Impact of ATG16L1, FAM126A and INPP5K depletion on early, intermediate and late stages of IAV infection (related to Fig 4).
<p><b>A, B.</b> A549 cells were treated with the indicated siRNA for 48 h and subsequently infected with WSN at a MOI of 5 PFU/cell, in the presence (+) or absence (−) of cycloheximide (CHX). (A) At 6 hpi, total cell lysates were prepared and the steady-state levels of viral protei...
محفوظ في:
| المؤلف الرئيسي: | |
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| مؤلفون آخرون: | , , , , , , , , , , , |
| منشور في: |
2025
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| الموضوعات: | |
| الوسوم: |
إضافة وسم
لا توجد وسوم, كن أول من يضع وسما على هذه التسجيلة!
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| الملخص: | <p><b>A, B.</b> A549 cells were treated with the indicated siRNA for 48 h and subsequently infected with WSN at a MOI of 5 PFU/cell, in the presence (+) or absence (−) of cycloheximide (CHX). (A) At 6 hpi, total cell lysates were prepared and the steady-state levels of viral proteins were analyzed by western blot, to control for the efficiency of CHX treatment (no viral protein expression). (B) At 6 hpi, total RNA were extracted and the levels of NP-mRNAs and NP-vRNAs were analyzed by strand-specific RTqPCR. The results of three independent experiments, labeled #1, #2, and #3 in (A), are shown. Dotted line: background for mRNA detection in mock-infected cells. Two-way ANOVA with Dunnett’s multiple comparison test; using the siNT without/with CHX samples as a reference for the other without/with CHX samples, respectively, revealed no statistically significant differences. <b>C, D.</b> A549 cells were treated with the indicated siRNA for 48 h and subsequently infected with WSN at a MOI of 5 PFU/cell or mock-infected. At 5 hpi, total cell lysates were prepared and the steady-state levels of viral proteins were analyzed by western blot. (C) Cropped blots of three independent experiments, labeled #1, #2, and #3, are shown. (D) The signals for HA, NP ad NS1 were normalized over the α-tubulin signal (α-Tub) and expressed as percentages (100%: NT siRNA). The data shown are the mean ± SD of the three independent experiments. *: <i>p</i>-value < 0.05, **: <i>p</i>-value < 0.01, ***: <i>p</i>-value < 0.001 (two-way ANOVA with Dunnett’s multiple comparison test, reference: siNT). <b>E.</b> A549 cells were treated with the indicated siRNA for 48 h and subsequently infected with WSN at a MOI of 5 PFU/mL. At 3 or 4 hpi, the culture medium was extensively washed away four times to remove the viral input and replaced with fresh medium. One hour later, i.e., at 4 or 5 hpi, respectively, the supernatants were collected. The last wash (residual input) and collected supernatants were titrated by a plaque assay. The titers were normalized over the control condition (NT siRNA). The data shown are the mean ± SD of the three independent experiments. *: <i>p</i>-value < 0.05, **: <i>p</i>-value < 0.01, ***: <i>p</i>-value < 0.001 (two-way ANOVA with Dunnett’s multiple comparison test, reference: siNT). The dotted lines represent the average ratio value for residual inputs. <b>F.</b> A549 cells were infected with WSN at a MOI of 5 PFU/cell for 4 h, or mock-infected. Fixed cells were stained for PI4P using the SNAP-SidC probe. Nuclei were stained with DAPI (blue), and cells were imaged with a confocal microscope. Scale bar: 10 µm. <b>G.</b> A549 cells treated as in (F) were analyzed with the Fiji software to determine the mean intensity of the PI4P signal per cell. Each dot represents one cell, and the data from three independent experiments are shown (black, gray and blue dots). The median and standard deviation values are represented (115–144 cells per condition). <b>****</b>: <i>p</i>-value < 0.0001, unpaired <i>t</i> test. The data underlying this figure can be found at h<i>tt</i>ps://zenodo.org/records/15682874 (raw images), <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.3002958#pbio.3002958.s013" target="_blank">S4 File</a> (uncropped western blots) and <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.3002958#pbio.3002958.s015" target="_blank">S6 File</a> (graphs raw data).</p> <p>(TIF)</p> |
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