EMBO/soda-nel huggingface dataset:
FATE1 antagonizes calcium- and drug-induced apoptosis by uncoupling ER and mitochondria (2016)
Figure 2

Figure 2. Protein domains directing localization of FATE1 and its association with MAM.

A. Structure of the FATE1 protein. Two predicted coiled-coil domains (CC1 and CC2) and a C-terminal transmembrane region (TM) are present. Purple, Mff-homology region. The position of L151 is indicated.

B. Subcellular localization of EGFP-fusions of full-length (FL) FATE1 and its mutants: N-terminal (aa. 1-124), C-terminal (aa.125-183), 1-162, transmembrane domain (aa. 155-183) and L151D (green) transfected in H295R/TR SF-1 cells. Mitochondria were stained by anti-TOM20 antibody (red) and DNA by DAPI (blue). Scale bars, 10 μm.

C. EMD and Flag-tagged FATE1, but not the other OMM protein TOM20, were coimmunoprecipitated from Dox-treated H295R/TR N-Flag FATE1 cells. Control immunoprecipitations were performed with anti-myc (α-myc) and non-immune rabbit IgG (IgG-R) antibodies.

D. Confocal fluorescence microscopy showed colocalization of cytoplasmic EMD, as revealed by immunofluorescence (red), with ER, stained by ER-GFP BacMam (green). Scale bars, 10 μm.

E. Mic60/mitofilin and Flag-tagged FATE1 were coimmunoprecipitated from Dox-treated H295R/TR N-Flag FATE1 cells. Control immunoprecipitations were performed with anti-myc (α-myc) antibody.

F. Mitochondrial localization of Mic60/mitofilin (green) and FATE1 (red) in Dox-treated H295R/TR SF-1 cells. Scale bar, 10 μm.

G. Left, triple immunofluorescence labelling of endogenous FATE1 (green), ER labelled by calreticulin (red) and mitochondria labelled by HSP60 staining (blue) in Dox-treated H295R/TR SF-1 cells. One area showing close apposition of red, green and blue staining (white signals) is shown at higher magnification. Scale bar, 10 μm. Right, graph showing quantification of FATE1 signal in total mitochondria (white histogram) vs. ER-mitochondria contact sites (red histogram) (mean ± SEM; n=13). **p<0.01.

H. H295R/TR SF-1 cells were fractioned into crude mitochondria (CM), endoplasmic reticulum (ER), purified mitochondria (PM) and mitochondria-associated membranes (MAM) fractions. H, total homogenate. The localization of SERCA2, SOAT1, VDAC1, GRP75, S1R, EMD, Mic60/mitofilin, FATE1 and TOM20 proteins in the different fractions was revealed by immunoblot.
FATE1 antagonizes calcium- and drug-induced apoptosis by uncoupling ER and mitochondria (2016)
Figure 3

Figure 3. Effects of FATE1 expression on ER-mitochondria contacts and mitochondrial shape.

A. H295R/TR N-Flag FATE1 cells were transfected with D1ER marker for ER (green) and mtRFP marker for mitochondria (red). Manders' coefficient for green-red signals colocalization in basal (white histograms) and Dox-treated (black histograms) cells is shown on the right (mean ± SEM; n=12). ***p<0.001. Scale bars, 10 μm.

B. Effect of FATE1 expression on ER-mitochondria distance in H295R/TR N-Flag FATE1 cells measured using a split-GFP probe [29]. Quantification is shown in the graph. White histogram, number of fluorescent objects/cell in basal conditions; black histogram, number of fluorescent objects/cell in Dox-treated cells (mean ± SEM; n=6 with 90 cells analyzed in total). **p<0.01. Scale bars, 5 μm.

C. Transmission electron microscopy images of H295R/TR N-Flag-FATE1 cells cultured in basal conditions or treated with Dox. ER-mitochondria contacts are indicated by white arrowheads. Scale bars, 200 nm. Right, quantification of the number of contacts normalized by the number of mitochondria and the percentage of mitochondria with ER contact sites is shown in basal conditions (white histograms) and after Dox treatment (black histograms) of cells (mean ± SEM; n=38 for basal and 47 for Dox-treated cells). ***p<0.001.

D. Transmission electron microscopy images of H295R/TR SF-1 cells cultured in basal conditions or treated with Dox. ER-mitochondria contacts are indicated by white arrowheads. Scale bars, 200 nm. Right, quantification of the number of contacts normalized by the number of mitochondria and the percentage of mitochondria with ER contact sites is shown in basal conditions (white histograms) and after Dox treatment (black histograms) of cells (mean ± SEM; n=41 for basal and 42 for Dox-treated cells). ***p<0.001.

E. Mitochondrial shape (BacMam Mitochondria-RFP; in red) in H295R/TR N-Flag FATE1 cells shown by fluorescence confocal microscopy in basal conditions and after Dox treatment. FATE1 (green), DAPI (blue). A higher magnification of merged green and red signal is shown in the insets. Scale bars, 10 μm.

F. Mitochondrial fragmentation index in H295R/TR N-Flag FATE1 cells after Dox treatment (red histogram) compared to cells cultured in basal conditions (white histogram) (mean ± SEM; n=111 for basal and 101 for Dox-treated cells). ***p<0.001.

G. Mitochondrial fragmentation index in H295R/TR SF-1 cells after Dox treatment (green histogram) compared to cells cultured in basal conditions (white histogram). (mean ± SEM; n=37 for basal and 49 for Dox-treated cells). ***p<0.001.

H. Mitochondrial membrane potential (∆Ψ) measured by TMRM fluorescence after Dox treatment of H295R/TR N-Flag FATE1 cells (red histogram) compared to cells cultured in basal conditions (white histogram). Mean ± SEM is shown. (mean ± SEM; n=3 with 12 replicates/experiment). ns, not significant.
FATE1 antagonizes calcium- and drug-induced apoptosis by uncoupling ER and mitochondria (2016)
Figure 4

Figure 4. FATE1 expression reduces mitochondrial Ca2+ uptake and apoptosis induced by pharmacological agents and mitotane.

A. Mitochondrial Ca2+ uptake. H295R/TR parental clone (white/grey histograms) H295R/TR SF-1 (white/green histograms), H295R/TR N-Flag FATE1 (white/red histograms) and H295R/TR FATE1 cells (white/violet histograms) (mean ± SEM; n=3-5 with 12 replicates/condition). **p<0.01; ***p<0.001; ns, not significant.

B. Cytosolic Ca2+ concentration. Histograms as in A (mean ± SEM; n=3-5 with 12 replicates/condition). ns, not significant.

C. Mitochondrial (left) and cytosolic (right) Ca2+ concentrations in Dox-treated H295R/TR SF-1 cells transfected with control (siC; white histograms) or FATE1-specific (siFATE1; green histograms) siRNAs (mean ± SEM; n=3-5 with 12 replicates/condition). ***p<0.001; ns, not significant.

D. Caspase 3/7 activity in H295R/TR N-Flag FATE1 cells cultured in basal conditions (white histograms) or in the presence of Dox (red histograms) treated with H2O2 (500 μM) C2-ceramide (50 μM) or staurosporine (STS) (1 μM). (mean ± SEM; n=5-8 with 3 replicates/condition). *p<0.05; **p<0.01; ns, not significant.

E. Caspase 3/7 activity in Dox-treated H295R/TR SF-1 cells transfected with control (siC; white histograms) or FATE1-specific (siFATE1; green histograms) siRNAs and treated with H2O2 or STS (mean ± SEM; n=5-8 with 3 replicates/condition). *p<0.05; ns, not significant.

F. Flow cytometric analysis of TUNEL staining in H295R/TR N-Flag FATE1 cells cultured in basal conditions or in the presence of Dox treated with vehicle (control) H2O2 (500 μM), C2-ceramide (50 μM) or staurosporine (STS) (1 μM).

G. Caspase 3/7 activity in H295R/TR N-Flag FATE1 cells cultured in basal conditions (white histograms) or in the presence of Dox (red histograms) and treated with mitotane (50 μM) (mean ± SEM; n=5-8 with 3 replicates/condition). **p<0.01.

H. Caspase 3/7 activity in Dox-treated H295R/TR SF-1 cells transfected with control (siC; white histograms) or FATE1-specific (siFATE1; green histograms) siRNAs and treated with mitotane (mean ± SEM; n=5-8 with 3 replicates/condition). *p<0.05.
FATE1 antagonizes calcium- and drug-induced apoptosis by uncoupling ER and mitochondria (2016)
Figure 5

Figure 5. Expression of FATE1 in tissues and correlation of its expression with ACC patients' survival.

A. Immunohistochemical analysis of FATE1 expression in normal adrenal cortex and adrenal hyperplasia, adenoma and carcinoma (ACC). Testis tissue was used as positive control. Scale bars, 200 µm (low magnification); 50 µm (high magnification).

B. Table summarizing the relative intensity of FATE1 IHC staining (H-score) in groups of normal, benign and malignant tissues. The highest FATE1 expression levels were found in ACC. n, number of samples studied for each category of tissues. NBST, normal and benign steroidogenic tissues.

C. Prognostic parameters in ACC patients. Results are presented as hazard ratios (HR) including 95% confidence interval (CI). The significance level was set at α=5% for all comparisons. The results for SF-1, FATE1 and combined SF-1/FATE1 expression levels are shown both in univariate and mutivariate (adjusted for age, sex and ENSAT tumour stage) analysis.

D. Overall survival of ACC patients according to FATE1 expression in their tumours, as measured by quantitative immunohistochemistry (low, green curve; high, red curve) in mutivariate analysis. p=0.008.

E. Diagram illustrating FATE1 effect on ER-mitochondria distance and Ca2+ influx into mitochondria. In the presence of FATE1 (right) at the level of MAM, coupling between ER and mitochondria and Ca2+ influx into mitochondria decrease, with consequent resistance to apoptosis triggered by oxidative stress and chemotherapeutic drugs.
Human SFI1 and centrin form a complex critical for centriole architecture and ciliogenesis. (2022)
Figure 1

Figure 1. SFI1 is a centriolar protein co-localizing with Centrin 2 and 3 at the distal tip of centrioles.

(A, B) Representative confocal images of cycling RPE-1 cells stained for SFI1 (green) and γ-Tubulin (magenta) (A) or SFI1 (green) and Centrin 2/3 (magenta) (B). Scale bar: 5µm. Dashed-line squares correspond to insets.

(C, D) Representative confocal images of expanded centrioles from RPE-1 cells stained for α/β-tubulin (αβTub, magenta) and SFI1 (green). Right panels show top view images across the centriolar length confirming the distal localization of SFI1 at centrioles. The white arrowhead indicates SFI1 distal dot at centrioles. Scale bars: 200 nm and 100 nm (right panels). Average position of SFI1 alongside the centriole is shown in (D).

(E, F) Representative confocal images of expanded centrioles from RPE-1 cells stained for α/β-tubulin (αβTub, magenta) and Centrin 2/3 (Cetn2/3, grey). Right panels show top view images across the centriolar length confirming the distal localization of Centrin2/3 at centrioles. The white arrowhead indicates Centrin2/3 distal dot at centrioles. Scale bars: 200 nm and 100 nm (right panels). Average position of Centrin2/3 alongside the centriole is shown in (F).

(G, H) Representative confocal images of expanded centrioles from RPE-1 cells stained for α/β-tubulin (αβTub, magenta) and Centrin 3 (Cetn3, cyan). Right panels show top view images across the centriolar length confirming the distal localization of Centrin 3 at centrioles. The white arrowhead indicates Centrin 3 distal dot at centrioles. Scale bars: 200 nm and 100 nm (right panels). Average position of Centrin 3 alongside the centriole is shown in (H).

(I, J) Representative confocal images of expanded centrioles from RPE-1 cells stained for α/β-tubulin (αβTub, magenta), SFI1 (green) and Centrin 2/3 (Cetn3, grey). Right panels show top view images confirming the distal localization of SFI1 and Centrin 2/3 at centrioles. Scale bars: 200 nm and 100 nm (right panels). Average position of SFI1 and Centrin2/3 alongside the centriole is shown in (J).

(K, L) Position of SFI1 and Centrin signals at the distal centriolar region in nm either from separated stainings (K) or co-stainings (L).

Data information: Average +/- SD, N, statistical analysis: (D) N=42 centrioles from 3 independent experiments. (F) N=26 centrioles from 3 independent experiments. (H) N=25 centrioles from 3 independent experiments. (J) N=54 centrioles from 3 independent experiments. (K) SFI1= 376 +/- 18 nm; Centrin2/3: 355 +/- 36 nm; Centrin3: 359 +/- 37 nm. N = 41, 25, 24 centrioles for SFI1, Centrin 2/3 and Centrin 3 respectively, from 2 independent experiments. One-way ANOVA followed by Bonferroni post-hoc test (SFI1 vs Cetn2/3 p=0.0196, SFI1 vs Cetn3 p=0.0702, Cetn2/3 vs Cetn3 p=0.999). (L) SFI1 = 316.3 +/- 43.8 nm; Centrin 2/3: 351.8 +/- 42.6 nm. N=53 centrioles from 3 independent experiments. Unpaired t-test (SFI1 vs Cetn2/3 **p<0.0001).
Human SFI1 and centrin form a complex critical for centriole architecture and ciliogenesis. (2022)
Figure 6

Figure 6. SFI1 depletion impacts the distal appendage CEP164 protein organization.

(A- D) Representative widefield images of expanded centrioles during ciliogenesis from siCT (A, B) and siSFI1 (C, D) RPE-1 treated cells. Cells were stained for α/β-tubulin (magenta) and CEP164 (red hot). Note the abnormal organization of the distal appendage protein CEP164 in siSFI1-treated cells. Scale bar: 250 nm.

(E-H) Representative widefield images of expanded centrioles during ciliogenesis from siCT (E, F) and siSFI1 (G, H) RPE-1 treated cells. Cells were stained for α/β-tubulin (magenta) and CEP90 (cyan). Scale bar: 250 nm.

(I) Frequency distribution of the number of dots per centriole formed by CEP164 in indicated conditions.

(J) Frequency distribution of the number of dots per centriole formed by CEP90 under the indicated conditions.

(K) Percentage of cells presenting abnormal CEP164 and CEP90 under the indicated conditions.

(L) Model of Centrin/SFI1 localization and function at the distal end of human centrioles.

Data information : Average +/- SD, N, statistical analysis: (I) siCT= 0-7 dots: 0%, 8 dots: 9.5%, 9 dots: 90.5%. siSFI1= 0 dot: 3.6%, 1-2 dots: 0%, 3 dots: 1.8%, 4 dots: 5.4%, 5 dots: 14.3%, 6 dots:17.9%, 7 dots: 14.3%, 8 dots: 17.9%, 9 dots: 25. N=21 and 56 centrioles for siCT and siSFI1 respectively from 3 independent experiments. Mann-Whitney test (***p<0.0001). (J) siCT= 0-7 dots: 0%, 8 dots: 35.7%, 9 dots: 64.3%. siSFI1= 0-7 dots: 0%, 8 dots: 21.4%, 9 dots: 78.6%. N=14 and 28 centrioles for siCT and siSFI1 respectively from 3 independent experiments. Mann-Whitney test (p=0.459). (K) siCT= CEP164: 4.2%+/- 3.6, CEP90: 7.8% +/- 4.4. siSFI1= CEP164: 52.8% +/- 2.5, CEP90: 11.4% +/- 3.7. N=3 independent experiments (>50 centrioles/experiments). Two-way ANOVA followed by Sidak's multiple comparison (CEP164 siCT vs siSFI1, ***p<0.0001; CEP90 siCT vs siSFI1, p=0.607).

Expanded View Figure Legends
Ronin governs the metabolic capacity of the embryonic lineage for post-implantation development (2021)
Figure 1

Figure 1. Ronin depletion induces quiescent state in ESC.

Western blot analysis of Ronin depletion upon treatment of Ronin fl/del Cre-ERT2 cells with 4OHT.

Brightfield images of Ronin fl/del and Ronin del/del ESC.

Cell count of Ronin fl/del and Ronin del/del ESC. Data represent mean ± SD, n = three independent experiments.

Annexin V cell death assay of Ronin fl/del and Ronin del/del cultured for 6 days. Two-tailed unpaired Student's t test, data represent mean ± SD, n = three independent experiments.

FACS plot of cell cycle distribution of day 6 Ronin del/del and Ronin fl/del, determined by EdU assay.

Quantification of the cell cycle distribution determined by EdU assay. Data represent mean ± SD, n = three independent experiments.

Ronin fl/del and Ronin del/del ESC stained for Ronin (left panel) or Oct4 (right panel). The nuclei are counterstained with DAPI.

Data information: Scale bars (B), 100 μm; (G), 10 μm. See also Figure EV1.
Ronin governs the metabolic capacity of the embryonic lineage for post-implantation development (2021)
Figure 2

Figure 2. The quiescent state in Ronin-deficient ESC is reversible.

Western blot analysis of ecto-Ronin expression Ronin del/del + TET-ON Ronin-FLAG ESC cultured in the presence of Dox or without Dox for 3 days. The endogenous levels of Ronin are compared to wild-type (WT) E14 ESC.

Average growth rate per day (k) of ecto-Ronin (Ronin del/del TET-ON Ronin-FLAG) ESC cultured continuously in the presence of Dox (+ Dox); without Dox for 5 passages (-Dox) or grown without Dox for 3 passages and then switched to Dox-containing medium for another 2 passages (-Dox -> +Dox). Ordinary one-way Anova, error bars represent median with interquartile range, technical replicates (+ Dox, n = 13), (- Dox n = 13), (-Dox -> +Dox, n = 7) of three independent experiments.

Tumour growth of Ronin fl/del, ecto-Ronin and Ronin del/del teratomas, n teratomas for each genotype = 3.

Brightfield images of teratomas derived from Ronin fl/del and ecto-Ronin ESC.

Haematoxylin/eosin staining of paraffin sections of Ronin fl/del and ecto-Ronin teratomas.

In vitro differentiation of Ronin fl/del, ecto-Ronin (grown in the presence of Dox) and Ronin del/del ESC.

Data information: Scale bars (E), 50 μm; (F), 40 μm.
Ronin governs the metabolic capacity of the embryonic lineage for post-implantation development (2021)
Figure 3

Figure 3. Transcriptional analysis of Ronin ko ESC.

Principal component analysis (PCA) of Ronin fl/del, Ronin del/del (day 4) and Ronin del/del (day 8) transcriptomes; n = 3 independent RNA-seq datasets for each condition.

Volcano plot of gene expression in Ronin del/del day 4 vs. Ronin fl/del (left panel) and Ronin del/del day 8 vs. Ronin fl/del ESC (right panel) with annotated naïve and primed pluripotency markers.

Expression and binding of Ronin to core, naïve and primed pluripotency markers.

Gene set enrichment analysis (GSEA) plot of Il6/Jak/Stat3 signalling, day 8 Ronin ko ESC.

Expression and binding of Ronin to Il6/Lif signalling-related genes.

K-means clustering of 5 major groups of genes in Ronin fl/del, Ronin del/del (day 4) and Ronin del/del (day 8) transcriptomes.

Gene ontology (GO) enrichment analysis of the predefined gene clusters - molecular function (left panel) and cellular component (right panel).

See also Figure EV2.
Ronin governs the metabolic capacity of the embryonic lineage for post-implantation development (2021)
Figure 4

Figure 4. Ronin tunes the expression of genes encoding ribosomal proteins.

Volcano plot of gene expression in Ronin del/del day 8 vs. Ronin del/del day 4 ESC.

GO enrichment analysis of Ronin del/del day 8 vs. Ronin del/del day 4 downregulated genes. BP - biological process, CC - cellular component, MF - molecular function.

Expression and binding of Ronin to genes encoding cytoplasmic ribosomal proteins.

Binding of Ronin to the promoters of genes encoding cytoplasmic ribosomal proteins.

GO enrichment analysis of genes that are directly bound by Ronin. BP - biological process, CC - cellular component, MF - molecular function.

Analysis of nascent protein synthesis using OP-puro assay. Ronin fl/del cells treated with protein synthesis inhibitor (cycloheximide) are used as negative control. The nuclei are counterstained with DAPI.

Quantification of the OP-puro signal, normalised to DAPI. Ordinary one-way Anova data represent mean ± SD; n = three independent experiments for each genotype.

Data information: Scale bar (F), 15 μm. See also Figure EV3.
Ronin governs the metabolic capacity of the embryonic lineage for post-implantation development (2021)
Figure 5

Figure 5. Ronin controls mitochondrial cristae formation and function.

Expression and binding of Ronin to structural and regulatory factors of the mitochondrial cristae.

Binding of Ronin to the promoters of mitochondrial cristae genes.

Transmission electron microscopy analysis of mitochondrial structure in Ronin fl/del and Ronin del/del ESC. Mitochondria analysed, n > 150 for each condition.

Flow cytometry analysis of Ronin fl/del and Ronin del/del stained for MitoTracker and TMRE.

Quantification of the TMRE signal determined by the flow cytometry analysis. Ordinary one-way Anova, data represent mean ± SEM, n = three independent experiments.

OCR measurement using the Seahorse mitochondrial stress test assay. FCCP - Carbonyl cyanide-4 (trifluoromethoxy) phenylhydrazone; Rot - Rotenone; AA - Antimycin, data represent mean ± SD, three independent experiments.

Quantification of the OCR analysis. Kruskal-Wallis test, data represent mean ± SD, n = 16 technical replicates per genotype in three independent experiments.

Data information: Scale bars (C), 1 μm;
Ronin governs the metabolic capacity of the embryonic lineage for post-implantation development (2021)
Figure 6

Figure 6. Expression of Ronin and mitochondrial morphology in early embryos.

Blastocyst (E4.5) and diapause embryos stained for Ronin, Sox2 and Sox17. Egg cylinder stage (E5.5) embryo stained for Ronin and Sox17. Nuclei are counterstained with DAPI.

Transmission electron microscopy analysis of mitochondrial morphology in the epiblast of E4.5, diapause and E5.5 embryo. Mitochondria analysed, E4.5 (n mitochondria = 55), diapause (n mitochondria = 62), E5.5 (n mitochondria = 65).

Data information: Scale bar (A), 20 μm; (B), 500 μm. See also Figure EV4.
Ronin governs the metabolic capacity of the embryonic lineage for post-implantation development (2021)
Figure 7

Figure 7. Ronin function is essential for the post-implantation growth and morphogenesis of the embryonic lineage.

Schematic representation of the generation of chimeric embryos.

Schematic representation of the induction of diapause and reactivation of chimeric embryos.

E4.5 chimeric embryos stained for tdTomato and DAPI.

Quantification of donor cells' contribution in E4.5 blastocysts, Mann-Whitney test, data represent mean ± SD; Ronin fl/del n embryos = 26, Ronin del/del n embryos = 27.

Diapause embryos stained for tdTomato and DAPI.

Quantification of donor cells' contribution in diapause embryos, unpaired Student's t test, data represent mean ± SD, Ronin fl/del n embryos = 4, Ronin del/del n embryos = 12.

E5.5 chimeric embryos stained for tdTomato and DAPI.

Quantification of donor cells' contribution in E5.5 embryos, unpaired Student's t test, data represent mean ± SD, Ronin fl/del n embryos = 21, Ronin del/del n embryos = 15.

Reactivated E5.5 chimeric embryos stained for tdTomato and DAPI.

Quantification of donor cells' contribution in reactivated E5.5 embryos, Mann-Whitney test, data represent mean ± SD, Ronin fl/del n embryos = 17, Ronin del/del n embryos = 12.

E7.5 chimeric embryos stained for tdTomato and DAPI.

Quantification of donor cells' contribution in E7.5 embryos, data represent mean ± SD, Ronin fl/del n embryos = 14, Ronin del/del n embryos = 8.

E5.5 chimeric embryos stained for Podocalyxin, tdTomato, E-cad and DAPI.

Ronin fl/del and Ronin del/del (day 4) cells grown in 3D culture conditions for 48 h and stained for apical polarity markers - Podocalyxin and Par6, as well as Phalloidin, E-cad and tdTomato.

Quantification of Par6 positive and negative ESC clumps of Ronin fl/del (n = 44) and Ronin del/del (n = 13) cells grown in 3D culture conditions for 48 h.

Data information: Scale bars, (C), (E), (G), (I), 25 μm; (K), 100 μm; (M), 20 μm; (N), 10 μm.