Extended Data Fig. 1: SIVmac infection- mediated SAMHD1 depletion does not induce immune activation in THP-1, EA.hy926, HEK293T and HeLa cells.

A, Detection of endogenous STING and cGAS expression levels in THP-1, U937, HFF, and EA.hy926 cells (n = 2 independent biological experiments). B, THP-1 cells were treated with PMA (100 nM) for 4h and subsequently treated with 2’3’-cGAMP (4 μg/mL) for 12h. Total RNA was prepared and analyzed for the transcriptional level of the indicated genes by RT-qPCR (n = 3 independent biological experiments). Means and standard deviations are presented. The statistical significance analyses were performed using a two-sided unpaired t-test. C,D, THP-1 cells were infected with an equal amount of SIVmac or SIVmacΔVpx virus for 12h. Cell lysates were analyzed by immunoblotting using SAMHD1 and CAp27 antibodies (C) (n = 3 independent biological experiments). Total RNA was prepared and analyzed for the indicated genes by RT-qPCR (D) (n = 3 independent biological experiments). Means and standard deviations are presented (B, D). The statistical significance analyses were performed using a two-sided unpaired t-test. E, EA.hy926 cells were transfected with ISD (2 μg/mL) for 12h, and total RNA was prepared and analyzed for the indicated genes by RT-qPCR (n = 3 independent biological experiments). Means and standard deviations are presented. The statistical significance analyses were performed using a two-sided unpaired t-test. F,G, EA.hy926 cells were infected with an equal amount of SIVmac or SIVmacΔVpx virus for 12h. RT- qPCR (F) and immunoblotting (G) were performed as described in C,D (n = 3 independent biological experiments). Means and standard deviations are presented. The statistical significance analyses were performed using a two-sided unpaired t-test. H, Endogenous SAMHD1 and p-SAMHD1 protein expression levels in THP-1, U937, and EA.hy926 cells (One representative immunoblotting result out of n = 2 independent biological experiments is shown.). I,K, HEK293T cells or J,L, HeLa cells were infected with an equal amount of SIVmac or SIVmacΔVpx virus for 12h. Immunoblotting and RT- qPCR were performed as described in C,D (n = 3 independent biological experiments). Means and standard deviations are presented (K, L). The statistical significance analyses were performed using a two-sided unpaired t-test. M, HEK293T, HeLa, EA.hy926, THP-1, CD4+ T cells, and MDM cells were infected with equal amounts of SIVΔEnv GFP or SIVΔEnvΔVpx GFP virus for 48h. The infected cells were then harvested, and GFP-positive cells were tested by flow cytometry (n = 3 independent biological experiments). Means and standard deviations are presented. The statistical significance analyses were performed using two-sided unpaired t-test.