EMBO/soda-nel huggingface dataset:
The breast cancer oncogene IKKε coordinates mitochondrial function and serine metabolism (2020)
Figure 5

Figure 5. The SBP is primarily regulated by an IKKε-mediated transcriptional response

(A) qRT-PCR analysis of PHGDH, PSAT1 and PSPH mRNA levels in a panel of IKBKE (IKKε)-silenced breast cancer cell lines. Data are expressed as fold changes, relative to levels in a non-silenced control of each cell line and normalised to β-Actin (n≥3 biological replicates).

(B) Representative western blot showing levels of IKKε and the SBP enzymes in IKBKE (IKKε)-silenced ZR-75-1, T47D, MDA-MB-468 and MCF7 breast cancer cell lines.

(C-E) Levels of the SBP enzymes in a panel of IKBKE (IKKε)-silenced breast cancer cell lines. (C) PHGDH, (D) PSAT1 and (E) PSPH levels in indicated cell lines normalised to Vinculin. Densitometry analysis quantified single sample density as a percentage of total blot density per cell line prior to vinculin normalisation (n≥3 biological replicates).

(F) qRT-PCR analysis of PHGDH, PSAT1 and PSPH mRNA levels in ATF4-silenced ZR-75-1, T47D and MDA-MB-468 breast cancer cell lines. Data are expressed as fold changes, relative to levels in non-silenced control cells and normalised to β-Actin (n=3 biological replicates).

(G) Representative western blot showing levels of PHGDH, PSAT1 and PSPH in ATF4-silenced ZR-75-1, MDA-MB-468, MDA-MB-231, T47D and HCC1143 breast cancer cell lines.

Data Information: In (A, C-F), data are presented as mean ± SEM. *p<0.05, **p<0.01, ***p<0.001. In (A, F) one sample t-tests were performed using log transformed fold change values for all samples, except Sum44 PHGDH in (A), in which case a Wilcoxon Signed Rank was used. In (C-E) two-tailed paired t-tests were performed.­
The breast cancer oncogene IKKε coordinates mitochondrial function and serine metabolism (2020)
Figure 6

Figure 6. The pathophysiological role of IKKε-induced metabolic and gene expression alterations in breast cancer

(A) Correlation of change in OCR (ΔOCR) in a panel of IKBKE (IKKε)-silenced breast cancer cell lines (from Fig. 3C) and the change in cell confluency (Δconfluency) upon treatment of the panel of cell lines with NCT502 (from Fig. EV5A).

(B) Correlation of ΔOCR in a panel of IKBKE (IKKε)-silenced breast cancer cell lines (from Fig. 3C) and the Δconfluency upon treatment of the panel of cell lines with 6-Diazo-5-oxo-L-norleucine (DON) (from Fig. EV5C).

(C) Association between IKBKE (IKKε) and PSAT1 mRNA overexpression evaluated by a chi-squared independence test. The + sign indicates samples with significant (p<0.05) overexpression of IKBKE or PSAT1. Number and percentage of samples, as well as the Chi-square values are shown.

(D-I) Expression of IKBKE (IKKε) and the SBP enzymes PSAT1, PHGDH and PSPH in the METABRIC dataset. The expression of a proliferation related gene set and ATF4 are also shown. Samples were stratified by Pam50 intrinsic subtypes and ER status. Brown-Forsythe and Welch ANOVA test with unpaired t-test with Welch correction were applied. *p<0.05, **p< 0.01, ***p<0.001, ****p<0.0001.

(J) Association between IKBKE (IKKε) and ATF4 mRNA overexpression evaluated by chi-squared independence test. The + sign indicates samples with significant (p<0.05) overexpression of IKBKE or PSAT1. Number and percentage of samples, as well as the Chi-square values are shown.

(K, L) Correlation of IKBKE (IKKε) and PSAT1 (K) or IKBKE (IKKε) and ATF4 (L) expression in the ER negative sample subset.

Data Information: In (A-B) cell confluency was measured using the IncuCyte Zoom, ΔOCR was measured using Seahorse XF96e or XF24 analysis. Data are n≥3 biological replicates. In (A, B, K, L) linear regression, correlation coefficients (Pearson's correlation, Spearman's rho) significance of difference from slope = 0 are shown.

EXPANDED VIEW FIGURE LEGENDS
Human RAP1 specifically protects telomeres of senescent cells from DNA damage (2020)
Figure 1

Figure 1. RAP1 protects telomeres against DNA damage

(A) Western blotting showing the expression of RAP1 and TRF2 in MRC-5 cells of different population doublings (PD). Senescent cells (PD 72) were left in culture for at least 4 weeks before harvesting for analysis.

(B) ChIP analysis of young (PD 30) and senescent (PD 72) MRC-5 cells performed with either anti-RAP1, anti-TRF2 or an IgG antibody. The immunoprecipitated and input products were loaded into a slot blot membrane and hybridized with a telomere probe, stripped and hybridized to an Alu probe in order to determine unspecific binding. Quantification was performed by normalizing the telomere signal of the immunoprecipitated to that of the input. Data represent mean +/- SD of three biological replicates.

(C) Immunofluorescence detection of 53BP1 (red) and a FISH probe staining telomeres (green) in young (PD 26), pre-senescent (PD 66) and senescent (PD 72 + 4 weeks) MRC-5 fibroblasts. The upper graph shows the percentage of telomere co-localizing with 53BP1 (TIFs). Total DNA damage was measured by immunofluourescence detection of 53BP1. The lower graph shows the total number of 53BP1 foci per nucleus. Approximately 40-50 cells were analyzed per replicate and per condition. Bars represent SEM of 2 biological replicates.

Data information: Statistical analyses were performed using Mann-Whitney U test (*p < 0.05; **p < 0.001; ***p < 0.0001). Scale bar = 10 µm.
Human RAP1 specifically protects telomeres of senescent cells from DNA damage (2020)
Figure 2

Figure 2. RAP1 prevents telomere fusions in senescent cells

(A) Number of telomere fusions performed by PCR using 3 subtelomeric primers in the same reaction (16p1, 21q1 and XpYpM). The primer 16p1 is able to bind the subtelomeric region of 1p 9q, 12p, 15q, 16p, XqYq and the interstitial 2q14 region; the 21q1 primer binds the subtelomeric region of 1q, 2q, 5q, 6q, 6p, 8p, 10q, 13q, 19p, 19q, 21q, 22q and the interstitial 2q13 site. The PCR-fusion assay was performed in MRC-5 primary fibroblasts of different population doublings (PD) with or without depletion of RAP1 (shRAP1 or shControl respectively). Lentiviral infection was performed for 10 days. Data represent mean +/- SD of 3 biological replicates. Statistical analyses were performed using Mann-Whitney U test (**p < 0.001; ***p < 0.0001).

(B) Southern blotting showing the hybridization of the 16p probe for the conditions described in A. Number of fusions per 1 µg of DNA is shown at the bottom of the gels.

(C-D) Number of fusions per 1 µg of DNA in MRC-5 senescent cells. Lentiviral transfections with shRNAs were carried out for 10 days while transfections with siRNAs for 6 days (two subsequent transfections of 3 days each). Representative southern blot membranes hybridized with the 16p probe are shown. Data represent mean +/- SD of 3 biological replicates. Statistical analyses were performed using Mann-Whitney U test (*p < 0.05; **p < 0.001; ***p < 0.0001).
Human RAP1 specifically protects telomeres of senescent cells from DNA damage (2020)
Figure 3

Figure 3. Telomere fusions in senescent cells contain short telomere arrays

(A) Distribution of the telomere repeat array found at the junction between two fused chromosomes of senescent cells transduced with lentiviral vectors expressing either shControl or shRAP1. Variant telomere repeats were included in the estimation of the telomere array length.

Data represent median +/- interquartile range (green lines) of 3 biological replicates. Statistical analysis was performed using Mann-Whitney U test ***p < 0.0001).

(B) Examples of DNA sequences found at the telomere fusion points of shControl and shRAP1 conditions. Telomere repeats are underlined in blue while variant telomere repeats are underlined in red. The sequence ID corresponds to the identifier in Table EV1.
Human RAP1 specifically protects telomeres of senescent cells from DNA damage (2020)
Figure 4

Figure 4. Telomere fusions in HeLa cells after RAP1 depletion

(A) RAP1 expression in HeLa cells treated with the telomerase inhibitor BIBR1532 for 25 days (20 µM final concentration). Fifteen days before cell harvesting, doxycycline (DOX; 1 µg/µl final concentration) was added to deplete the expression of RAP1.

(B) Telomere length analysis by southern blotting of the samples described in A. The size of the main intensity peak is indicated at the bottom of the gel.

(C) Number of fusions in HeLa cells after 25 days in culture. Cells were maintained with BIBR1532 during the whole period of the experiment, while doxycycline (1 µg/µl final concentration) and the infection with shControl, shLIG3 or shLIG4 was carried out for the last 15 days of the experiment. Data represent mean +/- SD of three biological replicates are shown. Statistical analyses were performed using Mann-Whitney U test (*p < 0.05; **p < 0.001; ***p < 0.0001).

(D) Examples of metaphase spreads hybridized with a telomeric PNA probe (green) when RAP1 was depleted (+DOX). Telomerase was inhibited with BIBR1532 for 25 days. Scale bar = 10 µm.

(E) Quantification of chromosome aberrations observed in RAP1 depleted cells (+DOX) or control (-DOX). Data represent mean +/- SD of three biological replicates. Approximately 15 metaphase spreads were analyzed per replicate with a total of 2300 chromosomes examined per condition. *p < 0.05; two-tailed Student's t test.
Human RAP1 specifically protects telomeres of senescent cells from DNA damage (2020)
Figure 5

Figure 5. RAP1 depletion in post-senescent MRC-5 cells

(A) Growth curve of MRC-5 fibroblasts. Cells were grown until senescence and after three weeks of no growth detection, lentiviral infections containing either shp21CIP1 or shp21CIP1 + shRAP1 sequences were performed. Cells were harvested 15 days post infection (dpi). Data represent mean +/- SD of three biological replicates measured after 8 and 15 dpi.

(B) Expression of p21CIP1 and RAP1 after 15 dpi of cells transduced with shControl, shp21CIP1 or shp21CIP1 + shRAP1.

(C) Dividing cells (magenta) were identified by EdU staining (1 µM EdU for 24 h). The percentage of dividing cells after 15 dpi is shown (n = 3). Scale bar = 10 µm.

(D) Senescence-associated β-galactosidase (SA-β-gal) staining in senescent and post-senescent fibroblasts (1 and 15 dpi) is shown. Percentage of SA-β-gal positive cells is indicated for each condition. Three biological replicates were performed, with approximately 300 cells analyzed per condition. Scale bar = 50 µm.

(E) Senescent cells, transduced with shp21CIP1 or shp21CIP1+shRAP1 for 15 days, were stained with CytoCalcein violet (live cells), Apopxin Green (apoptotic cells) and 7-aminoactinomycin D (7-AAD) (late apoptostic/necrotic cells) and visualized by flow cytometry.

(F) Quantification of apoptotic cells from the conditions described in E. Data represent mean +/- SD of three biological replicates (**P < 0.001; two-tailed Student's t test).
Transient rapamycin treatment during developmental stage extends lifespan in Mus musculus and Drosophila melanogaster (2022)
Figure 2

Figure 2 -Physical and physiological status of P4-P30 and P30-P60 rapamycin treated mice

(A) Upper panels: Representative pictures showing rapamycin effects on mice body size during P4-P30 treatment. Images of n=23 biological replicates (both for control and rapamycin treated mice) have been taken at P4, P14 and P30 (end of the treatment). Scale bar: 3 cm. Lower panels: Scatter dot plot indicating body weight of control and rapamycin treated mice during P4-P30 treatment. Data are indicated as mean + SD. Two-tailed Student's t-test; ***p-value<0.0005, n.s.-not significant.

(B) Upper panels: Representative pictures showing rapamycin effects on mice body size after P4-P30 treatment. Images of n=23 biological replicates (both for control and rapamycin treated mice) have been taken at P40, P100 and P150. Scale bar: 3 cm. Lower panels: Scatter dot plot indicating body weight of control and P4-P30 rapamycin treated mice after the treatment. Data are indicated as mean + SD. Two-tailed Student's t-test; ***p-value<0.0005, n.s.- not significant.

(C, D) Upper part, schematic illustration of the experimental procedure. Western blot analysis of S6 Ribosomal Protein and phospho-S6 Ribosomal Protein (Ser235/236) from whole-liver protein extracts of female and male at P8 (C) and P34 (D) treated mice. Mice were sampled after 4 days of Et-OH or rapamycin treatment.

(E) Schematic illustration of the different timepoints analyzed (left side) and frailty index box (right side) of P4-P30 and P30-P60 rapamycin treated mice. Red scatter dots indicate the frailty index of P4-P30 rapamycin mice at 7 months (n=20; biological replicates), 15 months (n=18; biological replicates) and 20 months (n=12; biological replicates). Blue scatter dots indicate the frailty index of P30-P60 rapamycin mice at 7 months (n=14; biological replicates), 15 months (n=9; biological replicates) and 20 months (n=7; biological replicates). Data are indicated as mean + SEM. Two-way ANOVA ***p-value<0.0005, n.s.- not significant.
Single-cell transcriptome analysis reveals thyrocyte diversity in the zebrafish thyroid gland (2020)
Figure 2

Figure 2: Single-cell RNA-Seq. of the zebrafish thyroid gland

(A) A t-SNE plot displaying the 6249 single-cells profiled in the zebrafish thyroid gland atlas. The colors represent cell clusters denoting a specific cell-type.

(B-D) Cluster #1 represents the thyrocytes that express tg, slc5a5 (NIS) and tpo. The color scale represents the normalized expression counts for each gene ranging from lowest (grey) to highest (red).
Single-cell transcriptome analysis reveals thyrocyte diversity in the zebrafish thyroid gland (2020)
Figure 4

Figure 4: Immunofluorescence-based visualization of cell-types surrounding zebrafish thyroid follicles

(A-D) Images show immunofluorescence labeling of thyroid gland from adult zebrafish. Transverse sections were utilized for imaging. The organ was isolated from tissue-specific transgenic lines to allow marking of a particular cell-type adjacent to the thyroid follicle. Blood vessels were marked using Tg(kdrl:EGFP) (A), macrophages using Tg(mpeg1.1:mCherry) (B) and stroma using Tg(col1a2:mCherry) (C). Thyrocytes were labeled with pax2amKO2 expression in (A) (described in Fig 7) and Tg(tg:nls-EGFP) expression in (B - C). (D) NFE was labeled using antibody against TP63 in sections of the thyroid gland isolated from Tg(tg:nls-EGFP) animals. Gills are marked based on their morphological appearance. DAPI labels nuclei. Scale bars: 10 µm (A - B), 50 µm (C - D).
Single-cell transcriptome analysis reveals thyrocyte diversity in the zebrafish thyroid gland (2020)
Figure 6

Figure 6: Thyrocytes display transcriptional heterogeneity

(A) Dot plot depicting expression entropy on Y-axis against average gene expression on X-axis for the thyrocyte population. Each dot depicts a gene, with red dots depicting genes that show statistically significant (p-value < 0.05) difference in entropy from expected value. Expected value is represented by black regression line. pax2a and ctsba are marked on the graph.

(B) t-SNE plot of the thyrocyte cluster with expression of tg, pax2a and ctsba. The color scale represents the normalized expression counts for each gene ranging from lowest (yellow) to highest (red).

(B') Histogram overlaid with density plots depicting the distribution of normalized gene expression for tg, pax2a and ctsba.
Single-cell transcriptome analysis reveals thyrocyte diversity in the zebrafish thyroid gland (2020)
Figure 7

Figure 7: pax2amKO2 knock-in line faithfully reports pax2a expression and knock-down

(A) Schematic of the knock-in strategy used to generate the pax2amKO2 line. Double strand break was induced between the penultimate codon and the STOP codon of pax2a gene using CRISPR/Cas9. DNA repair integrates the donor construct at the site of double strand break, resulting in a pax2a reporter line. The donor construct contains T2A-mKO2 reporter cassette flanked by left homology (LH) and right homology (RH) arms.

(B) Whole mount immunofluorescence of 9.5hpf pax2amKO2 embryos stained with anti-mKO2 antibody (red) and anti-PAX2A antibody (green). Anterior is to the left, and dorsal side is to the top. Scale bar: 0.1 mm.

(C) Whole mount immunofluorescence of 55 hpf pax2amKO2; Tg(tg:nls-EGFP) stained with PAX2A antibody (PAX2A-Ab) displays an overlap of mKO2 and PAX2A-Ab signal. The otic vesicle (OV), mid-hindbrain barrier (MHB), interneurons (IN) and thyroid gland (THY) is labelled. White dashed line represents the outline of the zebrafish larvae. Scale bar: 100 µm. Anterior to the right.

(D - F) Confocal microscopy imaging of a sagittal section of a 55 hpf pax2amKO2 (red); Tg(tg:nls-EGFP) (green) embryos immunostained with PAX2A antibody (cyan) showing co-localization of mKO2 and pax2a in the pronephros (D), thyroid gland (E) and mid-hindbrain barrier (F). In the thyroid gland, mKO2 (red), PAX2A-Ab (cyan) and thyrocyte-specific GFP (green) show co-localization. Scale bar: 50 µm.

(G , H) Snapshots from live imaging of 55 hpf pax2amKO2; Tg(tg:nls-EGFP) embryos injected with sgRNA targeting pax2a coding sequence. The anterior part of a representative control embryo (G) is shown alongside a representative crispant (H). Crispants display a strong reduction of mKO2 fluorescence, as well as an absence of GFP signal suggesting absence of thyroid (THY) tissue. White dashed line represents the outline of the zebrafish larvae. Scale bar: 100 µm. Anterior to the right.
Single-cell transcriptome analysis reveals thyrocyte diversity in the zebrafish thyroid gland (2020)
Figure 8

Figure 8: Immunofluorescence-based validation of thyrocyte heterogeneity

(A-D) Analysis of 3 mpf thyroid gland from pax2amKO2 zebrafish shows heterogeneity in pax2a reporter expression. (A) Whole mount confocal imaging of a 3 mpf pax2amKO2 thyroid labelled with mKO2 (red), E-cadherin (cyan, not shown in 'A' for clarity reasons) and DAPI (dark blue) for nuclear localisation. (B - D) Optical sections of three follicles, with mKO2-Low cells labelled with arrows. E-cadherin (B' - D') and DAPI (B'' - D'') staining shows that absence of mKO2 signal does not correspond to an absence of cells. Anterior to the bottom of the pictures.

(E,F) (E) Confocal image of thyroid gland section from Tg(tg:nls-EGFP) at 4 mpf stained with PAX2A antibody and DAPI. The dotted region is displayed at high magnification in

(F). Arrows marks thyrocytes displaying low PAX2A staining. Notably, PAX2A-Low thyrocytes display tg-driven EGFP expression, demonstrating their differentiated status. Data information: Scale bars: 250 µm (A), 50 µm (B - E), 10 µm (F).
Single-cell transcriptome analysis reveals thyrocyte diversity in the zebrafish thyroid gland (2020)
Figure 9

Figure 9: Transcriptome profiling of pax2amKO2-High and pax2amKO2-Low thyrocytes

(A - C) Cells from the thyroid gland of 5 mpf Tg(tg:nls-GFP); pax2amKO2 animals were labelled with calcein (live cell marker) and analysed using FACS. (A) A FACS plot showing calcein on Y-axis and GFP on X-axis. The box encompassing the GFP+ cells represents the thyrocyte population, which was gated for further analysis. (B) Histogram showing the distribution of GFP intensity in thyrocytes. (C) Histogram showing the distribution of mKO2 intensity in thyrocytes. Thyrocytes were selected by gating for GFP+ population. Horizontal lines indicate the mKO2-Low and mKO2-High expression level, with percentage values representing proportion of thyrocytes with mKO2-Low and mKO2-High expression.

(D , E) RNA-Seq. analysis of transcriptome isolated from pax2amKO2-High and pax2amKO2-Low subpopulations. (D) Volcano plot representing the differential expression of genes between the two thyrocyte subpopulations. Dots for pax2a, tg, mKO2 and EGFP are marked. Dots for genes that display significant differential expression (DESeq2, adjusted p-value < 0.05) are coloured. (E) Heatmap displaying the expression of selected genes in the two subpopulations. Biological replicates are represented by individual columns.

Expanded View Figure Legends
Bardet-Biedl Syndrome ciliopathy is linked to altered hematopoiesis and dysregulated self-tolerance (2020)
Figure 1

Figure 1. Basic characterization of Bbs4-deficient mouse models

(A) Relative expression of the BBSome subunits in the indicated organs and T cells measured by qPCR. CT values of the BBS genes were normalized to the geometric mean of the CT values of reference genes Gapdh, Tubb2a, and Eef1a1. The expression levels are normalized to those of brain (=1). Mean+SD. Three biological replicates (the result of each biological replicate is a median of three technical replicates).

(B) Immunoblot analysis of BBS4 expression in T and B lymphocytes isolated from the lymph nodes and spleen of C57Bl/6 mouse. β-actin staining served as a loading control. The identical immunoblot is shown in Fig EV4A. A representative experiment out of three biological replicates in total is shown.

(C) Schematic representation of mouse models of Bbs4 deficiency used in this study. Bbs4 GT, gene-trap allele interrupting the Bbs4 gene. Bbs4 FL, allele with floxed Bbs4 exon 6. Bbs4 KO and cKO alleles with deleted exon 6.

(D) Immunoblot analysis of BBS4 expression in testes and thymi lysates of Bbs4+/+ (WT), Bbs4GT/+, and Bbs4GT/GT mice. β-actin staining serves as a loading control. A representative experiment out of three biological replicates in total is shown.

(E) Immunoblot analysis of BBS4 expression in the brain lysates of Bbs4+/+, Bbs4GT/GT, and Bbs4KO/KO mice. β-actin staining served as a loading control. A representative experiment out of two biological replicates is shown.

(F) Hematoxylin and eosin staining of sections of seminiferous tubules from ~4 weeks old Bbs4+/+ (n=2 mice) and Bbs4GT/GT (n=4) males. Scale bar, 100 µm. Representative images are shown.

(G) Genotypic ratio of Bbs4+/+, Bbs4GT/+ or Bbs4KO/+ (Het), and Bbs4GT/GT (GT) or Bbs4KO/KO (KO) offspring at weaning from mating of Bbs4GT/+ (n=145 pups) or Bbs4+/KO (n=168 pups) parents. Binomial test was used for statistical comparison of the observed distribution to the expected Mendelian ratio.

(H) Bbs4+/+ and Bbs4GT/GT female littermates at 20 weeks of age. Representative litter out of seven in total.

(I) Bbs4+/+ and Bbs4KO/KO female littermates at 20 weeks of age. Representative litter out of five in total.

(J) Growth curves of Bbs4-deficient mice, mean ± SD is shown. Females: Bbs4+/+ (n=12 mice), Bbs4GT/GT (n=12), Bbs4KO/KO (n=6). Males: Bbs4+/+ (n=6 mice), Bbs4GT/GT (n=7), Bbs4KO/KO (n=5).

(K) Leptin concentration in blood plasma taken from young adult (7-8 weeks) or mid-age (14-20 weeks) mice. Young adult mice: Bbs4+/+ (n=4 mice), Bbs4KO/KO (n=3), analyzed in two independent experiments. Mean+SEM. Mid-age adult mice: Bbs4+/+ (n=7 mice), Bbs4KO/KO (n=5), Bbs4GT/GT (n=3), analyzed in 4 independent experiments. Kruskal-Wallis tests was used for the statistical analysis. Mean+SEM.