Supplementary MaterialsS1 Fig: Western blot quantifications for Fig 1 and mTORC1 activation in Rictor knockdown cells. or (C) HBEC30KT cells were infected AZ 3146 enzyme inhibitor with WSN for 6 h or 8 h, respectively, at MOI of 2 PFU/cell. A549 cells were infected with (D) Sh/1 (H7N9), (E) rSh/1 (recombinant Sh/1) and WSN (H1N1), (F) VSV-GFP, WSN (or treated with 5% serum for 7 h) for 6h at MOI of 2 PFU/cell. Immunoblot analyses were performed for detection of viral proteins (influenza computer virus M1 or VSV M) or host proteins (total and phosphorylated S6K and 4E-BP1). Total S6K serves as the loading control. The upper band in the S6K/p-S6K blots is usually p85 S6K, whereas the lower band is usually p70 S6K. Data are representative of three impartial experiments.(TIF) ppat.1006635.s003.tif (854K) GUID:?CBA75387-03A5-46BA-8955-41044F3E0B5D S4 Fig: Autophagy, M2, and IFN expression are not required for mTORC1 activation by influenza computer virus. (A) A549 cells were infected with wild-type PR8:WSN or PR8:WSNDeficientM2 at MOI of 2 PFU/cell for 6 h. (B) A549 cells were transfected with the indicated siRNAs for 48 h followed by contamination with WSN at MOI of 2 PFU/cell for 6 h. (C) and Gata3 MEFs were AZ 3146 enzyme inhibitor infected with WSN at MOI of 2 PFU/cell for 6 h. Immunoblot analyses were performed with antibodies against the depicted proteins. Total S6K serves as the loading control. Data are representative of three (A) or two (B,C) impartial experiments. (D) UV inactivation of WSN. WSN was UV-inactivated for 7 moments under UV light. WSN and UV-inactivated WSN (UV WSN) were subjected to both plaque assay and HA assay to confirm UV inactivation prior to contamination by assessing infectious computer virus (PFU/mL) and quantifying virions (HA unit/50 l). These assays were carried out each time WSN was UV-inactivated prior to contamination. (E) Poly(I:C) activation does not induce mTORC1 activiation. MEFs were non-treated or treated with rapamycin (250nM) or Torin (250nM) AZ 3146 enzyme inhibitor and transfected with high molecular excess weight (HMW) poly(I:C) at 1 g/ml for the indicated time points. Cell lysates were subjected to immunoblot analysis with the indicated antibodies. Mito70 was used as loading control. (F) As control for E, MEFs were also mock infected or infected with influenza A computer virus at MOI of 2 PFU/cell. Cell extracts were obtained at 8h post-infection and subjected to immunoblot analysis with the depicted antibodies. (G) MEFs were AZ 3146 enzyme inhibitor mock transfected or transfected with HMW poly(I:C) at 0.5 g/ml for 6 and 12h. Total RNA was extracted at the indicated time points post-transfection and the relative large quantity of mouse IFN was measured by real time PCR. Data from triplicate experiments were normalized to -Actin.(TIF) ppat.1006635.s004.tif (1.3M) GUID:?13A2303B-4A60-4A2E-8762-6CA27D357753 S5 Fig: Quantification of Fig 4A. Western blots shown in Fig 4A were quantified and normalized to respective controls, as depicted in this physique, using AZ 3146 enzyme inhibitor the ImageJ64 analysis.(TIF) ppat.1006635.s005.tif (3.9M) GUID:?30E9E7F4-B42F-45D3-BCEE-8F846DB7020C S6 Fig: Cell viability at multiple times during Torin1 treatment and viral replication. (A) A549 cells were treated with 0.1% DMSO or 250 nM Torin1 for the indicated occasions. Cell viability was determined by measuring ATP levels and calculated as a percent of the DMSO control. (B) A549 cells were infected with WSN at MOI of 2 PFU/cell for 1 h and then treated with 250 nM Torin1 or DMSO for an additional 9 h. QPCR was performed to measure viral mRNA levels. Mean and SD are shown, = 3, ***p 0.001. (C) A549 cells were infected for 24h with rSh/1 at MOI of 0.001 in the absence or presence of Torin. Viral titers were measured by plaque assay. Error bars are SEM, = 9, **p 0.01..