EMBO/soda-nel huggingface dataset:
Functional coordination of non-myocytes plays a key role in adult zebrafish heart regeneration (2021)
Figure 3

Figure 3. Transcriptome dynamics of fibroblasts and endothelial cells during zebrafish heart regeneration.

A-C, Zoom-in analysis of fibroblasts identified in Fig 2. A, tSNE plot colored by FB subpopulations. B, Dot plot showing expression of ECM genes in FB subpopulations. C, Bar plot showing relative proportion of FB subpopulations in FB at each time point. D-E, Expression of fn1a, col12a1a (D) and tagln (E) in FB at each time point. F, In situ hybridization showing temporal spatial expression patterns of postnb. The white boxed region is shown in zoom-in images at the bottom. Scale bar = 25 μm. White dashed lines outline the heart and the yellow dashed lines indicate approximate resection plane. A stands for arium, V stand for ventricle and OFT stands for outflow tract. G, Expression of tnfrsf1a and tgfbr2a in FB at each time point. H, Fluorescent in situ hybridization for postnb, tnfα, and tnfrsf1a in the injury area at 7 dpf, respectively. The white boxed regions are shown in their respective zoom-in images to the right. White dashed lines outline cardiac apex and the yellow dashed lines indicate approximate resection plane. Scale bar = 25 μm. I, tSNE plot colored by eEC subpopulations. Bar plot showing relative proportion of eEC subpopulations in eEC at each time point. J, Violin plots showing expression of cxcl18b, sele, atf3, and fosl1a in each eEC subpopulation. In panels E and I, "un" stands for uninjured. K, Fluorescent in situ hybridization for fosl1a at 7 dpi. The white boxed region is shown in a higher magnification image to the right for the injury area. cdh5 was used to label all EC cells and the red boxed region is shown in a higher magnification image to highlight a pan-EC cdh5 expression in green. White dashed lines outline the heart and the yellow dashed lines indicate approximate resection plane. Scale bar = 25 μm. A stands for arium, V stand for ventricle and OFT stands for outflow tract.
Functional coordination of non-myocytes plays a key role in adult zebrafish heart regeneration (2021)
Figure 4

Figure 4. Loss of kit function impairs heart regeneration.

A, Wildtype or kit mutant stained with AFOG at 14 days and 30 dpi. Sham-operated zebrafish hearts serve as control. Scale bar = 100 μm. B, Quantification of scar area at 30 dpi. N = 10 hearts. Data are presented as Mean ± SEM. P value calculated with two-tailed students' t test. ** P < 0.01. C, PCNA/Mef2 double staining showing the proliferating CMs in wildtype and mutant hearts at 7 dpi. The white boxed regions are shown in zoom-in images to their right. Blue dashed lines indicate approximate resection plane. White arrows point to the PCNA/Mef2 double positive nuclei. Scale bar = 50 μm. D, Quantification of % PCNA+ CMs (Mef2+) in C. N = 10 hearts. Data are presented as Mean ± SEM. P value calculated with two-tailed students' t test. ** P < 0.01. E-F, Analysis of bulk RNA-seq data of freshly isolated CMs at different time points during heart regeneration. E, Two representative gene sets from GSEA analysis, which are enriched in DEGs between mutant and wildtype CMs at 2 dpi. F, Heatmap showing expression levels of cell cycle regulators in CMs from wildtype and kit mutant hearts. G-H, Joint analysis of kit mutant nonCM scRNA-seq data from 0, 2, 7, and 14 dpi with LIGER. G, All nonCM visualized on tSNE. H, Bar plots showing comparison of nonCM cell type contribution between wildtype and kit mutant at each time point. I, Immunostaining for MC marker IB4 in WT (left) and mutant hearts (middle) at 7 dpi. Quantification of IB4+ cell number per unit area (right). The white boxed regions are shown in their respective zoom-in images to the right. White dashed lines indicate approximate resection plane. Scale bar = 50 μm. N = 6 hearts. Data are presented as Mean ± SEM. P value is calculated with two-tailed students' t test. * P < 0.05. In panels f and h, "un" stands for uninjured.
Functional coordination of non-myocytes plays a key role in adult zebrafish heart regeneration (2021)
Figure 5

Figure 5. Loss of kit function altered transcriptome dynamics of cardiac nonCM during zebrafish heart regeneration.

A-G, Joint analysis of WT and kit mutant MC. A, tSNE plot colored by WT and kit mutant MC subpopulations. B, Representative GO terms of genes upregulated in MC1 (WT, red bars) or KMC1 (kit mutant, blue bars) from the uninjured hearts, respectively. Dotted lines indicate P= 0.05. C, Bar plots showing proportion of each WT and kit mutant MC subpopulation in total nonCM at each time point. D, F, Violin plots showing the expression of il1b in MC1 (D), and ctsd expression in MC (F). E, G, Expression of il1b (E) and ctsd (G) in WT and kit mutant hearts determined by qRT-PCR. H-N, Joint analysis of WT and kit mutant FB. H, tSNE plot colored by WT and kit mutant FB subpopulations. I, Bar plots showing proportion of each WT and kit mutant FB subpopulation in total nonCM at each time point. J, L, Violin plots showing expression of col12a1a in FB3 (J) and mif expression in FB (L). K, M, Expression of col12a1a (K) and mif (M) in WT and kit mutant hearts determined by qRT-PCR. N, Bar plots showing relative proportion of each WT and kit mutant eEC subpopulation in eEC at each time point. O, Violin plots showing expression of junba in eEC. P, Expression of junba in WT and kit mutant hearts determined by qRT-PCR. Q, Representative image of hearts from 7 dpi tcf21:nucGFP transgenic fish treated by PBS liposome, Clodronate liposome and Dex, respectively. The red boxes mark the peripheral area, and the white boxes mark the injury area and are shown in their respective zoom-in images to the right.. White dashed lines indicate approximate resection plane. Scale bar = 50 μm. R, Quantification of the number of tcf21:nucGFP positive cells in the boxed areas in U. N = 5 hearts. Data are presented as Mean ± SEM. Two-tailed students' t test. *** P < 0.001. Data information: E, G, K, M, P. Mean ± SEM shown. N = 3. P value calculated with two-tailed students' t test. * P < 0.05, ** P < 0.01, *** P < 0.001. In panels (C-G, I-P), "un" stands for uninjured. Clod lipo stands for clodronate liposomes. Dex for dexamethasone.
53BP1-mediated recruitment of RASSF1A to ribosomal DNA breaks promotes local ATM signaling (2022)
Figure 1

Figure 1. RASSF1A recruitment at double strand breaks.

(A) HeLa cells were exposed to 5 Gy ionizing radiation (γIR), collected at the indicated time points and stained for RASSF1A and γH2AX. Images for representative intra-nuclear RASSF1A foci distribution are shown. Nucleolar boundaries are marked with dashed lines.

(B) Quantification of the number of cells with RASSF1A foci at the time points presented in (A). Error bars represent standard deviation and derive from 3 independent experiments.

(C) HeLa cells were treated with 5 Gy γIR and 2 h post treatment fixed and stained with the indicated antibodies.

(D) HeLa cells were irradiated with 5 Gy and 2 h post treatment stained for RASSF1A (R1A) and NUCLEOLIN (NCL).

(E) HeLa cells were treated with 50 ng/ml NCS for 30 min, washed and 2 h later fixed and stained with the indicated antibodies. The distance of RASSF1A+/γH2AX+ foci or RASSF1A-/γH2AX+ foci to the closest nucleolus (based on Fibrillarin (Fb) staining) was measured. Middle line represents the median and the boxes 25th and 75th percentiles. The whiskers mark the smallest and largest values.

(F) AsiSI expression in U2OS cells was induced by OHT and cells were stained for RASSF1A and γH2AX. Boxed areas are shown in higher magnification. Nucleolar boundaries are marked with dashed lines.

(G) Quantification of the number of cells with RASSF1A foci in (F). Error bars represent standard deviation and derive from 3 independent experiments.

(H) U2OS cells with stable integration of a Lac operator (LacO) sequence adjacent to an I-SceI endonuclease site were transfected with Lac Repressor (LacR)-mCherry and GFP or GFP-NLS-HA-I-SceI. 24 h post transfection cells were fixed and stained for 53BP1 or RASSF1A.

(I) Quantification of the LacO arrays enriched for 53BP1 or RASSF1A in (H). Error bars represent standard deviation and derive from 3 independent experiments.

(J) HeLa cells were transfected with I-PpoI endonuclease and stained for RASSF1A and γH2AX. Fluorescence intensity profiles of RASSF1A (green) and γH2AX (red) signals across the HeLa nuclei are shown. Position of line scan indicated by the yellow line.

Data information: DNA was stained with DAPI. Scale bars = 10 μm. P values in (B, G and I) were calculated using two-tailed student t-test. P value in (E) was calculated using a Mann-Whitney test. * Indicates P=0.05-0.01, ** indicates P= 0.01-0.001, *** indicates P ≤ 0.001
53BP1-mediated recruitment of RASSF1A to ribosomal DNA breaks promotes local ATM signaling (2022)
Figure 2

Figure 2. Phosphorylated RASSF1A at Serine 131 localizes at the rDNA DSBs.

(A) HeLa cells were transfected with I-PpoI mRNA and collected at the indicated time points. Cells were co-stained for RASSF1A (R1A) and NBS1. Boxed areas are shown in higher magnification. Nucleolar boundaries are marked with dashed lines.

(B) Quantification of (A). Error bars represent standard deviation and derive from 3 independent experiments.

(C) Assessment of RASSF1A localisation after induction of rDNA DSBs using the I-PpoI endonuclease in methanol fixed HeLa cells. Fluorescence intensity profile of RASSF1A (green) and LAMIN A/C (red) signals across the HeLa nuclei in the presence and absence of rDNA DSBs in control and I-PpoI treated cells. Position of line scan indicated by the yellow line.

(D) HeLa cells were transfected with the I-PpoI mRNA and 6 h later stained for RASSF1A-pS131 (R1A-pS131) and γH2AX. Fluorescence intensity profile of RASSF1A-pS131 (green) and γH2AX (red) signals across the HeLa nuclei are shown. Position of line scan indicated by the yellow line.

(E) Quantification of cells with RASSF1A-pS131 positive nucleolar caps 6 h post I-PpoI transfection. Error bars represent standard deviation and derive from 3 independent experiments. Representative images are shown in Fig EV3A.

(F) HeLa cells were treated with the ATMi or ATRi prior to transfection with I-PpoI mRNA. 6 h post mRNA transfection cells were stained for RASSF1A-pS131 (R1A-pS131).

(G) Quantification of (F). Error bars represent standard deviation and derive from 3 independent experiments.

(H) HeLa cells were treated or not with ATM inhibitor (ATMi) followed by I-PpoI mRNA transfection and 6 h later cell lysates were analyzed by western blot for the indicated antibodies.

(I) HeLa cells were treated or not with ATMi followed by I-PpoI mRNA transfection and 6 h later cells were fixed and RASSF1A recruitment at rDNA DSBs was assessed.

(J) Quantification of (I). Error bars represent standard deviation and derive from 3 independent experiments.

Data information: DNA was stained with DAPI. Scale bars = 10 μm. P values in (E, G and J) were calculated using two-tailed Student 's t-test. * Indicates P=0.05-0.01, ** indicates P= 0.01-0.001, *** indicates P ≤ 0.001
53BP1-mediated recruitment of RASSF1A to ribosomal DNA breaks promotes local ATM signaling (2022)
Figure 3

Figure 3. RASSF1A recruitment at rDNA DSBs depends on 53BP1.

(A) HeLa cells with I-PpoI induced rDNA breaks were stained with the indicated antibodies. Intra-nucleolar cap localization for RASSF1A and 53BP1 in correlation with other proteins was accessed. Boxed areas are shown in higher magnification.

(B) HeLa cells were transfected with I-PpoI mRNA and stained for RASSF1A and 53BP1. Fluorescence intensity profiles of RASSF1A (green) and 53BP1 (red) signals across the HeLa nuclei are shown. Position of line scan indicated by the yellow line.

(C) HeLa cells were treated with siRNAs against LUCIFERASE (siLUC) or 53BP1 (si53BP1), 48 h later transfected with I-PpoI mRNA, fixed and stained for RASSF1A (R1A) and γH2AX.

(D) Quantification of (C) and western blot analysis from siLUCIFERASE (siLUC) or si53BP1 treated cells for the indicated antibodies. Error bars represent standard deviation and derive from 3 independent experiments.

(E) HeLa cells were treated with siRNAs against LUSIFERASE (siLUC) or RNF8 (siRNF8), 48 h later transfected for I-PpoI mRNA, fixed and stained for RASSF1A and γH2AX.

(F) Quantification of (E) and western blot analysis from siLUCIFERASE (siLUC) or siRNF8 treated cells for the indicated antibodies. Error bars represent standard deviation and derive from 3 independent experiments.

(G) HeLa cells were treated with siRNA against 53BP1, or an ATM inhibitor (ATMi) followed by 5 Gy of ionizing radiation (γIR). Cells were fixed 2 h post treatment and stained for RASSF1A. Nucleolar boundaries are marked with dashed lines.

(H) Quantification of cells with RASSF1A foci in (G). Error bars represent standard deviation and derive from 3 independent experiments.

(I) Western blot analysis from total cell extracts with the indicated treatments and RASSF1A immuneprecipitates (IPs) with the indicated antibodies. IgG immunoprecipitation served as a control.

(J) Western blot analysis from total cell extracts and RASSF1A immunoprecipitates with the indicated treatments with the indicated antibodies. IgG immunoprecipitation served as a control.

(K) U2OS cells were transfected with FLAG-RASSF1A (FLAG-R1A), FLAG-RASSF1A131A (FLAG-R1A131A) or pcDNA3 and 24 h later transfected with the I-PpoI mRNA. Cell lysates were subjected to immunoprecipitation against FLAG. Western blot analysis of total cell extracts and the IPs is shown.

Data information: DNA was stained with DAPI. Scale bars = 10 μm. P values in (D, F and H) were calculated using two-tailed Student 's t-test. * Indicates P=0.05-0.01, ** indicates P= 0.01-0.001, *** indicates P ≤ 0.001
HOPS/TMUB1 retains p53 in the cytoplasm and sustains p53-dependent mitochondrial apoptosis (2019)
Figure 1

Figure 1 - Generation of Hops−/− mouse.

A Schematic diagram of gene targeting of Hops locus. Targeting vector containing lacZ and a ZEN-UB1 cassette was inserted into HOPS gene between exon 2 and 3, flanking with 2 kb 5′ - homology and 2 kb 3′ - homology.

B Hops−/− mouse genotyping and protein expression. Genotyping by PCR using genomic DNA showed a 242bp band for wild type (Hops+/+) allele and a 331bp band for mutant (Hops−/−) allele. NTC is Null Template Control (left panel). Genotyping by real time PCR confirmed the absence of Hops mRNA in Hops−/− mice (middle panel). Protein extracts from Hops+/+ and Hops−/− MEFs were analysed by Western Blot using anti-HOPS antibody and anti-α-tubulin antibody as loading control (right panel).

C Offspring distribution. Different HOPS genotypes at weaning age (approximately postnatal day 21) from crosses between homozygous knockout mice males and heterozygous females (n=1525 pups).

Data information: in B data are presented as mean ±SD. In C the χ2 test was performed to determine significant differences between the expected and observed ratios of Hops−/− to Hops+/− mice. P < 0.001, by two tailed Student's t-test.
HOPS/TMUB1 retains p53 in the cytoplasm and sustains p53-dependent mitochondrial apoptosis (2019)
Figure 2

Figure 2 - Hops−/− mice resistance to apoptosis.

A Reduced caspase-3 activation in Hops−/− mice upon apoptotic stimulus. Hops+/+ and Hops−/− mice were treated with etoposide for the indicated times and thymus, spleen, testis and liver were harvested and Tissue-Tek® O.C.T. embedded. Sections were immunostained with anti-cleaved caspase-3 antibody followed by appropriate secondary antibody labelling (green). The graphs display the relative positive cells number. Scale bars, 10µm. Cleaved caspase-3 and DAPI staining merged images were shown in Appendix Fig 1A-D.

B HOPS affects p53 increase after stress in vivo. Whole-cell homogenates from organs, were immediately prepared and immunoblotted with antibodies to detect p53 and GAPDH. The p53 levels were semi-quantified using GAPDH as loading control and relative p53 levels at time 0 were assumed as 1.

Data information: all the experiments were performed three times in three different mice per condition and a representative example is shown. In A, B data are presented as mean ±SD. ** P < 0.01; *** P < 0.001, by two tailed Student's t-test.
HOPS/TMUB1 retains p53 in the cytoplasm and sustains p53-dependent mitochondrial apoptosis (2019)
Figure 3

Figure 3 - HOPS acts on apoptosis induction.

A Thymocytes from Hops+/+ and Hops−/− mice were treated with dexamethasone (Dex) and the apoptotic cells were measured by flow cytometry. The flow cytometry analysis plots are shown on the left and the data represented as a histogram on the right.

B HOPS was pivotal for apoptosis induction. Hops+/+ and Hops−/− MEFs were subjected to etoposide (Eto) and apoptosis was analysed by flow cytometry (left) and showed as a graph (right).

C HOPS overexpression in MEFs promotes apoptosis. Hops+/+ and Hops−/− MEFs were transiently transfected with an empty vector (pEGFP-N1) or with a Hops encoding vector (pEGFP-N1-Hops). The apoptotic cells were measured by flow cytometry (left) on the GFP positive and negative populations and showed as a graph (right).

D HOPS affects p53 levels following stress in vitro. Hops+/+ and Hops−/− MEFs were treated with etoposide and harvested at the indicated time points. The p53 protein levels were semi-quantified using α-tubulin as loading control and relative p53 levels at 0 µM dosage were assumed as 1.

Data information: the experiments were performed three times in primary cells from three different mice per condition. The experiments in MEFs were performed three times. Representative panels are shown. In A, B, C data are presented as mean ±SD. * P < 0.05; ** P < 0.01; *** P < 0.001, by two tailed Student's t-test.
HOPS/TMUB1 retains p53 in the cytoplasm and sustains p53-dependent mitochondrial apoptosis (2019)
Figure 4

Figure 4 - HOPS role in p53 stability and p53 ubiquitination.

A HOPS loss and p53 half-life. Hops+/+ and Hops−/− MEFs untransfected or transfected with HOPS, were analysed by immunoblotting following cycloheximide treatment for the indicated time (left panel). The p53 protein levels were semi-quantified using GAPDH as loading control and relative p53 levels at time 0 were assumed as 100% (right panel).

B p19Arf-independent HOPS control of p53 half-life. p19Arf−/− MEFs untransfected (CTRL), transfected with empty vector (pSGV) or HOPS were treated with cycloheximide for the indicated times and subjected to immunoblot analysis with anti-p53, anti-HOPS and anti-GAPDH antibodies (left panel). The p53 protein levels were semi-quantified as above (right panel).

C HOPS and p53 ubiquitination. p53, HDM2, His-tagged ubiquitin were co-expressed in H1299 cells with HOPS or HOPS-G176A or HOPS-K126A mutants and treated with MG132. p53 ubiquitination rate was evaluated by elution using Ni2+-NTA His-bind resin followed by immunoblotting with anti-p53 and the indicated antibodies.

D HOPS depletion and HOPS-G176A mutant affect p53 ubiquitination. Hops+/+ and Hops−/− MEFs untransfected and Hops−/− MEFs transfected with HOPS or HOPS-G176A mutant were co-expressed with p53, HDM2 and His-tagged ubiquitin. Cells were treated with MG132 and the ubiquitinated p53 fraction was recovered as above. Immunodetection of ubiquitinated p53 was performed using anti-p53 antibody and the samples were tested with the indicated antibodies.

E HOPS-G176A mutant ability to stabilize p53. Hops−/− MEFs were transfected with HOPS-G176A, subjected to cycloheximide treatment for the indicated times and analysed by immunoblotting using the indicated antibodies.

Data information: all the experiments in MEFs were performed three times. Representative panels are shown. In A, B data are presented as mean ±SD.
HOPS/TMUB1 retains p53 in the cytoplasm and sustains p53-dependent mitochondrial apoptosis (2019)
Figure 5

Figure 5 - HOPS binding to p53.

A Protein binding in RKO cells. PLA showed HOPS/p53 interaction with or without etoposide treatment at the indicated time. Red spots showed a single interaction. DNA counterstaining was performed with DAPI. α-Tubulin-FITC antibody was used to identify the cellular edge. Background control was performed according to the manufacturer's instruction. Scale bars, 10µm. The graph represents the binding increase. Non treated cells binding was assumed as 1.

B Biochemical analysis of nuclear-cytoplasmic protein distribution. RKO cells were treated with etoposide, collected after 2 and 4 hours and processed to obtain nuclear and cytoplasmic fractions. Aliquots relative to the same cell number per fraction were immunoblotted using anti-HOPS and anti-p53 antibodies. GAPDH and Lamin B1 are purity controls of cytoplasmic and nuclear fraction respectively.

C HOPS and p53 binding in cytoplasm following apoptosis induction. RKO cells untreated or treated with etoposide as above were used to isolate cytoplasmic fractions. WCL and cytoplasmic fractions were immunoprecipitated with anti-HOPS antibody and subsequently immunoblotted using anti-p53 antibody.

Data information: all the experiments were performed three times, and representative examples are shown. In A data are presented as mean ±SD.
HOPS/TMUB1 retains p53 in the cytoplasm and sustains p53-dependent mitochondrial apoptosis (2019)
Figure 6

Figure 6 - HOPS, p53 and mitochondria.

A HOPS localization at mitochondria. RKO cells were stained by MitoTracker™ (red), immediately fixed and stained to visualize HOPS (green). Nuclei were DAPI stained (blue). Samples were analysed with fluorescent microscope and representative images were shown. Scale bars, 10µm.

B RKO cells were lysed and processed for mitochondria isolation. WCL, cytoplasm and mitochondria were analysed using anti-HOPS antibody. GAPDH, Lamin B1 and Tom20 are purity controls of mitochondrial fraction.

C HOPS and p53 translocation to mitochondria. RKO cells were treated with etoposide as indicated and processed for mitochondrial extraction and subsequent immunoblotting for p53, HOPS and the reported extraction purity controls.

D HOPS/p53 binding at mitochondria. RKO cells were treated with etoposide and subjected to mitochondrial purification, and subsequent immunoprecipitation using anti-HOPS antibody. Samples were immunoblotted to detect p53, HOPS and the reported controls.

E HOPS and p53 mitochondrial translocation in vivo. Hops+/+ and Hops−/− mice were treated with etoposide and thymus, spleen and testis were harvested. Organs were homogenized and mitochondrial fraction were isolated and analysed by immunoblotting versus p53. GAPDH and Tom20 are purity controls. p53 mitochondrial protein levels were semi-quantified using Tom20 as loading control and p53 levels at time 0 were assumed as 1.

F p53-dependent mitochondrial membrane potential (Δψm) induction in MEFs. Hops+/+ and Hops−/− MEFs were treated with etoposide and then incubated with JC-1 vital dye. The images were collected by fluorescent microscopy and representative images are shown (left panel). Scale bars, 10µm. Fluorescence was analysed by TECAN plate reader and plotted in the graph (right panel).

G p53 MOMP induction in HOPS deficiency. Mitochondria from Hops+/+ and Hops−/− MEFs, were purified and incubated with p53 recombinant protein. Cytochrome C release from mitochondria as metabolic marker was assayed by immunoblotting. Samples were tested to detect p53 as control and Tom20 as mitochondria structural marker.

Data information: assays involving animals were performed three times in three different mice per condition. Experiments on mitochondria from MEFs were performed five times. In E, F data are presented as mean ±SD. * P < 0.05; *** P < 0.001, by two tailed Student's t-test.