Y analyze and quantify the effects of perturbations with high confidence in all major steps of IAV entry, and in NP synthesis (Figure 3). The advantage of the single parameter approach is that one can interpret 1317923 the results intuitively. In Lammatory response are key determinants of the severity of the exacerbation contrast, machine learning does not require additional analysis steps by a computer Title Loaded From File vision expert, and the decision-making process is based solely on the expertise of the biologist. Our assay systems are sufficient to analyze the IAV entry pathway. In a modified form, they can be easily applied to other viruses and intracellular pathogens. They provide a platform to promote the understanding of dynamic biological processes through highcontent screening and will contribute to the discovery of anti-viral strategies that target host cell factors.siRNA TransfectionsiRNAs (AllStars, ATP6V1B2, ATP6AP2, ATP6V1A, CUL3, and CSE1L) were purchased from QIAGEN and reversetransfection was carried out with a final concentration 10 nM onto A549 cells in 24-well plates containing coverslips or 96-well optical-bottom Matrix plates (Thermo Scientific). The sequences of the above siRNAs are enlisted in the Table S3. LipofectamineFigure 3. Time-course of IAV entry as shown by individual assays. (a) Kinetics of IAV endocytosis in the `Pinda/perm HA’, `Pinda/HA’ and `perm Pinda/perm HA’ cells. Endocytosed IAV signal in the `Pinda/perm HA’cells peaks at 20 min post-infection. (b) Acidification time-course in the cells treated with AllStars negative and ATP6V1B2 siRNAs, and the cells treated with 50 nM Bafilomycin A1 (BafA1) to block endosomal acidification. The acidification signal in the AllStars negative siRNA-treated cells reaches the peak at 1 h post-infection. (c) Kinetics of viral fusion, which shows the dequenching signal from DiOC18(3) in the AllStars negative siRNA-treated cells peaks at 1.5 h post-infection. (d) Nucleocapsid uncoating time-course indicating the peak of M1 dispersal signal is at 3 h post-infection. (e) Nuclear import time course shows that the import plateaus at 3.5 h postinfection in the control cells. (f) Kinetics of infection (transcription and translation of NP), which shows that the optimal time for the detection of cells with newly synthesized NP is 8 h post-infection. Z’ factor values are represented by * – between 0 and 0.5; ** – between 0.5 and 0.8; and ***.0.8. doi:10.1371/journal.pone.0068450.gHigh-Content Analysis of IAV Entry EventsRNAiMax (Invitrogen) and D-MEM were mixed at a ratio 1:150. siRNAs were added, gently mixed, and incubated at room temperature (RT) for 1 h. Cells were trypsinized, counted and plated directly onto the siRNA-lipofectamine complex mixture. The number of cells plated in each well of the 24-well and 96-well plates was 12500 and 1500, respectively. Following transfection, the cells were kept in a 5 CO2 incubator at 37uC for 72 h, after which the entry assays were performed.Antibodies and ReagentsAnti-X31 rabbit polyclonal antibody (Pinda) and anti-HA monoclonal antibody (A1) specific for the post-acid conformation of HA have been previously described [6,7]. Hybridoma cell lines producing monoclonal antibody against IAV matrix protein (HB64), and nucleoprotein (HB65) were purchased from ATCC. Anti-ATP6V1B2 and anti-b actin antibodies were purchased from LifeSpan Biosciences and Sigma-Aldrich, respectively. R18 and SP-DiOC18(3) (Invitrogen) were re-suspended in EtOH and used at a final concentration of 0.4 mM and 0.2 mM, respectively. Labeling was performed as pre.Y analyze and quantify the effects of perturbations with high confidence in all major steps of IAV entry, and in NP synthesis (Figure 3). The advantage of the single parameter approach is that one can interpret 1317923 the results intuitively. In contrast, machine learning does not require additional analysis steps by a computer vision expert, and the decision-making process is based solely on the expertise of the biologist. Our assay systems are sufficient to analyze the IAV entry pathway. In a modified form, they can be easily applied to other viruses and intracellular pathogens. They provide a platform to promote the understanding of dynamic biological processes through highcontent screening and will contribute to the discovery of anti-viral strategies that target host cell factors.siRNA TransfectionsiRNAs (AllStars, ATP6V1B2, ATP6AP2, ATP6V1A, CUL3, and CSE1L) were purchased from QIAGEN and reversetransfection was carried out with a final concentration 10 nM onto A549 cells in 24-well plates containing coverslips or 96-well optical-bottom Matrix plates (Thermo Scientific). The sequences of the above siRNAs are enlisted in the Table S3. LipofectamineFigure 3. Time-course of IAV entry as shown by individual assays. (a) Kinetics of IAV endocytosis in the `Pinda/perm HA’, `Pinda/HA’ and `perm Pinda/perm HA’ cells. Endocytosed IAV signal in the `Pinda/perm HA’cells peaks at 20 min post-infection. (b) Acidification time-course in the cells treated with AllStars negative and ATP6V1B2 siRNAs, and the cells treated with 50 nM Bafilomycin A1 (BafA1) to block endosomal acidification. The acidification signal in the AllStars negative siRNA-treated cells reaches the peak at 1 h post-infection. (c) Kinetics of viral fusion, which shows the dequenching signal from DiOC18(3) in the AllStars negative siRNA-treated cells peaks at 1.5 h post-infection. (d) Nucleocapsid uncoating time-course indicating the peak of M1 dispersal signal is at 3 h post-infection. (e) Nuclear import time course shows that the import plateaus at 3.5 h postinfection in the control cells. (f) Kinetics of infection (transcription and translation of NP), which shows that the optimal time for the detection of cells with newly synthesized NP is 8 h post-infection. Z’ factor values are represented by * – between 0 and 0.5; ** – between 0.5 and 0.8; and ***.0.8. doi:10.1371/journal.pone.0068450.gHigh-Content Analysis of IAV Entry EventsRNAiMax (Invitrogen) and D-MEM were mixed at a ratio 1:150. siRNAs were added, gently mixed, and incubated at room temperature (RT) for 1 h. Cells were trypsinized, counted and plated directly onto the siRNA-lipofectamine complex mixture. The number of cells plated in each well of the 24-well and 96-well plates was 12500 and 1500, respectively. Following transfection, the cells were kept in a 5 CO2 incubator at 37uC for 72 h, after which the entry assays were performed.Antibodies and ReagentsAnti-X31 rabbit polyclonal antibody (Pinda) and anti-HA monoclonal antibody (A1) specific for the post-acid conformation of HA have been previously described [6,7]. Hybridoma cell lines producing monoclonal antibody against IAV matrix protein (HB64), and nucleoprotein (HB65) were purchased from ATCC. Anti-ATP6V1B2 and anti-b actin antibodies were purchased from LifeSpan Biosciences and Sigma-Aldrich, respectively. R18 and SP-DiOC18(3) (Invitrogen) were re-suspended in EtOH and used at a final concentration of 0.4 mM and 0.2 mM, respectively. Labeling was performed as pre.
