EMBO/soda-nel huggingface dataset:
SIRT5 impairs aggregation and activation of  MAVS through catalyzing lysine desuccinylation (2020)
Figure 3

Figure 3. MAVS is succinylated at lysine 7 and SIRT5 mediates its desuccinylation.

A-B MAVS could be succinylated in vitro. GST-MAVS (A) extracted from E.coli or Flag-tagged MAVS (B) protein purified from HEK293T cells was incubated with the indicated concentrations of succinyl-CoA. Protein succinylation was detected with anti-pan-succinyl-lysine antibody; GST-MAVS or Flag-MAVS was stained by Coomassie blue.

C The succinylated residue in MAVS was identified by mass spectrometry analysis.

D Sequence alignment of partial MAVS (1-30 amino acids) from human, macaque, cow, pig, dog, mouse and rat. The red box indicates a conserved lysine (K7).

E Dot-blot assay for the specificity of anti-succ-K7-MAVS antibody. Equal amounts of succinyl-peptides or the control peptides were immunoblotted with the indicated dilutions of anti-succ-K7-MAVS antibody.

F-G GST-MAVS (F) extracted from E.coli or Flag-MAVS (G) purified from HEK293T cells was incubated with the indicated concentrations of succinyl-CoA. The succinylation of MAVS at lysine 7 was detected by anti-succ-K7-MAVS antibody.

H Disruption of SIRT5 in H1299 cells enhanced succinylation of MAVS at Lys 7 compared to that in the SIRT5-intact H1299 cells (SIRT5 +/+) (3.2 vs. 1.0). The cell lysates from SIRT5-deficient H1299 cells or the SIRT5-intact H1299 cells were immunoprecipitated with anti-MAVS antibody or mouse IgG control, followed by immunoblotting with anti-succ-K7-MAVS antibody.

I Reconstitution of WT SIRT5 in SIRT5-deficient H1299 cells (SIRT5 -/-) caused a significant reduction in succinylation of MAVS at Lys 7 (1.0 vs. 0.2); but overexpression of SIRT5-H158Y in SIRT5 -/- H1299 cells has no effect on the reduction in succinylation of MAVS at Lys 7 (1.0 vs. 1.2). The SIRT5 -/- H1299 cells were transfected with Flag-SIRT5 or Flag-SIRT5-H158Y, followed by immunoprecipitating with anti-MAVS antibody or mouse IgG control, and immunoblotting with anti-succ-K7-MAVS antibody.

J-K Knockout of Sirt5 increased Mavs succinylation in mouse livers (J) and lungs (K). Proteins extracted from livers (J) and lungs (K) of Sirt5 KO and the wildtype littermates (n = 3 per group) were detected by the indicated antibodies. MAVS succinylation was determined by anti-succ-K7-MAVS antibody, and anti-pan-succinyl-lysine antibody was used as positive controls.

Data information: IP, immunoprecipitation; TCL, total cell lysates.
SIRT5 impairs aggregation and activation of  MAVS through catalyzing lysine desuccinylation (2020)
Figure 4

Figure 4. SIRT5 negatively regulates cellular antiviral response.

A-C Quantitative real time PCR analysis (qPCR) of IFNβ (A), CXCL10 (B) and IFIT1 (C) mRNA in HEK293T cells transfected with the control plasmid (Myc empty) or the plasmid expressing Myc-SIRT5 (Myc-SIRT5) for 24 h, followed by infection with or without SeV (SeV or UI) for 8h.

D-F qPCR analysis of IFNβ (D), CXCL10 (E), and IFIT1 (F) mRNA in SIRT5-deficient or WT H1299 cells (SIRT5 -/- or SIRT5 +/+) infected with or without SeV (SeV or UI) for 8 h.

G-I qPCR analysis of IFNβ (G), CXCL10 (H) and IFIT1 (I) mRNA in SIRT5-deficient or WT H1299 cells (SIRT5 -/- or SIRT5 +/+) infected with or without VSV (VSV or UI) for 8h.

J qPCR analysis of IFNβ mRNA in HEK293T cells transfected with the control plasmid (Myc empty), or the plasmid expressing Myc-SIRT5 (Myc-SIRT5), or its enzyme-deficient mutant H158Y (Myc-SIRT5-H158Y) for 24 h, followed by stimulation with or without poly(I:C) for 8 h.

K qPCR analysis of IFNβ mRNA in SIRT5-deficient H1299 cells (SIRT5-/-) transfected with the control plasmid (Myc empty) or the plasmid expressing Myc-SIRT5 (Myc-SIRT5) or its enzyme-deficient mutant H158Y (Myc-SIRT5-H158Y) for 24 h, followed by infection with (+) or without (-) SeV for 8 h.

L SDD-AGE analysis of MAVS aggregates in HEK293T cells transfected HA-MAVS together with WT Myc-SIRT5 or its enzyme-deficient mutant (Myc-SIRT5-H158Y) after infected with SeV for 12h. SDS-PAGE immunoblotting was used as a loading control.

M-N IFN-β promoter activity (M) and ISRE reporter activity (N) in HEK293T cells co-transfected Myc-SIRT5 together with HA-MAVS or HA-MAVS-K7R.

O-Q SIRT5-deficient or WT H1299 cells (SIRT5 -/- or SIRT5 +/+) were infected with VSV-GFP viruses (MOI = 0.1) for 12 h, and viral infectivity was detected by fluorescence microscopy (O), western blot analysis (P), or flow cytometry analysis (n = 3) (Q).

Data information: UI, un-infected. The graphs represent fold-induction relative to the untreated cells. All data are presented as the mean values based on three independent experiments, and error bars indicate S.E.M. (unpaired two-tailed Student's t test).
SIRT5 impairs aggregation and activation of  MAVS through catalyzing lysine desuccinylation (2020)
Figure 5

Figure 5. Sirt5 deficiency potentiates the host innate antiviral immune response.

A-F qPCR analysis of Ifnβ (A), Ifnα1 (B), Ifnα4 (C), Ifit1 (D), Cxcl10 (E), and Cxcl11 (F) mRNA in WT or Sirt5-deficient MEF cells (Sirt5 +/+ or Sirt5 -/-) infected with or without SeV (SeV or UI) for 8h.

G WT or Sirt5-deficient MEF cells (Sirt5 +/+ or Sirt5 -/-) were infected with or without SeV for the indicated times, and the cell lysates were analyzed by immunoblotting for monomeric (Monomer) and dimeric (Dimer) Irf3 (top; native-PAGE), phosphorylated Irf3 (p-Irf3), total Irf3, Sirt5, and GAPDH (bottom).

H WT or Sirt5-deficient MEF cells (Sirt5 +/+ or Sirt5 -/-) were infected with or without SeV for 8 h, the interaction between endogenous Mavs and Rig-I was revealed by immunoprecipitation using anti-Mavs antibody, followed by immunoblotting with anti-Rig-I antibody.

I SDD-AGE analysis of Mavs aggregates in WT or Sirt5-deficient MEF cells (Sirt5 +/+ or Sirt5 -/-) infected with SeV for 12 h. SDS-PAGE immunoblotting was used as a loading control.

J-O qPCR analysis of Ifnβ (J), Ifnα1 (K), Ifnα4 (L), Ifit1 (M), Cxcl10 (N), and Cxcl11 (O) mRNA in WT or Sirt5-deficient MEF cells (Sirt5 +/+ or Sirt5 -/-) infected with or without VSV (VSV or UI) for 8 h.

P-R WT or Sirt5-deficient MEF cells (Sirt5 +/+ or Sirt5 -/-) were infected with or without VSV-GFP viruses for 12 h, and viral infectivity was detected by fluorescence microscopy (P), western blot analysis (Q), and flow cytometry analysis (n = 3) (R).

Data information: UI, un-infected. The graphs represent fold-induction relative to the untreated cells. All data are presented as the mean values based on three independent experiments, and error bars indicate S.E.M. (unpaired two-tailed Student's t test).
SIRT5 impairs aggregation and activation of  MAVS through catalyzing lysine desuccinylation (2020)
Figure 6

Figure 6. Sirt5 deficiency potentiates antiviral immune response in bone marrow-derived dendritic (BMDC) cells.

A-G qPCR analysis of Ifnβ (A), Ifnα1 (B), Ifnα4 (C), Ifit1 (D), Rig-I (E), Cxcl10 (F), and Cxcl11 (G) mRNA in WT or Sirt5-deficient BMDC cells (Sirt5 +/+ or Sirt5 -/-) infected with or without SeV (Sev or UI) for 8 h.

H-N qPCR analysis of Ifnβ (H), Ifnα1 (I), Ifnα4 (J), Ifit1 (K), Rig-I (L), Cxcl10 (M) and Cxcl11 (N) mRNA in WT or Sirt5-deficient BMDC cells (Sirt5 +/+ or Sirt5 -/-) infected with or without VSV viruses (VSV or UI) for 8 h.

O Confocal fluorescence microscopy image of replication in WT or Sirt5-deficient BMDC cells (Sirt5 +/+ or Sirt5 -/-) after infected with VSV-GFP viruses for 12 h (MOI = 1) (fluorescence, upper; bright-field, bottom). Scale bar = 75 µm.

Data information: UI, un-infected. The graphs represent fold-induction relative to the untreated cells. All data are presented as the mean values based on three independent experiments, and error bars indicate S.E.M. (unpaired two-tailed Student's t test).
SIRT5 impairs aggregation and activation of  MAVS through catalyzing lysine desuccinylation (2020)
Figure 7

Figure 7. Pivotal role of Sirt5 in Mavs desuccinylation and antiviral immune response in vivo.

A-B VSV infection increased Mavs succinylation in mouse livers (A) and lungs (B). Proteins in the livers (A) and lungs (B) of WT littermates injected intraperitoneally with (+) VSV (1 × 107 plaque-forming units (PFU) per mouse) or PBS control (-) (n = 3 per group) were detected with the indicated antibodies. Mavs succinylation was determined by anti-succ-K7-MAVS antibody. Relative succinylation level was quantified (right panel).

C-D Knockout of Sirt5 increased Mavs succinylation in mouse livers (C) and lungs (D) upon VSV infection. Proteins in the livers (C) and lungs (D) of Sirt5-deficient or WT littermates (Sirt5 -/- or Sirt5 +/+) injected intraperitoneally with VSV (1 × 107 plaque-forming units (PFU) per mouse)) (n = 3 per group) for 24 h were detected with the indicated antibodies. Mavs succinylation was determined by anti-succ-K7-MAVS antibody. Relative succinylation level was quantified (right panel).

E Survival (Kaplan-Meier curve) of WT mice (n = 7) and Sirt5-deficient mice (n = 7) injected intraperitoneally with a high dose of VSV (5 × 107 PFU per mouse) and monitored for 10 d.

F ELISA assay of Ifnβ in serum from the WT mice (n = 4) and Sirt5-deficient mice (n = 4) injected intraperitoneally with VSV (1 × 107 plaque-forming units (PFU) per mouse)) or PBS control for 24 h.

G Hematoxylin-and-eosin-stained images of lung sections from mice in F. Scale bar = 50 µm.

H-O qPCR analysis of Ifnβ (H), Ifnα1 (I), Ifit1 (J), Isg15 (K), Cxcl10 (L), Cxcl11 (M), Rig-I (N), and Irf7 (O) mRNA in the lungs of WT or Sirt5-deficient mice (Sirt5 +/+ or Sirt5 -/-) injected intraperitoneally with VSV (1 × 107 plaque-forming units (PFU) per mouse) or PBS control for 24 h.

P qPCR analysis of VSV mRNA in the lungs of WT or Sirt5-deficient mice (Sirt5 +/+ or Sirt5 -/-) injected intraperitoneally with VSV (1 × 107 plaque-forming units (PFU) per mouse) or PBS control for 24 h.

Data information: The graphs represent fold-induction relative to the untreated WT mice. PBS, phosphate buffer saline. All data are presented as the mean values based on three independent experiments, and error bars indicate S.E.M. (unpaired two-tailed Student's t test or two-way ANOVA analysis).

Expanded View Figure Legends
Muscle derived GDF15 drives diurnal anorexia and systemic metabolic remodeling during mitochondrial stress (2020)
Figure 1

Figure 1. Muscle mitochondrial stress promotes GDF15 as a myokine

A Schematic representation of HSA-Ucp1-transgenic (TG) mouse line as model of compromised skeletal muscle-specific mitochondrial OXPHOS capacity via respiratory uncoupling.

B Uncoupled (LEAK) mitochondrial respiration of soleus (SOL) and extensor digitorum longus (EDL) muscle of wild type (WT) vs. Ucp1-TG (TG) mice (WT n=9, TG n=5).

C Relative mRNA expression in quadriceps (Quad) of ISR components (n=8 per genotype).

D, E Representative immunoblots of ISR component eIF2α and quantification of phospho-eIF2α (p-eIF2aSer51) relative protein expression in quadriceps skeletal muscle (WT n=3, TG n=4).

F, G Multi-tissue transcriptomic profiling of Gdf15 gene expression. Heatmap is shown as raw ct expression values (n=4 per genotype). Quantification of Gdf15 mRNA expression in TG mice is shown as fold change compared to WT littermates (WT n=5, TG n=4).

H Skeletal muscle (Quad and Gastroc) GDF15 protein content normalized to total protein content (WT n=9, TG n=18).

I Ex vivo secretion of GDF15 from SOL and EDL muscle of WT vs. TG mice (n=6 per genotype) after 2h incubation normalized to muscle wet weight (mg).

J Mouse GDF15 plasma levels in post-absorptive state of male and female WT vs. TG mice at 20wks of age (male WT n=8, TG n=9; female WT n=5, TG n=6).

K Mouse GDF15 plasma levels in post-absorptive state of male WT vs. TG mice fed low-fat diet (LFD) or high-fat diet (HFD) at 24wks of age (LFD WT n=6, TG n=5; HFD WT n=5, TG n=5).

L Mouse GDF15 plasma levels in post-absorptive state of young (10wks) vs. old (95wks) male WT and TG mice (young WT n=10, TG n=13; old WT n=13, TG n=13).

M Representative scheme of in vitro study design in C2C12 myocytes.

N Gdf15 gene expression of differentiated C2C12 muscle cells treated with vehicle control (Ctrl) or chemical mitochondrial uncoupler (FCCP, 1µM vs. 5µM) for 5hrs (n=3 biological replicates)

Data information: Circulating plasma parameters are expressed as interleaved box and whiskers (min to max) plots, all other data are expressed as means + SEM; P-value calculated by unpaired t-test (A-L) or one-way ANOVA with Tukey's post-hoc test (N); *p<0.05, **p<0.01, ***p<0.001.
Muscle derived GDF15 drives diurnal anorexia and systemic metabolic remodeling during mitochondrial stress (2020)
Figure 2

Figure 2. GDF15-independent muscle wasting and mitochondrial integrated stress response

A Genotyping PCR panel of Gdf15 and HSA-Ucp1 loci shown for wild type (WT), Gdf15-KO (KO), Ucp1-TG (TG) and Ucp1-TGxGdf15-KO (TGxKO) mice.

B Relative mRNA expression in quadriceps (Quad) of Ucp1 and Gdf15 in 20wks male WT (n=8), TG (n=7) and TGxKO (n=6) mice.

C Plasma mGDF15 levels at 20wks of age (WT n=9, TG n=10, TGxKO n=9).

D Voluntary wheel running (VWR) activity shown hourly over 24-hrs at 15 wks of age (n=4 per genotype).

E Grip strength at 10 - 20wks of age (WT n=15, TG n=14, TGxKO n=11).

F Skeletal muscle mass relative to body lean mass of quadriceps (Quad) and gastrocnemius (Gastroc) from mice at 95wk of age (WT n=8, TG n=5, TGxKO n=5).

G, H Representative H&E histological staining of tibialis anterior (TA) muscle (G) (scale bars represent 50 µm), and cross-sectional area (CSA) of myofibers (H) at 95wks (WT n=12, TG n=8, TGxKO n=8).

I Representative traces of oxygen consumption rate (OCR) during substrate-uncoupler-inhibitor-titration (SUIT) protocol for mitochondrial respiratory capacity in permeabilized mouse soleus muscle fibers, PM (Pyruvate+Malate; LEAK respiration), ADP (OXPHOS capacity), Cyt c (cytochrome c, integrity of outer mt-membrane), G (glutamate;), S (succinate), U (Uncoupler, FCCP), Rot (rotenone), Ama (antimycin A; less than 2% residual oxygen consumption, ROX).

J-L Mitochondrial respiratory capacity (oxygen consumption rate, OCR) from oxidative soleus (SOL) (J) and glycolytic extensor digitorum longus (EDL) muscle fibers (K), and NetOXPHOS control ratio of 95wks old mice (L) (n=5per genotype).

M Quadriceps (Quad) relative mRNA expression of ISR components at 95wks of age (WT n=8, TG n=5, TGxKO n=5).

N Representative immunoblots of ISR component eIF2α in quadriceps (Quad) muscle of 95wks old mice.

O Gastrocnemius enzyme activity of NQO1 and GPX in 95wks old male mice (WT n=8, TG n=5, TGxKO n=5).

Data information: Circulating plasma parameters are expressed as interleaved box and whiskers (min to max) plots, all other data are expressed as means + SEM; P-value calculated by one-way ANOVA with Tukey's post-hoc test; *p<0.05, **p<0.01, ***p<0.001.
Muscle derived GDF15 drives diurnal anorexia and systemic metabolic remodeling during mitochondrial stress (2020)
Figure 3

Figure 3. Genetic ablation of GDF15 drives adiposity during mitochondrial dysfunction

A-C Body mass (A), body lean mass (B) and body fat mass (C) development.

D-F Body mass (D), body lean mass (E) and body fat mass (F) during aging at 20wks, 45wks and 95wks of age.

G, H Subcutaneous white adipose tissue (sWAT) (G) and epididymal white adipose tissue (eWAT) (H) mass development at 10wks, 20wks, 45wks and 95wks of age.

Data information: Data are from male wild type (WT), Ucp1-TG (TG), and Ucp1-TGxGdf15-KO (TGxKO) mice at 10wks (WT n=6, TG n=7, TGxKO n=5), 20wks (WT n=10, TG n=10, TGxKO n=10), 45wks (WT n=5, TG n=6, TGxKO n=7) and 95wks (WT n=8, TG n=5, TGxKO n=5). All data are expressed as means + SEM; P-value calculated by one-way ANOVA with Tukey's post-hoc test; *p<0.05, **p<0.01, ***p<0.001.
Muscle derived GDF15 drives diurnal anorexia and systemic metabolic remodeling during mitochondrial stress (2020)
Figure 4

Figure 4. Genetic ablation of GDF15 abolishes white adipose tissue browning and insulin sensitivity during mitochondrial dysfunction

A, B Representative H&E histological staining of sWAT at 20wks of age (scale bars represent 50 μm) (A) and relative mRNA expression profile in sWAT of male mice (WT n=7, TG n=8, TGxKO n=8) (B).

C, D Representative immunoblots (C) and quantification (D) of UCP1 protein expression in sWAT of male mice at 20wks of age (WT n=4, TG n=6, TGxKO n=4).

E Relative mRNA expression profile in eWAT (WT n=7, TG n=8, TGxKO n=8).

F Plasma FGF21 levels from male mice at 20wks of age (n=10 per genotype).

G Plasma Leptin levels from male mice at 20wks of age (n=5 per genotype).

H Post-absorptive plasma insulin levels at 20wks of age (WT n=8, TG n=9, TG n=9).

I-K Blood glucose (I) and insulin levels (J) with total area under the curve (AUC) of insulin (K) during oral glucose tolerance test (oGTT) at 17wks of age (WT n=8, TG n=11, TG n=11).

Data information: Data are from male wild type (WT), Ucp1-TG (TG), and Ucp1-TGxGdf15-KO (TGxKO) mice. Circulating plasma parameters are expressed as interleaved box and whiskers (min to max) plots, all other data are expressed as means + SEM; P-value calculated by one-way ANOVA with Tukey's post-hoc test; *p<0.05, **p<0.01, ***p<0.001.
Muscle derived GDF15 drives diurnal anorexia and systemic metabolic remodeling during mitochondrial stress (2020)
Figure 5

Figure 5. Muscle-derived GDF15 promotes diurnal anorexia during mitochondrial dysfunction

A-F Physical activity (A, B), energy expenditure (C, D), total assimilated energy (E, F) of male mice shown hourly over 24-hrs and as day and night time.

G Table showing mean values of assimilated energy (kJ) at day/night time and per 24-hrs.

H Energy balance calculated as delta of assimilated energy (AE) and energy expenditure (EE) and energy balance (J) per 24-hrs.

I Table showing mean values of energy balance (∆AE-EE, kJ) at day/night time and per 24-hrs.

J-L Respiratory quotient (RQ) shown hourly over 24-hrs (J), RQ amplitude (K) and metabolic flexibility via percentage relative cumulative frequency (PRCF) (L).

Data information: Data shown are from male wild type (WT), Ucp1-TG (TG), and Ucp1-TGxGdf15-KO (TGxKO) mice at 17-18wks of age (WT n=12, TG n=11, TG n=10) and shown as means + SEM; P-value calculated by one-way ANOVA with Tukey's post-hoc test; *p<0.05, **p<0.01, ***p<0.001.
Muscle derived GDF15 drives diurnal anorexia and systemic metabolic remodeling during mitochondrial stress (2020)
Figure 6

Figure 6. Diurnal variation of muscle-derived GDF15 during mitochondrial dysfunction

A Relative quadriceps (Quad) mRNA expression of the ISR components Atf4, Atf5, Atf6 and Chop (WT day n=9, WT night n=5, TG day n=20, TG night n=12).

B, C Representative immunoblots of ISR component eIF2α (B) and quantification of phospho-eIF2α (p-eIF2aSer51) relative protein expression (C) in skeletal muscle (Gastroc, WT day n=4, WT night n=5, TG day n=9, TG night n=9).

D, E Relative mRNA expression of Rev-erba (D) and Gdf15 (E) in quadriceps, liver and eWAT (WT day n=9, WT night n=5, TG day n=20, TG night n=12).

F Skeletal muscle (Quad) GDF15 protein content normalized to total protein content (WT day n=4, WT night n=5, TG day n=14, TG night n=12)

G Circulating GDF15 plasma levels (WT day n=9, WT night n=5, TG day n=18, TG night n=12).

H Graphical summary showing muscle mitochondrial stress induced secretion and diurnal variation of GDF15 to drive a daytime-restricted anorexia, affecting in turn metabolic flexibility, adiposity, WAT browning and systemic insulin sensitivity. Parts of this cartoon were created using Servier Medical Art (http://smart.servier.com).

Data information: Data shown are from male wild type (WT) vs. Ucp1-TG (TG) mice sacrificed at day (10am) or night (10pm). Circulating plasma parameters are expressed as interleaved box and whiskers (min to max) plots, all other data are expressed as means + SEM; P-value calculated by one-way ANOVA with Tukey's post-hoc test; *p<0.05, **p<0.01, ***p<0.001.
Single-cell transcriptomics reveals a senescence-associated IL-6/CCR6 axis driving radiodermatitis (2022)
Figure 2

Figure 2. IL-6 signaling deficiency reduces alopecia and radiodermatitis in irradiated mice.

Relative expression and co-localization of IL6 mRNA and IL-6 Signaling Scoring in integrated skin cells by blended UMAPs.

Quantification by real-time qPCR analysis of Il6 mRNA in the skin of naïve and irradiated (15Gy) WT mice at indicated times, (n=3-6).

Quantification of ventral hair loss area 21 days post-IR (15Gy) in WT and IL-6-/- mice, (n=11).

Photographic images showing hair loss (area demarked by yellow dashed line) 21 days post-IR (15 Gy) in wild type (WT) and IL-6-/- mice.

Histochemical (H&E) and immunostaining (red) showing skin morphology, and T cell (CD3+) and neutrophil (Ly6b+) infiltration in WT or IL-6-/- mice before (naïve), and 14 days (H&E, Ly6b+), or 21 days (CD3+) post-IR. Infiltrating CD3+ T cells (yellow arrows) within degenerating hair follicles (HF; dashed lines) are indicated. Scale bars, 100 μm.

Quantification of epidermal thickness at 14 days post-IR (n = 5-8).

Kinetics of neutrophil (Ly6b+) (n = 3-9) and T cell (CD3+) (n = 3) infiltration.

Quantification by real-time qPCR analysis of Tnfa, Ccl3, and Ifng mRNAs in whole skin of WT and IL-6-/- mice at indicated times post-IR (15Gy), (n = 5-7).

Data information: Data represent mean ± SD (B,C,F,H) or mean ± SD (G). *P<0.05, **P<0.01, **P<0.01, ****P<0.001 by one-way ANOVA with Dunnet's multiple comparisons test (B), two-tailed Mann-Whitney test (C,F,H), or two-way ANOVA with Bonferroni's posthoc multiple comparisons test (G); five independent experiments. (See also Fig EV3 and Appendix Figs S7 and S9.)
Single-cell transcriptomics reveals a senescence-associated IL-6/CCR6 axis driving radiodermatitis (2022)
Figure 3

Figure 3. Blockade of STAT3 activation reduces IRIAD.

Western blot analysis shows phosphorylated STAT3 (p-Stat3), Akt (p-Akt)), Erk1 and Erk2 (p-Erk1/2)), and β-Actin in the skin of WT, IL-6-/-, and IL-1R-/- mice at indicated times (days) post-IR (15Gy).

Histochemical (H&E) and immunostaining (red) staining of T cells (CD3+), neutrophils (Ly6b+) and phosphorylated Stat3 (p-Stat3) (red nuclear staining, black arrows) in skin thin sections prepared at 14 days post-IR from mice treated by daily topical application of acquosum (control), ruxolitinib or tofacitinib following irradiation (15 Gy). Scale bar, 100 μm. (CD3 and Ly6b immunostaining images from Tofacitinib treated mice represent seperate analyses performed on serial thin sections.)

Quantification of epidermal thickness, T cells (CD3+) and neutrophils (Ly6b+), and p-STAT3+ cells in (B), (n = 4).

Photographic images showing hair loss in mice at 14 days post-IR (15 Gy) following treatment by daily topical application of acquosum (carrier control) or JAK inhibitors, ruxolitinib or tofacitinib. Chest and neck hairs of mice in all groups were clipped prior to irradiation to enhance drug absorption.

Quantification of the area of ventral hair loss (in pixels) in mice in (D), (n = 4).

Data information: Data represent mean ± SD. *P<0.05, **P<0.01, by two-tailed Student's t test; two independent experiments. (See also Appendix Fig S8.)
Single-cell transcriptomics reveals a senescence-associated IL-6/CCR6 axis driving radiodermatitis (2022)
Figure 4

Figure 4. IL-1R deficiency reduces irradiation-induced alopecia and radiodermatitis in mice.

UMAP plots depicting relative expression and localization of Il1a and Il1b mRNAs in irradiated and naïve skin-derived cell clusters.

Photographic images showing hair loss 14 days post-IR (15 Gy) in WT and IL-1R-/- mice.

Quantification of ventral hair loss area 14 days post-IR (15Gy) in WT and IL-1R-/- mice, (n = 4).

Histochemical (H&E) and immunostaining (red) showing morphology and neutrophil (Ly6b) infiltration in the skin of mice in (B). Scale bar, 50 μm.

Quantification of epidermal thickness, neutrophils (Ly6b) and T cells (CD3+) in specimens in (D), (n = 4).

Data information: Data represent mean ± SD. *P<0.05, by two-tailed Mann-Whitney test (C) or Student's t test (E); five independent experiments. (See also Appendix Fig S9.)