Fig. 2: Investigation of the function of ALCAM on HAdV-C5/B7-GFP infection in HEK-293A cells. | Nature Communications

Fig. 2: Investigation of the function of ALCAM on HAdV-C5/B7-GFP infection in HEK-293A cells.

From: ALCAM is an entry factor for severe community acquired Pneumonia-associated Human adenovirus species B

Fig. 2

a Analysis of ALCAM transcriptomic expression in the blood samples from SCAP patients with different pathogen infections. FPKM, Fragments Per Kilobase of exon model per Million mapped fragments. N.A., no available identifiable pathogen information. Others, all pathogens excluding HAdV. All analyzed SCAP patients. Data are presented as mean ± standard deviation (SD) (n ≥ 3) from different patients. Statistical analyses are performed between adenovirus group and other groups using two-tailed unpaired Student’s t test. Evaluation of the effects of ALCAM knockdown on HAdV-C5/B7-GFP infection in HEK-293A cells, as determined by GFP fluorescence using flow cytometry (b) or viral L3 DNA levels using qPCR quantification (c). Assays are performed at 48 h post infection. NC, negative control using scrambled siRNA. Rplp0 is used as an internal control for qPCR. Data are presented as mean ± SD (n = 3) from three independent biological replicates. The significant difference between siRNA-NC and ALCAM-siRNA groups is analyzed by two-tailed unpaired Student’s t test. d Flow chart showing the procedure of competition assay. The cell populations to be compared are highlighted in red boxes. Flow cytometry analysis of HAdV-C5/B7-GFP infection in the mixed population of WT cells with non-targeting (e) or ALCAM knockout (f) cells at 24 h, 48 h and 72 h post infection. ALCAM KO mix, ALCAM knockout on mixed population without isolation of single clones. g Flow cytometry analysis of HAdV-C5/B7-GFP infection in non-targeting sgRNA, ALCAM-/- and ALCAM overexpression-rescued cells at 48 h post infection. h Flow cytometry analysis of HAdV-C5/B7-GFP infection in the mixed population of WT cells with non-targeting sgRNA or ALCAM knockout single clone at 24 h and 48 h post infection in the competition asssay. For all competition experiments, WT group is used as an internal control, and non-targeting sgRNA and ALCAM-/- groups are normalized to WT. For e-h, data are presented as mean ± SD (n = 3) from three independent biological replicates. For (e, f) the significant difference is determined using two-way ANOVA with Sidak’s multiple comparisons test. The p values between WT and ALCAM KO mix at 48 h post infection is 0.000098. For (a–c and g, h), the significant difference is analyzed by two-tailed unpaired Student’s t test unless noted otherwise. Source data are provided as a Source Data file.

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