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