Journal: bioRxiv

Article Title: Nanchangmycin regulates FYN, FAK and ERK to control the fibrotic activity of hepatic stellate cells

doi: 10.1101/2021.10.08.463221

Figure Lengend Snippet: A. Expression of house-keeping genes in HSCs was plotted by RPKM as previously quantified . Three pairs of samples were used in this analysis: ceramide treatment vs control, nortriptyline treatment vs control, nortriptyline treatment vs control in TGF-β treated HSCs . Each dot represents the result from one sample, and bars represent mean ± standard deviation (sd). The value of sd is indicated above each mRNA. The fold change (fc) of EMC7 , VCP , PSMB2 and VPS29 in inactivated HSCs (ceramide or nortriptyline-treated) compared to corresponding control groups is indicated below the dots. Two groups of reference mRNAs were analyzed -- commonly used ones (left) and genes that are expressed uniformly across tissues . Red arrows indicate samples with sd of 0.15 or less. Blue arrows highlight the reference mRNAs with fold change of no more than 10% in inactivated HSCs and were selected for further analysis. GAPDH , which is used routinely as a reference control was also included. B. Quantification of expression of EMC7 , GAPDH , GUSB , POLR2A , and PSMB2 from HSC cDNA samples (left: n=14, right: n=53) that were reverse transcribed from equal amounts of total input RNA. All samples were normalized to the mean value of its own control group before they were combined for each of the reference mRNAs. Each dot represents the result from one sample, and bars represent mean ± standard deviation (sd) of all the tested samples. The value of sd is indicated above each mRNA.

Article Snippet: Reverse transcription was performed using iScript gDNA Clear cDNA Synthesis Kit (BIO-RAD, cat# 1725035) with 1 μg total RNA input, and quantitative real-time PCR was performed using TaqMan Universal PCR Master Mix (Applied Biosystems, cat# 4305719) and TaqMan Real-time PCR Assays for specific genes listed below.

Techniques: Expressing, Control, Standard Deviation, Reverse Transcription

Journal: bioRxiv

Article Title: Nanchangmycin regulates FYN, FAK and ERK to control the fibrotic activity of hepatic stellate cells

doi: 10.1101/2021.10.08.463221

Figure Lengend Snippet: A-B. FYN was depleted in HSCs with two siRNAs (si1 and si2). The expression levels of COL1A1 ( A ) and FYN ( B ) were analyzed by qPCR after 72 hours in comparison to a non-targeting control siRNA (NTC). Error bars represent mean ± SEM (n=3) for each of three donor lines. ** indicates p < 0.01, *** indicates p < 0.001, and **** indicates p < 0.0001 (one-way ANOVA test). C. HSCs were treated with NCMC or 1-Naphthyl PP1 (PP1) for 48 hours. COL1A1 level was analyzed by qPCR. Error bars represent mean ± SEM (n=3 for donor 1, and n=6 for donor 3). Data are representative of three independent experiments for donor 1 and experiment for donor 3. **** indicates p < 0.0001 (one-way ANOVA test). D. Effect of FYN-depletion on collagen deposition in ECM. Top: representative images. Scale bar represents 100 µm. Bottom: quantified results. Error bars represent mean ± SEM (n=6). Data are representative of two independent experiments. ** indicates p < 0.01, and **** indicates p < 0.0001 (one-way ANOVA test). E. HSCs transduced with control virus or virus containing the cDNA encoding dominant negative mutant FYN (FYN-DN) were treated with DMSO or 100 nM NCMC for 48 hours. Expression of COL1A1 was quantified by qPCR. Error bars represent mean ± SEM (n=3). Data are representative of three independent experiments). ns indicates not significant (p > 0.05), * indicates p < 0.05, ** indicates p < 0.01, and **** indicates p < 0.0001 (two-way ANOVA test). F. Phospho-ERK and phospho-FAK levels were determined by Western blot in control HSCs and HSCs overexpressing DN-FYN. Left: representative Western blot results. Right: quantified results. Representative of two independent experiments. This figure has four supplements.

Article Snippet: Reverse transcription was performed using iScript gDNA Clear cDNA Synthesis Kit (BIO-RAD, cat# 1725035) with 1 μg total RNA input, and quantitative real-time PCR was performed using TaqMan Universal PCR Master Mix (Applied Biosystems, cat# 4305719) and TaqMan Real-time PCR Assays for specific genes listed below.

Techniques: Expressing, Comparison, Control, Transduction, Virus, Dominant Negative Mutation, Western Blot

Journal: Cell host & microbe

Article Title: Antiretroviral therapy reveals triphasic decay of intact SIV genomes and persistence of ancestral variants.

doi: 10.1016/j.chom.2023.01.016

Figure Lengend Snippet: Figure 4. Comparison of gag ddPCR and IPDA quantification of SIV DNA (A) Correlation between gag+ SIV DNA copies and intact proviruses quantified using the IPDA. Individual datapoints represent the number of copies detected using either an amplicon in gag (y-axis)65 or the IPDA (x-axis).20 Each point is the geometric mean of 3 replicates. Intact proviruses are corrected for shearing (DSI) and env+2LTR circles. The correlation between the two variables was calculated using Pearson’s coefficient. Data from one animal, T624, were excluded from this analysis due to failure of the gag amplicon resulting from mutations or deletion. (B) Correlation between gag+ SIV DNA copies and intact proviruses, with gag values corrected using the same env+2LTR correction factor applied to the IPDA data, calculated using Pearson’s coefficient. (C) Comparison of the decay of SIV gag copies and intact proviruses for the animals in cohort 18–02. IPDA data are corrected for env+2LTR circles and DNA shearing. Vertical lines represent the standard deviations. See also Figure S4.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Bacterial and virus strains SIVmac251 swarm 56 Dr. Dan Barouch, BIDMC N/A Biological samples N/A SIVmac251-infected Rhesus macaque PBMCs Dr. Dan Barouch, BIDMC N/A Chemicals, peptides, and recombinant proteins Platinum TaqDNA Polymerase High Fidelity ThermoFisher Scientific Cat # 11304011 SuperScript III Reverse Transcriptase ThermoFisher Scientific Cat # 18-080-044 dNTP Mix (10 mM each) ThermoFisher Scientific Cat # 18-427-088 UltraPure 1M Tris-HCl Buffer (pH 8.0) ThermoFisher Scientific Cat # 15-568-025 RNaseOUT Recombinant Ribonuclease Inhibitor ThermoFisher Scientific Cat # 10777019 Critical commercial assays QIAamp DNA Mini Kit Qiagen Cat # 51304 CD4+ T Cell Isolation Kit, Non-Human Primate Miltenyi Biotec Cat # 130-092-144 2X ddPCR Supermix for Probes (no dUTPs) Bio-Rad Cat # 186-3024 Deposited data SIV – plasma RNA sequences This paper Genbank: OQ168641-OQ168979 SIV – non-defective proviral DNA sequences This paper Genbank: OQ168980-OQ170751 SIV – hypermutated (defective) proviral sequences This paper Genbank: OQ170752-OQ170785 Experimental models: Organisms/strains Rhesus macaque (Macaca mulatta) infected with SIVmac251 Indian origin Animals T523, T530, T537, T544, T545, T623, T624, T625, T627 & T628 Oligonucleotides Oligonucleotides, standard desalting (Sanger sequencing & ddPCR) – see STAR Methods and Table S8 IDT N/A FAM/VIC labeled probes with MGB quencher, HPLC purified (see Table S8) Applied Biosystems N/A Unlabeled competition probes w/MGB quencher, HPLC purified (see Table S8) Applied Biosystems N/A FAM/HEX labeled probes w/MGB-NFQ quencher, HPLC purified (see Table S8) IDT N/A Recombinant DNA Synthetic double-stranded DNA controls (gBlocks) – see STAR Methods IDT N/A Software and algorithms QuantaSoft Analysis-Pro Bio-Rad http://www.bio-rad.com/en-us/ product/qx200-droplet-digitalpcr-system?ID=MPOQQE4VY Geneious Prime Dotmatics https://www.geneious.com/prime/ MEGA v7.0 & v11 MEGA https://www.megasoftware.net/ (Continued on next page) Cell Host & Microbe 31, 356–372.e1–e5, March 8, 2023 e1

Techniques: Comparison, Amplification