s011.docx (17K) GUID:?40F3B4F7-CA92-409F-A05B-71478C64619B S7 Table: List of primers. (DOCX) ppat.1008057.s012.docx (34K) GUID:?A563B34C-9DDF-4919-84AB-B37D5D5ABDC0 Data Availability StatementRelevant data are within the manuscript and its Pomalidomide-PEG4-C-COOH Supporting Information documents, and less than https://github.com/hilldr/astrovirus Abstract Human being astroviruses (HAstV) are understudied positive-strand RNA viruses that cause gastroenteritis mostly in children and the elderly. Three clades of astroviruses, vintage, MLB-type and VA-type have.