EMBO/SourceData huggingface dataset:
Figure 5(A) Kaplan-Meier curve representing distal metastasis-free survival of a cohort of breast cancer patients (N = 1609) as a function of their primary tumor's CFL1 expression levels (Data from KMPlot (Gyorffy et al., 2010)). Patients' primary tumors' CFL1 expression levels were classified as low (blue) or high (red) expression.(B,C) Membrane and membrane-associated proteins were purified from cells transfected with siRNA targeting PTPRN2 (B) or PLCβ1 (C) or control siRNA. Fractions were subjected to Western blot analysis for CFL1 and EGFR levels. Right, densitometry analysis of CFL1 levels normalized to EGFR levels.(B,C) Membrane and membrane-associated proteins were purified from cells transfected with siRNA targeting PTPRN2 (B) or PLCβ1 (C) or control siRNA. Fractions were subjected to Western blot analysis for CFL1 and EGFR levels. Right, densitometry analysis of CFL1 levels normalized to EGFR levels.(D,E) Membrane and membrane-associated proteins were purified from cells overexpressing PTPRN2 (D), PLCβ1 (E) or a control vector. Fractions were subjected to Western blot analysis for CFL1 and EGFR levels. Right, densitometry analysis of CFL1 levels normalized to EGFR levels.(D,E) Membrane and membrane-associated proteins were purified from cells overexpressing PTPRN2 (D), PLCβ1 (E) or a control vector. Fractions were subjected to Western blot analysis for CFL1 and EGFR levels. Right, densitometry analysis of CFL1 levels normalized to EGFR levels.(F) LM2 cells transfected with siRNA targeting PTPRN2, PLCβ1, or control siRNA were partially permeabilized and incubated with biotin-actin monomers. Cells were stained for incorporated biotin-actin monomers using Streptavidin-555 (red) and DAPI (blue). Right, quantification of mean fluorescence intensity of incorporated biotin-actin monomers. N = 100 cells/group. Scale bar, 20 μm.
Figure 6(A) LM2 cells transfected with siRNA targeting PTPRN2, PLCβ1, or control siRNA were stained with phalloidin (red) and DAPI (blue) and analyzed using fluorescence microscopy. Right, mean fluorescence intensity quantification of whole cell phalloidin signal. N = 40 cells/group. Scale bar, 10 μm.(B) Mean fluorescence intensity quantification of whole cell phalloidin signal in MDA-MB-231 cells overexpressing PTPRN2, PLCβ1, or control vector. N = 40 cells/group.(C,D) MDA-MB-231 cells were transfected with siRNAs targeting CFL1 or a control siRNA in the setting of control or PTPRN2 overexpression and subjected to the migration (C) or invasion (D) assays. N = 5 inserts/group.(E) MDA-MB-231 cells were transfected with siRNAs targeting CFL1 or a control siRNA in the setting of control or PLCβ1 overexpression were subjected to the migration assay. N = 5 inserts/group.(F) Bioluminescence imaging quantification of 40,000 MDA-MB-231 cells overexpressing PTPRN2 or control vector and transfected with control siRNA or siRNA targeting CFL1. Right, H&amp;amp;E staining of representative lung sections. N = 5 mice/group.(G,H) MDA-MB-231 cells overexpressing PTPRN2 (G) or PLCβ1 (H) were transfected with siRNA targeting the 3'UTR of CFL1 to deplete endogenous CFL1. Cells were further transfected with plasmids encoding either GFP-CFL1-WT or GFP-CFL1-Lck and subjected to the migration assay. N = 5 inserts/group.
Figure 1A. In situ hybridization for GemC1 and McIdas mRNA was performed on sagittal sections of E10.5 dpc mouse brain. GemC1 and McIdas mRNA is specifically detected in the developing choroid plexuses of the ventricles. Lower panels show higher magnifications. Scale bars, 100 µm.B. In situ hybridization on sagittal (upper) and coronal (middle and lower) sections of the airways of E15.5 dpc mouse embryos, depicting specific GemC1 and McIdas mRNA expression in the respiratory epithelium of the nasal and oral cavity and in the upper bronchial tree in the mouse lungs. Inserts show higher magnification images. Scale bars, 200 µm.C. MTECs were cultured ex-vivo and transitioned to ALI culture condition. Quantitative real time PCR was used to measure the expression levels of GemC1, McIdas, FoxJ1 and Geminin mRNA levels at different time points during multiciliogenesis. Values indicate expression levels (+/- standard error, s.e.) relative to ALI DAY 1 (marked by *), following normalization against the house-keeping HPRT mRNA. qPCR data were analyzed by the REST-MCS beta software from at least three independent experiments.
Figure 2A. and B. MTEC cultures were infected with a lentivirus expressing either GFP-GemC1 (GemC1) or GFP as a control and two days later transitioned to ALI culture conditions. Cells harvested at different time points during differentiation were co-stained with antibodies against GFP (green) to mark infected cells and endogenous McIdas(A) or Foxj1(B) (red). Arrows indicate infected cells that are McIdas or Foxj1 positive. Scale bars, 10 µm.C. and D. The percentage of infected cells expressing McIdas(C) or Foxj1(D) at each time point was quantified. Data are presented as the mean values of at least 10 independent fields obtained from at least two independent experiments for each condition. Error bars indicate ± SEM. *p<0.05, ***p<0.001. P-values were calculated by the non-parametrical two-tailed Mann-Whitney test.
Figure 3MCIDAS (A) regulatory elements (see materials and methods) cloned upstream of the luciferase gene were co-transfected into HEK293T cells with vectors expressing GemC1, McIdas and Geminin, as indicated or an empty vector (-) as a control. All luciferase experiments (A, B, D) were normalized for transfection efficiency with an expression vector for Renilla luciferase. Fold induction is the ratio between the normalized luciferase activity induced by the expression constructs and that induced by the empty expression vector. Data are the mean values of at least three independent experiments and error bars indicate ± SEM. **p<0.01, ***p<0.001. P-values were calculated by the non-parametrical two-tailed Mann-Whitney test. Abbreviations: RLF: Relative Luciferase Fold induction.FOXJ1 (B, D) regulatory elements (see materials and methods) cloned upstream of the luciferase gene were co-transfected into HEK293T cells with vectors expressing GemC1, McIdas and Geminin, as indicated or an empty vector (-) as a control. All luciferase experiments (A, B, D) were normalized for transfection efficiency with an expression vector for Renilla luciferase. Fold induction is the ratio between the normalized luciferase activity induced by the expression constructs and that induced by the empty expression vector. Data are the mean values of at least three independent experiments and error bars indicate ± SEM. **p<0.01, ***p<0.001. P-values were calculated by the non-parametrical two-tailed Mann-Whitney test. Abbreviations: RLF: Relative Luciferase Fold induction.McIdasmRNA levels after McIdas RNAi were assessed by qPCR (C).FOXJ1 (B, D) regulatory elements (see materials and methods) cloned upstream of the luciferase gene were co-transfected into HEK293T cells with vectors expressing GemC1, McIdas and Geminin, as indicated or an empty vector (-) as a control. In D, McIdas siRNAs or a control siRNA were also co-transfected. All luciferase experiments (A, B, D) were normalized for transfection efficiency with an expression vector for Renilla luciferase. Fold induction is the ratio between the normalized luciferase activity induced by the expression constructs and that induced by the empty expression vector. Data are the mean values of at least three independent experiments and error bars indicate ± SEM. **p<0.01, ***p<0.001. P-values were calculated by the non-parametrical two-tailed Mann-Whitney test. Abbreviations: RLF: Relative Luciferase Fold induction.
Figure 4A. and B. MCIDAS (A) and FOXJ1 (B) regulatory elements cloned upstream of the luciferase gene were co-transfected into HEK293T cells with vectors expressing GemC1, McIdas, Geminin and E2F5, as indicated, or an empty vector (-) as a control. Luciferase values were normalized against co-transfected Renilla luciferase and depicted as fold induction over the control empty vector. Data shown are mean values of at least three independent experiments and error bars indicate ± SEM. **p<0.01, ***p<0.001. P-values were calculated by the non-parametrical two-tailed Mann-Whitney test. Abbreviations: RLF: Relative Luciferase Fold induction.C. and D. Chromatin immunoprecipitation of MCIDAS and FOXJ1 promoter fragments by GemC1. HEK293T cells were co-transfected with vectors expressing GemC1-GFP (GemC1) or GFP as a control, E2F5 and DP1, together with the regulatory elements of either MCIDAS (C) or FOXJ1 (D). Following chromatin immunoprecipitation, DNA fragments of the MCIDAS and FOXJ1 promoters were detected in immunoprecipitates by qPCR. Data from a representative experiment are shown as fold enrichment of MCIDAS and FOXJ1 promoter fragments in anti-GFP immunoprecipitates over control IgG immunoprecipitates. At least two independent experiments were performed.
Figure 5B. GemC1KO/KO mice compared to their wild type littermates (GemC1KO/WT) at P5.C. Survival plot of mice homozygous for the GemC1 deletion (GemC1KO/KO, n=8) and control wild type or heterozygous littermates GemC1 (GemC1WT, n=14). GemC1 knock-out mice exhibit markedly reduced growth after birth and die within the first postnatal week.
Figure 6A., B. Transverse sections from trachea isolated from GemC1WT/WT (upper) or GemC1KO/KO (lower) P0 mice, immunostained with antibodies against acetylated y-tubulin (ACT, cilia marker, red), pericentrin (PCNT, marker for nascent centrioles, green), γ tubulin (centriole marker, green) and E-cadherin (marker for cell boundaries, blue). Serial images have been merged to show the complete trachea section (left). Inserts show higher magnification images for acetylated y-tubulin and pericentrin staining in GemC1WT/WT and GemC1KO/KO trachea, respectively. Scale bars, 10 µm.C. McIdas and Foxj1 specific antibodies were used for immunofluorescence on trachea from GemC1WT/WT (upper) or GemC1KO/KO (lower) P0 mice. Arrows show representative positive cells for McIdas (green) and FoxJ1 (red) in the GemC1WT/WT mice. DNA was stained with Draq-5 (blue). Scale bars, 10 µm.D. Transverse sections from P0 GemC1WT/WT (upper) or GemC1KO/KO (lower) trachea, immunostained with antibodies against Keratin 5 (Krt5, basal cell marker, green), and acetylated y-tubulin (ACT, cilia marker, red). DNA was stained with Draq-5 (blue). Serial images have been merged to show the complete trachea section on the left. Higher magnification images are shown on the right. Scale bars, 10 µm.E. Transverse sections from P0 GemC1WT/WT (left two images) or GemC1KO/KO (right two images) trachea, immunostained with antibodies against Muc5ac (marker for mucus-expressing cells, red) and E-cadherin (marker for cell boundaries, green). DNA was stained with Draq-5 (blue). Serial images have been merged to show the complete trachea section (left). Higher magnifications from the same section are shown on the right. Scale bars, 10 µm.
Figure 1C. Representative flow cytometric profiles of thymocytes from 6-week old Rb∆K4 and control mice for CD3-, CD4- and CD8-positive cell populations. Bottom, average of CD3-, CD4- and CD8 single- and double-positive cells in Rb∆K4 vs. control thymocytes showing no significant (n.s.) difference by student's t-test. Bars represent mean ±SD (standard deviation); n=6 biological replicates).D. Short telomeres in Rb∆K4 mice. Images of splenocyte metaphase spreads hybridized with the telomere repeat probe (pink). Note shorter telomeres in Rb∆K4 splenocytes (arrows). Bottom, quantitative analysis of 10 splenocyte metaphases from one pair of Rb∆K4 and control littermate; (P<0.001 by two-tailed Mann-Whitney-Wilcoxon test).
Figure 2C. Left, representative flow cytometric analysis for CD3, CD4 and CD8, showing near normal T cell development in 10-day-old Rb∆K7 mice. Right, average of CD3, CD4 and CD8 single- and double-positive cell populations in Rb∆K7 vs control thymocytes with no significant difference (n.s.) by student's t-test. Results are presented as mean ±SD, n=4 biological replicates).D. Left, images showing apparent normal division of retinal progenitors in Rb∆K7 mice. Mice were labeled with BrdU for 2 hrs; retinal sections from indicated genotypes were stained with DAPI (blue), Ki67 (green), PH3 (red) or BrdU (red) to mark nuclei, all dividing cells, mitotic or S-phase cells, respectively. Right, quantification of staining showing no significant difference among the genotypes. Bars denote mean ±SD; n=3 biological replicates). Scale bar: 20 μm.E. Left, top, western blot analysis of Rb∆K7 thymocytes with an antibody that recognizes both hyper-phosphorylated (ppRb) and hypo-phosphorylated pRb species. Right, phospho-specific immunoblots showing absence of phosphorylation at upstream Thr350 and Ser601 or the substituted Ser773Ala site in Rb∆K7 thymocytes. Right, immuno-precipitation (IP)-Western blot analysis demonstrating phosphorylation of pRb∆K7 on Ser243/Thr246 (top) and Ser605 (bottom). n.s., non-specific band.
Figure 3B. Increased resistance of Rb∆K7 vs. control MEFs to CDK2 inhibition (P=0.008 by two-tailed unpaired student's t-test). Effect of a pan CDK inhibitor was near significance (P=0.07). Bars represent mean ±SD, n=3 independent MEFs.C. Left, representative flow cytometric analysis with 7-AAD and AnnexinV, showing a significant decrease in apoptosis in Rb∆K7 MEFs relative to control. Right, significant differences in average percentage of live (7-AAD-:AnnexinV-) vs. apoptotic (7-AAD+:AnnexinV+) cells. Bars represent mean ±SD; P value calculated by two-tailed unpaired student's t-test; n=3 independent MEFs.
Figure 4F-I. Delayed hair re-growth in eight-month old Rb∆K7 mice two weeks post hair plucking (F). (G) Abnormal skin histology in Rb∆K7 mice relative to control. FCL, subcutaneous fat cell layer, T, telogen; A, anagen. (H-I) Representative immunostaining for keratin 14 and PCNA showing reduced expression in Rb∆K7 hair follicles vs controls. Scale bar, 50 μm.J. Hyperglycemia in Rb∆K7 but not Rb∆K4 mice. Blood glucose in 10-12-month old fasting Rb∆K7 (left, n=11) or 18-month old fasting Rb∆K4 (right, n=15) mice versus control littermates (n=6 and n=8, respectively). Mean ±SD; P values by two-tailed unpaired student's t-test.
Figure 5B. Glucose tolerance Test (GTT) demonstrating progressive defect in clearance of blood glucose in Rb∆K7 mice. 6-7 week (left) or 6 month (right) old fasting Rb∆K7 mice and control littermates (n=5-6 each per group) were injected with glucose, and blood glucose was determined at indicated intervals. Error bars represent SD.F. Loss of pancreatic islet area in Rb∆K7 mice correlates with reduced proliferation and H3K36me3 histone marks, but not with increased cell death. TUNEL analysis showing apoptosis in 10 day-old Rb∆K7 islets is even lower than in control littermates. Arrow points to a rare apoptotic cell in control islet. Serine800/804 phosphorylation of pRb in β-cells from 10 day-old control but not Rb∆K7 islets with a Ser-to-Ala substitution at this site. BrdU incorporation and PH3 staining showing reduced S-phase and mitotic cells, respectively, in 2- and 3-week old Rb∆K7 vs control islets. Reduced H3K36me3 staining in pancreatic islets from 3-week old Rb∆K7 mice compared to control littermates (n>3 for each experiment). Scale bar, 100 μm.H. Reduced ability of Rb∆K7 β-cell to re-enter the cell cycle in response to pregnancy (top) or mitogenic (bottom) signals. Top, cell proliferation in P14.5 pregnant Rb∆K7 islet β-cell vs. control was determined by double immunostaining with BrdU and insulin (left); quantification (right). Results were normalized for the number of BrdU/Insulin positive cells in control islets. An independent experiment using ki67 to quantify cell cycle re-entry top. Bottom, cell proliferation in Rb∆K7 β-cell vs. control islets following Exendin4 treatment was determined by immunostaining for Ki67; results were normalized for the number of positive cells in control islets. Mean ±SD, P values by two-tailed unpaired student's t-test. Scale bar, 100 μm.I. Representative immunostaining showing induction of PDX1 (islet), SOX9 (pancreatic epithelium) and MCM4 (islet) expression in Rb∆K7 vs control mice. Scale bar, 100 μm.
Figure 6D. Representative image of a pancreatic islet from a 9-month old Rb∆K7 mouse immunestained for the SASP marker IL-6 (red) and insulin (green). DAPI was used to stain nuclei (blue). Arrows point to IL-6+, insulin+ pancreatic β-cells. Scale bar, 20 μm.E. Representative immunostaining for the DDR marker γ-H2A in islets from 3-week old Rb∆K7 and control mice (left); statistical analysis (right; n=5 each). Arrows point to pancreatic γ-H2A-positive Rb∆K7 islet cells. Error bars represent SD; P value calculated by one-way ANOVA, Tukey's multiple comparisons test. Scale bar, 100 μm.H. Vitamin C diet increases 5hmC expression and improves β-cell morphology and islet size in Rb∆K7 mice. Scale bar, 100 μm. I. Vitamin C diet reduces the difference in γ−H2A+ β-cells in Rb∆K7 vs control pancreatic islets relative to regular diet by 3.74 folds. Bars indicate mean ±SD. Adjusted P values by one-way ANOVA, Tukey's multiple comparisons test. J. Vitamin C diet increases pancreatic islet cell cycle re-entry during pregnancy of Rb∆K7 mice relative to control pregnant mice (no statistical difference, n.s.) or relative to pregnant Rb∆K7 vs control mice fed on regular diet (P<0.0001) by 3.8 folds. Bars represent mean ±SD. Adjusted P values by one-way ANOVA, Tukey's multiple comparisons test.
Figure 7B. Left, top, effect of retrovirus-mediated transduction of under-phosphorylated Rb∆K11 (rRb∆K11) on Rb-deficient mammary tumor cell proliferation as determined by MTT assay (n=4-5 technical replicates). Left, bottom, quantification of SA-βGAL in rRb∆K11-transduced cells. Bars represent mean ±SD; P value calculated by two-tailed unpaired student's t-test. Right, representative images of SA-βGAL staining showing robust senescence (arrows) in rRb∆K11-transduced cells. Original magnification: 200x.F. Vitamin C reduces cellular senescence induced by retroviral-mediated over-expression of Rb∆K11. Top - images of SA-βGAL staining in human diploid cells, IMR90, transduced with control or rRb∆K11 virus with or without vitamin C added at the time of infection. Arrows point to SA-βGAL-positive cells. Bottom left, quantification of SA-βGAL staining (bars represent mean ±SEM; n=4 biological replicates). P values by one-way ANOVA, Tukey's multiple comparisons test. Bottom right, dot-blot analysis with antibody for 5hmC on DNA extracted from IMR90 cells treated or not with vitamin C. Original magnification: 200x.
Figure 1Associations between CSF T-tau, Ng and NFL (panels A-C). Black squares: CN (n=109), red circles: MCI (n=187), blue triangles: AD (n=93). Associations are shown for Spearman correlations. Aβ42 and T-tau were measured using the the INNOBIA AlzBio3 kit (Fujirebio, Ghent, Belgium), Ng was measured using an in-house immunoassay for Ng (Portelius et al, 2015) and NFL was measured using the NF-light® ELISA kit (Uman Diagnostics, Umeå, Sweden).Associations between CSF T-tau, Ng and NFL and between these biomarkers and CSF A42 (panels D-F). Black squares: CN (n=109), red circles: MCI (n=187), blue triangles: AD (n=93). Associations are shown for Spearman correlations. Aβ42 and T-tau were measured using the the INNOBIA AlzBio3 kit (Fujirebio, Ghent, Belgium), Ng was measured using an in-house immunoassay for Ng (Portelius et al, 2015) and NFL was measured using the NF-light® ELISA kit (Uman Diagnostics, Umeå, Sweden).
Figure 1(B) GST, GST-MCM2N (human) and GST-Spt5N (yeast) were mixed as indicated with yeast histones H3-H4, in the presence of glutathione agarose beads (see STAR method for further details). The input and pulldown samples were analysed by SDS-PAGE before staining with colloidal Coomassie blue. The asterisks denote truncations of GST-Spt5N. (C) Similar experiment to (B) but with histones H2A-H2B. (D) Similar experiment to (B) to compare histone binding by GST-MCM2N and GST-MCM2N-2A (with Y81A Y90A mutations of conserved residues).(H). Budding yeast cells expressing fusions of Spt5N and Spt5N-3A to Protein A were grown in parallel with cells expressing the equivalent wild type and mutant versions of fission yeast Spt5N (Sp Spt5N and Sp Spt5N-3A - the latter contained the F146A, E150A and V153A mutations).
Figure 2(C) Yeast cells were generated that expressed Protein A-tagged versions of wild type Spt5 (full length or the indicated truncations), or full length Spt5-3A, under the control of the GAL1,10 promoter. Cells were grown in medium containing galactose and the ProteinA-tagged Spt5 proteins were isolated from cell extracts on magnetic beads coated with IgG. Association of the tagged Spt5 proteins with the elongating form of Pol II was monitored by immunoblotting, using antibodies specific to the indicated phosphorylation sites in the C-Terminal Domain repeats of the Rpb1 subunit of Pol II.(E) Serial dilutions of control cells or spt5-AID cells, expressing the indicated versions of Spt5 from the GAL1,10 promoter, were grown on the indicated medium.
Figure 3(B) Immunoblot analysis at the indicated times for the samples described in (A).(C) Cells from the end of the experiment described in (A) were fixed and processed for ChIP-Seq, using antibodies specific for phosphorylated Serine 5 in the C-Terminal Domain of the Pol II catalytic subunit Rpb1 (CTD-Ser5P). For each of the three indicated strains, the histograms represent the normalized read density (DNA sequence read count per million reads, or RPM), across a sample gene body.
Figure 4(A) Samples from the experiment described in Figure 3A were also processed for MNase-Seq. For each of the three indicated strains, the histograms represent the normalized DNA sequence read count per million reads (RPM), across a sample gene body.(C) Samples from the same experiment were further processed for ChIP-Seq using antibodies to histone H3K4me3. For each of the three indicated strains, the normalised read count per million reads is presented across a sample gene body.
Figure 1 Decreasing ∆ψm from ∆III to ρ0 cells. WT and mutant yeast cells were cultured to log phase and stained with TMRM for FACS analysis.  Decreasing proliferation rates from ∆III to ρ0 cells. Cells were cultured from 0.05 OD600 in SCD. OD600 was measured every 2 hours.  Transcriptome analysis of OXPHOS mutants. WT and mutant yeast cells were cultured to log phase. RNAseq was performed with three biological replicates per genotype (Dataset EV1). All the transcripts and four categories of transcripts (gene lists in Table EV1B) are shown. Significantly changed genes (P value < 0.05; fold change > 1.5 or < 0.67) are highlighted with dashed lines; numbers of significantly changed genes are indicated. qRT-PCR analysis of selected transcripts in ρ0 cells.
Figure 2 SILAC analysis of mitochondrial proteome of ∆IV and ρ0 cells. Equal amount of WT cells labeled with heavy amino acids and unlabeled mutant cells were mixed to extract mitochondrial fraction for mass-spectrometry analysis. Three biological replicates were prepared for each pair of strains (Dataset EV2). 568 high fidelity mitochondrial proteins (gene/protein list in Table EV1A) in ∆IV cells and 572 such proteins in ρ0 cells were quantitatively measured. Significantly changed proteins (P value < 0.05; fold change > 1.5 or < 0.67) are highlighted with dashed lines; numbers of significantly changed proteins are indicated.  Western blot analysis of representative mitochondrial proteins endogenously tagged with HA. The selection markers were removed by the Cre-loxP system. Whole cell lysates were extracted and equal amount of proteins was loaded for western blot. Polysome fractionation analysis of OXPHOS mutants. Polysomes were fractionated by sucrose gradients (10%-50%), and the gradients were separated and measured at A254.