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Fig. EV1 Characterization of monocytic Occludin expression. (A) Schematic representation of a CRISPR/Cas9 lentiviral construct used for the generation of OCLN KO THP-1 cells with indicated target sequences. (B) Western Blot analysis of OCLN expression in indicated cell lines. Actin is used as a loading control. The hCMEC/D3 and THP-1 cells express OCLN (two bands), the THP-1 OCLN KO cells do not show OCLN, although non-specific bands of lower size and very weak intensity can be observed. As controls, NIH 3T3 do not express OCLN and THP-1 WT transduced with EGFP-OCLN express both endogenous and overexpressed OCLN. (C) Western Blot analysis of OCLN expression in THP-1 OCLN KO cells. GAPDH is used as a loading control. EGFP-OCLN can be seen with both anti-Occludin and anti-GFP antibody (green arrow at EGFP). EGFP-OCLN-?C and EGFP-CAAX can be seen with anti-GFP antibody only (red and yellow arrows, respectively). (D) Mean fluorescence intensity of EGFP-CAAX, EGFP-OCLN, or EGFP-OCLN-?C expressed by THP-1 OCLN KO cells measured by flow cytometry. The data were normalized to EGFP-CAAX obtained from n = 3 individual experiments. Each symbol?s color corresponds to an individual experiment. (E) Western Blot showing endogenous OCLN expression in human primary monocytes and exogenous expression of EGFP-OCLN in the same donors (2 donors). GAPDH is used as a loading control. (F) Immunofluorescence images of primary monocytes transduced with EGFP-OCLN, EGFP-CAAX, or non-transduced, fixed and stained with anti-OCLN antibody (cyan) and DAPI (yellow). EGFP is in magenta. Scale bar: 2 ?m. (G) Efficiency of transduction of primary monocytes from 2 donors performed in two technical replicates measured by flow cytometry. (H) Mean fluorescence intensity of EGFP-CAAX, EGFP-OCLN or EGFP-OCLN-?C expressed by primary monocytes. The data were obtained from n = 6 monocyte donors normalized to EGFP-CAAX of each experiment. Each symbol?s color corresponds to an individual monocyte donor. In (D, G, H), the data are presented as mean ± SEM. Source data are available online for this figure.