SUMOylation of chromatin-associated proteins occurs at the active <i>VSG-</i>ES in the bloodstream form.

<p>(A) Schema of tagged cell lines used in ChIP experiments. SALR cell line: <i>firefly luciferase</i> gene (FLuc) inserted downstream of the active <i>VSG221</i>-ES promoter and SILR: FLuc gene inserted downstream of an inactive <i>VSG</i>-ES. <i>Renilla</i>-luciferase reporter gene (RLuc) was inserted in the tubulin locus of both cell lines, as RNA pol II control. Fragments amplified by qPCR using ChIPed DNA are indicated (primers are listed in the <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004545#ppat.1004545.s010" target="_blank">Table S1</a>). (B), (C) TbRPA1 ChIP analysis in SALR and SILR shows the occupancy of RNA pol I in these cell lines. High TbRPA1 enrichment is found in SALR along the active <i>VSG</i>-ES at FLuc gene, pseudo-VSG (pseVSG) and the active <i>VSG221</i> in contrast to inactive <i>VSGs</i>. Similar occupancy is detected in SILR except for the FLuc gene inserted in an inactive <i>VSG-ES</i> promoter of the BES5 in this cell line. The RNA pol I occupancies between FLuc SALR and FLuc SILR were found significantly different (p-value<0.01). Sequences present in rDNA promoter and 18S gene transcribed by RNA pol I were analyzed as positive controls and RNA pol II or pol III transcribed genes as negative controls. (D), (E) ChIP analysis using anti-TbSUMO mAb in SALR and SILR cell lines. Enrichment of SUMOylated proteins is found at the active <i>VSG</i>-ES chromatin from the promoter to the telomeric <i>VSG221</i>. SUMO ChIP levels between active (FLuc SALR) and inactive (FLuc SILR) reporters are significantly different (p-value<0.01). Similarly, SUMO enrichment on the active <i>VSG221</i> versus the inactive <i>VSGs</i> (<i>121</i>, <i>JS1</i> and <i>VO2</i>) is significantly different (p-values<0.05). ChIP analyses show the average from at least three independent experiments with standard error (SE). Data are represented as percentage of input immunoprecipitated (% input) after background subtraction of the negative control ChIP using the pre-bleed antiserum. Tubulin (Tub), Splicer Leader (SL), EP Procyclin promoter (EP pro), Procyclin gene (EP cds), rDNA promoter (rDNA pro), rDNA spacer (rDNA sp).</p

SUMOylation of chromatin-associated proteins by TbSIZ1 is important for efficient recruitment of RNA pol I and <i>VSG</i>-ES expression.

<p>(A) Reduced <i>VSG</i>-ES chromatin SUMOylation upon TbSIZ1 depletion. ChIP analysis carried out in TbSIZ1 depleted cells (48 h) and parental cell line (SALR) shows that SUMOylated chromatin is significantly reduced along all active <i>VSG</i>-ES, from the upstream promoter to the active <i>VSG221</i> gene. (B) Reduced RNA pol I occupancy upon TbSIZ1 depletion. Occupancy of RNA pol I was determined by TbRPA1 ChIP. Statistical analysis shows a significant difference of TbRPA1 levels between parental and TbSIZ1 depleted cells at the active <i>VSG</i>-ES. Data from three independent TbSIZ1 RNAi clones and parental controls are represented as percentage of input immunoprecipitated after background subtraction of the pre-bleed antiserum ChIP. The results show the average from at least three independent experiments with standard error (SE). Statistical analysis (Student's t-test), *p<0.05, **p<0.01. (C) Reduced RNA pol I occupancy results in reduced <i>VSG</i>-ES derived transcripts. Quantitative RT-PCR analysis shows reduced amounts of <i>FLuc</i> reporter gene and <i>VSG221</i> mRNA without significant effect in rDNA transcripts or RNA pol II derived transcripts <i>RLuc</i> and myosin. Results are the average from three independent clones. Data were normalized with U2 mRNA, transcribed by RNA pol III. Statistical analysis (Student's t-test) *p<0.05, **p<0.01.</p

TbRPA1 is SUMOylated in <i>T. brucei</i>.

<p>(A) Immunoprecipitation (IP) of SUMOylated proteins revealed that TbRPA1 is SUMOylated. Nuclear fraction from BF cell extracts was lysed in urea containing buffer. SUMOylated proteins were immunoprecipitated with anti-TbSUMO mAb or unspecific antiserum (mock) and probed with anti-TbRPA1 (arrow). As control the same blot was probed with anti-TbSUMO (bottom) (B) Reciprocal IP experiment was performed using anti-TbRPA1 antiserum and probed with anti-TbSUMO mAb. As control the same blot was probed with anti-TbRPA1 (bottom). Loading information: input (Inp): 0.3%, IPs: 50%. (C) TbRPA1 is SUMOylated in a TbSIZ1 dependent manner. Immunoprecipitation experiments were carried out using protein extracts from the parental cell line and compared with a TbSIZ1 depleted extract (48 h RNAi induced). SUMOylated proteins were immunoprecipitated with anti-TbSUMO mAb or anti-TOR4, as control. Western blot with anti-TbRPA1 shows a reduction of the SUMOylated TbRPA1 detected upon TbSIZ1 depletion (arrowhead). Loading information: input 2× (Inp 2×): 0.1%, input 1× (Inp 1×): 0.05%, IPs: 50%.</p

Chromatin upstream of active <i>VSG</i>-ES promoter is highly enriched for SUMOylated proteins.

<p>(A) Schema of <i>VSG</i>-ES promoter region mapping indicating fragments amplified by qPCR (See <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004545#ppat.1004545.s010" target="_blank">Table S1</a> and <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004545#ppat-1004545-g003" target="_blank">Figure 3A</a>). (B) ChIP analysis of sequences upstream of the <i>VSG</i>-ES promoters using anti-TbRPA1 and anti-TbSUMO antibodies. Fragments 5 and 6 are highly enriched for SUMOylated proteins and the fragments 1–4 show moderate enrichment. In contrast, TbRPA1 is no detected in the region upstream and enriched at the promoter and downstream sequences. PCR fragment 4 from genomic and ChIPed DNA was cloned and sequenced showing that SUMO-ChIPed chromatin corresponds to the active <i>VSG221</i>-ES (See <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004545#ppat.1004545.s005" target="_blank">Figure S5B</a>). (C) Schema of ribosomal DNA (rDNA) promoter showing mapped fragments amplified by qPCR (primers listed in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004545#ppat.1004545.s010" target="_blank">Table S1</a>). (D) Chromatin upstream of the rDNA promoter is not significantly SUMOylated. ChIP analysis of rDNA promoter region shows no SUMO enrichment detected above background. The results show the average from at least three independent experiments with standard error (SE). Data are represented as percentage of input immunoprecipitaded (% input) after background subtraction of the pre-bleed antiserum ChIP.</p

Expression pattern and subcellular localization of <i>T. brucei</i> SUMOylated proteins.

<p>(A) Multiple bands corresponding to SUMO conjugated proteins are reduced upon TbSUMO knockdown. Western blot analysis of SUMOylated proteins with a monoclonal antibody generated against TbSUMO (mAb 1C9H8). Whole cell extracts from bloodstream trypanosomes were prepared in the presence of 20 mM NEM and separated in 4–20% precast polyacrylamide gels (Bio-Rad). (5×10⁶ cells/lane); Parental, uninduced (−) and induced 48 h (+). Anti-tubulin was used as a loading control. (B) Expression pattern of SUMOylated proteins in two developmental forms of <i>T. brucei</i>. Bloodstream form (BF) and insect procyclic form (PF) total cell extracts were prepared in the presence of 20 mM NEM and analyzed by Western blot using the mAb 1C9H8. (C) SUMO conjugated proteins are diffusely distributed in the nucleoplasm including a Highly SUMOylated Focus (HSF) (arrowhead) in the bloodstream form of the parasite. Double indirect three-dimensional immunofluorescence (3D-IF) analysis was carried out using the anti-TbSUMO mAb 1C9H8 (green). (C′) Higher magnification of the nucleus showing anti-SUMO and DAPI fluorescence signals. DNA in the nucleus (N) and the kinetoplast (K) was stained with DAPI (blue). Statistical analysis showed the presence of a highly SUMOylated focus in 74.9% of the cells (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004545#ppat.1004545.s002" target="_blank">Figure S2B</a>). (D) SUMOylated proteins in the nucleus of procyclic form are diffusely distributed in many foci. 3D-IF analysis was carried out using the anti-TbSUMO mAb (green). (D′) Higher magnification of the nucleus showing anti-SUMO and DAPI fluorescence signals. DNA was stained with DAPI (blue). Scale bars 1 µm. Maximum intensity projections of deconvolved two-channel 3D stack are shown.</p

NAIP expression in monocytes, M0-, M1- and M2-macrophages.

<p>A, B: The protein level of NAIP was assessed by western blotting of cell extracts from undifferentiated monocytes and M0 or polarized M1/M2 THP-1 macrophages. HSC-70 was used as loading control. A: Representative western blot of one of three experiments with similar results. B: Quantification of NAIP protein abundance in western blots normalized to HSC-70 of three independent experiments. C: NAIP mRNA expression of NAIP was was analysed by RT-qPCR using primers that span exon 4 or between exons 16 and 17 of NAIP. Values are normalized to internal controls (HPRT1 and GAPDH) and presented as mean values ± s.d. of fold-change of three independent experiments. *P<0.05 ***P< 0.001 (ANOVA with Bonferroni post hoc).</p

NAIP expression in polarized primary human macrophages.

<p>A: NAIP mRNA levels from PBMC-derived monocytes or M1 and M2 macrophages were analyzed by RT-qPCR. Values are normalized to internal controls (HPRT1 and GAPDH) and presented as mean values ± s.d. of fold-change of three independent experiments. B: Protein levels of NAIP in the indicated cell populations were assessed by western blotting. Representative western blot from samples of one of the donors. Graph represents the quantification of the NAIP protein, normalized to HSC-70 of experiments from four different healthy donors. C: Representative confocal microscopy images of monocytes, M1 and M2 macrophages from volunteer 1. Note the amoeboid-like shape in M1 and M2 macrophages. Bar, 10<i>μ</i>m. D: Mean fluorescence intensity in monocytes and M1 and M2 macrophages from two healthy donors. 10 randomly selected cells in each case were analyzed for their mean fluorescence intensity per cell area using the <i>ImageJ</i> program. *Significant difference (P = 0.0163), **significant difference (P = 0.0037), ***significant difference (P = 0.0005).</p

cIAP1 and cIAP2 expression in unpolarized (THP-1/monocytes and M0-) and polarized M1- and M2-macrophages.

<p>A, B: Polarized and unpolarized THP-1 cell proteins were extracted and cIAP1/2 protein abundance was assessed by western blotting using the RIAP1 antibody. HSC-70 was used as loading control. A: Representative western blot of one of three experiments with similar results. B: Quantification of cIAP1 and cIAP2 protein abundance in western blots normalized to HSC-70 of three independent experiments. C: Total RNA was extracted, reverse transcribed and cIAP1 and cIAP2 mRNA were analysed by RTq-PCR. Values are normalized to internal controls (HPRT1 and GAPDH) and presented as mean values ± s.d. of fold-change of three independent experiments. *P<0.05 ***P< 0.001 (ANOVA with Bonferroni post hoc).</p

Smac mimetics modulate IAP expression during macrophage polarization.

<p>THP-1 M0 macrophages were treated with 1<i>μ</i>M LCL161 for 24 h prior to polarization into M1 or M2. A, B: The levels of NAIP, cIAP1 and cIAP2 were determined by western blotting. HSC-70 was used as loading control. A: Representative western blot from one of three experiments with similar results. B: Quantification of NAIP, cIAP1 and cIAP2 protein abundance normalized to the corresponding HSC-70 in three independent experiments. C: Determination of NAIP, cIAP1 and cIAP2 mRNA levels by RT-qPCR. NAIP mRNA expression was evaluated using primers spanning exon 4 or between exons 16 and 17. Values are normalized to internal controls (HPRT1 and GAPDH) and presented as mean values ± s.d. of fold-change of three independent experiments. *P<0.05 **P<0.01 ***P< 0.001 (ANOVA with Bonferroni post hoc).</p

SMCs alters the markers expression profile of polarized macrophages.

<p>THP-1 M0 macrophages were treated with 1<i>μ</i>M LCL161 for 24 hours and then induced to polarized into M1 or M2 states. A: CD206, CXCL10 and CD163 polarization marker genes were examined by semi-quantitative RT-PCR. mRNA expression was normalized to GAPDH in each group and then calculated as fold change against the expression of the control group. Data represent the mean and standard deviation of three independent experiments. B: Cells were subsequently analyzed by flow cytometry for the expression of the macrophage markers CD14 and CD11b and of the M1 (CD86) and M2 (CD206) polarization markers. Data represents mean MFI of three independent experiments. *P<0.05 *** P<0.0001 (ANOVA with Bonferroni post Hoc).</p