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. Author manuscript; available in PMC: 2019 Sep 1.

Published in final edited form as: Mol Immunol. 2018 Aug 9;101:450–460. doi: 10.1016/j.molimm.2018.08.005

Figure 1. — (A) SAMHD1 KO clones were prepared by using the CRISPR-Cas9 system. Three different THP-1 SAMHD1 KO clones (SAMHD1 KO 1, 2 and 1-3) were identified by Western blotting using antibodies against SAMHD1 (upper panel). THP-1 clones that underwent the CRISPR-Cas9 protocol but did not lose expression of SAMHD1 (Controls 1 and 2) were also identified (upper panel). In addition, a wild type THP-1 cell line (WT) was included. As a loading control, samples were also analyzed for GAPDH expression (upper panel). (B) Transcript levels of MXA and IFIT1 were analyzed by qRT-PCR in the indicated THP-1 SAMHD1 KO clones, and were compared to THP-1 control cells using a specific set of primers for each indicated gene (lower panel). Transcript levels were normalized to actin, and fold-induction relative to the THP-1 Control 1 is shown. P < 0.05 (*), P < 0.01 (**) or P < 0.001 (***) using two tailed Student’s t-test are shown. (C) Similarly the transcript levels of IFNβ were measured. Transcript levels were normalized to actin, and fold-induction relative to the THP-1 Control 1 is shown. (D) MXA, MXB and ISG15 protein levels were analyzed by Western blotting in the indicated THP-1 SAMHD1 KO clones. Expression levels for THP-1 Control clones are also shown. As a loading control, samples were also analyzed for GAPDH. Quantification of band intensities normalized to GAPDH is shown. Experiments were repeated at least three times and a representative example is shown. (E) Protein levels of MXA, MXB and ISG15 were also analyzed in PMA-treated THP-1 SAMHD1 KO clones. Quantification of band intensities normalized to GAPDH is shown. Experiments were repeated at least three times and a representative example is shown.

Figure 1. — (A) SAMHD1 KO clones were prepared by using the CRISPR-Cas9 system. Three different THP-1 SAMHD1 KO clones (SAMHD1 KO 1, 2 and 1-3) were identified by Western blotting using antibodies against SAMHD1 (upper panel). THP-1 clones that underwent the CRISPR-Cas9 protocol but did not lose expression of SAMHD1 (Controls 1 and 2) were also identified (upper panel). In addition, a wild type THP-1 cell line (WT) was included. As a loading control, samples were also analyzed for GAPDH expression (upper panel). (B) Transcript levels of MXA and IFIT1 were analyzed by qRT-PCR in the indicated THP-1 SAMHD1 KO clones, and were compared to THP-1 control cells using a specific set of primers for each indicated gene (lower panel). Transcript levels were normalized to actin, and fold-induction relative to the THP-1 Control 1 is shown. P < 0.05 (*), P < 0.01 (**) or P < 0.001 (***) using two tailed Student’s t-test are shown. (C) Similarly the transcript levels of IFNβ were measured. Transcript levels were normalized to actin, and fold-induction relative to the THP-1 Control 1 is shown. (D) MXA, MXB and ISG15 protein levels were analyzed by Western blotting in the indicated THP-1 SAMHD1 KO clones. Expression levels for THP-1 Control clones are also shown. As a loading control, samples were also analyzed for GAPDH. Quantification of band intensities normalized to GAPDH is shown. Experiments were repeated at least three times and a representative example is shown. (E) Protein levels of MXA, MXB and ISG15 were also analyzed in PMA-treated THP-1 SAMHD1 KO clones. Quantification of band intensities normalized to GAPDH is shown. Experiments were repeated at least three times and a representative example is shown.