Cross-correlation by DC-FCCS.

<p>Cross-correlation by DC-FCCS.</p

CENP-C/H/I/K/M/T/W/N/L and hMis12 but not CENP-S/X participate in complex formation in the nucleoplasm of living human interphase cells outside centromeres

<div><p>Kinetochore proteins assemble onto centromeric chromatin and regulate DNA segregation during cell division. The inner kinetochore proteins bind centromeres while most outer kinetochore proteins assemble at centromeres during mitosis, connecting the complex to microtubules. Here, we measured the co-migration between protein pairs of the constitutive centromere associated network (CCAN) and hMis12 complexes by fluorescence cross-correlation spectroscopy (FCCS) in the nucleoplasm outside centromeres in living human interphase cells. FCCS is a method that can tell if in living cells two differently fluorescently labelled molecules migrate independently, or co-migrate and thus are part of one and the same soluble complex. We also determined the apparent dissociation constants (K_d) of the hetero-dimers CENP-T/W and CENP-S/X. We measured co-migration between CENP-K and CENP-T as well as between CENP-M and CENP-T but not between CENP-T/W and CENP-S/X. Furthermore, CENP-C co-migrated with CENP-H, and CENP-K with CENP-N as well as with CENP-L. Thus, in the nucleoplasm outside centromeres, a large fraction of the CENP-H/I/K/M proteins interact with CENP-C, CENP-N/L and CENP-T/W but not with CENP-S/X. Our FCCS analysis of the Mis12 complex showed that hMis12, Nsl1, Dsn1 and Nnf1 also form a complex outside centromeres of which at least hMis12 associated with the CENP-C/H/I/K/M/T/W/N/L complex.</p></div

Protein-protein co-migration of CCAN and Mis12 proteins.

<p>The degree of co-migration (corrected) in the nucleoplasm outside centromeres of human interphase cells is color-coded (red dashed arrows: no or hardly detectable co-migration, green arrow: 5–30%, blue arrows: 30–60%, black arrows: above 60% co-migration). Please note: these green, blue or black arrows do not necessarily indicate direct protein-protein interaction; the labeled proteins co-migrate in a complex: their interaction might be either direct or mediated by a third (or more) protein.</p

DC-FCCS of EGFP-(s)-CENP-T and mCherry-(s)-CENP-S.

<p>A) Displayed are G versus lag time. Red: FCS- or autocorrelation-curve G (τ) for mCherry, green: FCS- or autocorrelation-curve G (τ) for EGFP, black: cross-correlation-curve G (τ), AC = autocorrelation. CC = cross-correlation, A(AC) = amplitude of autocorrelation curve, A(CC) = amplitude of cross-correlation curve. The cross-correlation analyses are amplified in inserts a. Count rates are displayed over 1 sec (inserts b; green = EGFP and red = mCherry). For the pair EGFP-(s)-CENP-T and mCherry-(s)-CENP-S no indication for complex formation in the nucleoplasm was detected (A(CC)/A(AC_mCherry) = 0%). The cross-correlation analysis (with a magnified scale of G (τ); insert a) resulted in a correlation of 1.001, whereas the autocorrelations yielded 1.322 for EGFP-(s)-CENP-T and 1.106 for mCherry-(s)-CENP-S. This ratio indicates that no nucleoplasmic CENP-T and -S are part of a common complex. B) Localization of cotransfected EGFP-(s)-CENP-T (EGFP) and mCherry-(s)-CENP-S (mCherry) in living human HEp-2 cells which were used for FCCS analysis. White bar = 10 μm. A cell nucleus is displayed showing co-localisation at centromeres (merge) and weak fluorescence in the nucleoplasm. Two locations of the same size and shape, a centromere (spot 1) and the centromere-free position of an FCCS measurement, as shown in Fig 4A (spot 2), in the nucleoplasm were selected for fluorescence intensity analysis. For the analyzed centromere in spot 1 the ratios of nucleoplasmic to centromeric fluorescence intensities was 1:43 for EGFP-(s)-CENP-T and 1:33 for mCherry-(s)-CENP-S. The concentrations of nucleoplasmic proteins, estimated by FCCS, was 6 nM for EGFP-(s)-CENP-T and 14 nM for mCherry-(s)-CENP-S.</p

Cross-correlation by SW-FCCS.

<p>Cross-correlation by SW-FCCS.</p

SW-FCCS analysis of CENP-T and CENP-W.

<p>(a): ACF curves of EGFP-(s)-CENP-W (green) and mCherry-(s)-CENP-T (red), and CCF curves (blue, purple) in the nucleoplasm of interphase HEK293 cells. The data show cross-correlation between CENP-T and CENP-W, indicating interaction. (b) and (c): <i>K</i>_<i>d</i> determination using Scatter plot (b) and a histogram (c) of multiple SW-FCCS measurements to determine an effective <i>K</i>_<i>d</i> of the interaction. (d): ACF curves of EGFP-(s)-CENP-T^∆N (green) and mCherry-(s)-CENP-W (red), and CCF curves (blue, purple) in the nucleoplasm of interphase HEK293 cells. The data show reduced cross-correlation between CENP-T^∆N and CENP-W. (e) and (f): <i>K</i>_<i>d</i> determination using Scatter plot (e) and a histogram (f) of multiple SW-FCCS measurements to determine the effective <i>K</i>_<i>d</i> of this interaction between the histone-fold domain of CENP-T (CENP-T^∆N) and CENP-W. A defined interaction is detected by both, the linear fit of the scatter plot as well as the log-normal fit of the histogram.</p

Effect of long term quiescence induction (100 or 150 days) in MRC-5 fibroblasts maintained at 20% O₂.

<p>(<b>A</b>) Growth curve of 3 independent MRC-5 fibroblast cell lines (control with no quiescence induction, and cell lines where quiescence was induced for 100 or 150 days respectively by contact inhibition and then maintained in culture till they approached senescence) maintained in culture at 20% O₂ as triplicates from an early PD until senescence at late PDs. Each growth curve is measured in triplicate. Data points of all measurements are displayed (not the mean). (<b>B & C</b>) Percentage of SA-β gal positive cells at different time points of their growth in culture in the control MRC-5 fibroblast cell line and in the cell lines where quiescence was induced for 100 or 150 days respectively. Fig. 2 B and C are plotted with PDs and days in the y-axis respectively. Each curve is measured in triplicate, the mean value is displayed with error bar (± S.E). (<b>D</b>) The blots show the protein expression levels of p16, p21, p27, Cyclin D1, Cyclin D2, Ki-67 and γH2A.X in MRC-5 fibroblast cell lines (subjected to different culture conditions of 100 or 150 days quiescence by contact inhibition and no quiescence induction) maintained in culture at 20% O₂ until they approached senescence at late PD. The up or down-regulation was signified by the presence or absence of the bands in Western Blots. (<b>E, F, G, H, I, J, K</b>) Comparison of mean fold change of protein expression levels of p16 (E), p21 (F), p27 (G), Cyclin D1 (H), Cyclin D2 (I), Ki-67 (J) and γH2A.X (K) in MRC-5 cell lines where quiescence was induced for 100 or 150 days by contact inhibition respectively compared to controls at corresponding span of time in culture. Cell lines were maintained at 20% O₂ as triplicates. The bars indicate the mean ± S.D. *** p<0.001 - significantly different compared to fibroblasts with PD assigned 1. n = 3.</p

Raman and Infrared Spectroscopy Distinguishing Replicative Senescent from Proliferating Primary Human Fibroblast Cells by Detecting Spectral Differences Mainly Due to Biomolecular Alterations

Cellular senescence is a terminal cell cycle arrested state, assumed to be involved in tumor suppression. We studied four human fibroblast cell strains (BJ, MRC-5, IMR-90, and WI-38) from proliferation into senescence. Cells were investigated by label-free vibrational Raman and infrared spectroscopy, following their transition into replicative senescence. During the transition into senescence, we observed rather similar biomolecular abundances in all four cell strains and between proliferating and senescent cells; however, in the four aging cell strains, we found common molecular differences dominated by protein and lipid modifications. Hence, aging induces a change in the appearance of biomolecules (including degradation and storage of waste) rather than in their amount present in the cells. For all fibroblast strains combined, the partial least squares-linear discriminant analysis (PLS-LDA) model resulted in 75% and 81% accuracy for the Raman and infrared (IR) data, respectively. Within this validation, senescent cells were recognized with 93% sensitivity and 90% specificity for the Raman and 84% sensitivity and 97% specificity for the IR data. Thus, Raman and infrared spectroscopy can identify replicative senescence on the single cell level, suggesting that vibrational spectroscopy may be suitable for identifying and distinguishing different cellular states <i>in vivo</i>, e.g., in skin

Additional file 6: Table S5. of Hormetic effect of rotenone in primary human fibroblasts

Common significantly up- and down-regulated pathways in primary human fibroblast strains independent of their cell origin. Pathways significantly (p < 0.05) up- and down-regulated on low dose rotenone treatment in both cell strains (MRC-5 and HFF). (DOCX 26 kb

Additional file 1: Table S1. of Hormetic effect of rotenone in primary human fibroblasts

Apoptosis induction by higher concentrations of rotenone treatment in MRC-5 fibroblasts. Numbers of days MRC-5 fibroblasts were maintained in culture before induction of apoptosis (as detected by phosphorylation of p38) in response to different concentrations of rotenone. (DOCX 15 kb