Fig. 2: Caspases inhibitors treatment did not protect H7N9-infected monocytes from cell death.

Purified CD14⁺ monocytes inoculated with A/Anhui/1/2013 (H7N9) virus at MOI of 2, after 1 h viral absorption at 37 °C, the following caspase inhibitors were added to the culture individually and further incubated: 20 µM of caspase-3 inhibitor (z-DEVD-FMK, C3i), caspase-8 inhibitor (z-IETD-FMK, C8i) or caspase-9 inhibitor (z-LEHD-FMK, C9i) and pan-caspase inhibitor IDN6556 (IDN, 10 µM). a Flow cytometry assay determined percentage of cleaved caspase-3 expressing cells in H7N9-infected monocytes treated or untreated with caspase inhibitors at 12 and 24hpi (n = 6 donors). b Cell viability determined by CellTiter-Glo luminescent assay at 12 and 24hpi (n = 6 donors). c Percentages of cleaved caspase-3 expressing cells in H7N9-infected monocytes treated or untreated with IDN at 12hpi (n = 8 donors). Error bars indicate standard error of the mean. ***p < 0.001 when compared with untreated control by one-way ANOVA. d Western blot determined cleavage of caspase-8, -9, -3 in H7N9-infected monocytes treated or untreated with IDN at 3, 6 and 12hpi. β-actin protein was used as the control for sample loading. e Representative images of TUNEL labelled (green) and DAPI (blue) stained mock- or H7N9-infected monocytes treated or untreated with IDN at 12hpi. Original magnification of 200x. f Cell viability of H7N9-infected with or without IDN treatment at 12, 24 and 48hpi (n = 4 donors). Error bars indicate standard error of the mean. ***p < 0.001 when compared with untreated control by one-way ANOVA. g Morphology examination of infected monocytes with or without IDN treatment at 12hpi. Representative images of immunofluorescence stained influenza A NP protein (green, upper) and bright field (BF, lower) images of the cells. Original magnification 400x