EMBO/soda-nel huggingface dataset:
SIRT7 activates quiescent hair follicle stem cells to ensure hair growth in mice (2020)
Figure 1

Figure 1. Sirt7 promotes HF telogen-to-anagen transition

(A) The hair coats of Sirt7−/− and Sirt7+/+ mice were clipped on P60 in the mid second telogen phase. Images were captured at P60, P120 and P150.

(B) The postnatal day when the hair coat recovered by 25%, 50%, 75% and 100% in shaved Sirt7−/− and Sirt7+/+ mice. n = 6 mice/genotype. Box-and-whisker plots: mid-line, median; box, 25th and 75th percentiles; whiskers, minimum and maximum.

(C) H&E staining of the skin in unshaved Sirt7−/− and Sirt7+/+ mice on P90 and P130.

(D) Representative immunofluorescence images showing Ki67 and p-cadherin (Pcad, labeling HG cells) expression in Sirt7−/− and Sirt7+/+ HFs on P74 and P90. The HF boundary and dermal papilla (DP) is denoted by the dotted and dashed lines, respectively.

(E) Percent Ki67 positive cells in each hair germ in D; n = 3 mice and 10 HGs were counted in each mouse.

(F) Telogen duration in Sirt7−/− and Sirt7+/+ mice. n = 6 mice per genotype. Box-and-whisker plots: mid-line, median; box, 25th and 75th percentiles; whiskers, minimum and maximum.

(G) The hair coats of Sirt7-TG and WT mice were clipped on P45 and images were captured on P56 and P90.

(H) The postnatal day when the hair coat recovered by 25%, 50%, 75% and 100% in shaved Sirt7-TG and WT mice. n = 6 mice per genotype. Box-and-whisker plots: mid-line, median; box, 25th and 75th percentiles; whiskers, minimum and maximum.

(I) Representative immunofluorescence images showing Ki67 and Pcad expression in Sirt7-TG and WT HFs at P60 and P65. The HF boundary and DP is noted by the dotted and dashed lines, respectively.

(J) The percentage of Ki67 positive cells in each HG; n = 3 mice and 10 HGs were counted in each mouse.

(K) H&E staining of skin tissues in unshaved Sirt7-TG and WT mice on P65.

(L) Telogen duration in Sirt7-TG and WT mice. n = 6 mice per genotype. Box-and-whisker plots: mid-line, median; box, 25th and 75th percentiles; whiskers, minimum and maximum.

(M) Abstracted HF cycling in Sirt7−/−, Sirt7+/+, Sirt7-TG and WT mice mentioned above. The red star indicates anagen entry in Sirt7−/− and Sirt7-TG mice; the black arrows show the date of doxycycline feeding.

Data information: Scale bar, 50 μm for immunofluorescence images; 200 μm for H&E staining images. The data represent the means ± s.e.m. **P < 0.01, ***P < 0.001, determined by Student's t-test.
SIRT7 activates quiescent hair follicle stem cells to ensure hair growth in mice (2020)
Figure 2

Figure 2. Sirt7 deletion delays HFSC activation

(A-B) IHC labeling of Sirt7 and Ki67 expression in the skin at different HF phases in the second hair cycle and in the depilation-induced hair cycle in WT mice. P56, P69, P74 and P84 indicate the postnatal day in the second hair cycle. Pd, post depilation. Dashed line indicates bulge (Bu) of a hair follicle and solid line indicates the Ki-67 positive staining region.

(C) The hair coats of Sirt7f/f and Sirt7f/f;K15-Cre mice were clipped at P60 and images were captured on P60, P125 and P150.

(D) The postnatal day of hair coat recovery by 25%, 50% 75% and 100% in shaved Sirt7f/f and Sirt7f/f;K15-Cre mice. n = 6 mice per genotype. Box-and-whisker plots: mid-line, median; box, 25th and 75th percentiles; whiskers, minimum and maximum.

(E) H&E staining of the skin in Sirt7f/f and Sirt7f/f;K15-Cre mice at P100.

(F) Representative immunofluorescence images showing Ki67/Pcad expression (upper) and Cd34/Ki67 expression (lower) in Sirt7fl/fl and Sirt7f/f;K15-Cre HFs on P56, P74 and P85. The white dashed line indicates bulge of hair follicle.

(G) The percentage of Ki67-positive cells per HG; n = 3 mice and 10 HGs were counted in each mouse.

(H) Ki67-positive cells per bulge; n = 3 mice and 10 bulges were counted in each mouse.

(I) Schematic illustrating the HF cycle in Sirt7f/f and Sirt7f/f;K15-Cre mice after mifepristone-induced Cre activation. The arrow indicates the time of mifepristone treatment (details in methods); the red star indicates the end of the telogen phase in Sirt7f/f;K15-Cre HFs.

Data information: Scale bar, 50 μm for IHC and immunofluorescence images, 200 μm for H&E staining images. The data represent the means ± s.e.m. *P <0.05, **P < 0.01, ***P < 0.001, obtained by Student's t-test.
SIRT7 activates quiescent hair follicle stem cells to ensure hair growth in mice (2020)
Figure 3

Figure 3. Altered expression of Nfatc1 in Sirt7-modified mice

(A) Representative colonies formed by Sirt7+/+, Sirt7-/- and Sirt7-TG bulge SCs that were puriﬁed from telogen-phase HFs and allowed to grow for 2 weeks.

(B-C) Quantiﬁcations of colony number and size in (A); n = 3 mice per genotype.

(D-E) Immunoblot analysis of the indicated proteins in HFSC colonies from Sirt7+/+, Sirt7-/- and Sirt7-TG mice (D) and Nfatc1 protein levels were quantified using Image J Plus 6.0 (E, n = 3 mice for each group).

(F) Immunoblot analysis of the NFATc1 expression level in human keratinocyte HaCaT cells treated with SIRT7 siRNAs.

(G) Immunoblot analysis of the NFATc1 expression in the presence of ectopic SIRT7 expression in HaCaT cells.

(H) NFATc1 luciferase activity was monitored in HEK293T cells after the indicated treatments (n=3). The relative luciferase activity was determined by dual luciferase reporter assay.

(I-J) IHC analyses of Nfatc1 expression in hair follicles of Sirt7f/f, Sirt7f/f ;K15-Cre and Sirt7-TG mice at the indicted time points. The black dashed line indicates the bulge (Bu) of hair follicle.

(K-L) qPCR analysis of Nfatc1-targeted genes in HFSCs colonies cultured from Sirt7+/+, Sirt7-/-and Sirt7-TG mice, n=3 for each genotypes.

Data information: Scale bar, 50 μm for the IHC images. The data represent the means ± s.e.m. *P < 0.05, **P < 0.01, ***P < 0.001, determined by Student's t-test.
SIRT7 activates quiescent hair follicle stem cells to ensure hair growth in mice (2020)
Figure 4

Figure 4. SIRT7 deacetylates NFATc1 at K612

(A-B) Immunoblot analysis of the interactions between ectopic HA-SIRT7 and FLAG-NFATc1in HEK293T cells.

(C) Co-immunoprecipitation (CoIP) between endogenous NFATc1 and SIRT7 in HEK293T cells.

(D) Immunoblots showing endogenous NFATc1 pull-down by GST-SIRT7 in HEK293T cells.

(E) Immunoblot analysis of HA-SIRT7 WT and various mutants in the anti His-NFATc1 immunoprecipitates. ∆N indicates N-terminal deletion; ∆C indicates C-terminal deletion; AC indicates N-terminal and C-terminal deletion.

(F) Immunoblot analysis of HA-SIRT7 levels in truncated FLAG-NFATc1 immunoprecipitates. N indicates the N-terminal domain; C indicates C-terminal domain.

(G-H) Immunoblot analysis of the interactions between endogenous NFATc1 and SIRT7 in keratinocyte HaCaT cells.

(I-J) Immunoblot analysis of NFATc1 acetylation levels in response to the indicated treatments.

(K-L) Immunoblot analysis of NFATc1-WT/K612R acetylation based on SIRT7 deacetylation assays in vitro (K) and in vivo (L).

(M-N) Immunoblots showing NFATc1 pan phosphorylation (probing to anti S/T antibody) and acetylation levels under the indicated treatments.

(O) Immunoblot analysis of NFATc1 phosphorylation among K612 mutants, noting that K612R represents deacetylation while K612Q hyperacetylation.
SIRT7 activates quiescent hair follicle stem cells to ensure hair growth in mice (2020)
Figure 5

Figure 5. SIRT7 promotes NFATc1 degradation via PA28γ

(A) Representative immunoblots showing NFATc1 degradation in HEK293T cells stably overexpressing NFATc1 in the presence of cycloheximide (CHX), with or without SIRT7 overexpression.

(B) Quantification of NFATc1 levels in (A) from 3 independent experiments.

(C) Immunoblots of Nfatc1 protein levels in Sirt7+/+ and Sirt7-/- keratinocytes.

(D) Quantification of Nfatc1 levels in (C) from 3 independent experiments.

(E-F) Immunoblots (E) and related quantitative data (F, n=3) showing NFATc1 protein levels in HEK293T cells overexpressing wildtype HA-SIRT7 or a SIRT7 enzyme-dead mutant (H187Y).

(G-H) Immunoblots (G) and quantitative data (H, n=3) showing NFATc1 levels in HEK293T cells transfected with NFATc1-CA, with or without ectopic SIRT7, treated with MG132.

(I-J) Immunoblots (I) and related quantitative data (J, n=3) showing NFATc1-WT and NFATc1-K612R protein turnover in HEK293T cells.

(K-L) Immunoblots showing the interaction (by co-immunoprecipitation) between ectopic NFATc1/PA28γ in HEK293T cells (K) or endogenous NFATc1/PA28γ in HaCaT cells (L) after manipulating SIRT7 levels.

(M-N) Immunoblot (M) and quantitative data (N, n=3) analysis of NFATc1 levels upon overexpression of HA-SIRT7 with or without PA28γ oligo siRNAs treatment.

(O) Co-immunoprecipitation between NFATc1 and PA28γ in HEK293T cells cotransfected with WT HA-SIRT7 or an enzyme-dead mutant (H187Y).

Data information: The data represent the means ± s.e.m. *P < 0.05, **P < 0.01, ***P < 0.001, determined by Student's t-test.
SIRT7 activates quiescent hair follicle stem cells to ensure hair growth in mice (2020)
Figure 6

Figure 6. Cyclosporin A abrogates HF cycling delay induced by Sirt7 deletion

(A) IHC staining of Nfatc1 expression in HFs on P84 in Sirt7+/+ and Sirt7-/- mice with or without Cyclosporin A (CsA) treatment. The black dashed line indicates bulge (Bu) of hair follicle.

(B) Quantitative analysis of Nfatc1 levels in (A, n=10 hair follicles) using ImageJ plus 6.0.

(C) Immunofluorescent staining of Ki67 and Pcad at P84 in Sirt7+/+ and Sirt7-/- HFs with or without CsA treatment.

(D) The percentage of Ki67-positive cells per HG in (C, n=10 hair follicles).

(E) H&E staining of the skin in the indicated treatment groups at P100.

(F) Representative images showing haircoat recovery after the indicated treatments. The dashed circle indicates the shaved area; the dotted line indicates the hair growth boundary.

Data information: Scale bars, 50 μm for the IHC and immunofluorescence images and 200 μm for the H&E images. The data represent the means ± s.e.m. ***P < 0.001, determined by Student's t-test.
SIRT7 activates quiescent hair follicle stem cells to ensure hair growth in mice (2020)
Figure 7

Figure 7. Sirt7 associates to aging-related hair dysfunction

(A) Immunoblot analysis of Sirt7 expression in young (3M) and aged (18M) HFSCs. #1, #2, #3 represented individual mice. Below, quantification of Sirt7 protein levels; n=3 mice for each group; the Y-axis represents the relative band intensity (measured with Image J®) normalized to the signal in young mice.

(B-D) Immunofluorescence and IHC staining of Nfatc1 and Sirt7 in HFs of young (3 months) and old (18 months) mice (B), and the relative levels of Sirt7 and Nfatc1-positive cells in the bulge (C-D). The white/black dashed line indicates bulge (Bu) of hair follicle.

(E) Images were captured of the hair coats of Sirt7+/+ and Sirt7-/- mice at 15 months. The yellow dashed lines indicate hair thinning or hair loss.

(F) Immunofluorescence and IHC staining of Nfatc1 and Sirt7 in HFs of 15-month-old Sirt7+/+ and Sirt7-/- mice. The white/black dashed line indicates bulge (Bu) of hair follicle.

(G) Quantification of Nfatc1-positive cells per bulge in (F, n=10 hair follicles).

(H) Images captured of the skin in the plucked regions (blue dotted lines) of WT (3 months), aged WT (20 months) and aged Sirt7-TG mice (20 months), 14 days after depilation. Pigmentation of the skin indicates hair regrowth. n = 3 mice per genotype.

(I) Hair re-growth scores were analyzed for young WT, old WT and old Sirt7-TG mice in (H), n=3 mice for each group.

(J) The hair coat recovery score (described in methods) after the back hair of old WT and old Sirt7-TG mice was shaved. n = 3 mice per genotype.

(K) Images captured of the hair coats of WT and Sirt7-TG mice at 28 months-of-age. The yellow dashed lines indicate the hair thinning or hair loss regions.

Data information: Scale bars, 50 μm for the IHC and immunofluorescence images. The data represent the means ± s.e.m. *P < 0.05, **P < 0.01, ***P < 0.001, determined by Student's t-test.
ALS-linked KIF5A ΔExon27 mutant causes neuronal toxicity through gain-of-function (2022)
Figure 1

Figure 1. ALS-associated KIF5A ΔExon27 is prone to form cytoplasmic aggregates.

(A) Schematic illustration of KIF5A WT and ΔExon27 showing the motor, stalk, and cargo domains (left). Several ALS-associated KIF5A variants disrupt the C-terminal tail by skipping exon 27 during mRNA splicing and produce a neopeptide of aberrant 39 amino acids (red) replacing the normal tail of 34 amino acids (right). (B) HEK293T cells were transfected with KIF5A WT-GFP or ΔExon27-GFP for 24 and 48 hours. Nuclei were stained with DAPI, three biological replicates, n = 200 cells per experiment. Scale bar: 20 µm. (C) Co-staining of G3BP1, p62, and Ubiquitin with KIF5A ∆Exon27 granules, three biological replicates, n = 250 cells per experiment. Scale bar: 20 µm. (D-E) Western blot analysis of RIPA soluble-fraction and RIPA-insoluble, urea-soluble fraction of proteins prepared from HEK293T cells expressing empty plasmid (control), FLAG-KIF5A WT, FLAG-ΔExon27. Insoluble/soluble KIF5A fractions were detected with anti-FLAG and actin was used as a loading control, three biological replicates. Statistical analysis was performed using student t-test (*p < 0.05, **p<0.01, ***p<0.001). Data represent the mean ± s.d.
ALS-linked KIF5A ΔExon27 mutant causes neuronal toxicity through gain-of-function (2022)
Figure 2

Figure 2. ΔExon27 interacts with WT KIF5A to form aggregates and causes neuronal toxicity

(A) Expression of KIF5A WT and ΔExon27 tagged with GFP and mApple, respectively, in HEK293T cells. Images were taken 48 hours after transfection, two biological replicates. Scale bar: 20 µm. (B) Pull down analysis showing the association of ΔExon27 with WT KIF5A, two biological replicates. (C) Staining of mouse endogenous KIF5A in N2a cells transfected with ΔExon27-GFP using an antibody generated with a peptide corresponding to the C-terminus of KIF5A WT (amino acids 1007-1027), which does recognize ΔExon27. ΔExon27 granules are highlighted with arrowheads. The nuclei of cells expressing ΔExon27 are indicated with asterisk, two biological replicates. Scale bar: 20 µm. (D) Mouse primary neurons transfected with KIF5A WT-GFP and ΔExon27-GFP were probed using anti-GFP antibody 24 hours after transfection. Neurons were identified by microtubule associated protein 2 (MAP2) and nuclei were stained with DAPI, three biological replicates, n = 200 cells per experiment. Scale bar: 20 µm. (E) 5-day-old mouse cortical neurons were transfected with mApple together with either GFP (n=150), KIF5A WT-GFP (n = 160), or ΔExon27-GFP (n = 135), three biological replicates. Images were taken every 24 hours after transfection. Survival of neurons was analyzed by Kaplan-Meier survival analysis (***p < 0.0001, log-rank test).
ALS-linked KIF5A ΔExon27 mutant causes neuronal toxicity through gain-of-function (2022)
Figure 5

Figure 5. Expression of endogenous mutant KIF5A proteins and increased KIF5A inclusions in three patient-derived iMN cell lines

(A) Sanger sequencing chromatograms of cDNA from control and patient-derived iMNs (P1-P3) showing the effect of mutations on KIF5A transcripts. While the c.3020+2T>C mutation causes exon 27 skipping, the c.2993-1G>A mutation results in an aberrant transcript with a splice acceptor shifted only by a single nucleotide, leading to a frameshift but containing exon 27. The respective protein product predicted for both mutations comprise the same C-terminal aberrant 39 amino acid end. (B) Western blot analysis for validation of the custom mutant-specific antibody using transfected HEK293T cells. While an antibody against the total KIF5A (lower panel) recognized both wild type and mutant protein, the custom antibody targeting the aberrant "neopeptide" detected ΔExon27 specifically (upper panel). NT: No transfection. (C) Mutant KIF5A proteins are endogenously expressed in iMNs, detected by using the custom mutant specific antibody directed against the C-terminal aberrant 39 amino acid sequence. CTL: Control, P1: Patient 1, P2: Patient 2, P3: Patient 3, two independent experiments. (D) Representative images of KIF5A distribution (arrowheads) in control and patient iMNs. Scale bar: 20 µm. (E) Abundance of KIF5A inclusions after normalizing to total neuron numbers. Graph represents data from n=146 control 1 (CTL1), n = 134 control 2 (CTL2), n = 154 patient 1 (P1), n = 162 patient 2 (P2) and n = 133 patient 3 (P3) iMNs from 3 individual experiments. Bars indicate mean ± s.d. Statistical analysis was performed using Mann Whitney Wilcoxon Test (*p<0.05).
Mitochondrial transfer from MSCs to T cells induces Treg differentiation and restricts inflammatory response (2019)
Figure 1

Figure 1 - MSCs efficiently transfer mitochondria in a dose-dependent manner to T and B lymphocytes.

A Schematic representation of the experimental design used for co-culture experiments.

B Representative confocal microscopy of human PBMCs stained with DAPI (white arrows) co-cultured with previously labeled UC-MSC with MitoTracker-Green (top image) and specific antibody for human CD90 (bottom image).

C Percentage of mitochondrial transfer (MitoT) on total human CD45+ cells after 24 hrs co-culture with MitoTracker-Green labeled bone marrow (BM), menstrual (Mens) or umbilical cord (UC)-MSCs (MSC:PBMC ratio 1:25) (n=3 biological replicates, with 3 different donors of BM, Mens and UC-MSCs).

D FACS analysis of MitoT on T CD3+ (n=4), B CD19+ (n=4) and natural killer (NK) CD56+ (n=3) cells after 24 hrs co-culture with MitoGreen labeled UC-MSCs (ratio 1:25) (Biological replicates, with different donors of UC-MSCs).

E FACS analysis of MitoT on CD3+CD4+ or CD3+CD8+ T cell populations after 24 hrs co-culture with labeled UC-MSCs (ratio 1:25) (n=3 biological replicates, with 3 different donors of UC-MSCs).

F Representative FACS plots of MitoT on CD45+CD3+ T cells at increasing amounts of UC-MSC.

G Percentage of MitoT on CD45+CD3+ human cells after 24 hrs co-culture with increasing ratios of UC-MSC:PBMCs (n=5 biological replicates, with 5 different UC-MSCs and 5 different PBMCs).

H FACS analysis of MitoT on CD45+CD3+ T cells after short times of co-culture with UC-MSCs at two different temperatures, at a ratio 1:25 (n=3 biological replicates, with 3 different UC-MSCs and 3 different PBMCs).

I Mean fluorescence intensity (MFI) of MitoTracker-Green+ UC-MSCs at baseline or after 72 hours PBMC co-culture. Representative FACS plot (left panel) and average of MFI (n=4 biological replicates, with 4 different UC-MSCs) (right panel).

J Confocal microscopy of human PBMCs after co-culture with MitoTracker-Green stained UC-MSCs. Images showed MSC-mitochondria (green) inside the CD3+ T cells (red).

K qPCR analysis of human β2-microglobulin (B2M), human specific mitochondrial (Mito) and mouse specific (β-actin) gene expression levels in UC-MSCs, mouse derived spleen cells or FACS sorted CD45+ mouse cells after 24 hrs co-culture with UC-MSCs (n=3 biological replicates). n/d = not detected.

L In vivo experimental design for MitoT in Balb/c mice transplanted with MitoTracker Green labeled MSCs.

M FACS analysis of in vivo MitoT from UC-MSCs to Balb/c mouse mesenteric lymph node and spleen cells, 24 hours after intraperitoneal transplantation of 5*106 MitoTracker-labeled MSCs or without cells (control) (n=1).

Data information: In C-E, G-I and K, graphs show mean ± SEM and statistical analysis by Student's t-test.
Mitochondrial transfer from MSCs to T cells induces Treg differentiation and restricts inflammatory response (2019)
Figure 2

Figure 2 - CD3+ T cells are permissive to artificial MitoT (Mitoception) resulting in an increase of their mitochondrial content.

A Schematic representation of the experimental design of the mitoception protocol.

B Confocal microscopy images of human CD3+ T cells with endogenous MT-labeled with MitoTracker Red CMXRos (white arrows, left panel) or after mitoception with MitoTracker Green labeled-MT from UC-MSCs (white asterisk, right panel).

C FACS analysis of MitoT on CD3+ human T cells after mitoception with increasing amounts of UC-MSC MT corresponding to the same cell number ratios used in co-culture experiments (n=4 biological replicates, with 4 different donors of UC-MSCs). Graph shows mean ± SEM and statistical analysis by Student's t-test.

D Representative transmission electron microscopy images of FACS-sorted human CD3+MitoTneg and CD3+MitoTpos cells following mitoception with UC-MSC MT. Red arrows show human mitochondria.

E Quantification of human MT in sorted CD3+ MitoTneg and MitoTpos cells after mitoception with UC-MSCs, by transmission electron microscopy (n=25 independent cells analyzed per group). Graph shows mean ± SEM and statistical analysis by Student's t-test.
Mitochondrial transfer from MSCs to T cells induces Treg differentiation and restricts inflammatory response (2019)
Figure 4

Figure 4 - Efficient MitoT to T cells underlies functional changes including enhanced immunosuppression through regulatory T cell (Treg) induction.

A Representative bright-field microscopy images of FACS-sorted CD4+ naïve MitoTneg (upper panel) and MitoTpos (lower panel) cells after 5 days in culture with plain media (no cytokines added). Red arrows are indicative of activation/proliferation clusters.

B Representative FACS plots of Treg (CD127lowCD25+FoxP3+) populations after T cell differentiation on media with or without cytokines and antibody supplements (anti-CD3, IL-2 and TGF-b1).

C FACS analysis of Treg induction after 5-7 days differentiation of FACS-sorted CD4+ naïve MitoTneg and MitoTpos cells in plain (n=5) or differentiation media (n=6) (Biological replicates, with different donors of PBMCs).

D ICOS expression on FACS-sorted CD4+ naïve MitoTneg and MitoTpos cells after 5-7 days in culture with differentiation media. Representative FACS plot (left panel) and bar graph (right panel) of ICOS high population (n=4 biological replicates).

E CCR7 expression on sorted CD4+ naïve MitoTneg and MitoTpos cells after 5-7 days in culture with differentiation media. Representative FACS plot (left panel) and bar graph (right panel) of CCR7+ population (n=4 biological replicates).

F FACS analysis of Th1 (CD4+IFNγ+) induction after 5-7 days differentiation of FACS-sorted CD4+ naïve MitoTneg and MitoTpos cells in plain media (n=3 biological replicates). n/s= not significant.

Data information: In C-F graphs show mean ± SEM and statistical analysis by Student's t-test.