Figure 1THP - 1 derived macrophages were infected with the wildtype ( Wt ) and ΔF2 H5N1 ( VN ) virus at MOI 10 . Viral protein expression for PB1 - F2 ( A ) was detected by immunoblotting at 24 h post - infection ( hpi ) Equal loading was confirmed by detection of beta actin . B THP - 1 derived macrophages were infected with the wildtype ( Wt ) and ΔF2 H5N1 ( VN ) virus at MOI 10 . Viral protein expression for NP ( B ) was detected by immunoblotting at indicated time points , respectively . Equal loading was confirmed by detection of beta actin . ( C ) THP - 1 derived macrophages were infected with the wildtype ( Wt ) and ΔF2 H5N1 ( VN ) virus at MOI 10 . Levels of viral M1 mRNA expression were detected by RT q ‑ PCR at 16 hpi . The mean ± standard deviation of three independent biological samples is shown . Statistical analysis was performed by students t - test . ( D ) THP - 1 derived macrophages were infected with VN and PR8 Wt and ΔF2 strains for 24 h at MOI 10 . Levels of IL - 1β were quantified by ELISA from the supernatants . The mean ± standard deviation of seven independent experiments is shown . Statistical analysis was performed by one - way ANOVA , p - values are indicated . ( E ) THP - 1 control and knockout cells for NLRP3 were mock treated or infected with the VN Wt and ΔF2 strains for 24 h at MOI 10 and IL - 1β levels were assessed by ELISA from the supernatants . The mean ± standard deviation of five independent experiments is shown . Statistical analysis was performed by one - way ANOVA , p - values are indicated . ( F ) Immunoblot detection for NLRP3 on control and NLRP3 knockout THP - 1 cells . Equal loading was confirmed by specific blotting against beta actin . Representative blot of three independent repeats is shown . ( G ) Human primary macrophages differentiated from PBMCs from two healthy donors were infected with H5N1 Wt and ΔF2 for 5 h at MOI 10 . Levels of IL - 1β were measured by ELISA in technical replicates . The mean of two independent experiments is shown . Statistical analysis was performed by one - way ANOVA , p - values are indicated . ( H ) THP - 1 derived macrophages were infected with a human H3N2 IAV strain ( Wyo ) Wt and ΔF2 for 24 h at MOI 4 . Levels of IL - 1β were measured by ELISA from the supernatants . The mean ± standard deviation of four independent experiments is shown . Statistical analysis was performed by one - way ANOVA , p - values are indicated . ( I ) Detection of viral matrix protein M1 by immunoblotting in cells infected with the H3N2 Wt and ΔF2 strains . ( J ) Human primary macrophages isolated and differentiated from PBMCs from three healthy donors were infected with H3N2 Wt and ΔF2 for 24 h at MOI 4 . Levels of IL - 1β were measured by ELISA . The mean ± standard deviation of three independent experiments is shown . Statistical analysis was performed by one - way ANOVA , p - values are indicated . Primary human alveolar macrophages from BALF of five pediatric patients ( patients are color coded ) were infected with the human H3N2 strain ( Wyo ) Wt and ΔF2 for 16 h at MOI 1 . ( K ) Levels of IL - 1β were measured by ELISA from the supernatants . The mean ± standard deviation is shown . Primary human alveolar macrophages from BALF of five pediatric patients ( patients are color coded ) were infected with the human H3N2 strain ( Wyo ) Wt and ΔF2 for 16 h at MOI 1 . Levels of viral M1 mRNA ( L ) , Mx1 mRNA ( M ) and IP10 mRNA ( N ) expression were detected by RT q ‑ PCR . The mean ± standard deviation for cells from four patients are shown . Individiual patients are color coded .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1THP-1 derived macrophages were infected with the wildtype (Wt) and ΔF2 H5N1 (VN) virus at MOI 10. Viral protein expression for PB1-F2 (A) was detected by immunoblotting at 24 h post-infection (hpi) Equal loading was confirmed by detection of beta actin.B THP-1 derived macrophages were infected with the wildtype (Wt) and ΔF2 H5N1 (VN) virus at MOI 10. Viral protein expression for NP (B) was detected by immunoblotting at indicated time points, respectively. Equal loading was confirmed by detection of beta actin.(C) THP-1 derived macrophages were infected with the wildtype (Wt) and ΔF2 H5N1 (VN) virus at MOI 10. Levels of viral M1 mRNA expression were detected by RT q‑PCR at 16 hpi. The mean ± standard deviation of three independent biological samples is shown. Statistical analysis was performed by students t-test.(D) THP-1 derived macrophages were infected with VN and PR8 Wt and ΔF2 strains for 24 h at MOI 10. Levels of IL-1β were quantified by ELISA from the supernatants. The mean ± standard deviation of seven independent experiments is shown. Statistical analysis was performed by one-way ANOVA, p-values are indicated.(E) THP-1 control and knockout cells for NLRP3 were mock treated or infected with the VN Wt and ΔF2 strains for 24 h at MOI 10 and IL-1β levels were assessed by ELISA from the supernatants. The mean ± standard deviation of five independent experiments is shown. Statistical analysis was performed by one-way ANOVA, p-values are indicated.(F) Immunoblot detection for NLRP3 on control and NLRP3 knockout THP-1 cells. Equal loading was confirmed by specific blotting against beta actin. Representative blot of three independent repeats is shown.(G) Human primary macrophages differentiated from PBMCs from two healthy donors were infected with H5N1 Wt and ΔF2 for 5 h at MOI 10. Levels of IL-1β were measured by ELISA in technical replicates. The mean of two independent experiments is shown. Statistical analysis was performed by one-way ANOVA, p-values are indicated.(H) THP-1 derived macrophages were infected with a human H3N2 IAV strain (Wyo) Wt and ΔF2 for 24 h at MOI 4. Levels of IL-1β were measured by ELISA from the supernatants. The mean ± standard deviation of four independent experiments is shown. Statistical analysis was performed by one-way ANOVA, p-values are indicated.(I) Detection of viral matrix protein M1 by immunoblotting in cells infected with the H3N2 Wt and ΔF2 strains.(J) Human primary macrophages isolated and differentiated from PBMCs from three healthy donors were infected with H3N2 Wt and ΔF2 for 24 h at MOI 4. Levels of IL-1β were measured by ELISA. The mean ± standard deviation of three independent experiments is shown. Statistical analysis was performed by one-way ANOVA, p-values are indicated.Primary human alveolar macrophages from BALF of five pediatric patients (patients are color coded) were infected with the human H3N2 strain (Wyo) Wt and ΔF2 for 16 h at MOI 1. (K) Levels of IL-1β were measured by ELISA from the supernatants. The mean ± standard deviation is shown.Primary human alveolar macrophages from BALF of five pediatric patients (patients are color coded) were infected with the human H3N2 strain (Wyo) Wt and ΔF2 for 16 h at MOI 1. Levels of viral M1 mRNA (L), Mx1 mRNA (M) and IP10 mRNA (N) expression were detected by RT q‑PCR. The mean ± standard deviation for cells from four patients are shown. Individiual patients are color coded.


Figure 2 ( A - D ) THP - 1 cells were infected with the wildtype ( Wt ) and ΔF2 H5N1 ( VN ) virus at MOI 10 . Flow cytometry results for annexin V single positive ( A and C ) and annexin V and zombie dye double positive ( B and D ) cells . Percentage of positive cells are depicted at 12 hpi or 24hpi . ( E and F ) THP - 1 cells were infected with the wildtype ( Wt ) and ΔF2 H5N1 ( VN ) virus at MOI 10 . LDH release measured from supernatants of mock treated or infected cells at 12 hpi ( E ) or 24hpi ( F ) . G THP - 1 Caspase 1 - / - cells were infected with the wildtype ( Wt ) and ΔF2 H5N1 ( VN ) virus at MOI 10 . LDH release measured from supernatants of mock treated or infected cells at 12 hpi or 24hpi . n = 3 for 12hpi , n = 5 for 24hpi . Medians are indicated , statistical analysis was performed by one - way ANOVA .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2(A-D) THP-1 cells were infected with the wildtype (Wt) and ΔF2 H5N1 (VN) virus at MOI 10. Flow cytometry results for annexin V single positive (A and C) and annexin V and zombie dye double positive (B and D) cells. Percentage of positive cells are depicted at 12 hpi or 24hpi.(E and F) THP-1 cells were infected with the wildtype (Wt) and ΔF2 H5N1 (VN) virus at MOI 10. LDH release measured from supernatants of mock treated or infected cells at 12 hpi (E) or 24hpi (F).G THP-1 Caspase 1 -/- cells were infected with the wildtype (Wt) and ΔF2 H5N1 (VN) virus at MOI 10. LDH release measured from supernatants of mock treated or infected cells at 12 hpi or 24hpi. n=3 for 12hpi, n=5 for 24hpi. Medians are indicated, statistical analysis was performed by one-way ANOVA.


Figure 3 ( A ) THP - 1 cells infected with VN Wt and ΔF2 virus at a MOI 10 for 24 h or treated with 6 . 7 μM of nigericin ( nig ) for 45 min were analyzed by immunoblotting . Detection of the full - length gasdermin D . Equal loading was confirmed by beta actin . One representative blot of five independent repeats is shown . ( B ) Quantification of band intensity for the gasdermin D full - length isoform . The mean ± standard deviation five independent experiments is shown . Statistical analysis was performed by the paired two ‑ tailed Student ' s t test . , The asterisk ( * ) marks a significant difference when compared to the mock group * * p < 0 . 01 . ( C ) THP - 1 cells infected with VN Wt and ΔF2 virus at a MOI 10 for 24 h or treated with 6 . 7 μM of nigericin ( nig ) for 45 min were analyzed by immunoblotting . Detection of cleaved gasdermin D N - terminal and C - terminal isoforms by immunoblot in supernatants ; one representative experiment of three independent repeats is shown . ( D ) Quantification of band intensity for N - terminal gasdermin D isoform band . Three independent experiments are shown . Statistical analysis was performed by the paired two ‑ tailed Student ' s t test . , * p < 0 . 05 . ( E ) Quantification of band intensity for C - terminal gasdermin D isoform band . Three independent experiments are shown . Statistical analysis was performed by the paired two ‑ tailed Student ' s t test . , * p < 0 . 05 . ( F ) THP - 1 cells infected with VN Wt and ΔF2 virus at a MOI 10 for 24 h or treated with 6 . 7 μM of nigericin ( nig ) for 45 min were analyzed by immunoblotting . Pro - caspase - 1 and active caspase - 1 were detected by immunoblot in cell lysates ( cell , upper panels ) and supernatants ( sup , lower panels ) from the same experiment . One representative experiment of three independent repeats is shown . ( G ) Quantification of band intensity for active caspase - 1 band . Five independent experiments are shown . Statistical analysis was performed by the paired two ‑ tailed Student ' s t test . * p < 0 . 05 . ( H ) Levels of total pro - caspase - 1 were quantified on pellets and respective supernatants . Three independent experiments are shown . Statistical analysis was performed by the paired two ‑ tailed Student ' s t test .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3(A) THP-1 cells infected with VN Wt and ΔF2 virus at a MOI 10 for 24 h or treated with 6.7 μM of nigericin (nig) for 45 min were analyzed by immunoblotting. Detection of the full-length gasdermin D. Equal loading was confirmed by beta actin. One representative blot of five independent repeats is shown. (B) Quantification of band intensity for the gasdermin D full-length isoform. The mean ± standard deviation five independent experiments is shown. Statistical analysis was performed by the paired two‑tailed Student's t test., The asterisk (*) marks a significant difference when compared to the mock group ** p < 0.01. (C) THP-1 cells infected with VN Wt and ΔF2 virus at a MOI 10 for 24 h or treated with 6.7 μM of nigericin (nig) for 45 min were analyzed by immunoblotting. Detection of cleaved gasdermin D N-terminal and C-terminal isoforms by immunoblot in supernatants; one representative experiment of three independent repeats is shown. (D) Quantification of band intensity for N-terminal gasdermin D isoform band. Three independent experiments are shown. Statistical analysis was performed by the paired two‑tailed Student's t test., * p < 0.05. (E) Quantification of band intensity for C-terminal gasdermin D isoform band. Three independent experiments are shown. Statistical analysis was performed by the paired two‑tailed Student's t test., * p < 0.05. (F) THP-1 cells infected with VN Wt and ΔF2 virus at a MOI 10 for 24 h or treated with 6.7 μM of nigericin (nig) for 45 min were analyzed by immunoblotting. Pro-caspase-1 and active caspase-1 were detected by immunoblot in cell lysates (cell, upper panels) and supernatants (sup, lower panels) from the same experiment. One representative experiment of three independent repeats is shown. (G) Quantification of band intensity for active caspase-1 band. Five independent experiments are shown. Statistical analysis was performed by the paired two‑tailed Student's t test. * p < 0.05. (H) Levels of total pro-caspase-1 were quantified on pellets and respective supernatants. Three independent experiments are shown. Statistical analysis was performed by the paired two‑tailed Student's t test.


Figure 4 ( A ) Western blot analysis of whole cell lysate ( input ) and Flag - specific immunoprecipitation ( IP ) from 293T cells co - transfected with expression plasmids encoding V5 - NLRP3 and Flag ZsGreen - PB1 - F2 from indicated viral strains for 24 h Representative blot of three independent repeats is shown . ( B ) Quantification of the NLRP3 IP band was normalized to the respective NLRP3 input band ( n = 3 independent biological replicates , normalized to the respective input ) . Mean values + - SD are indicated . ( E ) 293T cells co transfected with expression plasmids encoding different truncation mutants for Flag Zsgreen - PB1 - F2 from VN ( H5N1 ) and V5 - NLRP3 were processed as described in ( A ) . Representative blot of three independent repeats is shown .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4(A) Western blot analysis of whole cell lysate (input) and Flag-specific immunoprecipitation (IP) from 293T cells co-transfected with expression plasmids encoding V5-NLRP3 and Flag ZsGreen-PB1-F2 from indicated viral strains for 24 h Representative blot of three independent repeats is shown. (B) Quantification of the NLRP3 IP band was normalized to the respective NLRP3 input band (n=3 independent biological replicates, normalized to the respective input). Mean values +- SD are indicated. (E) 293T cells co transfected with expression plasmids encoding different truncation mutants for Flag Zsgreen-PB1-F2 from VN (H5N1) and V5-NLRP3 were processed as described in (A). Representative blot of three independent repeats is shown.


Figure 5 ( B ) 293T cells were co - transfected with expression plasmids encoding for the indicated V5 - tagged domains of NLRP3 for 24h . Whole cell lysate ( left blot ) and flag specific IP ( right blot ) were analyzed by immunoblotting . Equal loading was confirmed by blotting against tubulin . Representative blots of two independent experiments are shown . ( C ) 293T cells were co ‑ transfected with expression plasmids encoding Flag - NLRP3 , V5 ‑ PB1 - F2 and V5 ‑ ASC for 24h . Whole cell lysate and flag specific IP were analyzed by immunoblotting for indicated antigens . Equal loading of whole cell lysate was assured by blotting against beta actin . Representative blot of two independent repeats is shown . ( D ) 293T cells were co - transfected with expression plasmids encoding for Flag - NLRP3 , PB1 - F2 or NEK7 or combinations thereof as indicated . Whole cell lysate and Flag IP were analyzed by immunoblotting . Equal loading was confirmed by blotting against beta actin . Representative blots of three independent experiments are shown . Right panel : Quantification of IP bands ' intensity relative to their input . The mean ± standard deviation of three independent experiments is shown . Statistical analysis was performed by paired two ‑ tailed Student ' s t test ( * p < 0 . 05 ) . ( E ) As in D : 293T cells were co - transfected with expression plasmids encoding for Flag - NLRP3 - LRR , PB1 - F2 or NEK7 or combinations thereof as indicated . Whole cell lysate and NLRP3 - LRR IP were analyzed by immunoblotting . Equal loading was confirmed by blotting against beta actin . Representative blots of three independent experiments are shown . ( F ) Quantification of IP bands ' intensity relative to their input . The mean ± standard deviation of three independent experiments is shown . Statistical analysis was performed by paired two ‑ tailed Student ' s t test ( * p < 0 . 05 ) . G ) As in D and E : 293T cells were co - transfected with expression plasmids encoding for Flag - NLRP3 - LRR , PB1 - F2 or V5 - NLRP3 - Pyrin or combinations thereof as indicated . Whole cell lysate and NLRP3 - LRR IP were analyzed by immunoblotting . Equal loading was confirmed by blotting against tubulin . Representative blots of three independent experiments are shown .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5(B) 293T cells were co-transfected with expression plasmids encoding for the indicated V5-tagged domains of NLRP3 for 24h. Whole cell lysate (left blot) and flag specific IP (right blot) were analyzed by immunoblotting. Equal loading was confirmed by blotting against tubulin. Representative blots of two independent experiments are shown.(C) 293T cells were co‑transfected with expression plasmids encoding Flag-NLRP3, V5‑PB1-F2 and V5‑ASC for 24h. Whole cell lysate and flag specific IP were analyzed by immunoblotting for indicated antigens. Equal loading of whole cell lysate was assured by blotting against beta actin. Representative blot of two independent repeats is shown.(D) 293T cells were co-transfected with expression plasmids encoding for Flag-NLRP3, PB1-F2 or NEK7 or combinations thereof as indicated. Whole cell lysate and Flag IP were analyzed by immunoblotting. Equal loading was confirmed by blotting against beta actin. Representative blots of three independent experiments are shown. Right panel: Quantification of IP bands' intensity relative to their input. The mean ± standard deviation of three independent experiments is shown. Statistical analysis was performed by paired two‑tailed Student's t test (*p< 0.05).(E) As in D: 293T cells were co-transfected with expression plasmids encoding for Flag-NLRP3-LRR, PB1-F2 or NEK7 or combinations thereof as indicated. Whole cell lysate and NLRP3-LRR IP were analyzed by immunoblotting. Equal loading was confirmed by blotting against beta actin. Representative blots of three independent experiments are shown. (F) Quantification of IP bands' intensity relative to their input. The mean ± standard deviation of three independent experiments is shown. Statistical analysis was performed by paired two‑tailed Student's t test (*p< 0.05). G) As in D and E: 293T cells were co-transfected with expression plasmids encoding for Flag-NLRP3-LRR, PB1-F2 or V5-NLRP3-Pyrin or combinations thereof as indicated. Whole cell lysate and NLRP3-LRR IP were analyzed by immunoblotting. Equal loading was confirmed by blotting against tubulin. Representative blots of three independent experiments are shown.


Figure 1 ( C ) 293T cells were transfected with empty vector ( EV ) , a vector encoding FLAG - tagged full length CYLD ( wt ) , or constructs encoding FLAG - tagged CYLD fragments 1 - 581 ( F1 ) lacking the C - terminus , or 581 - 956 ( F2 ) lacking the N - terminus , as indicated , followed by FLAG - IP . The blot was probed with antibodies recognizing SPATA2 , FLAG and tubulin . Endogenous SPATA2 was co - immunoprecipitated to comparable levels by CYLD and the C - terminal CYLD protein fragment , containing the USP domain . ( D ) CYLD - / - MEF were infected with retrovirus encoding FLAG - CYLD as indicated and CYLD was purified by FLAG - IP . The blot was probed with antibodies recognizing CYLD , SPATA2 and actin . ( E ) 293T cells were transfected with empty vector ( EV ) , a vector encoding FLAG - tagged full length SPATA2 or a construct encoding the FLAG - tagged N - terminal part of SPATA2 ( NT ) , as indicated , followed by FLAG - IP . The blot was probed with antibodies recognizing CYLD , SPATA2 and tubulin . M , marker lane .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1(C) 293T cells were transfected with empty vector (EV), a vector encoding FLAG-tagged full length CYLD (wt), or constructs encoding FLAG-tagged CYLD fragments 1-581 (F1) lacking the C-terminus, or 581-956 (F2) lacking the N-terminus, as indicated, followed by FLAG-IP. The blot was probed with antibodies recognizing SPATA2, FLAG and tubulin. Endogenous SPATA2 was co-immunoprecipitated to comparable levels by CYLD and the C-terminal CYLD protein fragment, containing the USP domain.(D) CYLD-/- MEF were infected with retrovirus encoding FLAG-CYLD as indicated and CYLD was purified by FLAG-IP. The blot was probed with antibodies recognizing CYLD, SPATA2 and actin.(E) 293T cells were transfected with empty vector (EV), a vector encoding FLAG-tagged full length SPATA2 or a construct encoding the FLAG-tagged N-terminal part of SPATA2 (NT), as indicated, followed by FLAG-IP. The blot was probed with antibodies recognizing CYLD, SPATA2 and tubulin. M, marker lane.


Figure 2 ( A ) 293T cells were transfected with a vector encoding V5 - tagged full length SPATA2 , along with empty vector ( EV ) a vector encoding FLAG - tagged HOIP wild - type ( wt ) or a vector encoding a FLAG - tagged PUB domain mutant of HOIP ( N102A ) . After FLAG - IP , the blot was probed with antibodies recognizing HOIP , CYLD , V5 and actin . ( B ) 293T cells were transfected with empty vector or a vector encoding FLAG - tagged HOIP along with either V5 - tagged SPATA2 ( wt ) or a V5 - tagged PIM mutant ( PIMmut ) of SPATA2 . After V5 - IP , the blot was probed with antibodies recognizing FLAG , CYLD , V5 and tubulin . ( C ) 293T cells were transfected with a vector encoding FLAG - tagged HOIP and increasing amounts of V5 - tagged SPATA2 . After FLAG - IP , the blot was sequentially probed with antibodies recognizing OTULIN , HOIP , V5 and tubulin . ( D ) 293T cells were transfected with empty vector , FLAG - SPATA2 or FLAG - HOIP . After FLAG - IP , the blot was probed with antibodies recognizing OTULIN , CYLD , FLAG and actin . ( E ) 293T cells were transfected with empty vector , V5 - tagged SPATA2 ( wt ) or a V5 - tagged PUB domain mutant PUBmut of SPATA2 . After V5 - IP , the blot was probed with antibodies recognizing CYLD , FLAG , V5 and tubulin . ( F ) A549 cells were treated with FLAG - TNF ( 2µg / ml ) for the indicated time . The purified TNF - RSC was sequentially probed with antibodies recognizing SPATA2 , CYLD , RIPK1 , TRADD , SHARPIN , TNF and tubulin as indicated .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2(A) 293T cells were transfected with a vector encoding V5-tagged full length SPATA2, along with empty vector (EV) a vector encoding FLAG-tagged HOIP wild-type (wt) or a vector encoding a FLAG-tagged PUB domain mutant of HOIP (N102A). After FLAG-IP, the blot was probed with antibodies recognizing HOIP, CYLD, V5 and actin.(B) 293T cells were transfected with empty vector or a vector encoding FLAG-tagged HOIP along with either V5-tagged SPATA2 (wt) or a V5-tagged PIM mutant (PIMmut) of SPATA2. After V5-IP, the blot was probed with antibodies recognizing FLAG, CYLD, V5 and tubulin.(C) 293T cells were transfected with a vector encoding FLAG-tagged HOIP and increasing amounts of V5-tagged SPATA2. After FLAG-IP, the blot was sequentially probed with antibodies recognizing OTULIN, HOIP, V5 and tubulin.(D) 293T cells were transfected with empty vector, FLAG-SPATA2 or FLAG-HOIP. After FLAG-IP, the blot was probed with antibodies recognizing OTULIN, CYLD, FLAG and actin.(E) 293T cells were transfected with empty vector, V5-tagged SPATA2 (wt) or a V5-tagged PUB domain mutant PUBmut of SPATA2. After V5-IP, the blot was probed with antibodies recognizing CYLD, FLAG, V5 and tubulin.(F) A549 cells were treated with FLAG-TNF (2µg/ml) for the indicated time. The purified TNF-RSC was sequentially probed with antibodies recognizing SPATA2, CYLD, RIPK1, TRADD, SHARPIN, TNF and tubulin as indicated.


Figure 3 ( A ) A549 cells were infected to express a CRISPR / Cas9 system targeting luciferase ( as control , indicated wt ) or the spata2 gene ( ko ) . Mixed cell cultures were treated with FLAG - huTNF ( 2µg / ml ) for the indicated time . The purified TNF - RSC was sequentially probed with antibodies recognizing SPATA2 , CYLD , HOIP , SHARPIN , TRADD , TNF and tubulin as indicated . ( B ) MEF were infected to express a CRISPR / Cas9 system targeting luciferase ( wt ) or the spata2 gene ( ko ) . Single clones were generated , which were treated with FLAG - TNF ( 2µg / ml ) for the indicated time . The purified TNF - RSC was sequentially probed with antibodies recognizing M1 - linked ubiquitin , CYLD , RIPK1 , and tubulin as indicated .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3(A) A549 cells were infected to express a CRISPR/Cas9 system targeting luciferase (as control, indicated wt) or the spata2 gene (ko). Mixed cell cultures were treated with FLAG-huTNF (2µg/ml) for the indicated time. The purified TNF-RSC was sequentially probed with antibodies recognizing SPATA2, CYLD, HOIP, SHARPIN, TRADD, TNF and tubulin as indicated.(B) MEF were infected to express a CRISPR/Cas9 system targeting luciferase (wt) or the spata2 gene (ko). Single clones were generated, which were treated with FLAG-TNF (2µg/ml) for the indicated time. The purified TNF-RSC was sequentially probed with antibodies recognizing M1-linked ubiquitin, CYLD, RIPK1, and tubulin as indicated.


Figure 4FLAG - tagged CYLD or catalytically inactive FLAG - CYLD ( C598A ) , respectively , were stably expressed in CYLD - / - MEF , followed by infection with control retrovirus , or retrovirus encoding SPATA2 , and purified by FLAG - IP . Purified CYLD or the inactive mutant were added to recombinant di - ubiquitin for 0 , 20 or 40 min at 37 ° C as indicated . After electrophoresis , the gel was cut and the lower part was silver stained to visualize mono - and di - ubiquitin , while the upper part was transferred to a membrane and probed for FLAG and V5 . In ( A ) , the experiment was done with M1 - linked di - ubiquitin substrate , in ( B ) K63 - linked di - ubiquitin substrate was subjected to the assay , and in ( C ) K48 - linked di - ubiquitin substrate was analyzed .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4FLAG-tagged CYLD or catalytically inactive FLAG-CYLD (C598A), respectively, were stably expressed in CYLD-/-MEF, followed by infection with control retrovirus, or retrovirus encoding SPATA2, and purified by FLAG-IP. Purified CYLD or the inactive mutant were added to recombinant di-ubiquitin for 0, 20 or 40 min at 37°C as indicated. After electrophoresis, the gel was cut and the lower part was silver stained to visualize mono-and di-ubiquitin, while the upper part was transferred to a membrane and probed for FLAG and V5. In (A), the experiment was done with M1-linked di-ubiquitin substrate, in (B) K63-linked di-ubiquitin substrate was subjected to the assay, and in (C) K48-linked di-ubiquitin substrate was analyzed.


Figure 5 ( A ) MEF expressing CRISPR / Cas9 targeting luciferase ( wt ) or single clones generated from MEF expressing CRISPR / Cas9 targeting the spata2 gene ( ko ) were treated with mTNF ( 10 ng / ml ) as indicated . The blots were probed with antibodies recognizing P - IkBαIkBα , P - p38 , p38 , P - JNK , JNK , P - p65 , p65 and tubulin . ( B ) 15P - 1 Sertoli cells expressing CRISPR / Cas9 targeting luciferase ( as control ) or single clones generated from 15P - 1 Sertoli cells expressing CRISPR / Cas9 targeting the spata2 gene were treated with mTNF ( 10 ng / ml ) as indicated . The blots were probed with antibodies recognizing P - IkBα , IkBα , P - p38 , p38 , P - JNK , JNK , P - p65 , p65 and tubulin . ( C ) TAK1 - / - MEF were infected to express a CRISPR / Cas9 system targeting luciferase or the spata2 gene , from which single clones were generated . The cells were treated with mTNF ( 10 ng / ml ) or mTNF along with Necrostatin - 1 ( Nec - 1 ) for 1 - 3 h as indicated . The blot was sequentially probed with antibodies recognizing PARP , cleaved caspase - 3 and tubulin . ( D ) . The same cells as used in ( C ) were treated as before with TNF ( 10 ng / ml ) or TNF along with Necrostatin - 1 ( Nec - 1 ) for one and 2 h as indicated . Caspase activity was determined using the fluorogenic substrate DEVD - AMC . ( E ) Cells from the experiment as shown in ( D ) were analyzed for apoptosis by Annexin V staining after 2 h . In ( D ) and ( E ) , a representative experiment is shown with error bars referring to technical replicates . ( F ) Cells as described before were treated with mTNF ( 10 ng / ml ) for the indicated time and lysates were subjected to immunoprecipitation with an anti - FADD antibody . The immunoprecipitates were subjected to Western blotting and probed for RIPK1 , FADD and actin .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5(A) MEF expressing CRISPR/Cas9 targeting luciferase (wt) or single clones generated from MEF expressing CRISPR/Cas9 targeting the spata2 gene (ko) were treated with mTNF (10 ng/ml) as indicated. The blots were probed with antibodies recognizing P-IkBαIkBα, P-p38, p38, P-JNK, JNK, P-p65, p65 and tubulin.(B) 15P-1 Sertoli cells expressing CRISPR/Cas9 targeting luciferase (as control) or single clones generated from 15P-1 Sertoli cells expressing CRISPR/Cas9 targeting the spata2 gene were treated with mTNF (10 ng/ml) as indicated. The blots were probed with antibodies recognizing P-IkBα, IkBα, P-p38, p38, P-JNK, JNK, P-p65, p65 and tubulin.(C) TAK1-/-MEF were infected to express a CRISPR/Cas9 system targeting luciferase or the spata2 gene, from which single clones were generated. The cells were treated with mTNF (10 ng/ml) or mTNF along with Necrostatin-1 (Nec-1) for 1-3 h as indicated. The blot was sequentially probed with antibodies recognizing PARP, cleaved caspase-3 and tubulin.(D). The same cells as used in (C) were treated as before with TNF (10 ng/ml) or TNF along with Necrostatin-1 (Nec-1) for one and 2 h as indicated. Caspase activity was determined using the fluorogenic substrate DEVD-AMC.(E) Cells from the experiment as shown in (D) were analyzed for apoptosis by Annexin V staining after 2 h. In (D) and (E), a representative experiment is shown with error bars referring to technical replicates.(F) Cells as described before were treated with mTNF (10 ng/ml) for the indicated time and lysates were subjected to immunoprecipitation with an anti-FADD antibody. The immunoprecipitates were subjected to Western blotting and probed for RIPK1, FADD and actin.


Figure 1Electron microscopy showing two adjacent mitochondria ( one intact and one ruptured ) in a DM platelet . a . Lower powered view of the single DM platelet . b . The enlarged inset highlights the ruptured mitochondria ( with loss of cristae morphology ) next to the intact mitochondria . c . Black dash line indicates mitochondria membrane and red dash line indicate phagophore formation . ( M - mitochondria ) Immunoprecipitation ( Meng et al ) of MsrB2 in HC and DM human platelets followed by MsrB2 and LC3I / II western . LC3 and MsrB2 immuno - EM analysis of HC and DM platelets . 15 nm dots indicate immunogold - labeled LC3 clusters and 5nm dots indicate immunogold - labeled MsrB2 clusters . No significant clusters were found in HC ( a ) platelets . Representative areas of clusters of gold labeling in DM patients ( b - d ) are presented . The nonparametric t test was performed for comparisons of 2 groups . Analysis was performed with Prism software ( GraphPad Software , Inc , La Jolla , CA ) . A difference of P < 0 . 05 was considered significant .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1Electron microscopy showing two adjacent mitochondria (one intact and one ruptured) in a DM platelet. a. Lower powered view of the single DM platelet. b. The enlarged inset highlights the ruptured mitochondria (with loss of cristae morphology) next to the intact mitochondria. c. Black dash line indicates mitochondria membrane and red dash line indicate phagophore formation. (M- mitochondria)Immunoprecipitation (Meng et al) of MsrB2 in HC and DM human platelets followed by MsrB2 and LC3I/II western.LC3 and MsrB2 immuno-EM analysis of HC and DM platelets. 15 nm dots indicate immunogold-labeled LC3 clusters and 5nm dots indicate immunogold-labeled MsrB2 clusters. No significant clusters were found in HC (a) platelets. Representative areas of clusters of gold labeling in DM patients (b-d) are presented. The nonparametric t test was performed for comparisons of 2 groups. Analysis was performed with Prism software (GraphPad Software, Inc, La Jolla, CA). A difference of P<0.05 was considered significant.


Figure 2Western blot analysis of MsrB2 and LC3 I / II in Meg - 01 cell after shMsrB2 transfection ( 72 hrs ) . Cells were then treated with H2O2 ( 1 mM for 1 hr ) alone or with NAC ( 100 μM for 30 min ) . GAPDH was used as the loading control . Quantification and analysis of individual groups . GAPDH served as the loading control . ( shMsrB2 vs . shCon ; * p = 0 . 024 , shMsrB2 / H2O2 vs . shCon / H2O2 ; * p = 0 . 0295 , shMsrB2 / H2O2 / NAC vs . shCon / H2O2 / NAC * * p = 0 . 0100 , n = 3 ) Western blot analysis of MsrB2 , pp53 ( S15 ) and LC3 I / II in Meg - 01 cell after 25 mM HG treatment for 24 hrs . GAPDH was used as the loading control . Quantification analysis on individual groups . GAPDH served as the loading control . ( MsrB2 : shCon / HG vs . shCon ; * * p = 0 . 0017 , shMsrB2 / HG vs . shCon / HG ; * p = 0 . 0189 , pp53 : shMsrB2 / HG vs . shCon / HG ; * p = 0 . 0317 , LC3II : shCon / HG vs . shCon ; * p = 0 . 0215 , shMsrB2 / HG vs . shCon / HG ; * p = 0 . 0158 , n = 3 ) The nonparametric t test was performed for comparisons of 2 groups . Analysis was performed with Prism software ( GraphPad Software , Inc , La Jolla , CA ) . A difference of P < 0 . 05 was considered significant .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2Western blot analysis of MsrB2 and LC3 I/II in Meg-01 cell after shMsrB2 transfection (72 hrs). Cells were then treated with H2O2 (1 mM for 1 hr) alone or with NAC (100 μM for 30 min). GAPDH was used as the loading control. Quantification and analysis of individual groups. GAPDH served as the loading control. (shMsrB2 vs. shCon; *p=0.024, shMsrB2/H2O2 vs. shCon/H2O2; *p=0.0295, shMsrB2/H2O2/NAC vs. shCon/H2O2/NAC **p=0.0100, n=3)Western blot analysis of MsrB2, pp53(S15) and LC3 I/II in Meg-01 cell after 25 mM HG treatment for 24 hrs. GAPDH was used as the loading control. Quantification analysis on individual groups. GAPDH served as the loading control. (MsrB2: shCon/HG vs. shCon; **p=0.0017, shMsrB2/HG vs. shCon/HG; *p=0.0189, pp53: shMsrB2/HG vs. shCon/HG; *p=0.0317, LC3II: shCon/HG vs. shCon; *p=0.0215, shMsrB2/HG vs. shCon/HG; *p=0.0158, n=3) The nonparametric t test was performed for comparisons of 2 groups. Analysis was performed with Prism software (GraphPad Software, Inc, La Jolla, CA). A difference of P<0.05 was considered significant.


Figure 3Levels of reactive oxygen species ( ROS ) quantified in the platelets using the fluorescent dye DCFH - DA . ( ROS ; * p = 0 . 0139 , MsrB2 fl / fl n = 5 , MsrB2 - / - n = 6 ) C . Representative image and quantification of mitochondrial apoptosis , measured in freshly isolated platelets from the MsrB2 knockouts and age matched floxed mice . Results are expressed as % apoptotic cells . ( TMRE ; * p = 0 . 0118 , MsrB2 fl / fl n = 5 , MsrB2 - / - n = 5 ) Representative images showing the intraplatelet expression of LC3 and active caspase - 3 in CD41 stained platelets from the KO mice using fluorescent labelled antibodies against the respective proteins of interest . Magnification : 100X . Rightmost panel represents zoom in on an individual platelet showing the colocalization and expression of LC3 and active caspase - 3 . Graph indicates platelets positive for active caspase - 3 signal . The y ‐ axis indicates percentage of caspase - 3 positive cells from total cells calculated from n = 10 independent images per sample . ( active caspase - 3 signal ; * * p = 0 . 0003Intracellular caspase - 3 activity measured in freshly isolated platelets using the chromophore Ac - DEVD - pNA . Experiments performed in a minimum of n = 6 samples per group . Values expressed as Mean ± SEM . ( caspase - 3 activity ; * p = 0 . 0157 ) and H . Western blot analysis of Parkin , LC3I / II , MsrB2 and active caspasee3 in platelet after cell penetration peptide injection in chow and HFD mice . Control peptide ( CP ) , LIF peptide ( Foroud et al ) . Quantification analysis of individual band intensity . ( Parkin ; * p = 0 . 02964 , LC3II ; * p = 0 . 03200 , MsrB2 ; * * p = 0 . 0059 , Active caspase3 ; * p = 0 . 0368 vs . chow or HFD group , each group n = 3 ) . Actin served as the loading control . The nonparametric t test was performed for comparisons of 2 groups . Analysis was performed with Prism software ( GraphPad Software , Inc , La Jolla , CA ) . A difference of P < 0 . 05 was considered significant .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3Levels of reactive oxygen species (ROS) quantified in the platelets using the fluorescent dye DCFH-DA. (ROS; *p=0.0139, MsrB2 fl/fl n=5, MsrB2 -/- n=6)C. Representative image and quantification of mitochondrial apoptosis, measured in freshly isolated platelets from the MsrB2 knockouts and age matched floxed mice. Results are expressed as % apoptotic cells. (TMRE; *p=0.0118, MsrB2 fl/fl n=5, MsrB2 -/- n=5)Representative images showing the intraplatelet expression of LC3 and active caspase-3 in CD41 stained platelets from the KO mice using fluorescent labelled antibodies against the respective proteins of interest. Magnification: 100X. Rightmost panel represents zoom in on an individual platelet showing the colocalization and expression of LC3 and active caspase-3.Graph indicates platelets positive for active caspase-3 signal. The y‐axis indicates percentage of caspase-3 positive cells from total cells calculated from n = 10 independent images per sample. (active caspase-3 signal ; **p=0.0003Intracellular caspase-3 activity measured in freshly isolated platelets using the chromophore Ac-DEVD-pNA. Experiments performed in a minimum of n=6 samples per group. Values expressed as Mean ± SEM. (caspase-3 activity ; *p=0.0157)and H. Western blot analysis of Parkin, LC3I/II, MsrB2 and active caspasee3 in platelet after cell penetration peptide injection in chow and HFD mice. Control peptide (CP), LIF peptide (Foroud et al). Quantification analysis of individual band intensity. (Parkin; *p=0.02964, LC3II; *p=0.03200, MsrB2; **p=0.0059, Active caspase3; *p=0.0368 vs. chow or HFD group, each group n=3). Actin served as the loading control. The nonparametric t test was performed for comparisons of 2 groups. Analysis was performed with Prism software (GraphPad Software, Inc, La Jolla, CA). A difference of P<0.05 was considered significant.


Figure 4Immunoprecipitation ( Meng et al ) of MsrB2 in HC and DM platelets , followed by western blot analysis of precipitated Parkin and modified Parkin . Immunoprecipitation ( Meng et al ) of Parkin in HC and DM platelets , followed by Western blot analysis of precipitated Parkin and MsrB2 . Confocal microscopy was used to corroborate the Western analysis using triple staining for Parkin , LC3 and MsrB2 in HC and DM platelets . Arrows indicate sites of colocalization of Parkin , LC3 and MsrB2 as determined by Volocity software ( PerkinElmer , USA ) . Graphical quantification of colocalization between Parkin , LC3 and MsrB2 signal . Signal intensity of each group was converted to fold and compared with HC values ( colocalization of parkin , LC3 and MsrB2 ; * * p = 0 . 0006 ) . Immunoprecipitation ( Meng et al ) of Parkin in HC and DM platelets . Western blot analysis of MetO Parkin using specific Parkin and MetO antibodies . The detection of MetO Parkin , was performed w / o β - mercaptoethanol ( NonRe ) SDS sample buffer . In vitro assay . Parkin oxidation was induced with 1mM H2O2 then incubated with MsrB2 protein at 37ºC for 2hrs . Western blot analysis of Parkin and MsrB2 was performed . ( red arrow , low MsrB2 expression ; blue arrow , high MsrB2 expression ) . The nonparametric t test was performed for comparisons of 2 groups . Analysis was performed with Prism software ( GraphPad Software , Inc , La Jolla , CA ) . A difference of P < 0 . 05 was considered significant .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4Immunoprecipitation (Meng et al) of MsrB2 in HC and DM platelets, followed by western blot analysis of precipitated Parkin and modified Parkin.Immunoprecipitation (Meng et al) of Parkin in HC and DM platelets, followed by Western blot analysis of precipitated Parkin and MsrB2.Confocal microscopy was used to corroborate the Western analysis using triple staining for Parkin, LC3 and MsrB2 in HC and DM platelets. Arrows indicate sites of colocalization of Parkin, LC3 and MsrB2 as determined by Volocity software (PerkinElmer, USA). Graphical quantification of colocalization between Parkin, LC3 and MsrB2 signal.Signal intensity of each group was converted to fold and compared with HC values (colocalization of parkin, LC3 and MsrB2; **p=0.0006).Immunoprecipitation (Meng et al) of Parkin in HC and DM platelets. Western blot analysis of MetO Parkin using specific Parkin and MetO antibodies. The detection of MetO Parkin, was performed w/o β-mercaptoethanol (NonRe) SDS sample buffer.In vitro assay. Parkin oxidation was induced with 1mM H2O2 then incubated with MsrB2 protein at 37ºC for 2hrs. Western blot analysis of Parkin and MsrB2 was performed. (red arrow, low MsrB2 expression; blue arrow, high MsrB2 expression). The nonparametric t test was performed for comparisons of 2 groups. Analysis was performed with Prism software (GraphPad Software, Inc, La Jolla, CA). A difference of P<0.05 was considered significant.


Figure 5Western blot analysis of Methionine Sulfoxidation ( MetO ) , Methionine sulfide reductase B2 ( MsrB2 ) and Parkin in human Healthy Control ( HC ) and Diabetic Mellitus ( DM ) platelets . P . C . is a BSA - MetO positive control ( Cayman Chemical Co . ) . Quantitation of western blot analysis shown in Fig 1C . Graphical representation and statistical analysis of HC ( n = 3 ) and DM ( n = 12 ) individuals . ( MetO ; * * p = 0 . 0003 , MsrB2 ; * p = 0 . 0186 , Parkin ; * * p = 0 . 0003 vs . HC ) . Western blot analysis of Parkin and Parkin aggregation in human Healthy Control ( HC ) ( n = 3 ) and Diabetic Mellitus ( DM ) ( n = 12 ) platelets . Graphical representation and statistical analysis of HC ( n = 3 ) and DM ( n = 12 ) individuals . ( modified parkin * p = 0 . 035 vs . HC ) . Actin served as the loading control . Parkin IP was performed in HC , HC plus H2O2 , and DM patients . MetO sites were analyzed by mass spectrometry using the Mascot program Representative mass spectrometry analysis demonstrating identification of MetO192 peptide on Parkin . The nonparametric t test was performed for comparisons of 2 groups . Analysis was performed with Prism software ( GraphPad Software , Inc , La Jolla , CA ) . A difference of P < 0 . 05 was considered significant .

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Figure 5Western blot analysis of Methionine Sulfoxidation (MetO), Methionine sulfide reductase B2 (MsrB2) and Parkin in human Healthy Control (HC) and Diabetic Mellitus (DM) platelets. P.C. is a BSA-MetO positive control (Cayman Chemical Co.). Quantitation of western blot analysis shown in Fig 1C. Graphical representation and statistical analysis of HC (n=3) and DM (n=12) individuals. (MetO; **p=0.0003, MsrB2; *p=0.0186, Parkin; **p=0.0003 vs. HC).Western blot analysis of Parkin and Parkin aggregation in human Healthy Control (HC) (n=3) and Diabetic Mellitus (DM) (n=12) platelets. Graphical representation and statistical analysis of HC (n=3) and DM (n=12) individuals. (modified parkin *p=0.035 vs. HC). Actin served as the loading control.Parkin IP was performed in HC, HC plus H2O2, and DM patients.MetO sites were analyzed by mass spectrometry using the Mascot program Representative mass spectrometry analysis demonstrating identification of MetO192 peptide on Parkin. The nonparametric t test was performed for comparisons of 2 groups. Analysis was performed with Prism software (GraphPad Software, Inc, La Jolla, CA). A difference of P<0.05 was considered significant.


Figure 6Immunoprecipitation ( Meng et al ) of MsrB2 in HC and DM platelets . Western blot analysis of precipitated MsrB2 , Parkin and ubiquitinated MsrB2 ( UB ) . Confocal microscopy in individual platelets in HC vs DM assessing for CoxIV ( mitochondria marker ) , ubiquitin and MsrB2 . Colocalization is demomstrated by arrows and quantitated . ( colocalization ubiquitin , MsrB2 ; * * p = 0 . 0003 vs . HC ) . MsrB2 ubiquitination assay after transient transfection of MsrB2 - GFP ( 1µg ) with RFP empty vector ( E . V . ) or RFP - Parkin ( 3µg ) in HEK293 . After transfection ( 48hrs ) , an IP was performed using GFP - Trap bead , followed by Western blot analysis using UB and MsrB2 and Parkin antibodies . The nonparametric t test was performed for comparisons of 2 groups . Analysis was performed with Prism software ( GraphPad Software , Inc , La Jolla , CA ) . A difference of P < 0 . 05 was considered significant .

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Figure 6Immunoprecipitation (Meng et al) of MsrB2 in HC and DM platelets. Western blot analysis of precipitated MsrB2, Parkin and ubiquitinated MsrB2 (UB).Confocal microscopy in individual platelets in HC vs DM assessing for CoxIV (mitochondria marker), ubiquitin and MsrB2. Colocalization is demomstrated by arrows and quantitated. (colocalization ubiquitin, MsrB2; **p=0.0003 vs. HC).MsrB2 ubiquitination assay after transient transfection of MsrB2-GFP (1µg) with RFP empty vector (E.V.) or RFP-Parkin (3µg) in HEK293. After transfection (48hrs), an IP was performed using GFP-Trap bead, followed by Western blot analysis using UB and MsrB2 and Parkin antibodies. The nonparametric t test was performed for comparisons of 2 groups. Analysis was performed with Prism software (GraphPad Software, Inc, La Jolla, CA). A difference of P<0.05 was considered significant.


Figure 7Western blot analysis of MsrB2 and Cytochrome C in isolated mitochondria after incubation w / o Cyclosporin A ( CsA ; 2 μM for 2 hrs . ) or w / CsA . ATPB served as the loading and negative control ( mitochondrial matrix protein ) . Western blot analysis of Parkin , LC3I / II , ubiquitinated MsrB2 in chow ( n = 4 ) and HFD ( n = 6 ) mouse platelets . Confocal microscopy was initially stained with Tubulin antibody ( Panel a . , Chow diet , or b . High Fat Diet ) to assess platelet architecture . This was followed by staining using ATPB ( mitochondria marker - green ) and MsrB2 antibodies ( Blue ) ( Panel c - e ) . There was loss of colocalization in the HFD mice supporting release ( blue ) from ruptured mitochondria ( green ) ( Panel d & e ) . The nonparametric t test was performed for comparisons of 2 groups . Analysis was performed with Prism software ( GraphPad Software , Inc , La Jolla , CA ) . A difference of P < 0 . 05 was considered significant .

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Figure 7Western blot analysis of MsrB2 and Cytochrome C in isolated mitochondria after incubation w/o Cyclosporin A (CsA; 2 μM for 2 hrs.) or w/ CsA. ATPB served as the loading and negative control (mitochondrial matrix protein).Western blot analysis of Parkin, LC3I/II, ubiquitinated MsrB2 in chow (n=4) and HFD (n=6) mouse platelets.Confocal microscopy was initially stained with Tubulin antibody (Panel a., Chow diet, or b. High Fat Diet) to assess platelet architecture. This was followed by staining using ATPB (mitochondria marker - green) and MsrB2 antibodies (Blue) (Panel c-e). There was loss of colocalization in the HFD mice supporting release (blue) from ruptured mitochondria (green) (Panel d & e). The nonparametric t test was performed for comparisons of 2 groups. Analysis was performed with Prism software (GraphPad Software, Inc, La Jolla, CA). A difference of P<0.05 was considered significant.


Figure 8Representative western blot analysis of MsrB2 , LC3I / II and parkin using Parkinson ' s disease ( PD ) platelets . Quantification analysis of individual band intensity . ( MsrB2 ; * p = 0 . 0255 , LC3II ; * p = 0 . 0101 vs . HC ( n = 5 ) and PD ( n = 12 ) ) . GAPDH served as the loading control . Platelet apoptosis ( Annexin V ) were measured by flow cytometry analysis in HC ( n = 9 ) and PD ( n = 13 ) platelets . ( * * p < 0 . 01 vs . HC ) . The nonparametric t test was performed for comparisons of 2 groups . Analysis was performed with Prism software ( GraphPad Software , Inc , La Jolla , CA ) . A difference of P < 0 . 05 was considered significant .

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Figure 8Representative western blot analysis of MsrB2, LC3I/II and parkin using Parkinson's disease (PD) platelets. Quantification analysis of individual band intensity. (MsrB2; *p=0.0255, LC3II; *p=0.0101 vs. HC (n=5) and PD (n=12)). GAPDH served as the loading control.Platelet apoptosis (Annexin V) were measured by flow cytometry analysis in HC (n=9) and PD (n=13) platelets. (**p<0.01 vs. HC). The nonparametric t test was performed for comparisons of 2 groups. Analysis was performed with Prism software (GraphPad Software, Inc, La Jolla, CA). A difference of P<0.05 was considered significant.


figf1a , Mice were infected for 21 days and liver granulomas analysed by transmission electron microscopy ( TEM ) . Shown are spacious vacuoles ( SLAPs ) containing multiple bacteria . Magnification , × 5 , 200 . Region 1 and 2 ( white boxes ) are enlarged in the lower panels . b , SCID mice were infected as in a and liver sections stained for LAMP1 ( green ) , bacteria ( red ) and DNA ( blue ) . Shown is a LAMP1 + SLAP . c , The percentage of bacteria in LAMP1 + compartments was quantified and characterized as either spacious or tight - fitting . Mean ± s . e . m . for three mice examined . The image in a ( from ref . 2 ) and the tissue sections were provided by E . Unanue .

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figf1a, Mice were infected for 21 days and liver granulomas analysed by transmission electron microscopy (TEM). Shown are spacious vacuoles (SLAPs) containing multiple bacteria. Magnification, ×5,200. Region 1 and 2 (white boxes) are enlarged in the lower panels.b, SCID mice were infected as in a and liver sections stained for LAMP1 (green), bacteria (red) and DNA (blue). Shown is a LAMP1+ SLAP.c, The percentage of bacteria in LAMP1+ compartments was quantified and characterized as either spacious or tight-fitting. Mean ± s.e.m. for three mice examined. The image in a (from ref. 2) and the tissue sections were provided by E. Unanue.


figf2a , RAW 264 . 7macrophages infected for 4 or 8 h were analysed by TEM . Shown are spacious vacuoles ( SLAPs ) containing multiple bacteria . The arrow indicates septum of dividing bacteria . Scale bars , 0 . 5 µm . b , The arrow indicates a LAMP1 + SLAP colocalizing with green fluorescent protein ( GFP ) - LC3 in cells infected for 4 h . The percentage of GFP - LC3 + ( c ) d , The arrowhead indicates a LAMP1 + SLAP devoid of cathepsin D in cells infected for 4 h . Scale bars , 5 µm . The percentage of GFP - LC3 + ( c ) or cathepsin D + ( e ) f , GFP - LC3 - transfected macrophages were infected with wild - type or D actA bacteria . Where indicated , chloramphenicol ( CM ) was added to the media at 3 h post infection . The number of bacteria per SLAP was quantified . Brackets indicate significant differences , and corresponding P values are shown . g , Macrophages were infected with wild - type bacteria for 7 h , pulsed with BrdU for 1 h , and stained for LAMP1 ( red ) , bacteria ( blue ) and BrdU ( green ) . Magnified images and the arrow indicate SLAPs containing actively replicating bacteria ( BrdU + ) . h , The percentage of BrdU + bacteria in SLAPs , compared to bacteria and PFA - killed bacteria , was quantified as in c .

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figf2a, RAW 264.7macrophages infected for 4 or 8 h were analysed by TEM. Shown are spacious vacuoles (SLAPs) containing multiple bacteria. The arrow indicates septum of dividing bacteria. Scale bars, 0.5 µm.b, The arrow indicates a LAMP1+ SLAP colocalizing with green fluorescent protein (GFP)-LC3 in cells infected for 4 h.The percentage of GFP-LC3+ (c)d, The arrowhead indicates a LAMP1+ SLAP devoid of cathepsin D in cells infected for 4 h. Scale bars, 5 µm.The percentage of GFP-LC3+ (c) or cathepsin D+ (e)f, GFP-LC3-transfected macrophages were infected with wild-type or D actA bacteria. Where indicated, chloramphenicol (CM) was added to the media at 3 h post infection. The number of bacteria per SLAP was quantified. Brackets indicate significant differences, and corresponding P values are shown.g, Macrophages were infected with wild-type bacteria for 7 h, pulsed with BrdU for 1 h, and stained for LAMP1 (red), bacteria (blue) and BrdU (green). Magnified images and the arrow indicate SLAPs containing actively replicating bacteria (BrdU+). h, The percentage of BrdU+ bacteria in SLAPs, compared to bacteria and PFA-killed bacteria, was quantified as in c.


figf3a , GFP - LC3 - transfected macrophages were infected for 4 h , and the percentage of infected cells exhibiting SLAPs was quantified . Mean ± s . e . m . for three independent experiments . P values for strains with significant differences from wild - type levels are shown . b , The arrow indicates a GFP - LC3 + SLAP with internal LLO expression in cells infected for 4 h . Scale bar , 5 µm . c , Arrowheads indicate GFP - LC3 + SLAPs devoid of Lysotracker Red in cells infected for 4 h . e , The arrow indicates a LAMP1 + SLAP colocalizing with v - ATPase staining in cells infected for 4 h . Scale bar , 5 µm . f , The percentage of v - ATPase + SLAPs was quantified as in Fig . 2c . Mean ± s . e . m . for three independent experiments . P values for conditions significantly different from PFA - killed bacteria are shown .

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figf3a, GFP-LC3-transfected macrophages were infected for 4 h, and the percentage of infected cells exhibiting SLAPs was quantified. Mean ± s.e.m. for three independent experiments. P values for strains with significant differences from wild-type levels are shown.b, The arrow indicates a GFP-LC3+ SLAP with internal LLO expression in cells infected for 4 h. Scale bar, 5 µm.c, Arrowheads indicate GFP-LC3+ SLAPs devoid of Lysotracker Red in cells infected for 4 h.e, The arrow indicates a LAMP1+ SLAP colocalizing with v-ATPase staining in cells infected for 4 h. Scale bar, 5 µm. f, The percentage of v-ATPase+ SLAPs was quantified as in Fig. 2c. Mean ± s.e.m. for three independent experiments. P values for conditions significantly different from PFA-killed bacteria are shown.


figf4a , Arrows indicate LAMP1 - wild - type bacteria with actin ' comet tails ' in cells infected for 6 h . The boxed region is magnified in the bottom left panels . The merged image for this region is shown at the bottom right . Scale bar , 5 µm . b , Macrophages were infected with IPTG - induced iLLO bacteria . Arrowheads indicate actin - iLLO bacteria within LAMP1 + vacuoles . c , Macrophages were infected with wild - type ( triangles ) or IPTG - induced iLLO ( circles ) bacteria , and the percentage of LAMP1 + bacteria was quantified . Mean ± s . e . m . for three independent experiments . d , Macrophages were infected as in c with or without IPTG induction . Intracellular bacterial replication was determined using a gentamicin - protection assay . Shown is fold replication compared to 2 h post infection . Clear triangles , wild type ; filled triangles , wild type + IPTG ; clear circles , iLLO ; filled circles , iLLO + IPTG . Mean ± s . e . m . or range for three ( wild type , wild type + IPTG , iLLO + IPTG ) or two ( iLLO - IPTG ) independent experiments , respectively . e , Wild - type or Atg5 - / - MEFs were infected with iLLO bacteria , and intracellular bacterial replication was determined as in d . Mean ± s . e . m . for three independent experiments . Clear circles , wild - type MEFs ; filled circles , wild - type MEFs + IPTG ; clear squares , Atg5 - / - MEFs ; filled squares , Atg5 - / - MEFs + IPTG . f , Wild - type ( circles ) or Atg5 - / - ( squares ) MEFs were infected as in e with IPTG induction . The percentage of LAMP1 + bacteria was quantified as in c . Mean ± s . e . m . for three independent experiments .

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figf4a, Arrows indicate LAMP1- wild-type bacteria with actin 'comet tails' in cells infected for 6 h. The boxed region is magnified in the bottom left panels. The merged image for this region is shown at the bottom right. Scale bar, 5 µm.b, Macrophages were infected with IPTG-induced iLLO bacteria. Arrowheads indicate actin- iLLO bacteria within LAMP1+ vacuoles.c, Macrophages were infected with wild-type (triangles) or IPTG-induced iLLO (circles) bacteria, and the percentage of LAMP1+ bacteria was quantified. Mean ± s.e.m. for three independent experiments.d, Macrophages were infected as in c with or without IPTG induction. Intracellular bacterial replication was determined using a gentamicin-protection assay. Shown is fold replication compared to 2 h post infection. Clear triangles, wild type; filled triangles, wild type + IPTG; clear circles, iLLO; filled circles, iLLO + IPTG. Mean ± s.e.m. or range for three (wild type, wild type + IPTG, iLLO + IPTG) or two (iLLO-IPTG) independent experiments, respectively.e, Wild-type or Atg5-/- MEFs were infected with iLLO bacteria, and intracellular bacterial replication was determined as in d. Mean ± s.e.m. for three independent experiments. Clear circles, wild-type MEFs; filled circles, wild-type MEFs + IPTG; clear squares, Atg5-/- MEFs; filled squares, Atg5-/- MEFs + IPTG.f, Wild-type (circles) or Atg5-/- (squares) MEFs were infected as in e with IPTG induction. The percentage of LAMP1+ bacteria was quantified as in c. Mean ± s.e.m. for three independent experiments. g


Figure 1100 nM paclitaxel treatment of human hair follicles ( HFs ) in organ culture for 24h does not significantly affect the total number of Ki - 67 + cells ( A ) ( S - phase ) in the hair matrix . Unpaired t - test performed using N of 9 - 12 HFs from 3 patients . 100 nM paclitaxel treatment of human hair follicles ( HFs ) in organ culture for 24h does not significantly affect the total number of EdU + cells ( B ) ( S - phase ) in the hair matrix . Unpaired t - test performed using N of 9 - 12 HFs from 3 patients . C ) 100 nM paclitaxel treatment ( 24h ) significantly ( p = < 0 . 0001 [ * * * * ] ) increased the number of mitotic phospho - histone H3 ( pH3 ) + cells in the hair matrix . Welch ' s t - test performed using N of 9 HFs from 3 patients . D ) 100 nM docetaxel treatment ( 24h ) significantly ( p = 0 . 0004 [ * * * ] ) increases the number of pH3 + cells in the hair matrix . Unpaired t - test performed using N of 8 - 9 HFs from 3 patients . E ) Representative immunofluorescence images highlight the effects of 24h 100 nM taxane treatment on ( i ) Ki - 67 expression [ paclitaxel ] ; ( ii ) EdU incorporation and pH3 immunoreactivity [ paclitaxel ] ; ( iii ) pH3 immunoreactivity [ docetaxel ] . 20 µm scale . Values plotted represent the mean number of positive cells counted per HF analysed . Error bars are standard error .

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Figure 1100 nM paclitaxel treatment of human hair follicles (HFs) in organ culture for 24h does not significantly affect the total number of Ki-67+ cells (A) (S-phase) in the hair matrix. Unpaired t-test performed using N of 9-12 HFs from 3 patients.100 nM paclitaxel treatment of human hair follicles (HFs) in organ culture for 24h does not significantly affect the total number of EdU+ cells (B) (S-phase) in the hair matrix. Unpaired t-test performed using N of 9-12 HFs from 3 patients.C) 100 nM paclitaxel treatment (24h) significantly (p= <0.0001 [****]) increased the number of mitotic phospho-histone H3 (pH3)+ cells in the hair matrix. Welch's t-test performed using N of 9 HFs from 3 patients.D) 100 nM docetaxel treatment (24h) significantly (p = 0.0004 [***]) increases the number of pH3+ cells in the hair matrix. Unpaired t-test performed using N of 8-9 HFs from 3 patients.E) Representative immunofluorescence images highlight the effects of 24h 100 nM taxane treatment on (i) Ki-67 expression [paclitaxel]; (ii) EdU incorporation and pH3 immunoreactivity [paclitaxel]; (iii) pH3 immunoreactivity [docetaxel]. 20 µm scale. Values plotted represent the mean number of positive cells counted per HF analysed. Error bars are standard error.


Figure 2The presence of micronucleated cells in the hair matrix in paclitaxel ( 100 nM , 24h ) hair follicles ( HF ) compared to vehicle is significant ( p = < 0 . 0001 [ * * * * ] ) . Mann Whitney U test performed using N of 12 - 13 HFs ( paclitaxel ) from 3 patients . The presence of micronucleated cells in the hair matrix in docetaxel treated ( 100 nM , 24h ) hair follicles ( HF ) compared to vehicle is significant ( p = < 0 . 0001 [ * * * * ] ) . Mann Whitney U test performed using N of 8 HFs ( docetaxel ) from 3 patients . C ) Hoechst 33342 staining of healthy cell nuclei comprising the hair matrix ( lined ) and dermal papilla in untreated ( vehicle ) human HFs . 20 µm scale . D ) Paclitaxel treatment ( 100 nM , 24h ) induces the formation of micronucleated bodies , as visualised by Hoechst 33342 staining ( arrows ) , localising to the proliferative region of the hair matrix . i - 20 µm scale ; ii - 10 µm scale . E ) 100 nM docetaxel treatment was also seen to promote the formation of micronucleated bodies ( arrows ) . 10 µm scale . Error bars are standard error .

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Figure 2The presence of micronucleated cells in the hair matrix in paclitaxel (100 nM, 24h) hair follicles (HF) compared to vehicle is significant (p = <0.0001 [****]). Mann Whitney U test performed using N of 12-13 HFs (paclitaxel) from 3 patients.The presence of micronucleated cells in the hair matrix in docetaxel treated (100 nM, 24h) hair follicles (HF) compared to vehicle is significant (p = <0.0001 [****]). Mann Whitney U test performed using N of 8 HFs (docetaxel) from 3 patients.C) Hoechst 33342 staining of healthy cell nuclei comprising the hair matrix (lined) and dermal papilla in untreated (vehicle) human HFs. 20 µm scale.D) Paclitaxel treatment (100 nM, 24h) induces the formation of micronucleated bodies, as visualised by Hoechst 33342 staining (arrows), localising to the proliferative region of the hair matrix. i - 20 µm scale; ii - 10 µm scale.E) 100 nM docetaxel treatment was also seen to promote the formation of micronucleated bodies (arrows). 10 µm scale. Error bars are standard error.


Figure 3A ) Nascent RNA synthesis , as detected by ethynyl uridine ( EU ) incorporation , is blocked within clusters of nuclei in the hair matrix ( arrows ) following paclitaxel treatment ( 100 nM , 24h ) . 20 µm scale . B ) Quantitative analysis highlights a significant ( p = < 0 . 0001 [ * * * * ] ) decrease in the number of EU + nuclei following 24h paclitaxel treatment . Welch ' s t - test performed using N of 11 - 12 hair follicles ( HFs ) from 3 patients . C ) Representative dual fluorescence stain highlights how EU incorporation in the hair matrix is blocked within the pH3 + cell population that accumulates in response to paclitaxel treatment 10 µm scale . D ) Cleaved caspase 3 expression in the hair matrix following 24h paclitaxel treatment . 20 µm scale . E ) 100 nM paclitaxel treatment significantly ( p = 0 . 0016 [ exact ] [ * * ] ) increases the number of cleaved caspase 3 + cells in the hair matrix after 24h . Mann Whitney U test performed using N of 16 - 18 HFs from 5 patients . Values plotted represent the mean number of positive cells counted per HF analysed . Error bars are standard error .

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Figure 3A) Nascent RNA synthesis, as detected by ethynyl uridine (EU) incorporation, is blocked within clusters of nuclei in the hair matrix (arrows) following paclitaxel treatment (100 nM, 24h). 20 µm scale.B) Quantitative analysis highlights a significant (p =<0.0001 [****]) decrease in the number of EU+ nuclei following 24h paclitaxel treatment. Welch's t-test performed using N of 11-12 hair follicles (HFs) from 3 patients.C) Representative dual fluorescence stain highlights how EU incorporation in the hair matrix is blocked within the pH3+ cell population that accumulates in response to paclitaxel treatment 10 µm scale.D) Cleaved caspase 3 expression in the hair matrix following 24h paclitaxel treatment. 20 µm scale.E) 100 nM paclitaxel treatment significantly (p = 0.0016 [exact] [**]) increases the number of cleaved caspase 3+ cells in the hair matrix after 24h. Mann Whitney U test performed using N of 16-18 HFs from 5 patients. Values plotted represent the mean number of positive cells counted per HF analysed. Error bars are standard error.


Figure 4Ai ) Paclitaxel treatment promotes mitotic arrest ( orange arrows / pH3 + cells ) and apoptosis ( white arrows / caspase 3 + cells ) , within the K15 + bulge and K15 + proximal bulb outer root sheath ( pbORS ) stem / progenitor cell compartments of the human hair follicle ( HF ) . 20 µm scale . Aii ) High magnification montage demonstrating mitotic arrest ( orange arrow ) and apoptosis / caspase 3 positivity ( white arrow ) within the K15 + bulge . 10 µm scale . pbORS - proximal bulb outer root sheath . B ) Representative immunofluorescence images of heightened cleaved caspase 3 immunoreactivity within the K15 + bulge following extended paclitaxel organ culture experiments 50 µm scale . EC - extended cultures . C ) Graph showing significantly ( p = 0 . 029 [ * * ] ) increased K15 / caspase 3 double + cells within the bulge following extended paclitaxel HF organ cultures . Welch ' s t - test performed using N of 8 - 9 HFs from 3 patients . Values plotted represent the mean number of positive cells counted per HF analysed . Error bars are standard error . D ) K15 + cells of the human HF bulge express Ki - 67 during extended organ culture experiments . Paclitaxel treatment did not significantly affect the number of bulge K15 / Ki - 67 double + cells . Unpaired t - test performed using N of 8 - 9 HFs from 3 patients . Values plotted represent the mean number of positive cells counted per HF analysed . Error bars are standard error . E ) Representative double immunofluorescence images of elevated γH2A . X immunoreactivity within the K15 + bulge following extended paclitaxel organ culture experiments ( see methods ) . 50 µm scale . EC - extended cultures . F ) γH2A . X analysis showing a significant ( p = 0 . 065 [ * * ] ) increase in the number of cells with DNA double - strand breaks in the K15 + bulge following extended organ culture experiments . Mann Whitney U test performed using N of 5 - 6 HFs from 2 patients . Values plotted represent the mean number of positive cells counted per HF analysed . Error bars are standard error .

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Figure 4Ai) Paclitaxel treatment promotes mitotic arrest (orange arrows/pH3+ cells) and apoptosis (white arrows/caspase 3+ cells), within the K15+ bulge and K15+ proximal bulb outer root sheath (pbORS) stem/progenitor cell compartments of the human hair follicle (HF). 20 µm scale. Aii) High magnification montage demonstrating mitotic arrest (orange arrow) and apoptosis/caspase 3 positivity (white arrow) within the K15+ bulge. 10 µm scale. pbORS - proximal bulb outer root sheath.B) Representative immunofluorescence images of heightened cleaved caspase 3 immunoreactivity within the K15+ bulge following extended paclitaxel organ culture experiments 50 µm scale. EC - extended cultures.C) Graph showing significantly (p = 0.029 [**]) increased K15/caspase 3 double+ cells within the bulge following extended paclitaxel HF organ cultures. Welch's t-test performed using N of 8-9 HFs from 3 patients. Values plotted represent the mean number of positive cells counted per HF analysed. Error bars are standard error.D) K15+ cells of the human HF bulge express Ki-67 during extended organ culture experiments. Paclitaxel treatment did not significantly affect the number of bulge K15/Ki-67 double+ cells. Unpaired t-test performed using N of 8-9 HFs from 3 patients. Values plotted represent the mean number of positive cells counted per HF analysed. Error bars are standard error.E) Representative double immunofluorescence images of elevated γH2A.X immunoreactivity within the K15+ bulge following extended paclitaxel organ culture experiments (see methods). 50 µm scale. EC - extended cultures.F) γH2A.X analysis showing a significant (p = 0.065 [**]) increase in the number of cells with DNA double-strand breaks in the K15+ bulge following extended organ culture experiments. Mann Whitney U test performed using N of 5-6 HFs from 2 patients. Values plotted represent the mean number of positive cells counted per HF analysed. Error bars are standard error.


Figure 5B ) Palbociclib - only and palbociclib plus paclitaxel dual treated HFs show marked reductions in Ki - 67 expression in the hair matrix beyond that seen with just 24h treatment Data also confirms that Ki - 67 expression is unaffected by paclitaxel treatment Analysis performed using N of 6 - 9 HFs per condition from 3 patients . Ordinary one - way ANOVA with multiple comparisons performed . Adjusted p values = 0 . 0001 [ * * * ] and 0 . 0001 [ * * * * ] respectively . Values plotted represent the mean number of positive cells counted per HF analysed . Error bars are standard error . C ) Data confirming that paclitaxel treatment significantly increases ( adjusted p value = 0 . 0001 [ * * * ] ) the number of pH3 + cells in the hair matrix This effect was not observed when paclitaxel treated hair follicles were pre - and co - incubated with palbociclib . Analysis performed using N of 6 - 9 HFs per condition from 3 patients Values plotted represent the mean number of positive cells counted per HF analysed . Error bars are standard error . D ) Cleaved caspase 3 analysis in hair follicles from a patient donor that showed apoptotic sensitivity to paclitaxel treatment ( adjusted p value = 0 . 0056 [ * * ] ) . Paclitaxel treated hair follicles pre - and co - incubated with palbociclib do not show enhanced cleaved caspase 3 expression , contrasting with paclitaxel - only treatment . Ordinary one - way ANOVA with multiple comparisons performed using N of 4 - 5 HFs per condition . Values plotted represent the mean number of positive cells counted per HF analysed . Error bars are standard error . E ) Immunofluorescence data representing how palbociclib and / or paclitaxel affect ( i ) Ki - 67 expression , ( ii ) pH3 immunoreactivity and ( iii ) cleaved caspase 3 expression . 20 µm scale .

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Figure 5B) Palbociclib-only and palbociclib plus paclitaxel dual treated HFs show marked reductions in Ki-67 expression in the hair matrix beyond that seen with just 24h treatment Data also confirms that Ki-67 expression is unaffected by paclitaxel treatment Analysis performed using N of 6-9 HFs per condition from 3 patients. Ordinary one-way ANOVA with multiple comparisons performed. Adjusted p values = 0.0001[***] and 0.0001 [****] respectively. Values plotted represent the mean number of positive cells counted per HF analysed. Error bars are standard error.C) Data confirming that paclitaxel treatment significantly increases (adjusted p value = 0.0001[***]) the number of pH3+ cells in the hair matrix This effect was not observed when paclitaxel treated hair follicles were pre- and co-incubated with palbociclib. Analysis performed using N of 6-9 HFs per condition from 3 patients Values plotted represent the mean number of positive cells counted per HF analysed. Error bars are standard error.D) Cleaved caspase 3 analysis in hair follicles from a patient donor that showed apoptotic sensitivity to paclitaxel treatment (adjusted p value = 0.0056 [**]). Paclitaxel treated hair follicles pre- and co-incubated with palbociclib do not show enhanced cleaved caspase 3 expression, contrasting with paclitaxel-only treatment. Ordinary one-way ANOVA with multiple comparisons performed using N of 4-5 HFs per condition. Values plotted represent the mean number of positive cells counted per HF analysed. Error bars are standard error.E) Immunofluorescence data representing how palbociclib and/or paclitaxel affect (i) Ki-67 expression, (ii) pH3 immunoreactivity and (iii) cleaved caspase 3 expression. 20 µm scale.


Figure 6B ) No significant differences in EdU incorporation were found in any experimental conditions compared to vehicle . DNA synthesis in the hair matrix resumed in the palbociclib - only and palbociclib plus paclitaxel dual treated conditions following washout Ordinary one - way ANOVA with multiple comparisons performed using N of 8 - 13 HFs from 3 patients . Values plotted represent the mean total number of cells counted per HF analysed . Error bars are standard error . C ) A trending increase in the number of cleaved caspase 3 + cells was seen in the hair matrix of HFs dual treated with paclitaxel and palbociclib following drug washout , but this was not significant , whereas paclitaxel - only treatment significantly increased the number of cleaved caspase 3 + cells in the hair matrix ( adjusted p value = 0 . 0003 [ * * * ] ) . Palbociclib - only treatment did not increase the number of cleaved caspase 3 + cells in the hair matrix following drug washout . Ordinary one - way ANOVA with multiple comparisons performed using N of 8 - 12 HFs from 3 patients . Values plotted represent the mean total number of cells counted per HF analysed . Error bars are standard error . D ) HFs treated with paclitaxel alone showed a significant ( adjusted p value = 0 . 0316 [ * ] ) increase in the number of pH3 + cells in the matrix after washout . A significant ( adjusted p value = 0 . 0278 [ * ] ) increase in the number pH3 + cells was seen in HFs dual treated with both paclitaxel and palbociclib following drug washout . Palbociclib treatment alone showed no significant change in the number of pH3 + hair matrix keratinocytes after drug washout . Ordinary one - way ANOVA with multiple comparisons performed using N of 8 - 12 HFs from 3 patients . Values plotted represent the mean total number of cells counted per HF analysed . Error bars are standard error . E ) Representative fluorescence images of EdU and pH3 / cleaved caspase 3 staining in paclitaxel - only and palbociclib plus paclitaxel dual treated HFs following drug washout . Where DNA synthesis has resumed following reversal of the G1 arrest , paclitaxel cytotoxicity is emergent . HS - hair shaft ; " wo " - washout . 20 µm scale .

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Figure 6B) No significant differences in EdU incorporation were found in any experimental conditions compared to vehicle. DNA synthesis in the hair matrix resumed in the palbociclib-only and palbociclib plus paclitaxel dual treated conditions following washout Ordinary one-way ANOVA with multiple comparisons performed using N of 8-13 HFs from 3 patients. Values plotted represent the mean total number of cells counted per HF analysed. Error bars are standard error.C) A trending increase in the number of cleaved caspase 3+ cells was seen in the hair matrix of HFs dual treated with paclitaxel and palbociclib following drug washout, but this was not significant, whereas paclitaxel-only treatment significantly increased the number of cleaved caspase 3+ cells in the hair matrix (adjusted p value = 0.0003 [***]). Palbociclib-only treatment did not increase the number of cleaved caspase 3+ cells in the hair matrix following drug washout. Ordinary one-way ANOVA with multiple comparisons performed using N of 8-12 HFs from 3 patients. Values plotted represent the mean total number of cells counted per HF analysed. Error bars are standard error.D) HFs treated with paclitaxel alone showed a significant (adjusted p value = 0.0316 [*]) increase in the number of pH3+ cells in the matrix after washout. A significant (adjusted p value = 0.0278 [*]) increase in the number pH3+ cells was seen in HFs dual treated with both paclitaxel and palbociclib following drug washout. Palbociclib treatment alone showed no significant change in the number of pH3+ hair matrix keratinocytes after drug washout. Ordinary one-way ANOVA with multiple comparisons performed using N of 8-12 HFs from 3 patients. Values plotted represent the mean total number of cells counted per HF analysed. Error bars are standard error.E) Representative fluorescence images of EdU and pH3/cleaved caspase 3 staining in paclitaxel-only and palbociclib plus paclitaxel dual treated HFs following drug washout. Where DNA synthesis has resumed following reversal of the G1 arrest, paclitaxel cytotoxicity is emergent. HS - hair shaft; "wo" - washout. 20 µm scale.