Journal: Blood Science

Article Title: BECN1 modulates hematopoietic stem cells by targeting Caspase-3-GSDME-mediated pyroptosis

doi: 10.1097/BS9.0000000000000051

Figure Lengend Snippet: Becn1 deficient HSCs show increased GSDME-mediated pyroptosis. (A–C) Representative flow cytometry plots (A) and histograms (B and C) showing cell viability of LSKs from WT and Becn1 vKO mice. Freshly isolated LSKs were cultured for 24 h before cell viability analysis by Annexin V (A and B) or DAPI (C). n = 3 repeats per group, data are shown as mean ± SD. (D) Representative western blot showing the level of RIPK3, MLKL, pMLKL, GSDMD, GSDME, and Caspase-3 (CASP3) in freshly isolated hematopoietic progenitor (c-Kit + ) cells from WT and Becn1 vKO mice. Cell lysates were subjected to immunoblot analysis using indicated antibodies. pMLKL, phosphorylated MLKL; GSDME-FL, full-length GSDME; GSDME-N, the N-terminal product of GSDME. (E) Representative western blot showing the level of RIPK3, MLKL, GSDME, and Caspase-3 in HSCs (CD34 − LSK) from WT and Becn1 vKO mice. Freshly isolated HSCs were cultured for 8 days. Cell lysates were subjected to immunoblot analysis using indicated antibodies. (F) Representative western blot showing the activation of Caspase-3 and GSDME following apoptotic drug treatment. WT c-Kit + cells were treated with ABT263 (10 μM) plus S63845 (10 μM) or mock treated for 5 h. Cell lysates were subjected to immunoblot analysis using indicated antibodies. (G) Representative western blot showing the level of Caspase-3 and GSDME in Caspase-3-shRNA or none target control (NTC) shRNA infected c-Kit + cells with or without apoptosis induction. WT c-Kit + cells were infected with lentivirus carrying an NTC shRNA or a Caspase-3-shRNA for 3 days and then treated with ABT263 (10 μM) plus S63845 (10 μM) or mock treated for 5 h. The infection rate was around 90% and the total cell population was collected for western blot analysis without further isolation. Cell lysates were subjected to immunoblot using indicated antibodies. (H) Representative western blot showing GSDME overexpression in LSKs. Freshly isolated 10 5 LSKs were infected with the full-length GSDME-cDNA or control vector for 3 days. Cell lysates of the total cell population were subjected to western blot using indicated antibodies. (I and J) 40,000 mCherry + cells were isolated from full-length GSDME-cDNA or control vector infected LSKs at 3 days post infection and transplanted into lethally irradiated recipients together with 4.6 × 10 5 competitor cells. Chimera in peripheral blood was evaluated every month until the third month. (I) The schematic diagram showing the experimental design for GSDME (full-length) overexpression transplantation. (J) The line plots depict changes in peripheral blood chimerism of donor-derived cells (CD45.2) in recipients at the indicated time points after transplantation. Data are shown as mean ± SD, n = 5 mice per group. (K) Model for regulation of BECN1 in HSCs cell death.

Article Snippet: Samples were subjected to 13.5% or 15% SDS-PAGE with rabbit-derived primary antibodies against Caspase-3 (1:800, CST, 9662S) and LC3 (1:3000, Sigma, L7543), 10% SDS-PAGE with antibodies against RIPK3 (1:1000, PROSCI, 2283), MLKL (1:1000, ANGENT, AP14272b), Phospho-MLKL (1:1000, Abcom, ab196436), GSDMD (1:1000, Abcam, ab209845), GSDME (1:1000, Abcam, ab215191) and BECN1 (1:700, Proteintech, 11306–1-AP).

Techniques: Flow Cytometry, Isolation, Cell Culture, Western Blot, Activation Assay, shRNA, Infection, Over Expression, Plasmid Preparation, Irradiation, Transplantation Assay, Derivative Assay

Journal: eLife

Article Title: RIPK1-RIPK3-MLKL-dependent necrosis promotes the aging of mouse male reproductive system

doi: 10.7554/eLife.27692

Figure Lengend Snippet: ( A, B ) Macroscopic features and weights of Ripk 3 +/+ (wild-type) and Ripk 3 -/- ( Ripk3 -knockout) male mice. Ripk 3 +/+ (4 Month, n = 16; 18 Month, n = 27) and Ripk 3 -/- (4 Month, n = 16; 18 Month, n = 27) male mice were photographed and weighed. Data represent the mean ±the standard error of the mean (s.e.m). **p<0.01, ***p<0.001. p values were determined with unpaired Student’s t -tests. NS, not significant. ( C, D ) Macroscopic features and weights of seminal vesicles. Mice were sacrificed at 18 months of age, and the seminal vesicles from Ripk 3 +/+ (n = 33) and Ripk 3 -/- (n = 30) mice were photographed and weighed. Data represent the mean ±s.e.m. ***p < 0.001. p values were determined with unpaired Student’s t -tests. ( E ) Serum testosterone levels of mice assayed using ELISA. Mice were sacrificed, and the testosterone levels in serum from Ripk 3 +/+ (4 Month, n = 9; 18 Month, n = 9) and Ripk 3 -/- (4 Month, n = 9; 18 Month, n = 9) mice were measured using an ELISA kit for testosterone. Data represent the mean ±s.e.m. *p<0.05, ***p<0.001. p values were determined with unpaired Student’s t -tests. NS, not significant. ( F, G ) H and E of testis sections from Ripk 3 +/+ and Ripk 3 -/- mice. Ripk 3 +/+ (4 Months, n = 10; 18 Months, n = 10) and Ripk 3 -/- (4 Months, n = 10; 18 Months, n = 10) mice were sacrificed and testes were harvested and stained with H and E in ( F ). The number of empty seminiferous tubules was counted based on H and E staining and quantification in ( G ), empty seminiferous tubules were counted in five fields per testis. Scale bar, 100 μm. Data represent the mean ± S.D. ***p < 0.001. p values were determined with unpaired Student’s t -tests. NS, not significant. ( H ) Summary of the fertility rates of Ripk 3 +/+ and Ripk 3 -/- mice. One male mice of a given age was mated with a pairs of 10-week-old wild-type female mice for 3 months; females were replaced every 2 weeks. The number of male mice with reproductive capacity was counted (see Materials and methods). p values were determined using chi-square tests. ( I ) Reproductive longevity. When Ripk 3 +/+ (n = 12) and Ripk 3 -/- (n = 12) male mice were 2 months old, they were continuously mated with a pairs of 10-week-old female mice until pregnancies ceased; females were replaced every 2 months. The ages of the males at which their last litter was sired was recorded (calculated as the age at birth of the litter less 21 days, see Materials and methods). Data represent the mean ± S.D. **p < 0.01. p values were determined with unpaired Student’s t -tests. DOI: http://dx.doi.org/10.7554/eLife.27692.002 10.7554/eLife.27692.003 Figure 1—source data 1. Summary of the fertility rates and mortality rates of the offspring of 4- or 18-month-old Ripk 3 +/+ and Ripk 3 -/- male mice. DOI: http://dx.doi.org/10.7554/eLife.27692.003 10.7554/eLife.27692.004 Figure 1—source data 2. Summary of the fertility rates and mortality rates of the offspring of 13-month-old Ripk 3 +/+ and Ripk 3 -/- male mice. DOI: http://dx.doi.org/10.7554/eLife.27692.004

Article Snippet: The Antibody against RIPK3 (#2283; WB, 1:1000; IHC, 1:100) were obtained from ProSci (San Diego, CA).

Techniques: Knock-Out, Enzyme-linked Immunosorbent Assay, Staining

Journal: eLife

Article Title: RIPK1-RIPK3-MLKL-dependent necrosis promotes the aging of mouse male reproductive system

doi: 10.7554/eLife.27692

Figure Lengend Snippet: ( A ) Mortality of offspring from Ripk 3 +/+ and Ripk 3 -/- male mice. One male mice of a given age was mated with a pairs of 10-week-old female wild-type mice for 3 months; females were replaced every 2 weeks. Litters were counted by date of birth of the pups; if a litter was born but did not survive, we counted the dead pups; if we were not able to count the pups, the number of pups was entered as ‘0’. Mortality of offspring from 4-month-old Ripk 3 +/+ (n = 5) and Ripk 3 -/- (n = 5) male mice; offspring from 18-month-old Ripk3 +/+ (n = 4) and Ripk 3 -/- (n = 15) male mice were calculated. Data represent the mean ± S.D. **p < 0.01, ***p < 0.001. P values were determined with unpaired Student’s t -tests. NS, not significant. ( B, C ) IHC of Ripk 3 +/+ and Ripk 3 -/- testes with 8-OHdG antibody. Mice were sacrificed, testes from Ripk 3 +/+ (4 Months, n = 6; 18 Months, n = 6) and Ripk 3 -/- (4 Months, n = 6; 18 Months, n = 6) mice were harvested and stained with 8-OHdG antibody in ( B ) (black arrows for sperm with 8-OHdG staining). 8-OHdG + sperm were counted in five fields per testis and quantification in ( C ). Scale bar, 100 μm. Data represent the mean ± S.D. ***p < 0.001. p values were determined with unpaired Student’s t-tests. DOI: http://dx.doi.org/10.7554/eLife.27692.011

Article Snippet: The Antibody against RIPK3 (#2283; WB, 1:1000; IHC, 1:100) were obtained from ProSci (San Diego, CA).

Techniques: Staining

Journal: eLife

Article Title: RIPK1-RIPK3-MLKL-dependent necrosis promotes the aging of mouse male reproductive system

doi: 10.7554/eLife.27692

Figure Lengend Snippet: ( A ) Immunofluorescence of testes from Ripk 3 +/+ and Ripk 3 -/- mice (2 weeks) with RIPK3 antibody. Counterstaining with DAPI, blue. Scale bar, 100 μm. ( B ) No p-MLKL signaling in the testes from aged Mlkl -/- mice. WT (18 Months, n = 3) and Mlkl -/- (18 Months, n = 3) mice were sacrificed and testes were harvested and stained with p-MLKL antibody (black arrows indicate cells with p-MLKL staining). Scale bar, 100 μm. DOI: http://dx.doi.org/10.7554/eLife.27692.014

Article Snippet: The Antibody against RIPK3 (#2283; WB, 1:1000; IHC, 1:100) were obtained from ProSci (San Diego, CA).

Techniques: Immunofluorescence, Staining

Journal: eLife

Article Title: RIPK1-RIPK3-MLKL-dependent necrosis promotes the aging of mouse male reproductive system

doi: 10.7554/eLife.27692

Figure Lengend Snippet: ( A ) RIPK3 expression in spermatogonia, Sertoli cells, and spermatocytes. Immunofluorescence in an 8-week-old testis with antibodies against RIPK3 (red), HSD3B1 (Leydig cells specific protein, green), GATA-1 (Sertoli cells specific protein, green), and UTF1 (spermatogonium specific protein, green). Scale bar, 100 μm. ( B ) RIPK3 expression in germ line cells and Sertoli cells. Primary testis cells were isolated from wild-type testes, Immunofluorescence of Leydig cells, Sertoli cells, spermatogonia, and primary spermatocytes with antibodies against RIPK3 (red), HSD3B1 (green), GATA-1 (green), UTF1 (green), and SMAD3 (primary spermatocytes specific protein, green). Counterstaining with DAPI, blue. Scale bar, 10 μm. ( C, D ) Immunohistochemistry (IHC) of testes from Ripk 3 +/+ and Ripk 3 -/- mice with phosphor-MLKL (p-MLKL) antibody. Ripk 3 +/+ (4 Months, n = 6; 18 Months, n = 6) and Ripk 3 -/- (4 Months, n = 6; 18 Months, n = 6) mice were sacrificed and testes were harvested and stained with p-MLKL antibody in ( C ) (black arrows indicate cells with p-MLKL staining). p-MLKL + cells were counted in five fields per testis and quantification in ( D ). Scale bar, 100 μm. Data represent the mean ± s.e.m. ***p<0.001. p values were determined with unpaired Student’s t -tests. NS, not significant. ( E ) Western blot analysis of RIPK1, RIPK3, MLKL, and p-MLKL levels in the testis after perfusion, each group is representative of at least three mice. GAPDH was used as loading control. The asterisk (*) indicates non-specific bands. ( F ) Immunofluorescence in an 18-month-old testis with antibodies against p-MLKL (red, purple arrows indicate spermatogonium with p-MLKL staining), HSD3B1, GATA-1, and UTF1. Scale bar, 50 μm. DOI: http://dx.doi.org/10.7554/eLife.27692.013

Article Snippet: The Antibody against RIPK3 (#2283; WB, 1:1000; IHC, 1:100) were obtained from ProSci (San Diego, CA).

Techniques: Expressing, Immunofluorescence, Isolation, Immunohistochemistry, Staining, Western Blot

Journal: eLife

Article Title: RIPK1-RIPK3-MLKL-dependent necrosis promotes the aging of mouse male reproductive system

doi: 10.7554/eLife.27692

Figure Lengend Snippet: ( A, B ) IHC of testis from Ripk +/+ and Ripk 3 -/- mice with Cleaved-Caspase-3 antibody. Mice were sacrificed, testes from Ripk 3 +/+ (4 Month, n = 6; 18 Month, n = 6) and Ripk 3 -/- (4 Month, n = 6; 18 Month, n = 6) mice were harvested and stained with Cleaved-Caspase-3 antibody in ( A ) (black arrows for Leydig cells with Cleaved-Caspase-3 staining). Cleaved-Caspase-3 + cells were counted in six fields per testis and quantification in ( B ). Scale bar, 100 μm. Data represent the mean ± s.e.m. *p < 0.05, ***p<0.001. p values were determined with unpaired Student’s t -tests. ( C, D ) IHC of testis from Ripk +/+ and Ripk -/- mice with Cleaved-Caspase-8 antibody. Mice were sacrificed, testes from Ripk +/+ (4 Month, n = 6; 18 Month, n = 6) and Ripk 3 -/- (4 Month, n = 6; 18 Month, n = 6) mice were harvested and stained with Cleaved-caspase-8 antibody in ( C ) (black arrows for Leydig cells with Cleaved-caspase-8 staining). Cleaved-caspase-8 + cells were counted in six fields per testis and quantification in ( D ). Scale bar, 100 μm. Data represent the mean ± s.e.m. *p<0.05, ***p<0.001. p values were determined with unpaired Student’s t -tests. ( E, F ) Caspase8 levels decrease during aging in empty seminiferous tubules. Immunofluorescence of testes from 4-month-old and 18-month-old wild-type mice with caspase8 and RIPK3 antibody in ( E ). The caspase8 levels were quantified in ( F ). Counterstaining with DAPI, blue. Scale bar, 100 μm. DOI: http://dx.doi.org/10.7554/eLife.27692.015

Article Snippet: The Antibody against RIPK3 (#2283; WB, 1:1000; IHC, 1:100) were obtained from ProSci (San Diego, CA).

Techniques: Staining, Immunofluorescence

Journal: eLife

Article Title: RIPK1-RIPK3-MLKL-dependent necrosis promotes the aging of mouse male reproductive system

doi: 10.7554/eLife.27692

Figure Lengend Snippet: ( A, B ) IHC of testis from Ripk 3 +/+ and Ripk 3 -/- mice with Cleaved-Caspase-3 antibody. Mice were sacrificed, testes from Ripk 3 +/+ (4 Month, n = 6; 36 Months, n = 6) and Ripk 3 -/- (4 Month, n = 6; 36 Months, n = 6) mice were harvested and stained with Cleaved-Caspase-3 antibody in ( A ) (black arrows for Leydig cells with Cleaved-Caspase-3 staining). Cleaved-Caspase-3 + cells were counted in six fields per testis and quantification in ( B ). Scale bar, 100 μm. Data represent the mean ± s.e.m. *p<0.05, ***p<0.001. p values were determined with unpaired Student’s t -tests. ( C ) Western blot analysis of RIPK3 and Cleaved-caspase-3 levels in the testis after perfusion; each group is representative of at least three mice. GAPDH was used as a loading control. DOI: http://dx.doi.org/10.7554/eLife.27692.016

Article Snippet: The Antibody against RIPK3 (#2283; WB, 1:1000; IHC, 1:100) were obtained from ProSci (San Diego, CA).

Techniques: Staining, Western Blot

Journal: eLife

Article Title: RIPK1-RIPK3-MLKL-dependent necrosis promotes the aging of mouse male reproductive system

doi: 10.7554/eLife.27692

Figure Lengend Snippet: ( A ) Western blot analysis of RIPK1, RIPK3, MLKL, and p-MLKL levels in testes after injection with TSZ. The 2-month-old wild-type male mice continuously feed with RIPA-56 (0 mg/kg, n = 6; 150 mg/kg, n = 3; 300 mg/kg, n = 3) for one week. Testes were injected with TSZ (see Materials and methods); 72 hr after the injection, mice were sacrificed and the testes were harvested. The proteins were extracted from testes and were analyzed with western blotting. GAPDH was used as a loading control. ( B–H ) 13-month-old wild-type male mice were feed with AIN93G (RIPA-56:0 mg/kg) or AIN93G-RIPA-56 (RIPA-56:300 mg/kg) for 2 months in SPF facility. Mice were weighed before and after feed with RIPA-56 in ( B ). Data represent the mean ± s.e.m. *p<0.05, ***p<0.001. p values were determined with unpaired Student’s t -tests. Mice were sacrificed and the seminal vesicles were photographed and weighed. Macroscopic features and weights of seminal vesicles form mice in ( C, D ). Data represent the mean ± s.e.m. ***p<0.001. p values were determined with unpaired Student’s t -tests. Testosterone levels in serum from mice were measured using a testosterone ELISA kit in ( E ). Data represent the mean ± s.e.m. **p<0.01. p values were determined with unpaired Student’s t -tests. The testes were harvested and stained with H and E in ( F ). The number of empty seminiferous tubules was counted based on H and E staining and quantification in ( G ). Empty seminiferous tubules were counted in five fields per testis. Scale bar, 100 μm. Data represent the mean ± S.D. **p<0.01. p values were determined with unpaired Student’s t -tests. The fertility rate of each RIPA-56-treated (0 mg/kg, n = 23; 300 mg/kg, n = 25) male mice was assessed by mating it with four 10-week-old wild-type female mice successively (see Materials and methods). The number of mice with reproductive capacity was counted in ( H ). p values were determined using chi-square tests. ( I–K ) 13-month-old wild-type male mice were feed with AIN93G (RIPA-56:0 mg/kg) or AIN93G-RIPA-56 (RIPA-56:300 mg/kg) for 5 months in SPF facility. The fertility rate of each RIPA-56-treated (0 mg/kg, n = 15; 300 mg/kg, n = 15) male mice was assessed by mating it with four 10-week-old wild-type female mice successively (see Materials and methods). The number of mice with reproductive capacity was counted in ( I ). p values were determined using chi-square tests. After fertility test, mice were sacrificed and testes were harvested and stained with p-MLKL antibody in ( J ) (black arrows indicate cells with p-MLKL staining). p-MLKL + cells were counted in five fields per testis and quantification in ( K ). Scale bar, 100 μm. Data represent the mean ± s.e.m. ***p<0.001. P values were determined with unpaired Student’s t -tests. DOI: http://dx.doi.org/10.7554/eLife.27692.022

Article Snippet: The Antibody against RIPK3 (#2283; WB, 1:1000; IHC, 1:100) were obtained from ProSci (San Diego, CA).

Techniques: Western Blot, Injection, Enzyme-linked Immunosorbent Assay, Staining

Journal: eLife

Article Title: RIPK1-RIPK3-MLKL-dependent necrosis promotes the aging of mouse male reproductive system

doi: 10.7554/eLife.27692

Figure Lengend Snippet: Western blot analysis of TNF-α, RIPK1, RIPK3, MLKL, Cleaved-caspase3 and p-MLKL levels in the testis after perfusion, each group is representative of at least three mice. GAPDH was used as loading control. DOI: http://dx.doi.org/10.7554/eLife.27692.023

Article Snippet: The Antibody against RIPK3 (#2283; WB, 1:1000; IHC, 1:100) were obtained from ProSci (San Diego, CA).

Techniques: Western Blot