Fig 2: Flow cytometric assessment of SDC expression and SARS-CoV-2’s and its spike protein’s uptake in human and murine cell lines. SDC expression of L929, RAW, and K562 cells was analyzed with imaging flow cytometry using fluorescently labeled SDC antibodies. (A,B) Representative flow cytometry histograms and fluorescent images showing SDC expression levels in L929, RAW, and K562 cells. Scale bar = 20 µm. (C) Detected SDC expression level cells were normalized to K562s. The bars represent the mean + SEM of three independent experiments. Statistical significance was assessed with analysis of variance (ANOVA). * p < 0.01; ** p < 0.001. (D–G) L929, RAW, and K562 cells and SDC4 transfectants (created in K562 cells) were incubated with either inactivated SARS-CoV-2 (at 1 MOI) or spike protein (50 nM) for 3 h at 37 °C. After incubation, the cells were washed, trypsinized, fixed, permeabilized, and treated with antibodies specific for the spike glycoprotein (and AF 488-labeled secondary antibodies). Cellular uptake of SARS-CoV-2 and spike was then analyzed with imaging flow cytometry. (D,E) Representative flow cytometry histograms and fluorescent images showing the intracellular fluorescence of SARS-CoV-2- and spike-treated cells. Scale bar = 20 µm. (F,G) Detected fluorescence intensities were normalized to SARS-CoV-2- or spike-treated K562 cells as standards. The bars represent the mean + SEM of four (F) and three (G) independent experiments. Statistical significance vs. standards was assessed with ANOVA. * p < 0.05; ** p < 0.01; *** p < 0.001.

Fig 3: Colocalization of SDC4 with SARS-CoV-2 in various organs of the mice. Liver, heart, lung, spleen, and brain samples of mice receiving i.v. administration of inactivated SARS-CoV-2. Tissue SDC4 is detected with an APC-labeled SDC4 antibody, while SARS-CoV-2 with an AF 488-labeled spike antibody. Scale bar = 20 µm. MOC ± SEM and PCC ± SEM for the overlap and colocalization of SARS-CoV-2 with SDC4 were calculated by analyzing 18 images from 3 samples of each animal.

Fig 4: Effect of SDC4 knockdown (KD) on the internalization of inactivated SARS-CoV-2 into L929 and RAW cells. SDC4 KD in L929 and RAW cells was performed using a lentiviral vector specific to mouse SDC4. (A) Western blot validation of SDC4 kD in L929 and RAW cells. ß-tubulin was used as a loading control. (B) SDC4 expression levels were also measured with imaging flow cytometry, as shown by the representative histograms and cellular images. Scale bar = 20 µm. Detected SDC4 levels of KD cells were normalized to WT cells as standards. (C,D) WT and SDC4 KD cells were incubated with inactivated SARS-CoV-2 (1 MOI) for 3 h at 37 °C. After incubation, the cells were washed, trypsinized, fixed, permeabilized, and treated with antibodies specific for the spike glycoprotein (and AF 488-labeled secondary antibodies). Cellular uptake of SARS-CoV-2 and spike was then analyzed with imaging flow cytometry. (C) Representative flow cytometry histograms and cellular images show the intracellular fluorescence of SARS-CoV-2-treated WT or SDC4 KD cells. Scale bar = 20 µm. (D) The effect of SDC4 KD on SDC4 expression and SARS-CoV-2 uptake expressed as percent decrease. The bars represent the mean + SEM of three independent experiments. Statistical significance vs. controls was assessed with ANOVA. * p < 0.05; ** p < 0.01; *** p < 0.001.