Characterization of cytoplasmic SMC3 protein.

<p>(<b>A</b>) Expression of mRNA of Smc1, Smc3 and Rad21 72 h after esiSMC1 treatment, compared to control (esiEGFP), was examined by real-time RT-PCR. Relative quantification of gene expression was achieved by normalization to ß-actin, (n = 3). (<b>B</b>) Effect of SMC1 knockdown on RAD21 protein in total RIPA extracts as monitored by IB with anti-Rad21 antibody 72 h post esiSMC1 or esiEGFP transfection. Bottom: quantification of IB from three independent experiments. (<b>C</b>) Sequential IP. IP #1 from nuclear and cytoplasmic extracts from esiSMC1-treated and control cells. The supernatant was used for IP#2 with anti-SMC3 antibody. Eluates were analyzed by IB using anti-RAD21, -SMC3, and -SMC1 antibodies. (<b>D</b>) Nuclear export was inhibited 70 h after esiSMC1 or control treatment by addition of LMB to a final concentration of 5 ng/mL for another 2 h. The localization of SMC3 (in red, top half) was examined by immunofluorescence. NFκB (in red, bottom half) was used to confirm the LMB effect. DNA was visualized by DAPI (in blue). (<b>E</b>) Immunoprecipitation of SMC3 from cytoplasm and nuclear extracts after LMB inhibition was performed and eluates examined by silver staining. Arrows indicate the positions of SMC1, SMC3 and RAD21. The asterisk indicates an unspecific band as specified by mass spectrometric analysis.</p

Transient down-regulation of endogenous human SMC1 in HeLa cells using specific esiRNA or siRNA impairs the nuclear localization of SMC3.

<p>(<b>A</b>) RIPA total cell extracts were prepared 72 h after treatment with three different concentrations of esiSMC1 and examined by IB using anti-SMC1 antibody. Mock transfected cells were used as negative control. The membrane was reprobed with anti-SMC3 antibody to confirm the specificity of esiSMC1 and equal loading. The percentages of SMC1 protein levels, normalized to SMC3 protein levels with respect to mock control set at 100%, are indicated. (<b>B</b>) Kinetics of recovery of SMC1 expression after treatment of cells with 750 ng/mL of esiSMC1 was analyzed by IB as described in A. (<b>C</b>) Quantification of SMC1 and SMC3 in RIPA total extracts of cells treated with 750 ng/mL of esiRNA and collected 72 h post transfection. Average of six independent experiments is shown. (<b>D</b>) IF microscopic analysis of SMC1 knockdown 72 h post esiRNA transfection by anti-SMC1 staining in red and DAPI in blue. Specific esiRNA against EGFP (esiEGFP) was used as a control. (<b>E</b>) IF microscopic analyses of esiSMC1- or esiEGFP-treated cells (72 h) using anti-SMC3 (red) and DAPI (blue). (<b>F</b>) Cytoplasmic and nuclear extracts from esiSMC1 or control treated cells were analyzed by IB using anti-SMC3. The membrane was reprobed with anti-SMC1. Anti-ß tubulin and Topo II antibodies were used to determine the purity of nuclear and cytoplasm extracts. (<b>G</b>) Quantification of results from four independent experiments that were performed as described in F. (<b>H</b>) Time course of SMC3 localization upon treatment of cells with siSMC1 (#1) as visualized by IF microscopy using anti-SMC3 (in red) and DAPI (in blue).</p

FRAP experiments using EGFP-msSMC1 (A) or EGFP-msSMC3 (B) as bleach substrates.

<p>The mobile fraction (recovery), the intensity of the EGFP-tagged protein signal (given as mean grey values), and tau (τ) as a value inversely correlating with mobility are shown; ns  =  non significant; p-values are shown within each graph.</p

Two-species system: knockdown of human SMC3 by siRNA and rescue by stably expressed EGFP-msSMC3.

<p>(<b>A</b>) Cells stably transfected with EGFP-msSMC3 were analyzed by IF microscopy. (<b>B</b>) Total cell (RIPA), cytoplasmic and nuclear extracts from cells stably expressing EGFP-msSMC3 collected 72 h after treatment with siSMC1 or siSMC3 were analyzed by IB using anti-EGFP, -SMC3 and -SMC1 antibodies. RIPA buffer was used for the final extraction step after the nuclear extraction with 250 mM ammonium sulfate. Topo II and Karyopherin ß1 were used as a loading control. Relative protein levels are shown at the bottom (representative of 3 experiments).</p

Imbalance of SMC1 and SMC3 Cohesins Causes Specific and Distinct Effects

<div><p>SMC1 and SMC3 form a high-affinity heterodimer, which provides an open backbone of the cohesin ring, to be closed by a kleisin protein. RNAi mediated knock-down of either one heterodimer partner, SMC1 or SMC3, is expected to cause very similar if not identical phenotypes. However, we observed highly distinct, protein-specific phenotypes. Upon knock-down of human SMC1, much of SMC3 remains stable, accumulates in the cytoplasm and does not associate with other cohesin proteins. Most of the excess nuclear SMC3 is highly mobile and not or only weakly chromosome-associated. In contrast, human SMC3 knock-down rendered SMC1 instable without cytoplasmic accumulation. As observed by differential protein extraction and in FRAP experiments the remaining SMC1 or SMC3 proteins in the respective SMC1 or SMC3 knock-down experiments constituted a cohesin pool, which is associated with chromatin with highest affinity, likely the least expendable. Expression of bovine EGFP-SMC1 or mouse EGFP-SMC3 in human cells under conditions of human SMC1 or SMC3 knock-down rescued the respective phenotypes, but in untreated cells over-expressed exogenous SMC proteins mis-localized. Paucity of either one of the SMC proteins causes RAD21 degradation. These results argue for great caution in interpreting SMC1 and SMC3 RNAi or over-expression experiments. Under challenged conditions these two proteins unexpectedly behave differently, which may have biological consequences for regulation of cohesin-associated functions and for human cohesin pathologies.</p></div

SMC3 knock-down by siRNA reduces the stability of SMC1.

<p>(<b>A</b>) Time course of reduction and recovery of SMC3 analyzed by IF microscopy with anti-SMC3 (in red) and DAPI (in blue). (<b>B</b>) IF analysis using antiSMC1 antibody (in red) and DAPI (in blue). (<b>C</b>) Quantification of three independent IB experiments using total cell extracts (RIPA) after siSMC3 treatment. (<b>D</b>) Expression of mRNA of Smc1, Smc3 and ß-actin 72 h after esiSMC1, siSMC1, or siSMC3 treatment, compared to control (esiEGFP), was examined by RT PCR and agarose gel electrophoresis. (<b>E</b>) Cytoplasmic, nuclear and total cell extracts (RIPA) were prepared 72 h after transfection with esiEGFP, esiSMC1, siSMC1, siSMC3, or non targeting siRNA control. Protein amounts were analyzed by IB using anti-SMC1, anti-RAD21 and anti-SMC3 antibodies. Topo II was used as a nuclear control and cPLA2 as a cytoplasmic loading control. (<b>F</b>) 48 h after siSMC3 or esiSMC1 treatment the cells were transfected with EGFP-bSMC1 for further 24 h. The localization of EGFP-bSMC1 was analyzed by IF microscopy using anti-EGFP antibody.</p

IF analysis of transiently over-expressed bovine SMC1 (EGFP-bSMC1) in HeLa cells.

<p>Anti-SMC1 antibody (red) and EGFP (green) were used. Nuclear staining was obtained using DAPI (blue) (<b>A</b>) 24 h after transfection of bovine SMC1 without any tag or N-terminally tagged EGFP-bSMC1. As a control an EGFP-empty vector was used. Anti-SMC1 antibody (red) stains both, tagged and non-tagged SMC1 proteins. (<b>B</b>) Stability of transiently overexpressed EGFP-bSMC1 (green) 24, 48 and 72 h after transfection.</p

CD11c expression and IL-12 production in single cell clones.

<p>De-iniDC single cell clones were stimulated with LPS or left untreated for 24 hours in the presence of the protein transport inhibitor Monensin. Afterwards, cells were stained for the surface marker CD11c, permeabilized and stained for intracellular IL-12. (A) CD11c expression (black) of LPS stimulated cells is displayed. (B) Intracellular IL-12 expression level of CD11c⁺ LPS stimulated (black) and non-stimulated cells (grey) are shown. Isotype control is displayed as grey, dotted curve (A, B).</p

Dendritic cell surface marker expression.

<p>(A) BM-DCs, iniDCs and 3-days cultured de-iniDCs were stained with antibodies against the dendritic cell subset markers CD11c, CD8α, CD11b, B220 and Ly6C. CD11c⁺ cells (black curve) were further gated for CD8α and CD11b, Ly6C and B220 (contour blots). Gates for CD8α and CD11b, Ly6C and B220 were set on the respective unstained control (red). (B) Immature and mature BM-DCs, iniDCs and de-iniDCs were stained for MHCII, CD40, and CD86. Dead cells (DAPI staining) and cell doublets were excluded. Histograms show the isotype control (grey, dotted), immature cells (grey) and LPS-matured cells (black). The result of one representative experiment is given.</p

Lentiviral vector mediated transgene expression in iniDCs.

<p>(A) RFP expression level was measured in untransduced (grey dotted) and lentiviral vector particle-transduced iniDCs before (grey) and after (black) puromycin selection. (B) Expression level of maturation markers MHCII, CD40 and CD86 were determined in transduced iniDCs and after their deinduction (de-iniDCs) using flow cytometry. Transduced iniDCs and de-iniDCs (grey) and LPS-stimulated transduced iniDCs and de-iniDCs (black) are shown. Isotype controls are displayed as grey dotted lines. One representative experiment out of 3 is shown.</p