Additional file 2: Figure S1. of Involvement of fatty acid synthase in dengue virus infection
Creators: name: Natthida Tongluan
name: Suwipa Ramphan
name: Phitchayapak Wintachai
name: Janthima Jaresitthikunchai
name: Sarawut Khongwichit
name: Nitwara Wikan
name: Supoth Rajakam
name: Sutee Yoksan
name: Nuttaporn Wongsiriroj
name: Sittiruk Roytrakul
name: Smith, Duncan
Publisher: name: Figshare
Year: 2017
Subjects: subject: Biochemistry
subject: Microbiology
subject: FOS: Biological sciences
subject: FOS: Biological sciences
subject: Cell Biology
subject: Molecular Biology
subject: 39999 Chemical Sciences not elsewhere classified
subject: FOS: Chemical sciences
subject: FOS: Chemical sciences
subject: Developmental Biology
subject: Cancer
subject: 110309 Infectious Diseases
subject: FOS: Health sciences
subject: FOS: Health sciences
subject: Plant Biology
subject: 60506 Virology

description: Real-time PCR validation of siRNA mediated gene silencing of fatty acid synthase (FASN) gene. HEK293T/17 cells were not treated (mock) or treated with a siRNA control (GFP) or treated with one of four siRNAs directed to FASN (FASN1 to FASN 4). On days 1 to 5 post transfection the level of FASN transcript was determined by real time PCR. Normalization expression data relative to actin is shown. Bars show mean +/−SD. (a) 1 day post transfection, (b) 2 days post transfection, (c) 3 days post transfection, (d) 4 days post transfection and (e) 5 days post transfection. Bars show mean +/−SD (*; p value &lt;0.05). Figure S2. Assessment of cell viability after siRNA transfection. HEK293T/17 cells were not treated (mock) or treated with a siRNA control (GFP) or treated with siRNAs directed to FASN (FASN1 and FASN 4). On day 2 post transfection cell viability was assessed by trypan blue staining and counting cells using a hemocytometer. Bars show mean +/−SD. Figure S3. Real-time PCR validation of siRNA mediated gene silencing of fatty acid synthase (FASN) gene. HEK293T/17 cells were not treated (mock) or treated with a siRNA control (GFP) or treated with one of four siRNAs directed to FASN (FASN1 to FASN 4). On day 2 post transfection (a) the level of FASN transcript was determined by real time PCR and (b) amplification product was run on an agarose gel and products visualized after ethidium bromide staining. Normalization expression data relative to actin is shown. Bars show mean +/−SD (*; p value &lt;0.05). Figure S4. Western analysis of FASN expression after siRNA treatment. HEK293T/17 cells were not treated (mock) or treated with a siRNA control (GFP) or treated with one of four siRNAs directed to FASN (FASN1 to FASN 4). On days 1 to 4 post transfection the level of FASN protein was determined by western blot analysis. Normalization expression data relative to actin is shown. Bars show mean +/−SD (* p value &lt;0.05; ** p value &lt;0.01). Figure S5. Determination of orlistat cytotoxicity to HEK293T/17 cells. HEK293T/17 cells were incubated with different concentrations of orlistat or not treated (−) for (a) 24 h or (b) 36 h followed by MTT cell viability assays. Data is derived from 8 replicates. Treatment with 5% DMSO was used as a positive control. Bars show mean +/−SD (*; p value &lt;0.05). Figure S6. The morphology of HEK293T/17 cells after orlistat treatment. HEK293T/17 cells were incubated with different concentrations of orlistat or not treated (mock) for (a) 24 h or (b) 36 h followed by observation under an inverted microscope. Magnification × 20. Figure S7. Evaluation of virucidal activity of orlistat. Stock DENV-2 was incubated with orlistat at concentrations of 1, 10, 20, 50 μM for 1 h and then used in the standard infection protocol. At 24 h.p.i (a) flow cytometry was performed to determine the percentage of infection and (b) supernatants were used to determine the virus titers. No deficit was observed in either percentage cell infection or virus titer. (PDF 2701 kb)