EMBO/soda-nel huggingface dataset:
The anti-apoptotic Bcl-2 protein regulates hair follicle stem cell function (2021)
Figure 1

Figure 1 - Differential requirement for Bcl-2 preventing suprabasal BSC loss

A Treatment scheme of experimental timeline and Immunofluorescence staining of telogen backskin hair follicles (HF) for Integrin α6 (Itgα6, red), active Caspase 3 (aCas3, green) and DAPI (blue) in control (Ctr) and Bcl-2 inhibitor (ABT-199) treated mice. B: bulge, CH: club hair, DP: dermal papilla, HF: hair follicle, HG: secondary hair germ. Scale bar, 50 µm

B Quantification of backskin HF with aCas3+ cells in bulge, hair germ and dermal papilla of Ctr and ABT-199 treated mice (n=4 mice biological replicates; mean ± standard deviation (SD); *** p<0.001, ns=not significant, two-way ANOVA test). nd=not detected.

C,D Treatment scheme, FACS plot (C) and quantification (D) of keratinocytes stained for CD34 and Itgα6 after 6 days of treatment with vehicle solution (Ctr) or ABT-199 (n=6 biological replicates; mean ± standard deviation (SD); ** p<0.01, ns=not significant, two-tailed unpaired Student's t-test). suprabasal (sbBSC, CD34+/Itgα6low), basal (bBSC, CD34+/Itgα6high) BSCs and n-B (non-bulge epidermal cells). ** p<0.005.
The anti-apoptotic Bcl-2 protein regulates hair follicle stem cell function (2021)
Figure 2

Figure 2 - Loss of suprabasal BSCs delays cyclic hair regeneration.

A qRT PCR for Bcl-2 mRNA expression in sorted n-B (non-Bulge), bBSC (basal BSCs) and sbBSC (suprabasal BSCs) (n=3 biological replicates; mean ± standard deviation (SD); **** p<0.0001, ns=not significant, two-way ANOVA test).

B Treatment scheme of experimental timeline and histology of backskin sections of control (Ctr) and ABT-199 treated mice. Scale bar, 200 µm.

C Treatment scheme and Hair-coat recovery monitored for 5 weeks following daily treatment (Pd50-Pd55) with vehicle solution (Ctr) and ABT-199.

D qRT-PCR for mRNA expression of the transcription factors Lef1 and Runx1 in epidermis of ABT-199 treated and control (Ctr) mice at P77 as shown in (B) (n=3 biological replicates; mean ± standard error of mean (SEM); ** p<0.01, ns=not significant, two-tailed unpaired Student's t-test).
The anti-apoptotic Bcl-2 protein regulates hair follicle stem cell function (2021)
Figure 3

Figure 3 - Suprabasal BSCs are prone to detachment-induced apoptosis.

A GO classification of differentially regulated genes in P50 suprabasal BSCs (CD34+/Itgα6low) compared to basal BSCs (CD34+/Itgα6high) clustered for biological process (clustering and statistical analysis done with Metascape, according to Zhou et al. 2019 (Zhou et al, 2019). Columns of interest in red.

B Functional classification of selected differentially expressed genes in sorted suprabasal BSC vs basal BSC after RNA-seq analysis (red: up-regulated genes, green: downregulated genes; fold change > 2, p < 0,05; statistical analysis done according to Wagle et al. 2015 (Wagle et al., 2015)).

C qRT-PCR for mRNA expression of the anoikis-related markers Bim and Bmf in sorted n-B (non-bulge), bBSC (basal BSCs) and sbBSC (suprabasal BSCs) (n=3 biological replicates; mean ± standard error of mean (SEM); * p<0.05, ns=not significant, two-way ANOVA test).
The anti-apoptotic Bcl-2 protein regulates hair follicle stem cell function (2021)
Figure 4

Figure 4 - Bcl-2 expression regulates size and architecture of SC compartment and delays hair-coat recovery.

A Histology of telogen backskin HF of Ctr and Bcl-2EOE mice (P60). B= bulge. Scale bar, 50 µm.

B Quantification of the bulge area in backskin at P60 (n=4; multiple measurements/biological replicate; mean ± standard deviation (SD); **** p<0.0001, two-tailed unpaired Student's t-test).

C FACS plot and quantification of keratinocytes stained for CD34 and Itgα6 in 8 week old control (Ctr) and Bcl-2EOE mice (n=3 biological replicates; mean ± standard deviation (SD); ns=not significant, two-tailed unpaired Student's t-test). suprabasal (sbBSC, CD34+/Itgα6low), basal (bBSC, CD34+/Itgα6high) BSCs.

D Immunofluorescence staining of telogen backskin HF for BrdU incorporation (1 h pulse, green) and DAPI (blue) in 8 week old control (Ctr) and Bcl-2EOE mice. Scale bar, 50 µm.

E Quantification of BrdU+ bulge cells of Ctr and Bcl-2EOE mice. (n=4 biological replicates; mean ± standard deviation (SD); ns=not significant, two-tailed unpaired Student's t-test).
The anti-apoptotic Bcl-2 protein regulates hair follicle stem cell function (2021)
Figure 5

Figure 5- Bcl-2 blocks apoptosis during catagen of the hair follicle cycle.

A Histology of P19 catagen backskin HF of control (Ctr) and Bcl-2EOE mice. RS: retracting strand, Scale bar, 50 µm.

B Analysis of length and width of retracting strands of control (Ctr) and Bcl-2EOE mice (n=4; multiple measurements/biological replicate; whiskers: min and max; boxes: median ± first and third quartile; **** p<0.0001, two-tailed unpaired Student's t-test).

C Itgα6 and aCas3 Immunofluorescence staining of catagen tailskin HF of control (Ctr) and Bcl-2EOE mice (n=4 biological replicates). Dashed lines: retracting strands. Scale bar, 100 µm.

D Quantification of aCas3+ cells in the RS of P19 Ctr and Bcl-2EOE mice (n=4 biological replicates; mean ± standard deviation (SD); **** p<0.0001, two-tailed unpaired Student's t-test). HF: hair follicle.

E Hair-coat recovery in Ctr and Bcl-2EOE mice following depilation (n=3 biological replicates).

F,G qRT-PCR for mRNA expression of the quiescence-related markers Bmp6 (F) and Fgf18 (G) in epidermis of control (Ctr) and Bcl-2EOE mice (P27), (n=4 biological replicates; mean ± standard error of mean (SEM); * p<0.05, *** p<0.001, two-tailed unpaired Student's t-test).
The anti-apoptotic Bcl-2 protein regulates hair follicle stem cell function (2021)
Figure 6

Figure 6 - Bcl-2 overexpression blocks BSC apoptosis and proliferation in K15ΔNLef1 mice.

A qRT-PCR for Bcl-2 mRNA expression in sorted (CD34+) BSCs of control (Ctr), K15ΔNLef1 and K15ΔNLef1;Bcl-2EOE mice (n=2 biological replicates).

B Immunofluorescence staining of epidermal tail whole mounts of control (Ctr), K15ΔNLef1 and K15ΔNLef1;Bcl-2EOE mice, stained for keratin 15 (Krt15, green), activated Caspase 3 (aCas3, red) and DAPI (blue). Note increased number of aCas+ cells in the bulge of K15ΔNLef1 mice (arrows). B: bulge, HG: secondary hair germ, SG: sebaceous gland. Scale bar, 100µm.

C Quantification of hair follicles (HF) with activated Caspase 3 (aCas3+), apoptotic cells in control (Ctr), K15ΔNLef1 and K15ΔNLef1;Bcl-2EOE mice (n=6 biological replicates; mean ± standard deviation (SD); *** p<0.001, two-way ANOVA test).

D Immunofluorescence staining of epidermal tail whole mounts of control (Ctr), K15ΔNLef1 and K15ΔNLef1;Bcl-2EOE mice, stained for keratin 15 (Krt15, green), BrdU (1 h pulse, red) and DAPI (blue). Note increased number of BrdU+ cells in the bulge of K15ΔNLef1 mice (arrows). B: bulge, HG: secondary hair germ, SG: sebaceous gland. Scale bar, 100µm

E Quantification of hair follicles (HF) with BrdU+, proliferating cells in bulge of control (Ctr), Bcl-2EOE, K15ΔNLef1 and K15ΔNLef1;Bcl-2EOE mice (n=6 biological replicates; mean ± standard deviation (SD); **** p<0.0001, multiple t-test comparisons). nd: not detected.
The anti-apoptotic Bcl-2 protein regulates hair follicle stem cell function (2021)
Figure 7

Figure 7 - Bcl-2 promotes SC-driven tumour initiation.

A Schematic of tumour experiment timeline in K15ΔNLef1 genetic background.

B,C Tumour incidence (B) and frequency (C) in Ctr, Bcl-2EOE, K15ΔNLef1 and K15ΔNLef1;Bcl-2EOE mice (n=10 biological replicates; mean ± standard error of mean (SEM)).

D Histology of sebaceous tumours of K15ΔNLef1 and K15ΔNLef1;Bcl-2EOE mice. Scale bar, 100µm.

Expanded View Figure Legends
The effects of proteasomal inhibition on synaptic proteostasis (2016)
Figure 1

Figure 1. Lactacystin strongly suppresses UPS-mediated protein degradation in living neurons.

A) Differential interference contrast (DIC) image of cortical neurons in culture. The arrowhead points to a neuron expressing Ub-R-GFP. B) GFP fluorescence before and after exposure to lactacystin (10µM). C) A neuron coexpressing Ub-R-GFP and CFP before (left) and 10 hours after exposure to lactacystin (right). Top row - GFP fluorescence; bottom row - CFP fluorescence. Bars: 50µm (A,B), 20 µm (C). D) Changes in GFP and CFP fluorescence following exposure to lactacystin at t=1 hour (vertical dashed line). Changes normalized to initial fluorescence in same cells. 9 neurons from 5 experiments; averages and SEM. E) Same as in D, except that here neurons were exposed only to carrier solution. 11 neurons from 5 experiments; averages and SEM (barely observable). F) Comparison of GFP fluorescence accumulation rates. Linear fits shown as black lines; fit parameters shown next to fits. Same data as in D, and E.
The effects of proteasomal inhibition on synaptic proteostasis (2016)
Figure 2

Figure 2. Proteasomal inhibition does not lead to generalized accumulations of synaptic proteins at synaptic sites.

A) Hippocampal neurons in primary culture immunolabeled against three synaptic proteins (the postsynaptic scaffold molecule Dlg4/PSD-95, the presynaptic active zone molecule RIM and the presynaptic vesicle protein SV2A) following 10 hour incubations in carrier solution (control, upper panels) or Lactacystin (10 μM; bottom panels). Scale bar, 10μm B) Average synaptic immunofluorescence following 10 hour incubations in Lactacystin or carrier solution. Numbers within bars represent the number of fields of view analyzed for each condition (~170 synapses/field of view, on average). Error bars, SEM; P values – two tailed unpaired t-test. Note that a statistically significant accumulation was observed only for RIM, whereas immunolabeling of all other proteins was either unchanged or reduced. C) Hippocampal neurons in primary culture were grown in the presence of Lactacystin (10 μM), fixed at two hour intervals and immunolabeled against Shank3/ProSAP2. Two representative fields of view at t=0 and t=24h. Scale bar, 10μm. D) Average synaptic Shank3/ProSAP2 immunofluorescence at increasing times in the presence of Lactacystin. Fluorescence was normalized to mean fluorescence at t=0. Numbers under data points reflect the numbers of fields of view analyzed at each time point. Error bars, SEM.
An oligomeric state-dependent switch in the ER enzyme FICD regulates AMPylation and deAMPylation of BiP (2019)
Figure 1

Figure 1 - Monomeric mutant FICD promotes BiP AMPylation.

A Immunoblot of endogenous BiP resolved by native-PAGE from lysates of CHO-K1 S21 wild-type (wt) or FICD-/- cells either transiently overexpressing wild-type FICD (high expression level; Hi) or mutant FICDE234G (E/G) or stably expressing recombinant wild-type FICD (low expression level; Lo). The cells in lanes 1-4 were mock transfected. Where indicated cells were exposed to cycloheximide (CHX; 100 µg/mL) for 3 h before lysis. Unmodified ('A') and AMPylated ('B') monomeric and oligomeric (II and III) forms of BiP are indicated. Immunoblots of the same samples resolved by SDS-PAGE report on FICD, total BiP and eIF2α (loading control). Data representative of four independent experiments are shown. See Fig EV1B-C.

B Wild-type FICD forms homomeric complexes in vivo. Immunoblots of orthogonally-tagged wild-type and Leu258Asp mutant FICD in the input cell lysate and following recovery by pull-down with streptavidin (recognising the AviTag) or anti-FLAG antibody. Proteins were detected with fluorescently-labelled streptavidin (StrepIR800) or FLAG antibody. Data representative of three independent experiments are shown.

C Immunoblot of endogenous BiP from transfected CHO-K1 S21 FICD-/- cells (as in A). Note that cells expressing monomeric FICDL258D accumulate AMPylated BiP. Data representative of three independent experiments are shown.

D Size-exclusion chromatography (SEC) analysis of wild-type and mutant FICD proteins (each at 20 µM). The elution times of protein standards are indicated as a reference. Note that the Leu258Asp mutation monomerises FICD, while Gly299Ser causes partial monomerisation. See Fig EV1D-E.

E Comparison of the signal-averaged sedimentation coefficients of wild-type (red) and monomeric mutant FICDL258D (blue), as measured by analytical ultracentrifugation. A fit for monomer-dimer association (solid red line), constrained using the average value for the monomeric protein (dashed line, 2.82 S, Sw,20 = 3.02 S), yielded a Kd of 1.2 nM with a 95% confidence interval between 1.1 to 1.4 nM and a value of 4.08 S for the dimer (Sw,20 = 4.36 S). The fitted data points are from three independent experiments. See Fig EV1F-G.

F Autoradiograph of BiP, AMPylated in vitro by the indicated FICD derivatives, with [α-32P]-ATP as a substrate and resolved by SDS-PAGE. Proteins in the gel were visualised by Coomassie staining. A representative result of three independent experiments is shown. The graph shows the quantified relative BiP-AMP signals and SD bars generated by wild-type (wt) FICD and the indicated monomeric mutants.
An oligomeric state-dependent switch in the ER enzyme FICD regulates AMPylation and deAMPylation of BiP (2019)
Figure 2

Figure 2 - Monomerising mutations de-repress FICD's AMPylation activity.

A Monomerising FICD mutations inhibit deAMPylation. Shown is a representative plot of data points and fit curves of the time-dependent deAMPylation of a fluorescent BiPV461F-AMPFAM by the indicated FICD proteins (at 7.5 µM) as detected by a change in fluorescence polarisation (FP). DeAMPylation rates calculated from independent experiments are given in Fig EV2A.

B-C Dimer interface mutants both AMPylate and deAMPylate BiP. Shown are representative autoradiographs of thin layer chromatography (TLC) plates revealing AMP produced from reactions containing [α-32P]-ATP and the indicated FICD enzymes in the presence or absence of the co-substrate BiP (arrow indicates direction of nucleotide migration). The radioactive signals were quantified and the AMP signals were normalised to the total nucleotide signal in each sample. Plotted below are mean values ± SD from at least three independent experiments. Unpaired t-tests were performed. See Fig EV2D.

D Cartoon depicting sequestration of AMPylated BiP by a covalently linked, disulphide-stapled, S-SFICDA252C-H363A-C421S dimer (trap). See Fig EV2E-H.

E Detection of the time-dependent accumulation of AMPylated BiPT229A-V461F in radioactive reactions, containing [α-32P]-ATP and the indicated FICD proteins, in the presence of excess trap. At the specified time-points samples were taken and analysed by SDS-PAGE. The autoradiograph (32P) illustrates the radioactive signals, which represent AMPylated BiP. Proteins were visualised by Coomassie staining. The radioactive signals were quantified and presented in the graph below. Mean values ± SD of three independent experiments are shown.
An oligomeric state-dependent switch in the ER enzyme FICD regulates AMPylation and deAMPylation of BiP (2019)
Figure 3

Figure 3 - Monomerisation by dilution enhances the AMPylation activity of wild-type FICD.

A Autoradiographs of in vitro reactions containing varying concentration of wild-type FICD protein and fixed concentrations of BiPT229A-V461F and [α-32P]-ATP as co-substrates, resolved by SDS-PAGE after the indicated incubation times. The proteins were visualised by Coomassie staining of the gel (bottom). The reactions shown on the right were performed in the presence of an excess of S-SFICDA252C-H363A-C421S (trap) to delay de-modification of BiP. Representative gels are shown, and similar results were observed in three independent experiments.

B As in (A) but with 0.2 µM of the indicated FICD variant. The radioactive signals were detected by autoradiography, quantified, and normalised to the signal in lane 6. The mean radioactive signals ± SD from three independent experiments are given. The proteins were visualised by staining with Coomassie. See Appendix Fig S1A-B.

C As in (A) but with dilutions of FICDC421S or covalently linked S-SFICDA252C-C421S. Reactions were preceded by a 16 h incubation of FICD in presence or absence of the reducing agent (DTT). Representative gels are shown of three independent experiments. See Appenidx Fig S1C.

D Forced dimerisation does not significantly alter deAMPylation rates. Time-dependent deAMPylation of fluorescent BiPV461F-AMPFAM by the indicated FICD proteins (at 7.5 µM) assayed by fluorescence polarisation (as in Fig 2A). A representative experiment (data points and fit curves) is shown and rates are given in Fig EV2A. See Appendix Fig S1D.

E Representative autoradiograph of thin layer chromatography (TLC) plates revealing AMP produced from reactions containing [α-32P]-ATP and the indicated FICD enzymes in the presence of the co-substrate BiP. AMP signals were normalised to the total nucleotide signal in each sample and the graph below plots mean values ± SD from at least three independent experiments.
An oligomeric state-dependent switch in the ER enzyme FICD regulates AMPylation and deAMPylation of BiP (2019)
Figure 4

Figure 4 - Residues connecting the FICD dimer interface with the inhibitory α-helix stabilise FICD and repress AMPylation.

A Ribbon diagram of the FICD dimer interface with monomers in purple and blue ribbons (PDB: 6I7G). Residues involved in a H-bond network linking the dimer interface to the αinh (as well as Gly299 and Glu234) are shown as green sticks. Sub-3.50 Å hydrogen bonds made by Asn236, Leu238 and Lys256 are depicted as dotted cyan lines.

B Size-exclusion chromatography elution profile of wild-type and mutant FICD proteins (each at 20 µM). Protein absorbance at 280 nm is plotted against elution time. The elution times of protein standards are indicated as a reference.

C Radioactive in vitro AMPylation reactions containing the indicated FICD proteins, [α-32P]-ATP, and BiPT229A-V461F were analysed by SDS-PAGE. The radioactive BiP-AMP signals were detected by autoradiography and proteins were visualised by Coomassie staining of the gel. See Fig EV3A.

D Representative autoradiograph of thin layer chromatography (TLC) plates revealing AMP produced from reactions containing [α-32P]-ATP and the indicated FICD enzymes in the presence of the co-substrate BiP. The radioactive signals were quantified and the AMP signals were normalised to the total nucleotide signal in each sample. The graph shows mean AMP values ± SD from three independent experiments.

E Melting temperatures (Tm) of the indicated FICD mutants (at 2 µM) were measured by differential scanning fluorimetry (DSF). Shown is the mean Tm ± SD of three independent experiments. The inset shows melt curves with their negative first derivatives from a representative experiment. See Fig EV3D.

F A plot of the melting temperature of the indicated FICD proteins in absence (Apo) or presence of nucleotides. Shown are the mean Tm values ± SD of three independent DSF experiments. Monomeric FICDL258D (mFICD) and FICDL258D-E234G (mFICDE/G) as well as dimeric wild-type FICD (dFICD) and FICDE234G (dFICDE/G) were tested. ADP and ATP concentrations in mM are given in parentheses. See Fig EV3E for K½ quantification.
An oligomeric state-dependent switch in the ER enzyme FICD regulates AMPylation and deAMPylation of BiP (2019)
Figure 6

Figure 6 - ATP destabilises the pre-AMPylation complex and the FICD dimer.

A BioLayer interferometry (BLI) derived association and dissociation traces of monomeric FICDL258D-H363A (mFICDH363A) or dimeric FICDH363A (dFICDH363A) from immobilised biotinylated BiPT229A-V461F in absence or presence of nucleotides. Unless indicated (*) BiP was saturated with ATP before exposure to FICD variants. A representative experiment of three independent repetitions is shown. See Fig EV6A-B.

B BLI dissociation traces of proteins as in (A). At t = 0 a pre-assembled complex of immobilised, ATP-saturated BiP and the indicated FICD proteins (associated without ATP) were transferred into a solution without or with ATP, as indicated. A representative experiment is shown and the biphasic dissociation kinetics are quantified in (C) and (D). Full association and dissociation traces are shown in Fig EV6C.

C Graph of the slow dissociation rates (koff,slow) of monomeric FICD from BiP:ATP as shown in (B). Bars represent mean values ± SD of three independent experiments.

D The ATP-induced fold change in the percentage of the dissociation phase attributed to a fast dissociation (%Fast), koff,fast, and koff,slow derived from the data represented in (B). Bars represent mean values ± SD of three independent experiments. See Fig EV6D.

E (i) BLI assay workflow used to derive FICD dimer off rate data presented in (ii) and Fig EV6E-F. (ii) Observed dimer off rates under different nucleotide conditions (5 mM, where applicable). ATP, but no ADP, significantly increases the dimer dissociation rate [n.s.: not significant by Tukey test]. Data shown is the mean ± SD of four independent experiments. See Fig EV6E-F.

F Validation of the homo-FRET dimerisation probe. The dimerisation sensitive TMR fluorescence of the labelled FRET dimer (2.5 nM) is de-quenched specifically by equilibration with excess unlabelled dimerisation-competent FICDH363A but not monomeric FICDL258D-H363A. Mean ± SD of three independent experiments.

G Homo-FRET measurement of nucleotide dependent modulation of the FICD monomer-dimer equilibrium. ATP increases and ADP decreases the proportion of monomeric FICD. * Plateaus were constrained to a shared best fit value. Data shown is the mean ± SD of four independent experiments.
RAB13 mRNA compartmentalisation spatially orients tissue morphogenesis (2020)
Figure 2

Figure 2. Clustering of RNAseq datasets defines an RNA motif enriched in 3'UTR sequences that target k 5 mRNAs to protrusions.

A. Diagram of k 5 mRNA 3'UTRs and relative positions of the RNA motif shared between transcripts.

B. RNA motif over-represented in k 5 mRNA 3'UTRs.

C. Frequency of mRNAs within each k-means cluster containing at least 2 of the RNA motif over-represented in k 5 mRNAs.

D. Scheme depicts the in vitro MS2 system strategy. CMV promoter-driven expression of MCP-GFPnls and hHBB-24xMS2-tagged RAB13 3'UTR. The visualisation of MCP-GFPnls bound to 24xMS2 allows the identification of the minimal region in the 3'UTR of RAB13 necessary for its localisation.

E. Left: representative subconfluent motile cells co-transfected with plasmids expressing Lyn-mCherry, MCP-GFPnls and 24xMS2-RAB13 3'UTR or 24xMS2. Right: percentage of cells with MCP-GFP localised to protrusions when co-transfected with full length or deletion versions of RAB13 3'UTR (n≥3 experiments).

F. Left: representative cells co-transfected with plasmids expressing Lyn-mCherry, MCP-GFPnls and 24xMS2-k 5 3'UTRs. Right: percentage of cells with MCP-GFP localised to protrusions when co-transfected with full length or deletion versions of k 5 3'UTR (n≥3 experiments).

Data information: white arrowheads indicate non-nuclear localisation of MCP-GFPnls; arrows indicate the orientation of RNA localisation; yellow dashed lines outline cell borders; scale bars = 20 µm (E,F); scale bars in insets = 5 µm (E) and 2 µm (F). For each k 5 mRNA 3'UTR, a diagram of the full length 3'UTR and the positions of the RNA motif is shown together with the respective RNAseq mapped reads from HUVEC protrusions; black arrowheads indicate predicted polyadenylation sites (E,F). Bar charts are presented as means±s.d.