Immunoblotting was performed using TCLs prepared from TREx-F3H3-K-Rta BCBL-1 cells; untreated, or treated with 20 ng/ml TPA and 0

Immunoblotting was performed using TCLs prepared from TREx-F3H3-K-Rta BCBL-1 cells; untreated, or treated with 20 ng/ml TPA and 0.2 g/ml Dox for the time period shown. anti-K-Rta antibody to confirm the induction of K-Rta in TREx-F3H3-K-Rta BCBL-1 cells after 0.2 g/ml Dox treatment for 12 hours. Anti–tubulin antibody was utilized for loading control. (B) ChIP assay for SUMO paralogs was performed using chromatin prepared from non-induced (0 hour) and 0.2 g/ml Dox-treated (12 hours) TREx-F3H3-K-Rta BCBL-1 cells Romidepsin (FK228 ,Depsipeptide) using anti-SUMO-1 or SUMO-2/3 antibody. SUMO-1 and SUMO-2/3 binding to promoters in SUMO-2/3 enrichment and H3K9me3-rich areas were analyzed by real-time qPCR. ChIP enrichment was computed by comparing values from Dox-treated samples to their non-induced settings. Rabbit IgG was used as bad antibody control and IgG enrichment is very low or not visible in both ChIP assays.(TIFF) ppat.1005051.s002.tiff (2.9M) GUID:?7E1EEC4A-E72F-4058-98C2-D13E1E7FC98F S3 Fig: Deposition of SUMO-1 and SUMO-2 modification about determined KSHV promoters representing high SUMO-2/3 enrichment or H3K9me3-rich regions during reactivation. (A) TCLs from non-induced (0 hour) and 0.2 g/ml Dox-treated (12 hours) TREx-MH-K-Rta BCBL-1 and TREx-MH-K-Rta-Flag-SUMO-1 and -Flag-SUMO-2 BCBL-1 cells were analyzed by immunoblotting using anti-Flag and anti-K-Rta antibodies. Anti–tubulin antibody was utilized for loading control. Ratio for each construct is the Flag/-tubulin transmission observed for Dox treatment at 12 hour using Dox at 0 hour arranged as 1.0. (B) ChIP was performed using chromatin prepared from cells treated as explained in (A) using anti-Flag antibody. Rabbit IgG was used as bad antibody control and enrichment is not visible in most cases. SUMO-1 and SUMO-2 binding to SUMO-2/3 enrichment and H3K9me3-rich regions Romidepsin (FK228 ,Depsipeptide) were analyzed by real-time qPCR using primer pairs as explained in Fig 2.(TIFF) ppat.1005051.s003.tiff (2.8M) GUID:?18D0F86D-F480-4EF3-8D6C-CFC27BC4BE9B S4 Fig: Knockdown SUMO-2/3 does not induce the expression of viral lytic genes located in the high SUMO-2/3 enrichment region in latent KSHV infected BCBL-1 cells. (A) pLenti4-shSUMO-2 and -3 were launched into TREx-BCBL-1 cells by lentiviral transduction. 0.2 g/ml of Dox was added to cells 48 hours after transduction to induce SUMO-2/3 knockdown. TCLs were collected 24 hours after Dox treatment and analyzed by immunoblotting using anti-SUMO-2/3 antibody. Anti–tubulin antibody was utilized for loading control. (B Romidepsin (FK228 ,Depsipeptide) and C) Total RNA isolated from cells treated as explained in (A) was reverse transcribed using oligo-d(T)18 primer. The manifestation Romidepsin (FK228 ,Depsipeptide) level of SUMO-2, SUMO-3 (B), and four viral genes representing SUMO-2/3 enrichment region as explained in Fig 1 (C) were quantified by real-time qPCR. All reactions were run in triplicate and normalized against GAPDH. Col13a1 The fold was computed by using non-induced settings as 1.0. **; P 0.005. NS; non-significant.(TIFF) ppat.1005051.s004.tiff (1.9M) GUID:?504C6FEA-82F4-4530-A2BD-3A52F3F17EC3 S5 Fig: SUMO-2/3 knockdown in SUMO-2/3 inducible knockdown BCBL-1 cells. TCLs isolated from non-induced (0 hour) and 0.2 g/ml Dox-induced (for 12, 24 and 48 hours) TREx-F3H3-K-Rta BCBL-1 and TREx-F3H3-K-Rta-shSUMO-2/3 BCBL-1 cells were subjected to immunoblotting analysis using anti-SUMO-2/3 antibody. Induction of K-Rta and manifestation of K-bZIP was confirmed by using anti-K-Rta and anti-K-bZIP antibodies. Anti-GAPDH antibody was utilized for loading control. Ratio for each cell line is the SUMO-2/3/GAPDH transmission observed for Dox treatment at 0 (for shSUMO-2/3), 12, 24, and 48 hour using TREx-F3H3-K-Rta BCBL-1 cells at 0 hour arranged as 1.0.(TIFF) ppat.1005051.s005.tiff (3.8M) GUID:?D2E906C7-E853-4690-B86E-BC7EC6F8A6F7 S6 Fig: Knockdown of SUMO-2/3 increases transactivation of KSHV lytic genes located in the SUMO-2/3 enrichment and high H3K9me3 region but not in latent genes located in SUMO-2/3 enrichment and high SUMO-1 region. (A) The manifestation level of three viral lytic genes representing the SUMO-2/3 enrichment and high H3K9me3 region were quantified by real-time qPCR using cDNA from Fig 4A. All reactions were run in triplicate and normalized against GAPDH. The fold switch was computed by comparing induced values to their non-induced settings. **; P 0.005. NS; non-significant. (B) The manifestation level of three viral latent genes representing the SUMO-2/3 enrichment and high SUMO-1 region were quantified as explained in (A).(TIFF) ppat.1005051.s006.tiff (1.3M) GUID:?CACF53C0-DD2B-4AE7-8EA5-7588796869DE S7 Fig: SUMO-2/3 knockdown does not switch histone marks within the KSHV genome during viral reactivation. (A) TREx-F3H3-K-Rta BCBL-1 and TREx-F3H3-K-Rta-shSUMO-2/3 BCBL-1 cells were treated as explained in Fig 4A. TCLs were collected and analyzed by immunoblotting using anti-SUMO-2/3, anti-K-Rta and anti-K-bZIP antibodies. Anti-GAPDH antibody was utilized for loading control. (B) ChIP assays were performed using chromatin prepared from cells treated as described in (A) using anti-H3K9me3, anti-H3K27me3, anti-H3K4me3, anti-H3K36me3, and anti-H3Ac.