EMBO/soda-nel huggingface dataset:
Hair follicle stem cell cultures reveal self-organizing plasticity of stem cells and their progeny (2016)
Figure 1

Figure 1. Establishment of a HFSC culture system

A. Representative FACS plots of freshly isolated epidermal cells (Epi d0) and day 14 (d14) cultures of epidermal cells in different growth conditions and media. KGM 2D: 2D in basal KGM; 3C: 3D-Matrigel in KGM + Y27632 + VEGF-A + FGF-2. B. CD34+α6+ cells were quantified by flow cytometry from d14 as in (A) (mean ±SD; n=3-5; *p ≤ 0.05; ***p ≤ 0.001, Kruskal-Wallis/Dunn’s post test). 3C 2D: 2D in KGM + Y27632 + VEGF-A + FGF-2; Y: 3D-Matrigel in KGM + Y27632; YV: Y + VEGF-A; YF: Y + FGF-2; 3C: Y + VEGF-A + FGF-2. C. Absolute numbers of CD34+α6+ cells from Epi d0 and d14 cultures as in (A-B). Fold enrichment over Epi d0 is shown (mean ±SEM, n=3-5; *p ≤ 0.05, Mann-Whitney U test). D. Epidermal cells grown for 14 days in the indicated conditions. 3C: 3D-Matrigel in KGM + Y27632 + VEGF-A + FGF-2. 3C 2D: 2D in 3C medium. Scale bars 30µm. E. Immunofluorescence analysis of 3C cultures showing the expression of the HFSC markers CD34 and SOX9 in a subset of cells. See also Appendix Appendix figure S1.
Hair follicle stem cell cultures reveal self-organizing plasticity of stem cells and their progeny (2016)
Figure 2

Figure 2. Cells in 3C cultures retain self-renewing capacity and multipotency

A. Colony forming assays show increased proliferative potential of cells cultured in 3C compared to freshly isolated epidermal cells (Epi d0; control) (mean ±SEM; n=5; *p ≤ 0.05, Mann-Whitney U-test). 3C: 3D-Matrigel in KGM + Y27632 + VEGF-A + FGF-2. B. Full thickness skin reconstitution assay with freshly isolated epidermal cells (Epi d0) or 3C cultures shows that cells cultured in 3C retain their self-renewal capacity and multipotency. A representative recipient of 4 mice/condition is shown. Right panel shows quantification of hair follicle density. Note that 3C cultures give rise to more hair compared to controls (mean ±SEM; n=4; *p ≤ 0.05, Mann-Whitney U test). C. Long-term culture of cells in 3C was performed as shown in the left panel. 3C cultures maintain a stable population of CD34+α6+ long term (data are shown using a Box-and-Whisker plot: box indicates 25th and 75th percentiles; error bars represent 10th and 90th percentiles; middle line is the median; n=3-5 independent experiments). p: passage. D. Full thickness skin reconstitution assay using 3C cells from passage 1 (p1) and passage 15 (p15) show that cells cultured in 3C maintain their multipotency and self-renewal capacity in long-term cultures. A representative recipient of 4 mice/condition is shown. See also Appendix figure S2.
Hair follicle stem cell cultures reveal self-organizing plasticity of stem cells and their progeny (2016)
Figure 3

Figure 3. Transcriptomes of cells cultured in 3C resemble in vivo HFSCs

A. Schematic workflow of the RNAseq experiment from FACS-purified CD34+α6+ HFSCs (CD34+α6+), cells cultured in 3C and freshly isolated epidermal cells (Epi d0). B. Heatmap and Euclidian distance dendrogram of quantified transcripts from RNAseq data generated as shown in panel A. 3C cultures cluster with purified CD34+α6+ HFSCs (n=3 biological replicates). C. Venn diagram of genes most significantly upregulated in 3C and in purified CD34+α6+ (log2FC >2, padj<0.05) together with published HFSC signature shows significant overlap between the 3 signatures (p= 0.00252451 and p=3.058928e-51, hypergeometric distribution). D. Schematic workflow of the RNAseq experiment from FACS-purified CD34+α6+ and CD34–α6+ cells from 3C cultures. E. Heatmap of selected genes differentially expressed in 3C-CD34+α6+ and 3C-CD34¯α6+ cells. Note upregulation of multiple key HFSC lineage identity transcription factors and bulge markers (triangles) in 3C-CD34+α6+ cells, and upregulation of genes implicated in lineage progression in 3C-CD34¯α6+ cells (circles). F. Venn diagram of genes upregulated in 3C-CD34+α6+ and in purified CD34+α6+ cells (log2FC >2, padj<0.05) together with a published HFSC signature shows significant overlap between the 3 groups (p= 1.381896e-09 and p= 0.01636903, hypergeometric distribution). G. RT-qPCR analyses of FACS-purified CD34+α6+ and CD34–α6+ cells from either 3C cultures or from freshly isolated epidermis show upregulation of HFSC identity in both freshly isolated and 3C-CD34+α6+ cells compared to freshly isolated CD34–α6+ progenitors. 3C- CD34–α6+ cells show intermediated expression (mean ±SEM; n=4; *p ≤ 0.05, ** p ≤ 0.01, Kruskal-Wallis). H. Both 3C-CD34+α6+ and 3C-CD34–α6+ cells show upregulation of a panel of cell cycle genes and genes shown to be enriched in HFSC progeny (mean ±SEM; n=4; *p ≤ 0.05, ** p ≤ 0.01, Kruskal-Wallis). See also Appendix figure S3 and Tables S1-S3.
Hair follicle stem cell cultures reveal self-organizing plasticity of stem cells and their progeny (2016)
Figure 4

Figure 4. 3C cultures evolve into a stable population equilibrium of HFSCs and non-HFSCs

A. FACS analysis of CD34+α6+ cells over time shows that 3C cultures reach a ~50:50 population equilibrium after 10 days of culture (mean ±SEM; n=3). B. Cells were EdU-labeled for 24 h (day 10–11) prior to analysis by flow cytometry to quantify proliferating cells. Percentage of EdU+ cells is indicated (n=4; mean ±SD; *p ≤ 0.05, Mann-Whitney U-test). C. Schematic illustration of the parameters used in the mathematical model. HFSCs (CD34+α6+) and non-HFSCs (CD34¯α6+) have a maximum proliferation rate of pSC,max and pP,max, respectively. They enter apoptosis with rates apSC and apP and are both degraded with a rate deg. Non-HFSCs can convert to HFSCs with rate tPSC and vice versa with rate tSCP. D. Simulated population proportions of CD34+α6+ HFSCs and CD34¯α6+ non-HFSCs derived from the mathematical model describing steady state cultures. Experimental data from (A) are shown as reference (in grey). See also Appendix figure S4 and Dataset EV4.
Hair follicle stem cell cultures reveal self-organizing plasticity of stem cells and their progeny (2016)
Figure 5

Figure 5. Bidirectional interconversion drives a dynamic self-organizing equilibrium between HFSCs and non-HFSCs

A. CD34+α6+ HFSCs and CD34¯α6+ non-HFSCs were FACS-purified from total epidermis (d0) to ≥98% purity post sorting (d0 post-sort), and grown 14 days in 3C cultures (d14 3C). A significant population of CD34+α6+ HFSCs and CD34¯α6+ non-HFSCs is generated from both purified populations after 14 days. A representative of 3 independent experiments is shown (mean ±SD). B-C. Time course analyses of 3C cultures established from FACS-purified populations of CD34+α6+ HFSCs (B) or CD34¯α6+ non-HFSCs (C) from 3C cultures. Both cultures reach population equilibrium between day 6 and 10 (n=4; mean ±SD). D-E. Simulated population proportions of CD34+α6+ HFSCs (D) and CD34¯α6+ non-HFSCs (E) from the mathematical model describing behavior of pure cell populations. The model parameters were estimated from the experimental data in B-C (shown in grey as reference). F. Schematic illustration of lineage tracing experiments. G. Lineage tracing of FACS-purified CD34+α6+ HFSCs and CD34¯α6+ non-HFSCs from EGFP¯ and EGFP+ 3C cultures, respectively. CD34+α6+ and CD34¯α6+ were mixed at the indicated ratios at d0 and EGFP expression was used to trace the cells of origin over time by flow cytometry. Presence of CD34+α6+ EGFP+ cells in cultures at d10 indicates non-HFSC to HFSC interconversion. H. Lineage tracing as in (F) shows that 3C cultures reach equilibrium between day 6 and 10 independent of the initial proportions of HFSCs and non-HFSCs. I. Quantification of the proportion of CD34+α6+ cells that maintain their fate over time (experiments with both EGFP+ or EGFP¯ labeled cells are indicated). J. Quantification of the proportion of CD34¯α6+ cells that maintain their fate over time (experiments with both EGFP+ or EGFP¯ labeled cells are indicated). Data in panels G-J show n=4; mean ±SD. See also Appendix figure S5.
Mitochondrial translocation of cyclin C stimulates intrinsic apoptosis through Bax recruitment (2019)
Figure 1

Fig 1. Oxidative stress-induced Bax activation requires cyclin C and the fission machinery.

A WT and Ccnc-/- MEF cultures were treated with 0.4 mM H2O2 as indicated, extracts prepared and immunoprecipitated with antibody recognizing the active conformation of Bax. The immunoprecipitates were subjected to Western blot analysis and probed for total Bax or cyclin C. Whole cell extracts (WCE) were subjected to Western blot analysis as indicated to control for protein concentrations in the extracts. Molecular weight markers (kDa) are indicated on the left.

B Cyclin C and Bax directly interact. Recombinant Bax and SUMO-His6-cyclin C were either incubated together or separately at room temperature then passed over Co++ resin. The elutions were subjected to Western blot analysis probing for Bax and cyclin C (lanes 4-6). Input controls for each sample (1/10) are included in lanes 1-3. Molecular weight markers (kDa) are indicated on the left.

C Mitochondrial relocalization of cyclin C is required for Bax activation. The experiment described in (A) was repeated except that H2O2 treated cells were treated with 1 µM pifithrin-µ (PFT) for 24 h as indicated. Molecular weight markers (kDa) are indicated on the left.

D Cyclin C is required for efficient Bax-Bcl-XL disassociation. Extracts prepared from MEF cells with the indicated genotypes following treatment with H2O2 (0.4 mM, 4 h) were immunoprecipitated with active conformation Bax antibodies. The immunoprecipitates were subjected to Western blot analysis probing with Bcl-XL antibodies. Western blot analysis of WCE was performed as input concentration controls. Molecular weight markers (kDa) are indicated on the left.

E The mitochondrial fission complex is required for cyclin C-dependent Bax activation. Extracts were prepared MEF cultures with the indicated genotypes treated with H2O2 (0.4 mM, 4 h). The extracts were treated as in A. Molecular weight markers (kDa) are indicated on the left.

F Drp1 is required for cyclin C-Bax interaction. Extracts were prepared from wild type or Drp1-/- MEF cultures treated or not with H2O2 and analyzed for the presence of Bax and cyclin C as described in A. Molecular weight markers (kDa) are indicated on the left.
Mitochondrial translocation of cyclin C stimulates intrinsic apoptosis through Bax recruitment (2019)
Figure 2

Fig 2. Ectopic nuclear release of cyclin C is sufficient to induce mitochondrial fragmentation and Bax recruitment in its active confirmation.

A Wild type MEF cells were fixed before (control) and following S-HAD treatment (10 µM, 4 h) then nuclei (DAPI), cyclin C (indirect immunofluorescence) and mitochondria (MitoTracker Red) were visualized by fluorescence microscopy. Merge panels present DAPI staining (blue) only. Zoom panels (4X) are indicated by the boxes in the merge panels. Green arrows indicate cyclin C and mitochondrial co-localization. Bar indicates 10 µM.

B Timecourse experiment with wild type and Ccnc-/- MEF cells treated with S-HAD (10 µM) for the times indicated (n=3). Error bars indicate SD. Asterisks indicate p values <0.05 from pretreatment control (Student's T-Test).

C S-HAD peptide activity requires cyclin C. The experiment described in A was repeated with Ccnc-/- MEF cultures. Bar indicates 10 µM. Results quantitated in B.

D S-HAD treatment increases Bax mitochondrial localization. Western blot analysis of the indicated proteins in nuclear and mitochondrial fractions before and following S-HAD treatment (10 µM, 4 h). Atp5A and fibrillarin levels controlled for mitochondrial and nuclear loadings, respectively. Molecular weight markers (kDa) are indicated on the left.

E Bax activation was monitored in wild-type MEF cultures treated with S-HAD (10 µM) for the times indicated. The anti-oxidant N-acetyl cysteine (NAC) was added (1 mM) 2 h prior to S-HAD treatment. Active Bax immunoprecipitates were subjected to Western blot analysis for Bax and cyclin C as indicated. Bax, cyclin C and ß-actin levels were monitored in extract preparations as input controls. Molecular weight markers (kDa) are indicated on the left.
Mitochondrial translocation of cyclin C stimulates intrinsic apoptosis through Bax recruitment (2019)
Figure 3

Fig 3. Cyclin C nuclear release alone is insufficient to induce PCD.

A Caspase activation was measured by fluorescent cell analysis in wild-type MEF cells treated with S-HAD (10 µM, 24 h) or cisplatin (CP, 30 µM, 16 h). n = 3. Error bars indicate SD with * and ** indicate p <0.05 and p<0.01, respectively using Student's T test.

B PARP cleavage was not induced by S-HAD treatment. PARP cleavage was monitored by Western blot analysis in Hela cells treated with S-HAD or cisplatin (CP) as described in (A). The blot was stripped and reprobed with ß-actin antibodies as a loading control. Molecular weight markers (kDa) are indicated on the left.

C Wild type and Ccnc-/- MEF cultures were treated with S-HAD and cisplatin (CP) as just described. Cells were imaged and examined for a "rounding up" phenotype diagnostic for death. See Fig. EV3 for quantitation.

D Cyclin C is required for normal Bax oligomerization in response to cisplatin. Whole cell extracts prepared from DSP crosslinked wild type and CCNC-/- MEF cells exposed to cisplatin (30 µM, 16 h) were probed for total Bax. Bax multimers are indicated. Molecular weight markers (kDa) are indicated on the left.

E S-HAD does not induce Bax oligomerization. Experiments described in D were repeated with cultures treated with S-HAD (10 µM, 24 h). Bax multimers are indicated. Molecular weight markers (kDa) are indicated on the left.

F S-HAD treatment sensitizes Hela cells to cisplatin. Hela cultures were treated with cisplatin (30 µM), S-HAD (10 µM) and N-acetyl cysteine (NAC, 1 mM) as indicated. Fluorescent cell analysis was used to quantitate the apoptotic (Annexin V positive) and necrotic (PI positive) population percentages, respectively. n = 3 independent cultures. Error bars indicate SD. Asterisk indicates p <0.05 (Student's T-test).
Mitochondrial translocation of cyclin C stimulates intrinsic apoptosis through Bax recruitment (2019)
Figure 4

Fig 4. Endogenous ROS enhances cyclin C-Bax interaction.

A S-HAD treatment induces mitochondrial ROS. Wild type and Ccnc-/- MEF cultures were treated with S-HAD (10 µM, 24 h), rotenone or vehicle control as indicated. Mitochondrial ROS was measured by MitoSox staining (4 µM, 15 min) and quantitative confocal imaging. Error bars indicate SD. n=3, asterisk indicates p<0.05 (Student's T-test), n.s., not significantly different.

B Quenching mitochondrial ROS reduces cyclin C-Bax interaction. Wild type and Ccnc-/- MEF cultures were treated with S-HAD (10 µM, 16 h) and 100 nM mitochondrial-specific reactive oxygen scavenger MitoTempol. Western blot analysis was conducted as described in Fig. 1. Molecular weight markers (kDa) are indicated on the left.

C Insulin treatment stimulates S-HAD induced Bax activation. Wild type and Ccnc-/- MEF cultures were grown with or without insulin (10 µg/ml) then treated with S-HAD (10 µM, 2 h). Mitochondrial and cytosolic fractions were prepared and probed for the indicated proteins. Atp5A and ß-actin levels control for mitochondrial and cytosolic loading, respectively. The two blots were prepared, processed and developed identically. Molecular weight markers (kDa) are indicated on the left.

D, E Model for Bax-mitochondrial association before (D) and after (E) PCD-inducing stress. In unstressed cells, Bax continuously samples the MOM but does not insert into the MOM and is removed by pro-survival proteins (Bcl-2, Bcl-XL) in a process termed retrotranslocation. In response to stress, cyclin C associates with Bax stabilizing its interaction with the MOM in a fission complex dependent manner. Increased dwell time makes Bax more efficiently targeted by BH-3 only proteins activated by stress. BH-3 only protein binding induces conformational changes promoting Bax MOM insertion, homodimer formation and oligomerization ultimately leading to PCD. See text for details.

Expanded View Figure Legends.
Cell-to-cell transmission of C9orf72 poly-(Gly-Ala) triggers key features of ALS/FTD (2020)
Figure 1

Figure 1. Cell-to-cell transmission of poly-GA causes cytoplasmic mislocalization of TDP-43.

(A-C) Primary hippocampal neurons were transduced with GFP or GA175-GFP (DIV4+4) and co-cultured with naïve primary neurons for 4 days. Endogenous TDP-43 and poly-GA aggregates in donor and receiver coverslips were analyzed by immunofluorescence. (A) Schematic representation of co-culture experiments. (B) Cytoplasmic TDP-43 immunostaining is elevated not only in poly-GA transduced neurons, but also in the non-transduced receiver cells. White and red arrows indicate cells with cytoplasmic TDP-43 in GFP positive and negative cells, respectively. (C) Automated quantification of cells with cytoplasmic TDP-43 in GFP or GA175-GFP transduced (donor), non-transduced (receiver) neurons. Cells with and without GFP signal were counted separately (indicated by +/­­−). Two groups (GFP negative donor, GFP positive receiver) were excluded due to very high GFP transduction rate and very low GFP transmission rate. n=4 biological replicates. In total 283 donor GFP, 273 donor GA175-GFP, 284 receiver GFP and 266 receiver GA175-GFP cells were analyzed. Scatter plot with bar-graphs of mean ± SD. One-way ANOVA with Tukey's multiple comparisons test. *** denotes p<0.001.

(D-G) Co-culture model in HeLa cells transfected with iRFP or GA175-iRFP in the donor compartment and TDP-43ΔNLS-GFP in donor and receiver compartment. (D) Immunofluorescence staining and (E) automatic quantification of TDP-43∆NLS aggregate number per cell, (F) in addition to filter trap assay of SDS-insoluble TDP-43ΔNLS-GFP aggregates compared in iRFP or GA175-iRFP transfected cells. In (E) n=3 biological replicates with 368 donor iRFP, 251 donor GA175-iRFP, 430 receiver iRFP and 328 GA175-iRFP cells were analyzed. Cells with and without GFP signal were analyzed separately (indicated by +/­­−). White and red arrows indicate cells with cytoplasmic TDP-43 in GFP positive and negative cells, respectively. Scatter plot with bar-graphs of mean ± SD. One-way ANOVA with Tukey's multiple comparisons test. (G) GFP mRNA expression levels were measured by qPCR. RNA levels were normalized to GAPDH, β-actin and β2-microglobulin mRNA. Bar-graphs of mean ± SD. Unpaired two-tailed t-test with Welch's correction. * denotes p<0.05, and ** denotes p<0.01.

Data information: Scale bars denotes 20 µm. Larger fields of view for Fig. 1B and 1D are shown in Appendix Fig S2A/E.
Cell-to-cell transmission of C9orf72 poly-(Gly-Ala) triggers key features of ALS/FTD (2020)
Figure 2

Figure 2. Anti-GA immunodepletion prevents the non-cell-autonomous effects of poly-GA on TDP-43 in conditioned media.

Rat primary hippocampal neurons were transduced with GFP or GA175-GFP. 2 days after transduction neurons were washed 3 times every 2 hours with conditioned media and then incubated for another 2 days. Cell supernatant was collected 2 days later, and immunodepleted with either control IgG or anti-GA antibody coupled beads. The immunodepleted supernatants were then collected, equilibrated to 37 °C, and finally put on receiver cells for 4 days.

(A) Confocal imaging showed anti-GA antibody treatment reduces poly-GA aggregates and TDP-43 mislocalization in hippocampal neurons. White and red arrows show cells with cytoplasmic TDP-43 in GFP-positive and -negative cells, respectively. Scale bar denotes 30 µm.

(B) Automated quantification of cells with cytoplasmic TDP-43 in GFP or GA175-GFP transduced (donor), non-transduced (receiver) neurons. Four groups were excluded due to very high GFP transduction rate (GFP-negative donor) and very low GFP transmission rate (GFP-positive receiver with IgG and anti-GA) and complete prevention of GA-RFP transmission of anti-GA immunodepletion (GA-GFP receiver with anti-GA). n=3 biological replicates. In total 280 donor GFP, 284 receiver GFP with IgG, 317 receiver GFP with anti-GA, 277 donor GA175-GFP, 294 receiver GA175-GFP with IgG, 311 receiver GA175-GFP with anti-GA cells were analyzed. Scatter plot with bar-graphs of mean ± SD. One-way ANOVA with Tukey's multiple comparisons test. *** denotes p<0.001.

(C) Immunoblotting of poly-GA immunoprecipitated from conditioned media using antibody-coupled beads and antibody leftover pre and post immunoprecipitation in the conditioned media.

(D) Poly-GA levels in conditioned media before and after immunodepletion with control IgG and anti-GA antibody were determined by immunoassay. n=3 biological replicates. Scatter plot with bar-graphs of mean ± SD. One-way ANOVA with Tukey's multiple comparisons test. ** denotes p<0.01, and *** denotes p<0.001.
Cell-to-cell transmission of C9orf72 poly-(Gly-Ala) triggers key features of ALS/FTD (2020)
Figure 3

Figure 3. Poly-GA inhibits the proteasome cell-autonomously and non-cell-autonomously.

(A-B) Double immunofluorescence of the proteasome subunit PSMC4 and poly-GA inclusions in spinal cord of GA149-CFP transgenic mouse and cortex of C9orf72 patients compared to controls. Scale bar denotes 20 µm.

(C,D) Co-culture model of HeLa cells transfected with iRFP or GA175-iRFP in the donor compartment and an UbG76V-GFP proteostasis reporter in donor and receiver compartment (48 h). (C) Separate analysis of both compartments by immunoblot and (D) immunoblot quantification. For quantitative analysis of immunoblots UbG76V-GFP was normalized to calnexin. n=3 biological replicates. Scatter plot with mean ± SD. One-way ANOVA with Tukey's multiple comparisons test. Red dashed line indicates the control's expression level. * denotes p<0.05, *** denotes p<0.001.

(E) GFP mRNA expression levels were measured by qPCR. RNA levels were normalized to GAPDH, β-actin and β2-microglobulin mRNA. Bar-graphs of mean ± SD. n=3 biological replicates. Unpaired two-tailed t-test with Welch's correction.

(F,G) Flow cytometry analysis of a UbG76V-GFP reporter cell line incubated 48h with conditioned media from RFP or GA175-RFP transfected cells upon immunodepletion of poly-GA or control depletion using unspecific IgG. n=3 biological replicates. Scatter plot with bar-graphs of mean ± SD. One-way ANOVA with Tukey's multiple comparisons test. * denotes p<0.05. (G) Comparisons of the corresponding histograms for compensated RFP and UbG76V-GFP fluorescence from one representative experiment that shows specific transmission of GA175-RFP associated with accumulation of UbG76V-GFP in cells incubated with GA175-RFP conditioned media. For flow cytometry analysis of co-culture experiments with direct antibody treatment see Fig EV3E-H.