Fig. 5.

ORC6 is important for LPS-induced NFκB activation in macrophages/monocytes. Stable THP-1 human macrophages with the specific ORC6 shRNAs (“shORC6-1/-4/-6”, encompassing three distinct sequences) or a scramble control non-sense shRNA (“shC”), the Cas9-expressing THP-1 macrophages with the lentiviral CRISPR-ORC6-KO construct (koORC6-Clo1 or koORC6-Clo2, representing two clones) or the control cells with the control construct (“koC”) as well as THP-1 macrophages with the ORC6-expressing lentiviral construct (oeORC6-Slc1 or oeORC6-Slc2, two stable clones) or the empty vector (“Vec”) were treated with or without LPS (100 ng/mL) for 2 h, NFκB activation was tested by p65 DNA-binding assay (A-C). The stable peripheral blood mononuclear cells (PBMCs) or bone marrow-derived macrophages (BMDMs) with the specific ORC6 shRNA or a scramble control non-sense shRNA (“shC”) were treated with or without LPS (100 ng/mL) for 2 h, NFκB activation was tested by p65 DNA-binding assay (D and E). The koC, koORC6Clo2, Vec or oeORC6-Slc1 THP-1 macrophages were treated with LPS (100 ng/mL) for 1 h, expression levels of listed proteins in total cell lysates were tested (F, H, I and K), and the AP-1 luciferase activity was also tested (G and J). The designation “C” denotes vehicle control treatment. Data were presented as mean ± standard deviation (SD, n = 5), with statistical significance indicated by *P < 0.05 compared to “C” and ^#P < 0.05 compared to LPS treatment in “shC”/“Vec”/“koC” cells. Non-statistically significant differences (P > 0.05) were denoted as “N. S.“. These experiments were repeated five times, yielding consistent results across replicates