Figure 1 ( A ) Schematic representation of the study design and Western blot analysis for TIP30 and GAPDH in hearts from TIP30 wild - type ( WT ) , heterozygous ( Het ) and homozygous knock - out ( KO ) mice under basal conditions . Quantification of heart weight ( HW ) / tibia length ( TL ) ratio ( B ) N = 4 to 18 mice / group , all 6 weeks after TAC or sham surgery . Quantification of lung weight ( LuW / TL ) ratio ( C ) N = 4 to 18 mice / group , all 6 weeks after TAC or sham surgery . Quantification of , echocardiographic fractional area change ( D ) N = 4 to 18 mice / group , all 6 weeks after TAC or sham surgery . Quantification of left ventricular enddiastolic area ( LVEDA ; E ) N = 4 to 18 mice / group , all 6 weeks after TAC or sham surgery . Quantification of dP / dt max and dP / dt min ( Millar catheter ; F , G ) N = 4 to 18 mice / group , all 6 weeks after TAC or sham surgery . Quantification of α - MHC transcript abundance N = 4 to 18 mice / group , all 6 weeks after TAC or sham surgery . Quantification of β - MHC transcript abundance N = 4 to 18 mice / group , all 6 weeks after TAC or sham surgery . Representative images of Sirius - red - stained heart sections ( scale bar : 1mm ) ( J ) fibrosis quantification ( K ) of indicated mice 6 weeks after TAC or sham surgery . Quantification of cardiomyocyte area of isolated adult cardiac myocytes ( N = 4 to 11 mice / group ) of indicated mice 6 weeks after TAC or sham surgery . * P < 0 . 05 . One - way ANOVA with Sidak ´ s multiple comparisons test . Microscopy images of heart sections of indicated mice 6 weeks after TAC surgery stained for Isolectin B4 ( green ) and WGA ( red , M ) quantification of capillaries per myocyte ( N ) . ( N = 5 to 7 mice / group , scale bar : 50µm ) . * * P < 0 . 01 . One - way ANOVA with Sidak ´ s multiple comparisons test . ( P ) Western blot analysis for TIP30 and GAPDH in hearts from TIP30 wild - type ( WT ) and heterozygous ( Het ) after AAV - TropT - TIP30 or AAV - Control ( AAV - Con ) injection followed by 6 weeks of TAC surgery . Quantification of HW / TL ratio ( Q ) ratio in AAV - Con or AAV - TropT - TIP30 treated Tip30 heterozygous ( Het ) or WT mice 6 weeks after TAC or sham surgery ( N = 5 to 11 mice / group ) . Quantification of LuW / TL ( R ) ratio in AAV - Con or AAV - TropT - TIP30 treated Tip30 heterozygous ( Het ) or WT mice 6 weeks after TAC or sham surgery ( N = 5 to 11 mice / group ) .

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Figure 1(A) Schematic representation of the study design and Western blot analysis for TIP30 and GAPDH in hearts from TIP30 wild-type (WT), heterozygous (Het) and homozygous knock-out (KO) mice under basal conditions.Quantification of heart weight (HW)/tibia length (TL) ratio (B) N=4 to 18 mice/group, all 6 weeks after TAC or sham surgery.Quantification of lung weight (LuW/TL) ratio (C) N=4 to 18 mice/group, all 6 weeks after TAC or sham surgery.Quantification of , echocardiographic fractional area change (D) N=4 to 18 mice/group, all 6 weeks after TAC or sham surgery.Quantification of left ventricular enddiastolic area (LVEDA; E) N=4 to 18 mice/group, all 6 weeks after TAC or sham surgery.Quantification of dP/dt max and dP/dt min (Millar catheter; F, G) N=4 to 18 mice/group, all 6 weeks after TAC or sham surgery.Quantification of α-MHC transcript abundance N=4 to 18 mice/group, all 6 weeks after TAC or sham surgery.Quantification of β-MHC transcript abundance N=4 to 18 mice/group, all 6 weeks after TAC or sham surgery.Representative images of Sirius-red-stained heart sections (scale bar: 1mm) (J)fibrosis quantification (K) of indicated mice 6 weeks after TAC or sham surgery.Quantification of cardiomyocyte area of isolated adult cardiac myocytes (N=4 to 11 mice/group) of indicated mice 6 weeks after TAC or sham surgery. *P<0.05. One-way ANOVA with Sidak´s multiple comparisons test.Microscopy images of heart sections of indicated mice 6 weeks after TAC surgery stained for Isolectin B4 (green) and WGA (red, M)quantification of capillaries per myocyte (N). (N=5 to 7 mice/group, scale bar: 50µm). **P<0.01. One-way ANOVA with Sidak´s multiple comparisons test.(P) Western blot analysis for TIP30 and GAPDH in hearts from TIP30 wild-type (WT) and heterozygous (Het) after AAV-TropT-TIP30 or AAV-Control (AAV-Con) injection followed by 6 weeks of TAC surgery.Quantification of HW/TL ratio (Q) ratio in AAV-Con or AAV-TropT-TIP30 treated Tip30 heterozygous (Het) or WT mice 6 weeks after TAC or sham surgery (N=5 to 11 mice/group).Quantification of LuW/TL (R) ratio in AAV-Con or AAV-TropT-TIP30 treated Tip30 heterozygous (Het) or WT mice 6 weeks after TAC or sham surgery (N=5 to 11 mice/group).


Figure 2 ( A ) Western blot for TIP30 and GAPDH in neonatal rat cardiomyocytes ( NRCM ) after adenoviral transduction with Ad . Control ( Ad . Con ) or Ad . TIP30 . Quantification of cardiomyocyte area ( B ) , N = 6 to 8 samples / group in NRCM transduced with Ad . Con or Ad . TIP30 and stimulated as indicated . ET - 1 : endothelin - 1 , FBS : fetal bovine serum , PE : phenylephrine . Quantification of protein / DNA ratio ( C ) , N = 9 samples / group in NRCM transduced with Ad . Con or Ad . TIP30 and stimulated as indicated . PE : phenylephrine . Quantification of Acta1 mRNA transcript abundance ( D ) , N = 3 samples / group in NRCM transduced with Ad . Con or Ad . TIP30 and stimulated as indicated . ET - 1 : endothelin - 1 PE : phenylephrine . Quantification of cell death with a 7 - AAD assay ( E ) , N = 7 samples / group in NRCM transduced with Ad . Con or Ad . TIP30 and stimulated as indicated . PE : phenylephrine . ( G ) Western blot for TIP30 and Actin in mouse hearts with AAV9 mediated overexpression of TIP30 ( AAV - TIP30 ) or from mice treated with a control AAV9 construct ( AAV - Con ) followed by 2 weeks of TAC surgery . Quantification of HW / TL ratio ( H ) , N = 8 to 13 mice / group 2 weeks after sham or TAC surgery in AAV - Con or AAV - TIP30 treated C57BL / 6 WT mice . * Quantification of cardiomyocyte area ( I ) , N = 4 to 5 mice / group 2 weeks after sham or TAC surgery in AAV - Con or AAV - TIP30 treated C57BL / 6 WT mice . ( J ) Serial echocardiography with quantification of echocardiographic fractional area change 2 , 4 and 6 weeks after sham or TAC surgery in AAV - Con or AAV - TIP30 treated C57BL / 6 WT mice ( N = 10 to 14 mice / group and time point ) .

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Figure 2(A) Western blot for TIP30 and GAPDH in neonatal rat cardiomyocytes (NRCM) after adenoviral transduction with Ad.Control (Ad.Con) or Ad.TIP30.Quantification of cardiomyocyte area (B), N=6 to 8 samples/group in NRCM transduced with Ad.Con or Ad.TIP30 and stimulated as indicated. ET-1: endothelin-1, FBS: fetal bovine serum, PE: phenylephrine.Quantification of protein/DNA ratio (C), N=9 samples/group in NRCM transduced with Ad.Con or Ad.TIP30 and stimulated as indicated. PE: phenylephrine.Quantification of Acta1 mRNA transcript abundance (D), N=3 samples/group in NRCM transduced with Ad.Con or Ad.TIP30 and stimulated as indicated. ET-1: endothelin-1 PE: phenylephrine.Quantification of cell death with a 7-AAD assay (E), N=7 samples/group in NRCM transduced with Ad.Con or Ad.TIP30 and stimulated as indicated. PE: phenylephrine.(G) Western blot for TIP30 and Actin in mouse hearts with AAV9 mediated overexpression of TIP30 (AAV-TIP30) or from mice treated with a control AAV9 construct (AAV-Con) followed by 2 weeks of TAC surgery.Quantification of HW/TL ratio (H), N=8 to 13 mice/group 2 weeks after sham or TAC surgery in AAV-Con or AAV-TIP30 treated C57BL/6 WT mice. *Quantification of cardiomyocyte area (I), N=4 to 5 mice/group 2 weeks after sham or TAC surgery in AAV-Con or AAV-TIP30 treated C57BL/6 WT mice.(J) Serial echocardiography with quantification of echocardiographic fractional area change 2, 4 and 6 weeks after sham or TAC surgery in AAV-Con or AAV-TIP30 treated C57BL/6 WT mice (N=10 to 14 mice/group and time point).


Figure 3 ( A ) Western blot analysis of GST - pulldown assays with GST - TIP30 fragments of the indicated length ( as : amino acids from N - terminus ) with Myc - tagged binding partners . ( B ) Western blot analysis of co - immunoprecipitation ( IP ) from HEK cells transfected with GST - TIP30 and eEF1A1 - Myc . ( C ) Confocal microscopy images of neonatal rat cardiomyocytes ( NRCM ) stained with antibodies for endogenous TIP30 ( green ) and endogenous eEF1A1 ( red ) . DAPI : blue . ( Scale bar : 20µm ) . ( D ) Microscopy images of isolated neonatal rat cardiomyocytes and subsequent Proximity Ligation Assay ( PLA ) . Red : endogenous TIP30 / eEF1A1 interaction ; Blue : DAPI . Control cells were stained for TIP30 / Myc - Tag interaction ( scale bar : 50µm ) . ( E ) Western blot analysis of co - immunoprecipitation ( IP ) for endogenous TIP30 and eEF1A1 protein in NRCM after stimulation with phenylephrine for 24 h . IgG HC - High chain of IgG molecule . Western blot analysis of GST - pulldown assays with GST or GST - tagged eEF1A1 ( GST - eEF1A1 ) and TIP30 - His full length protein ( TIP30 - His ) and TIP30 deletion mutants ΔN25 ( TIP30 - ΔN25 - His ) , ΔN52 ( TIP30 - ΔN52 - His ) , ΔC15 ( TIP30 - ΔC15 - His ) and Δ102 - 107 ( TIP30 - ΔN102 - 104 - His , G ) Western blot analysis of GST - tagged eEF1A1 and GST - Control ( H ) . ( J ) Quantitative Real - Time PCR analysis of eEF1A1 and eEF1A2 mRNA abundance in hearts from neonatal mice , adult wild - type mice ( Adult ) and adult wild - type mice 2 weeks after TAC surgery ( Adult TAC , N = 3 to 4 mice / group ) . * P < 0 . 05 , * * * P < 0 . 001 . One - way ANOVA with Sidak ´ s multiple comparisons test . ( K ) Western blot analysis for eEF1A1 , eEF1A2 and GAPDH in hearts from wild - type mice at the age of 7 days ( 7d ) and 8 weeks ( 8w ) as well as 2 weeks ( TAC 2w ) after TAC surgery . ( L ) Western blot analysis of co - immunoprecipitation ( IP ) with GST - TIP30 and eEF1A2 - His .

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Figure 3(A) Western blot analysis of GST-pulldown assays with GST-TIP30 fragments of the indicated length (as: amino acids from N-terminus) with Myc-tagged binding partners.(B) Western blot analysis of co-immunoprecipitation (IP) from HEK cells transfected with GST-TIP30 and eEF1A1-Myc.(C) Confocal microscopy images of neonatal rat cardiomyocytes (NRCM) stained with antibodies for endogenous TIP30 (green) and endogenous eEF1A1 (red). DAPI: blue. (Scale bar: 20µm).(D) Microscopy images of isolated neonatal rat cardiomyocytes and subsequent Proximity Ligation Assay (PLA). Red: endogenous TIP30/eEF1A1 interaction; Blue: DAPI. Control cells were stained for TIP30/Myc-Tag interaction (scale bar: 50µm).(E) Western blot analysis of co-immunoprecipitation (IP) for endogenous TIP30 and eEF1A1 protein in NRCM after stimulation with phenylephrine for 24 h. IgG HC - High chain of IgG molecule.Western blot analysis of GST-pulldown assays with GST or GST-tagged eEF1A1 (GST-eEF1A1) and TIP30-His full length protein (TIP30-His) and TIP30 deletion mutants ΔN25 (TIP30-ΔN25-His), ΔN52 (TIP30-ΔN52-His), ΔC15 (TIP30-ΔC15-His) and Δ102-107 (TIP30-ΔN102-104-His, G)Western blot analysis of GST-tagged eEF1A1 and GST-Control (H).(J) Quantitative Real-Time PCR analysis of eEF1A1 and eEF1A2 mRNA abundance in hearts from neonatal mice, adult wild-type mice (Adult) and adult wild-type mice 2 weeks after TAC surgery (Adult TAC, N=3 to 4 mice/group). *P<0.05, ***P<0.001. One-way ANOVA with Sidak´s multiple comparisons test.(K) Western blot analysis for eEF1A1, eEF1A2 and GAPDH in hearts from wild-type mice at the age of 7 days (7d) and 8 weeks (8w) as well as 2 weeks (TAC 2w) after TAC surgery.(L) Western blot analysis of co-immunoprecipitation (IP) with GST-TIP30 and eEF1A2-His.


Figure 4 ( A ) Western blot analysis of GST - pulldown assays with GST or GST - eEF1A1 and purified eEF1B2 - His . Purified TIP30 - His was added in indicated amounts . ( B ) Western blot analysis of anti - Myc immunoprecipitation ( IP ) in NRCM co - transduced with Ad . eEF1A1 - Myc and either control virus ( Ad . con ) or Ad . TIP30 . Endogenous eEF1B2 was detected . The IP input for eEF1B2 , eEF1A1 - myc and TIP30 is shown below . ( C ) Quantification of eEF1B2 abundance after eEF1A1 - myc IP under conditions shown in ( B ) ( D ) Microscopy images of NRCM after adenoviral transduction with Ad . TIP30 or control virus ( Ad . Con ) and stimulation with phenylephrine ( PE ) and subsequent Proximity Ligation Assay ( PLA ) . Red : eEF1A1 - eEF1B2 interaction ; Blue : DAPI . ( scale bar : 50µm ) . ( E ) Quantification of eEF1A1 - eEF1B2 interaction in conditions described in ( D ) ( F ) Microscopy images of adult mouse cardiomyocytes isolated from hearts 3 days ( d ) after TAC or sham surgery and subsequent PLA . Red : eEF1A1 - eEF1B2 interaction ; Blue : DAPI ( scale bar : 100µm ) . ( G ) Quantification of eEF1A1 - eEF1B2 interaction in adult mouse cardiomyocytes isolated from hearts 3 days after TAC or sham surgery and subsequent PLA ( N = 4 to 6 mice / group ) . *

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Figure 4(A) Western blot analysis of GST-pulldown assays with GST or GST-eEF1A1 and purified eEF1B2-His. Purified TIP30-His was added in indicated amounts.(B) Western blot analysis of anti-Myc immunoprecipitation (IP) in NRCM co-transduced with Ad.eEF1A1-Myc and either control virus (Ad.con) or Ad.TIP30. Endogenous eEF1B2 was detected. The IP input for eEF1B2, eEF1A1-myc and TIP30 is shown below. (C) Quantification of eEF1B2 abundance after eEF1A1-myc IP under conditions shown in (B) (D) Microscopy images of NRCM after adenoviral transduction with Ad.TIP30 or control virus (Ad.Con) and stimulation with phenylephrine (PE) and subsequent Proximity Ligation Assay (PLA). Red: eEF1A1-eEF1B2 interaction; Blue: DAPI. (scale bar: 50µm). (E) Quantification of eEF1A1-eEF1B2 interaction in conditions described in (D) (F) Microscopy images of adult mouse cardiomyocytes isolated from hearts 3 days (d) after TAC or sham surgery and subsequent PLA. Red: eEF1A1-eEF1B2 interaction; Blue: DAPI (scale bar: 100µm). (G) Quantification of eEF1A1-eEF1B2 interaction in adult mouse cardiomyocytes isolated from hearts 3 days after TAC or sham surgery and subsequent PLA (N=4 to 6 mice/group). *


Figure 5Western blot analysis for TIP30 , eEF1A1 and GAPDH in C57BL / 6 WT mice 3 days ( A , B ) , N = 4 mice / group , 2 weeks ( C , D ) , N = 4 to 8 mice / group and 6 weeks ( E , F ) , N = 4 to 8 mice / group after TAC or sham surgery and their quantification . KO denotes TIP30 homozygous knock - out . ( G , H ) Western blot for TIP30 , eEF1A1 and Actin in TIP30 Het mice 6 after TAC surgery and their quantification ( N = 4 mice / group ) . ( I , J ) Western blot for TIP30 , eEF1A1 and Actin 2 weeks after sham or TAC surgery in AAV - Con or AAV - TIP30 treated C57BL / 6 WT mice and their quantification ( N = 4 mice / group ) . A ratio of TIP30 and eEF1A1 expression was calculated for each condition .

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Figure 5Western blot analysis for TIP30, eEF1A1 and GAPDH in C57BL/6 WT mice 3 days (A, B), N=4 mice/group, 2 weeks (C, D), N=4 to 8 mice/group and 6 weeks (E, F), N=4 to 8 mice/group after TAC or sham surgery and their quantification. KO denotes TIP30 homozygous knock-out.(G, H) Western blot for TIP30, eEF1A1 and Actin in TIP30 Het mice 6 after TAC surgery and their quantification (N=4 mice/group).(I, J) Western blot for TIP30, eEF1A1 and Actin 2 weeks after sham or TAC surgery in AAV-Con or AAV-TIP30 treated C57BL/6 WT mice and their quantification (N=4 mice/group). A ratio of TIP30 and eEF1A1 expression was calculated for each condition.


Figure 6Quantification of Tip30 and eEF1A1 mRNA transcript abundance in human failing hearts ( A ) , N = 6 to 8 hearts / group A ratio of Tip30 and eEF1A1 expression was calculatedQuantification of Tip30 and eEF1A1 mRNA transcript abundance in patients with Hypertrophic Cardiomyopathy ( HCM ; B , N = 4 to 8 hearts / group ) A ratio of Tip30 and eEF1A1 expression was calculatedQuantification of Tip30 and eEF1A1 mRNA transcript abundance in 6 months old mdx mice or WT mice ( C ) , N = 4 mice / group . A ratio of Tip30 and eEF1A1 expression was calculated ( E ) Echocardiographic diastolic left - ventricular wall - thickness in MDX mice at the age of 2 months at baseline , and 1 - 9 months ( mo ) after injection of AAV9 - Con or AAV9 - TIP30 ( TIP ; N = 6 to 7 mice / group ) . ( F ) Quantification of HW / TL ratio of 9mo old MDX mice , N = 5 to 6 mice / group .

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Figure 6Quantification of Tip30 and eEF1A1 mRNA transcript abundance in human failing hearts (A), N=6 to 8 hearts/group A ratio of Tip30 and eEF1A1 expression was calculatedQuantification of Tip30 and eEF1A1 mRNA transcript abundance in patients with Hypertrophic Cardiomyopathy (HCM; B, N=4 to 8 hearts/group) A ratio of Tip30 and eEF1A1 expression was calculatedQuantification of Tip30 and eEF1A1 mRNA transcript abundance in 6 months old mdx mice or WT mice (C), N=4 mice/group. A ratio of Tip30 and eEF1A1 expression was calculated(E) Echocardiographic diastolic left-ventricular wall-thickness in MDX mice at the age of 2 months at baseline, and 1-9 months (mo) after injection of AAV9-Con or AAV9-TIP30 (TIP; N=6 to 7 mice/group).(F) Quantification of HW/TL ratio of 9mo old MDX mice, N=5 to 6 mice/group.


Figure 7 ( A ) Western blot analysis of isolated neonatal rat cardiomyocytes ( NRCM ) after adenoviral transduction either with control adenovirus ( Con ) or Ad . TIP30 ( TIP30 ) followed by stimulation with phenylephrine ( PE , for 3 hours ) and puromycin incorporation ( for 30 min ) . ( B ) Quantification of the Western blot shown in ( A ) ( N = 3 samples / group ) . ( C ) Quantification of cell surface area of isolated neonatal mouse cardiomyocyte of Tip30 Het and WT mice treated with endothelin - 1 ( ET - 1 ) and Narciclasine ( Narci ) or without stimulation as indicated ( N = 6 samples / group ) . Quantification of HW / TL ratio ( E ) , N = 6 to 10 mice / group , in Tip30 Het or WT mice 2 weeks after TAC . Animals were treated with Narciclasine daily for 14 days after TAC as indicated . Quantification of , echocardiographic wall thickness ( F ) , N = 6 to 10 mice / group , in Tip30 Het or WT mice 2 weeks after TAC . Animals were treated with Narciclasine daily for 14 days after TAC as indicated . Quantification of cardiomyocyte area ( G ) , N = 5 mice / group in Tip30 Het or WT mice 2 weeks after TAC . Animals were treated with Narciclasine daily for 14 days after TAC as indicated . Quantification of fractional area change ( FAC , H , N = 6 to 10 mice / group ) in Tip30 Het or WT mice 2 weeks after TAC . Animals were treated with Narciclasine daily for 14 days after TAC as indicated . ( I ) Western blot analysis of puromycin incorporation in hearts of Tip30 Het and WT mice 3 days after TAC or sham surgery and daily Narciclasine ( Narci ) injection and their quantification ( N = 2 to 4 mice / group ) . Puromycin was injected 3 hours prior to sacrifice .

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 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 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 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 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

Figure 7(A) Western blot analysis of isolated neonatal rat cardiomyocytes (NRCM) after adenoviral transduction either with control adenovirus (Con) or Ad.TIP30 (TIP30) followed by stimulation with phenylephrine (PE, for 3 hours) and puromycin incorporation (for 30 min). (B) Quantification of the Western blot shown in (A) (N=3 samples/group). (C) Quantification of cell surface area of isolated neonatal mouse cardiomyocyte of Tip30 Het and WT mice treated with endothelin-1 (ET-1) and Narciclasine (Narci) or without stimulation as indicated (N=6 samples/group).Quantification of HW/TL ratio (E), N=6 to 10 mice/group, in Tip30 Het or WT mice 2 weeks after TAC. Animals were treated with Narciclasine daily for 14 days after TAC as indicated.Quantification of , echocardiographic wall thickness (F), N=6 to 10 mice/group, in Tip30 Het or WT mice 2 weeks after TAC. Animals were treated with Narciclasine daily for 14 days after TAC as indicated.Quantification of cardiomyocyte area (G), N=5 mice/group in Tip30 Het or WT mice 2 weeks after TAC. Animals were treated with Narciclasine daily for 14 days after TAC as indicated.Quantification of fractional area change (FAC, H, N=6 to 10 mice/group) in Tip30 Het or WT mice 2 weeks after TAC. Animals were treated with Narciclasine daily for 14 days after TAC as indicated.(I) Western blot analysis of puromycin incorporation in hearts of Tip30 Het and WT mice 3 days after TAC or sham surgery and daily Narciclasine (Narci) injection and their quantification (N=2 to 4 mice/group). Puromycin was injected 3 hours prior to sacrifice.


Figure 1 ( A ) After randomized , 8 - 12 weeks old and sex - matched Phb1F / F mice and Phb1MyeKO mice were administered with PBS or LPS ( 30 mg / kg ) for 12 hours via intraperitoneal injection . Serum was collected and ELISA assays were used to detect the level of IL - 1β ( n = 6 , 10 , 6 , and 12 mice for each group , respectively ) . Data presented as mean ± SEM . * p < 0 . 05 , * * p < 0 . 01 , * * * * p < 0 . 0001 ( Student ' s t - test ) . ( B ) BMDMs were extracted from Phb1F / F or Phb1MyeKO mice and stimulated by 20 % culture supernatant of L929 cells for 7 days . Differentiated BMDMs were stimulated with LPS ( 200 ng / ml ) for 6 hours and ATP ( 4 mM ) for 45 minutes , the cells were lysed and the culture medium was collected . Western blotting ( WB ) assays were used to detect protein levels in whole cell lysates ( Lys ) and culture medium ( Sup ) . ( C ) BMDMs were extracted from Phb1F / F or Phb1MyeKO mice and stimulated with 20 % culture supernatant of L929 cells for 7 days . Differentiated BMDMs were stimulated with LPS ( 200 ng / ml ) for 6 hours and ATP ( 4 mM ) for 45 minutes . ELISA assays were used to detect protein levels in the culture medium . Data of BMDMs from the separate mice ( n = 6 mice for each group ) and are presented as mean ± SEM . * * * p < 0 . 001 ( Student ' s t - test ) . ( D ) After randomized , 4 weeks old and sex - matched Phb1F / F mice and Phb1MyeKO mice were administered with PBS or LPS ( 300 mg / kg ) to induce septic shock via intraperitoneal injection . The life span of Phb1F / F mice ( n = 6 ) and Phb1MyeKO mice ( n = 7 ) was recorded . ( E ) PBMCs were extracted from normal controls and patients with sepsis ( n = 6 individuals ) . Intracellular mRNA levels were detected by real - time quantitative PCR ( RT - qPCR ) assays ( mean ± SEM ) . n . s . , no significance , * p < 0 . 05 , * * p < 0 . 01 ( Student ' s t - test ) . ( F ) PBMCs were extracted from normal controls and infant patients with sepsis . Protein levels in whole cell lysates were detected by WB assays . J774A . 1 cells were stimulated with LPS ( 200 ng / ml ) for 6 hours Intracellular mRNA levels were detected by RT - qPCR assays . Data are from four independent experiments for ( G ) ( mean ± SEM ) . n . s . , no significance , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 ( Student ' s t - test ) . J774A . 1 cells were stimulated with recombinant IL - 1β ( 100 pg / ml ) for 24 hours . Intracellular mRNA levels were detected by RT - qPCR assays . Data are from six independent experiments for ( H ) ( mean ± SEM ) . n . s . , no significance , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 ( Student ' s t - test ) . ( I ) After J774A . 1 cells were stimulated with LPS ( 200 ng / ml ) for 0 , 3 , 6 , 12 hours and ATP ( 4 mM ) for 45 minutes , the cells were lysed , and the culture medium was collected . Protein levels in whole cell lysates ( Lys ) and culture medium ( Sup ) were detected by WB assays . ( J ) After J774A . 1 cells with Phb1 or Phb2 knockdown were stimulated with LPS ( 200 ng / ml ) for 6 hours and ATP ( 4 mM ) for 45 minutes , the cells were lysed , and the culture medium was collected . Protein levels in whole cell lysates ( Lys ) and culture medium ( Sup ) were detected by WB assays . ( K ) After J774A . 1 cells with Phb1 or Phb2 knockdown were stimulated with LPS ( 200 ng / ml ) for 6 hours and ATP ( 4 mM ) for 45 minutes , the culture medium was collected . ELISA assays were used to detect IL - 1β levels in the culture medium . Data are from three independent experiments ( mean ± SEM ) . n . s . , no significance , * * * * p < 0 . 0001 ( 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 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 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 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 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 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 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 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 O O O O O O O O O O O O O O O O O O O O O O O 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

Figure 1(A) After randomized, 8-12 weeks old and sex-matched Phb1F/F mice and Phb1MyeKO mice were administered with PBS or LPS (30 mg/kg) for 12 hours via intraperitoneal injection. Serum was collected and ELISA assays were used to detect the level of IL-1β (n = 6, 10, 6, and 12 mice for each group, respectively). Data presented as mean ± SEM. * p < 0.05, ** p < 0.01, **** p< 0.0001 (Student's t-test).(B) BMDMs were extracted from Phb1F/F or Phb1MyeKO mice and stimulated by 20% culture supernatant of L929 cells for 7 days. Differentiated BMDMs were stimulated with LPS (200 ng/ml) for 6 hours and ATP (4 mM) for 45 minutes, the cells were lysed and the culture medium was collected. Western blotting (WB) assays were used to detect protein levels in whole cell lysates (Lys) and culture medium (Sup).(C) BMDMs were extracted from Phb1F/F or Phb1MyeKO mice and stimulated with 20% culture supernatant of L929 cells for 7 days. Differentiated BMDMs were stimulated with LPS (200 ng/ml) for 6 hours and ATP (4 mM) for 45 minutes. ELISA assays were used to detect protein levels in the culture medium. Data of BMDMs from the separate mice (n = 6 mice for each group) and are presented as mean ± SEM. *** p < 0.001 (Student's t-test).(D) After randomized, 4 weeks old and sex-matched Phb1F/F mice and Phb1MyeKO mice were administered with PBS or LPS (300 mg/kg) to induce septic shock via intraperitoneal injection. The life span of Phb1F/F mice (n = 6) and Phb1MyeKO mice (n = 7) was recorded.(E) PBMCs were extracted from normal controls and patients with sepsis (n = 6 individuals). Intracellular mRNA levels were detected by real-time quantitative PCR (RT-qPCR) assays (mean ± SEM). n.s., no significance, * p < 0.05, ** p < 0.01 (Student's t-test).(F) PBMCs were extracted from normal controls and infant patients with sepsis. Protein levels in whole cell lysates were detected by WB assays.J774A.1 cells were stimulated with LPS (200 ng/ml) for 6 hours Intracellular mRNA levels were detected by RT-qPCR assays. Data are from four independent experiments for (G) (mean ± SEM). n.s., no significance, ** p < 0.01, *** p < 0.001, **** p< 0.0001 (Student's t-test).J774A.1 cells were stimulated with recombinant IL-1β (100 pg/ml) for 24 hours. Intracellular mRNA levels were detected by RT-qPCR assays. Data are from six independent experiments for (H) (mean ± SEM). n.s., no significance, ** p < 0.01, *** p < 0.001, **** p< 0.0001 (Student's t-test).(I) After J774A.1 cells were stimulated with LPS (200 ng/ml) for 0, 3, 6, 12 hours and ATP (4 mM) for 45 minutes, the cells were lysed, and the culture medium was collected. Protein levels in whole cell lysates (Lys) and culture medium (Sup) were detected by WB assays.(J) After J774A.1 cells with Phb1 or Phb2 knockdown were stimulated with LPS (200 ng/ml) for 6 hours and ATP (4 mM) for 45 minutes, the cells were lysed, and the culture medium was collected. Protein levels in whole cell lysates (Lys) and culture medium (Sup) were detected by WB assays.(K) After J774A.1 cells with Phb1 or Phb2 knockdown were stimulated with LPS (200 ng/ml) for 6 hours and ATP (4 mM) for 45 minutes, the culture medium was collected. ELISA assays were used to detect IL-1β levels in the culture medium. Data are from three independent experiments (mean ± SEM). n.s., no significance, **** p < 0.0001 (Student's t-test).


Figure 2 ( A - B ) GFP - expressing HeLa cells with stable Phb1 knockdown were fixed and subjected to immunofluorescence ( IF ) analysis to detect mitochondria ( MitoTracker , red ; A ) or ΔΨm ( TMRM , red ; B ) . Images shown are representative of at least three independent experiments . White boxed regions in the panels in ( A ) are enlarged . Scale bar : 10 μm . ( C ) Flow cytometric analysis of ΔΨm ( TMRM ) . HeLa cells with stable Phb1 knockdown were treated with CsA ( 2 μM ) for 30 minutes or FCCP ( 4 μM ) for 5 minutes . ( D ) The mitochondrial respiratory capacity of J774A . 1 cells with or without Phb1 knockdown was measured with a Seahorse XFe96 analyzer . Data are from at least three independent experiments ( mean ± SEM ) . n . s . , no significance . * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 ( Student ' s t - test ) . ( E ) HeLa cells with stable PHB1 ablation were incubated with H2O2 ( 2 μM ) and CsA ( 2 μM ) for 30 minutes . Flow cytometric analysis of intracellular ROS levels ( DHE ) . ( F - G ) Confocal microscopy analyses of mitochondrial Ca2 + in time - series mode . HeLa cells stably expressing 4mt - RCaMPh with or without Phb1 knockdown were treated with CsA ( 2 μM ) for 30 minutes . During acquisition of fluorescence images ( F ) , cells were treated with histamine ( 200 μM ) . Traces of mitochondrial Ca2 + ( 4mt - RCaMPh ) dynamics are shown in ( G ) ( mean ± SEM ) . n = 16 , 16 , 15 , 16 ( from the top to the bottom ) . Scale bar : 10 μm . * p < 0 . 05 relative to Scramble group , # p < 0 . 05 relative to shPHB1 group ( 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 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 O O O O O O O O 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 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

Figure 2(A-B) GFP-expressing HeLa cells with stable Phb1 knockdown were fixed and subjected to immunofluorescence (IF) analysis to detect mitochondria (MitoTracker, red; A) or ΔΨm (TMRM, red; B). Images shown are representative of at least three independent experiments. White boxed regions in the panels in (A) are enlarged. Scale bar: 10 μm.(C) Flow cytometric analysis of ΔΨm (TMRM). HeLa cells with stable Phb1 knockdown were treated with CsA (2 μM) for 30 minutes or FCCP (4 μM) for 5 minutes.(D) The mitochondrial respiratory capacity of J774A.1 cells with or without Phb1 knockdown was measured with a Seahorse XFe96 analyzer. Data are from at least three independent experiments (mean ± SEM). n.s., no significance. ** p < 0.01, *** p < 0.001, **** p < 0.0001 (Student's t-test).(E) HeLa cells with stable PHB1 ablation were incubated with H2O2 (2 μM) and CsA (2 μM) for 30 minutes. Flow cytometric analysis of intracellular ROS levels (DHE).(F-G) Confocal microscopy analyses of mitochondrial Ca2+ in time-series mode. HeLa cells stably expressing 4mt-RCaMPh with or without Phb1 knockdown were treated with CsA (2 μM) for 30 minutes. During acquisition of fluorescence images (F), cells were treated with histamine (200 μM). Traces of mitochondrial Ca2+ (4mt-RCaMPh) dynamics are shown in (G) (mean ± SEM). n = 16, 16, 15, 16 (from the top to the bottom). Scale bar: 10 μm. * p < 0.05 relative to Scramble group, # p < 0.05 relative to shPHB1 group (Student's t-test).


Figure 3 ( A ) The co - localization between mtDNA ( SG - ALK , green ) and mitochondria ( MitoTracker , red ) was examined in J774A . 1 cells with or without knockdown of Phb1 or Phb2 . White boxed regions in the panels are enlarged . The white arrows indicate mtDNA outside mitochondria . Scale bar : 10 μm . ( B ) Quantitative analysis of data from ( A ) . Data are from ten , eight , and seven images from three independent experiments ( left to right , respectively ) ( mean ± SEM ) . n . s . , no significance , * * * p < 0 . 001 ( Student ' s t - test ) . ( C ) J774A . 1 cells with or without Phb1 knockdown were lysed , and DNA was extracted . qPCR assays were used to detect the mtDNA levels ( D - loop ) in the cytosol relative to nuclear DNA levels ( Tert ) in the whole cell lysates . Data are from six independent experiments ( mean ± SEM ) . * * * * p < 0 . 0001 ( Student ' s t - test ) . ( D ) BMDMs isolated from Phb1F / F mice and Phb1MyeKO mice were fixed and subjected to IF analysis to detect the co - localization between mitochondria ( MitoTracker , red ) and mtDNA ( SG - ALK , green ) . White boxed regions in the panels are enlarged . The white arrows indicate mtDNA outside mitochondria . Scale bar : 10 μm . ( E ) Quantitative analysis of data from ( D ) . Data are from ten and eight images from three independent experiments ( mean ± SEM ) . * * p < 0 . 01 ( Student ' s t - test ) . ( F ) PBMCs were extracted from Phb1F / F mice and Phb1MyeKO mice . Cells were lysed , and DNA was extracted . qPCR assays were used to detect the levels of mtDNA ( D - loop ) in the cytosol relative to the levels of nuclear DNA levels ( Tert ) in the whole cell lysates . Data of PBMC are from seven Phb1F / F mice and Phb1MyeKO mice , respectively ( mean ± SEM ) . * * p < 0 . 01 ( Student ' s t - test ) . ( G ) HeLa cells expressing TFAM - FLAG with or without Phb1 knockdown were incubated with CsA ( 2 μM ) for 30 minutes . Cells were fixed and subjected to IF analysis to detect the co - localization between mitochondria ( anti - TOMM20 , red ) and TFAM - FLAG ( anti - FLAG , green ) . White boxed regions in the panels are enlarged . The white arrows indicate mtDNA outside mitochondria . The yellow arrows indicate mtDNA inside mitochondria . Scale bar : 10 μm . ( H ) Quantitative analysis of data from ( G ) . Data from 30 , 27 , 25 , and 24 images from three independent experiments ( mean ± SEM ) . * * * * p < 0 . 0001 ( Student ' s t - test ) . ( I ) HeLa cells with or without Phb1 knockdown were incubated with H2O2 ( 5 mM ) for 20 minutes and CsA ( 4 μM ) for 12 hours . Cells were lysed , and DNA was extracted . qPCR assays were used to detect mtDNA levels ( ND1 ) in the cytosol and nuclear DNA levels ( 18S ) in the whole cell lysates . Data are from four independent experiments ( mean ± SEM ) . * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 ( Student ' s t - test ) . HeLa cells with or without Phb1 knockdown were incubated with VBIT - 4 ( 10 μM ) for 1 hour and subsequently lysed , and DNA was extracted . qPCR assays were used to detect mtDNA levels ( ND1 in HeLa cells in the cytosol relative to nuclear DNA levels ( TERT in HeLa cells in the whole cell lysates . Data are from three independent experiments ( mean ± SEM ) . n . s . , no significance , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 ( Student ' s t - test ) . J774A . 1 cells with or without Phb1 knockdown were incubated with VBIT - 4 ( 10 μM ) for 1 hour and subsequently lysed , and DNA was extracted . qPCR assays were used to detect mtDNA levels D - loop in J774A . 1 cells ) in the cytosol relative to nuclear DNA levels Tert in J774A . 1 cells ) in the whole cell lysates . Data are from three independent experiments ( mean ± SEM ) . n . s . , no significance , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 ( Student ' s t - test ) . ( L ) J774A . 1 cells with or without Phb1 knockdown were lysed . The whole cell lysates were immunoprecipitated by IgG or anti - ASC antibody and the resulting proteins were detected by WB assay . ( M ) BMDMs isolated from Phb1F / F mice and Phb1MyeKO mice were incubated with MCC950 ( 50 nM ) for 30 mins and were subsequently stimulated by LPS ( 200 ng / ml ) for 6 hours and ATP ( 4 mM ) for 45 minutes . Protein levels in whole cell lysates and culture medium were detected by WB assay .

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 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 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 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 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 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 O O O O O O O O O O O O O O O O O O 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 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 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 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

Figure 3(A) The co-localization between mtDNA (SG-ALK, green) and mitochondria (MitoTracker, red) was examined in J774A.1 cells with or without knockdown of Phb1 or Phb2. White boxed regions in the panels are enlarged. The white arrows indicate mtDNA outside mitochondria. Scale bar: 10 μm. (B) Quantitative analysis of data from (A). Data are from ten, eight, and seven images from three independent experiments (left to right, respectively) (mean ± SEM). n.s., no significance, *** p < 0.001 (Student's t-test).(C) J774A.1 cells with or without Phb1 knockdown were lysed, and DNA was extracted. qPCR assays were used to detect the mtDNA levels (D-loop) in the cytosol relative to nuclear DNA levels (Tert) in the whole cell lysates. Data are from six independent experiments (mean ± SEM). **** p < 0.0001 (Student's t-test).(D) BMDMs isolated from Phb1F/F mice and Phb1MyeKO mice were fixed and subjected to IF analysis to detect the co-localization between mitochondria (MitoTracker, red) and mtDNA (SG-ALK, green). White boxed regions in the panels are enlarged. The white arrows indicate mtDNA outside mitochondria. Scale bar: 10 μm. (E) Quantitative analysis of data from (D). Data are from ten and eight images from three independent experiments (mean ± SEM). ** p < 0.01 (Student's t-test).(F) PBMCs were extracted from Phb1F/F mice and Phb1MyeKO mice. Cells were lysed, and DNA was extracted. qPCR assays were used to detect the levels of mtDNA (D-loop) in the cytosol relative to the levels of nuclear DNA levels (Tert) in the whole cell lysates. Data of PBMC are from seven Phb1F/F mice and Phb1MyeKO mice, respectively (mean ± SEM). ** p < 0.01 (Student's t-test).(G) HeLa cells expressing TFAM-FLAG with or without Phb1 knockdown were incubated with CsA (2 μM) for 30 minutes. Cells were fixed and subjected to IF analysis to detect the co-localization between mitochondria (anti-TOMM20, red) and TFAM-FLAG (anti-FLAG, green). White boxed regions in the panels are enlarged. The white arrows indicate mtDNA outside mitochondria. The yellow arrows indicate mtDNA inside mitochondria. Scale bar: 10 μm.(H) Quantitative analysis of data from (G). Data from 30, 27, 25, and 24 images from three independent experiments (mean ± SEM). **** p < 0.0001 (Student's t-test).(I) HeLa cells with or without Phb1 knockdown were incubated with H2O2 (5 mM) for 20 minutes and CsA (4 μM) for 12 hours. Cells were lysed, and DNA was extracted. qPCR assays were used to detect mtDNA levels (ND1) in the cytosol and nuclear DNA levels (18S) in the whole cell lysates. Data are from four independent experiments (mean ± SEM). ** p < 0.01, *** p < 0.001, **** p < 0.0001 (Student's t-test).HeLa cells with or without Phb1 knockdown were incubated with VBIT-4 (10 μM) for 1 hour and subsequently lysed, and DNA was extracted. qPCR assays were used to detect mtDNA levels (ND1 in HeLa cells in the cytosol relative to nuclear DNA levels (TERT in HeLa cells in the whole cell lysates. Data are from three independent experiments (mean ± SEM). n.s., no significance, * p < 0.05, ** p < 0.01, *** p < 0.001 (Student's t-test).J774A.1 cells with or without Phb1 knockdown were incubated with VBIT-4 (10 μM) for 1 hour and subsequently lysed, and DNA was extracted. qPCR assays were used to detect mtDNA levels D-loop in J774A.1 cells) in the cytosol relative to nuclear DNA levels Tert in J774A.1 cells) in the whole cell lysates. Data are from three independent experiments (mean ± SEM). n.s., no significance, * p < 0.05, ** p < 0.01, *** p < 0.001 (Student's t-test).(L) J774A.1 cells with or without Phb1 knockdown were lysed. The whole cell lysates were immunoprecipitated by IgG or anti-ASC antibody and the resulting proteins were detected by WB assay.(M) BMDMs isolated from Phb1F/F mice and Phb1MyeKO mice were incubated with MCC950 (50 nM) for 30 mins and were subsequently stimulated by LPS (200 ng/ml) for 6 hours and ATP (4 mM) for 45 minutes. Protein levels in whole cell lysates and culture medium were detected by WB assay.


Figure 4 ( A ) BMDMs isolated from Phb1F / F mice and Phb1MyeKO mice were stimulated with LPS ( 200 ng / ml ) for 6 hours and ATP ( 4 mM ) for 45 minutes . Cells were fixed and subjected to IF analysis to detect the co - localization between NLRP3 ( anti - NLRP3 , red ) and mtDNA ( PicoGreen , green ) . White boxed regions in the panels are enlarged . The white arrows indicate mtDNA co - localized with NLRP3 . Scale bar : 10 μm . ( B ) Quantitative analysis of data from ( A ) . Data are from at least 14 images from three independent experiments ( mean ± SEM ) . * * * * p < 0 . 0001 ( Student ' s t - test ) . ( C ) J774A . 1 cells with or without Phb1 knockdown were stimulated with LPS ( 200 ng / ml ) for 6 hours and ATP ( 4 mM ) for 45 minutes . Cells were fixed and subjected to IF analysis to detect the co - localization between NLRP3 ( anti - NLRP3 , red ) and mtDNA ( PicoGreen , green ) . White boxed regions in the panels are enlarged . The white arrows indicate mtDNA co - localized with NLRP3 . Scale bar : 10 μm . ( D ) Quantitative analysis of data from ( C ) . Data are from at least six images from three independent experiments ( mean ± SEM ) . * p < 0 . 05 , * * p < 0 . 01 , * * * * p < 0 . 0001 ( Student ' s t - test ) . ( E ) J774A . 1 cells with or without Phb1 knockdown were stimulated with LPS ( 200 ng / ml ) for 6 hours and ATP ( 4 mM ) for 45 minutes . After cells were lysed , NLRP3 was immunoprecipitated by anti - NLRP3 antibody . mtDNA levels ( D - loop ) from immunoprecipitants were detected by qPCR in J774A . 1 cells with or without Phb1 knockdown . Data are from five independent experiments ( mean ± SEM ) . * * p < 0 . 01 , * * * * p < 0 . 0001 ( Student ' s t - test ) . ( F ) J774A . 1 cells with Phb1 knockdown were incubated with EtBr ( 150 ng / ml ) for 3 days , followed by LPS ( 200 ng / ml ) for 6 hours and ATP ( 4 mM ) for 45 minutes . WB was used to detect the protein levels in whole cell lysates and culture medium . ( G ) BMDMs isolated from Phb1F / F mice and Phb1MyeKO mice were treated with NAC ( 2 mM ) for 8 hours , LPS ( 200 ng / ml ) for 6 hours and ATP ( 4 mM ) for 45 minutes . WB assay was used to detect the protein levels in whole cell lysates and in the culture medium .

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 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 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 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 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 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

Figure 4(A) BMDMs isolated from Phb1F/F mice and Phb1MyeKO mice were stimulated with LPS (200 ng/ml) for 6 hours and ATP (4 mM) for 45 minutes. Cells were fixed and subjected to IF analysis to detect the co-localization between NLRP3 (anti-NLRP3, red) and mtDNA (PicoGreen, green). White boxed regions in the panels are enlarged. The white arrows indicate mtDNA co-localized with NLRP3. Scale bar: 10 μm. (B) Quantitative analysis of data from (A). Data are from at least 14 images from three independent experiments (mean ± SEM). **** p < 0.0001 (Student's t-test).(C) J774A.1 cells with or without Phb1 knockdown were stimulated with LPS (200 ng/ml) for 6 hours and ATP (4 mM) for 45 minutes. Cells were fixed and subjected to IF analysis to detect the co-localization between NLRP3 (anti-NLRP3, red) and mtDNA (PicoGreen, green). White boxed regions in the panels are enlarged. The white arrows indicate mtDNA co-localized with NLRP3. Scale bar: 10 μm.(D) Quantitative analysis of data from (C). Data are from at least six images from three independent experiments (mean ± SEM). * p <0.05, ** p < 0.01, **** p < 0.0001 (Student's t-test).(E) J774A.1 cells with or without Phb1 knockdown were stimulated with LPS (200 ng/ml) for 6 hours and ATP (4 mM) for 45 minutes. After cells were lysed, NLRP3 was immunoprecipitated by anti-NLRP3 antibody. mtDNA levels (D-loop) from immunoprecipitants were detected by qPCR in J774A.1 cells with or without Phb1 knockdown. Data are from five independent experiments (mean ± SEM). ** p < 0.01, **** p < 0.0001 (Student's t-test).(F) J774A.1 cells with Phb1 knockdown were incubated with EtBr (150 ng/ml) for 3 days, followed by LPS (200 ng/ml) for 6 hours and ATP (4 mM) for 45 minutes. WB was used to detect the protein levels in whole cell lysates and culture medium.(G) BMDMs isolated from Phb1F/F mice and Phb1MyeKO mice were treated with NAC (2 mM) for 8 hours, LPS (200 ng/ml) for 6 hours and ATP (4 mM) for 45 minutes. WB assay was used to detect the protein levels in whole cell lysates and in the culture medium.


Figure 5 ( A ) HeLa cells were incubated with H2O2 ( 5 mM ) for 20 minutes and then lysed . The whole cell lysate was immunoprecipitated by IgG , anti - AFG3L2 antibody or anti - SPG7 antibody . WB was used to detect the SPG7 or AFG3L2 levels , respectively , in the precipitated products . ( B ) J774A . 1 cells were stimulated with LPS ( 200 ng / ml ) for 6 hours and lysed . The whole cell lysate was immunoprecipitated by IgG , anti - AFG3L2 antibody or anti - SPG7 antibody . The respective levels of precipitated SPG7 or AFG3L2 were examined by WB . HeLa cells with or without Phb1 knockdown were lysed . The whole cell lysate was immunoprecipitated by IgG , anti - AFG3L2 antibody or anti - SPG7 antibody . The respective levels of precipitated SPG7 or AFG3L2 were examined by WB . J774A . 1 cells with or without Phb1 knockdown were lysed . The whole cell lysate was immunoprecipitated by IgG , anti - AFG3L2 antibody or anti - SPG7 antibody . The respective levels of precipitated SPG7 or AFG3L2 were examined by WB .

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 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 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

Figure 5(A) HeLa cells were incubated with H2O2 (5 mM) for 20 minutes and then lysed. The whole cell lysate was immunoprecipitated by IgG, anti-AFG3L2 antibody or anti-SPG7 antibody. WB was used to detect the SPG7 or AFG3L2 levels, respectively, in the precipitated products.(B) J774A.1 cells were stimulated with LPS (200 ng/ml) for 6 hours and lysed. The whole cell lysate was immunoprecipitated by IgG, anti-AFG3L2 antibody or anti-SPG7 antibody. The respective levels of precipitated SPG7 or AFG3L2 were examined by WB.HeLa cells with or without Phb1 knockdown were lysed. The whole cell lysate was immunoprecipitated by IgG, anti-AFG3L2 antibody or anti-SPG7 antibody. The respective levels of precipitated SPG7 or AFG3L2 were examined by WB.J774A.1 cells with or without Phb1 knockdown were lysed. The whole cell lysate was immunoprecipitated by IgG, anti-AFG3L2 antibody or anti-SPG7 antibody. The respective levels of precipitated SPG7 or AFG3L2 were examined by WB.


Figure 6 ( A ) J774A . 1 cells with knockdown of Phb1 alone , Afg3l2 alone , Spg7 alone , Phb1 + Afg3l2 , or Phb1 + Spg7 were fixed and subjected to IF analysis to assess the co - localization between mitochondria ( MitoTracker , red ) and mtDNA ( PicoGreen , green ) . White boxed regions in the panels are enlarged . The white arrows indicate mtDNA outside mitochondria . Scale bar : 10 μm . ( B ) Quantitative analysis of the data from ( A ) . Data are from at least 12 images from three independent experiments ( mean ± SEM ) . * * p < 0 . 01 , * * * * p < 0 . 0001 ( Student ' s t - test ) . ( C ) J774A . 1 cells with knockdown of Phb1 alone , Afg3l2 alone , Spg7 alone , Phb1 + Afg3l2 , or Phb1 + Spg7 were lysed , and DNA was extracted . qPCR was used to detect the mtDNA levels ( D - loop ) in the cytosol relative to the nuclear DNA levels ( Tert ) in the whole cell lysate . Data are from six independent experiments ( mean ± SEM ) . * * * * p < 0 . 0001 ( Student ' s t - test ) . ( D ) J774A . 1 cells with knockdown of Phb1 alone , Afg3l2 alone , Spg7 alone , Phb1 + Afg3l2 , or Phb1 + Spg7 were stimulated with LPS ( 200 ng / ml ) for 6 hours and ATP ( 4 mM ) for 45 minutes . The culture medium was collected and ELISA assays were used to detect IL - 1β levels in culture medium . Data are from four independent experiments ( mean ± SEM ) . * * * * p < 0 . 0001 ( Student ' s t - test ) . ( E ) J774A . 1 cells with Phb1 knockdown , Spg7 knockdown , or combined Phb1 and Spg7 knockdowns were stimulated with LPS ( 200 ng / ml ) for 6 hours and ATP ( 4 mM ) for 45 minutes . WB assay was used to detect the protein levels in whole cell lysates and culture medium . ( F ) J774A . 1 cells with Phb1 knockdown , Afg3l2 knockdown , and combined Phb1 and Afg3l2 knockdowns were stimulated with LPS ( 200 ng / ml ) for 6 hours and ATP ( 4 mM ) for 45 minutes . WB assay was used to detect the protein levels in whole cell lysates and culture medium . ( G ) J774A . 1 cells were stimulated with LPS ( 200 ng / ml ) for 6 hours and lysed . The whole cell lysate was immunoprecipitated by IgG or anti - SPG7 antibody . WB was used to detect the CYPD and VDAC levels in the precipitated products . ( H ) J774A . 1 cells with or without Phb1 knockdown were lysed . The whole cell lysates were immunoprecipitated by IgG or anti - SPG7 antibody . WB was used to detect the CYPD and VDAC levels in the precipitated products .

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 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 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 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 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

Figure 6(A) J774A.1 cells with knockdown of Phb1 alone, Afg3l2 alone, Spg7 alone, Phb1 + Afg3l2, or Phb1 + Spg7 were fixed and subjected to IF analysis to assess the co-localization between mitochondria (MitoTracker, red) and mtDNA (PicoGreen, green). White boxed regions in the panels are enlarged. The white arrows indicate mtDNA outside mitochondria. Scale bar: 10 μm. (B) Quantitative analysis of the data from (A). Data are from at least 12 images from three independent experiments (mean ± SEM). ** p < 0.01, **** p < 0.0001 (Student's t-test).(C) J774A.1 cells with knockdown of Phb1 alone, Afg3l2 alone, Spg7 alone, Phb1 + Afg3l2, or Phb1 + Spg7 were lysed, and DNA was extracted. qPCR was used to detect the mtDNA levels (D-loop) in the cytosol relative to the nuclear DNA levels (Tert) in the whole cell lysate. Data are from six independent experiments (mean ± SEM). **** p < 0.0001 (Student's t-test).(D) J774A.1 cells with knockdown of Phb1 alone, Afg3l2 alone, Spg7 alone, Phb1 + Afg3l2, or Phb1 + Spg7 were stimulated with LPS (200 ng/ml) for 6 hours and ATP (4 mM) for 45 minutes. The culture medium was collected and ELISA assays were used to detect IL-1β levels in culture medium. Data are from four independent experiments (mean ± SEM). **** p < 0.0001 (Student's t-test).(E) J774A.1 cells with Phb1 knockdown, Spg7 knockdown, or combined Phb1 and Spg7 knockdowns were stimulated with LPS (200 ng/ml) for 6 hours and ATP (4 mM) for 45 minutes. WB assay was used to detect the protein levels in whole cell lysates and culture medium.(F) J774A.1 cells with Phb1 knockdown, Afg3l2 knockdown, and combined Phb1 and Afg3l2 knockdowns were stimulated with LPS (200 ng/ml) for 6 hours and ATP (4 mM) for 45 minutes. WB assay was used to detect the protein levels in whole cell lysates and culture medium.(G) J774A.1 cells were stimulated with LPS (200 ng/ml) for 6 hours and lysed. The whole cell lysate was immunoprecipitated by IgG or anti-SPG7 antibody. WB was used to detect the CYPD and VDAC levels in the precipitated products.(H) J774A.1 cells with or without Phb1 knockdown were lysed. The whole cell lysates were immunoprecipitated by IgG or anti-SPG7 antibody. WB was used to detect the CYPD and VDAC levels in the precipitated products.


Figure 2 ( A ) Anti - acetyllysine Western blot analyses and total protein stains of E . coli cell extracts . Cellular protein extracts before and after induction of His6 - MBP - GNAT expression were separated on 12 % acrylamide gels and immunoblotted by using an anti - acetyllysine antibody or stained with Coomassie Dye . GNAT1 , 2 , 5 , 6 , 7 and 10 as well as His6 - MBP control were expressed in E . coli BL21 ( DE3 ) pLysS . GNAT3 and 4 were expressed in Rosetta ( DE3 ) . In the total protein stains , recombinant GNAT protein constructs were highlighted by blue arrowheads . Protein expression before ( ni ) and after ( i ) induction with IPTG is indicated . The luminescence signal , indicating acetylated proteins , was usually recorded after 40 - 120 seconds . Since MBP - GNAT4 expression resulted in a saturated signal , the luminescence was additionally recorded for 10 seconds ( indicated by an asterisk ) .

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

Figure 2(A) Anti-acetyllysine Western blot analyses and total protein stains of E. coli cell extracts. Cellular protein extracts before and after induction of His6-MBP-GNAT expression were separated on 12 % acrylamide gels and immunoblotted by using an anti-acetyllysine antibody or stained with Coomassie Dye. GNAT1, 2, 5, 6, 7 and 10 as well as His6-MBP control were expressed in E. coli BL21(DE3)pLysS. GNAT3 and 4 were expressed in Rosetta(DE3). In the total protein stains, recombinant GNAT protein constructs were highlighted by blue arrowheads. Protein expression before (ni) and after (i) induction with IPTG is indicated. The luminescence signal, indicating acetylated proteins, was usually recorded after 40-120 seconds. Since MBP-GNAT4 expression resulted in a saturated signal, the luminescence was additionally recorded for 10 seconds (indicated by an asterisk).


Figure 3 ( A ) Number of characterized substrates with significant increases of NTA yields . The NTA yields of all retrieved N - termini upon a given GNAT expression was divided into four categories and each column is the result of the sum of all categories : 50 - 100 % , dark green ; 20 - 50 % , light green ; 10 - 20 % , orange ; 5 - 10 % , red . The number of protein occurrence with a yield higher than 30 % is also displayed to visualize the most significant acetylation status of each GNAT . The respective overexpression level of each GNAT as assessed from gel electrophoresis ( + + + + is for very high expression , + + + for high , + + for medium , + for intermediate ) .

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

Figure 3(A) Number of characterized substrates with significant increases of NTA yields. The NTA yields of all retrieved N-termini upon a given GNAT expression was divided into four categories and each column is the result of the sum of all categories: 50-100%, dark green; 20-50%, light green; 10-20%, orange; 5-10%, red. The number of protein occurrence with a yield higher than 30% is also displayed to visualize the most significant acetylation status of each GNAT. The respective overexpression level of each GNAT as assessed from gel electrophoresis (++++ is for very high expression, +++ for high, ++ for medium, + for intermediate).


Figure 5 ( C ) Comparison of NTA yield of retrieved N - termini of proteins starting at position 1 or 2 . The majority of these proteins , corresponding mostly to cytosolic components , undergoing or not to N - terminal methionine excision , were not affected by inactivation of GNAT2 . For statistical analyses nsi - 1 and nsi - 2 were pooled and compared to the wild type . Two independent technical replicates of four biological replicates for each of the WT , nsi - 1 and nsi - 2 samples were analysed . Error bars are ± SD . ( D ) Comparison of NTA yield of retrieved N - termini of proteins starting at positions > 2 . Clear alteration of NTA yield was observed in nsi mutant lines in the pool of nuclear - encoded plastid proteins . nsi - 1 and nsi - 2 samples were treated as in D . Error bars are ± SD ( E ) Comparison of NTA yields of retrieved N - termini in plastid proteins . Similar variation as in panel D was observed when NTA of only plastid proteins was analyzed . ( F ) Volcano plot representing NTA analyses of nsi knockout lines ( treated together ) and wild type . For this analysis , the p - value was calculated using Excel ' s two - tailed t - test function , for two - sample with equal variance . The most impacted proteins are shown in green . N is related to the number of quantified N - termini .

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 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(C) Comparison of NTA yield of retrieved N-termini of proteins starting at position 1 or 2. The majority of these proteins, corresponding mostly to cytosolic components, undergoing or not to N-terminal methionine excision, were not affected by inactivation of GNAT2. For statistical analyses nsi-1 and nsi-2 were pooled and compared to the wild type. Two independent technical replicates of four biological replicates for each of the WT, nsi-1 and nsi-2 samples were analysed. Error bars are ± SD. (D) Comparison of NTA yield of retrieved N-termini of proteins starting at positions > 2. Clear alteration of NTA yield was observed in nsi mutant lines in the pool of nuclear-encoded plastid proteins. nsi-1 and nsi-2 samples were treated as in D. Error bars are ± SD (E) Comparison of NTA yields of retrieved N-termini in plastid proteins. Similar variation as in panel D was observed when NTA of only plastid proteins was analyzed.(F) Volcano plot representing NTA analyses of nsi knockout lines (treated together) and wild type. For this analysis, the p-value was calculated using Excel's two-tailed t-test function, for two-sample with equal variance. The most impacted proteins are shown in green. N is related to the number of quantified N-termini.


Figure 1 ( L ) Larval NSCs of control ( ctrl ; yw ) , pr - set7716 / Df , and UAS - pr - set7 induced with insc - Gal4 at 72h ALH were stained for Pr - set7 , Mira and DNA . ( M , N ) Quantifications of NSC diameters ( M ) at 24h ALH and EdU incorporation ( N ) for genotypes in ( O ) . In ( M ) , n = 152 for control ; n = 129 for pr - set7716 / Df . In ( N ) , for control , n = 601 , 13 BL at 24h ALH ; n = 988 , 14 BL at 48h ALH ; n = 813 , 9 BL at 72h ALH and n = 829 , 10 BL at 96h ALH . For pr - set7716 / Df , n = 566 , 12 BL at 24h ALH ; n = 808 , 13 BL at 48h ALH ; n = 1032 , 10 BL at 72h ALH and n = 825 , 9 BL at 96h ALH . Data information : Quantification data are presented in as mean ± SD M , N ) . n numbers in N ) were total NSCs counted with total brain lobes measured , while each brain lobe considered as one biological replicate . White arrows point to NSCs Statistical analyses were done comparing two different genotypes using Mann - Whitney test M , N ) . * * * P < 0 . 001 . Scale bars , 5 μm L ) ,

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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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(L) Larval NSCs of control (ctrl; yw), pr-set7716/Df, and UAS-pr-set7 induced with insc-Gal4 at 72h ALH were stained for Pr-set7, Mira and DNA. (M, N) Quantifications of NSC diameters (M) at 24h ALH and EdU incorporation (N) for genotypes in (O). In (M), n=152 for control; n=129 for pr-set7716/Df. In (N), for control, n=601, 13 BL at 24h ALH; n=988, 14 BL at 48h ALH; n=813, 9 BL at 72h ALH and n=829, 10 BL at 96h ALH. For pr-set7716/Df, n=566, 12 BL at 24h ALH; n=808, 13 BL at 48h ALH; n=1032, 10 BL at 72h ALH and n=825, 9 BL at 96h ALH. Data information: Quantification data are presented in as mean ± SD M, N). n numbers in N) were total NSCs counted with total brain lobes measured, while each brain lobe considered as one biological replicate. White arrows point to NSCs Statistical analyses were done comparing two different genotypes using Mann-Whitney test M, N). ***P < 0.001. Scale bars, 5 μm L),


Figure 2 ( C ) Larval NSCs of control ( ctrl ; yw ) , pr - set7716 / Df , pr - set7716 / Df with UAS - pr - set7 induced with insc - Gal4 , and pr - set7716 / Df with pr - set7 - BAC at 24h ALH were stained for H4K20me1 , Dpn , Mira and DAPI . Control : n = 108 ; pr - set7716 / Df : n = 87 ; pr - set7716 / Df with UAS - pr - set7 induced with insc - Gal4 : n = 167 and pr - set7716 / Df with pr - set7 - BAC : n = 104 . Data information : White arrows point to NSCs . Scale bars , 5 μm ( E ) Larval NSCs with FUCCI constructs induced with insc - Gal4 at 24h were stained for GFP , RFP , Dpn , and Prospero ( Pros ) . ( F ) Quantifications of quiescent NSCs in different cell - cycle stages from FUCCI flies ( induced with insc - Gal4 ) in control ( and insc - Gal4 > FUCCI ) and pr - set7716 / Df . Quiescent NSCs of insc - Gal4 > FUCCI : 76 . 1 % ± 1 . 5 % in G2 , 1 . 7 % ± 0 . 2 % in S and 22 . 2 % ± 1 . 3 % in G0 ( n = 343 ) . Quiescent NSCs of insc - Gal4 > FUCCI ; pr - set7716 / Df : 74 . 4 % ± 1 . 0 % in G2 , 0 . 9 % ± 0 . 2 % in S and 24 . 7 % ± 0 . 8 % in G0 ( n = 344 ) . Data information : Quantification data are presented in as mean ± SD Statistical analyses were done comparing two different genotypes using Mann - Whitney test ns : non - significant . White arrows point to NSCs . Scale bars , 5

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 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 O O O O O O O O O O O O O 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(C) Larval NSCs of control (ctrl; yw), pr-set7716/Df, pr-set7716/Df with UAS-pr-set7 induced with insc-Gal4, and pr-set7716/Df with pr-set7-BAC at 24h ALH were stained for H4K20me1, Dpn, Mira and DAPI. Control: n=108; pr-set7716/Df: n=87; pr-set7716/Df with UAS-pr-set7 induced with insc-Gal4: n=167 and pr-set7716/Df with pr-set7-BAC: n=104. Data information: White arrows point to NSCs. Scale bars, 5 μm(E) Larval NSCs with FUCCI constructs induced with insc-Gal4 at 24h were stained for GFP, RFP, Dpn, and Prospero (Pros). (F) Quantifications of quiescent NSCs in different cell-cycle stages from FUCCI flies (induced with insc-Gal4) in control (and insc-Gal4>FUCCI) and pr-set7716/Df. Quiescent NSCs of insc-Gal4>FUCCI: 76.1%±1.5% in G2, 1.7%±0.2% in S and 22.2%±1.3% in G0 (n=343). Quiescent NSCs of insc-Gal4>FUCCI; pr-set7716/Df: 74.4%±1.0% in G2, 0.9%±0.2% in S and 24.7%±0.8% in G0 (n=344). Data information: Quantification data are presented in as mean ± SD Statistical analyses were done comparing two different genotypes using Mann-Whitney test ns: non-significant. White arrows point to NSCs. Scale bars, 5 μm


Figure 3 ( D ) qPCR analysis of pr - set7 , cdk1 , ebd1 and arm in larval brains of control ( ctrl ; yw ) and pr - set7716 / Df ( 3R ) ED5644 ( 716 / Df ) at 24h ALH . n = 7 as biological replicates . Statistical analyses were done comparing between control ( GAPDH1 , GAPDH2 , Tbp1 and Rpl32 ) and pr - set7716 / Df , using Mann - Whitney test . The mRNA levels of control were normalized to 1 . Data are presented as mean ± SD calculated from all replicates . Data information : Data are presented in ( D , as mean ± SD . Statistical analyses were done comparing two different genotypes using Mann - Whitney test ( D * P < 0 . 05G ) Larval NSCs of control ( ctrl ; yw ) and pr - set7716 / Df at 24h ALH were stained for Ebd1 , Dpn and Mira . Yellow dotted boxes , zoomed - in area . White arrows point to NSCs . Data information : Scale bars , 15 μm ( G ) . ( H , I ) Quantifications of nuclear Ebd1 ( H ) and the corrected total cell fluorescence ( CCF ) ( I ) In ( H ) , n = 756 , 9 BL for control ; n = 765 , 9 BL for pr - set7716 / Df . In ( I ) , CCF = Integrated Density - ( Area of selected cell × Mean fluorescence of background readings ) . CCFs were 1102 . 2 ± 493 . 2 ( n = 99 ) for control ( ctrl ; yw ) and 144 . 0 ± 88 . 0 ( n = 98 ) for 716 / Df . Data information : Data are presented in , I , s mean ± SD . n numbers in ( H ) were total NSCs counted with total brain lobes measured , while each brain lobe was considered as one biological replicate . Statistical analyses were done comparing two different genotypes using Mann - Whitney test , H , I , * * * P < 0 . 001 .

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 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 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 O O

Figure 3(D) qPCR analysis of pr-set7, cdk1, ebd1 and arm in larval brains of control (ctrl; yw) and pr-set7716/Df(3R)ED5644 (716/Df) at 24h ALH. n=7 as biological replicates. Statistical analyses were done comparing between control (GAPDH1, GAPDH2, Tbp1 and Rpl32) and pr-set7716/Df, using Mann-Whitney test. The mRNA levels of control were normalized to 1. Data are presented as mean ± SD calculated from all replicates. Data information: Data are presented in (D, as mean ± SD. Statistical analyses were done comparing two different genotypes using Mann-Whitney test (D *P < 0.05G) Larval NSCs of control (ctrl; yw) and pr-set7716/Df at 24h ALH were stained for Ebd1, Dpn and Mira. Yellow dotted boxes, zoomed-in area. White arrows point to NSCs. Data information: Scale bars, 15 μm (G).(H, I) Quantifications of nuclear Ebd1 (H) and the corrected total cell fluorescence (CCF) (I) In (H), n=756, 9 BL for control; n=765, 9 BL for pr-set7716/Df. In (I), CCF = Integrated Density - (Area of selected cell × Mean fluorescence of background readings). CCFs were 1102.2±493.2 (n=99) for control (ctrl; yw) and 144.0±88.0 (n=98) for 716/Df. Data information: Data are presented in , I, s mean ± SD. n numbers in (H) were total NSCs counted with total brain lobes measured, while each brain lobe was considered as one biological replicate. Statistical analyses were done comparing two different genotypes using Mann-Whitney test , H, I, ***P < 0.001.


Figure 4 ( H ) Larval brains of control ( ctrl ; yw ) , cdk1 - Myc overexpression under insc - Gal4 , pr - set7716 / Df ( 3R ) ED5644 ( 716 / Df ) , and pr - set7716 / Df with cdk1 - Myc induced with insc - Gal4 at 24h ALH were labelled with Dpn and EdU . ( I , J ) Quantifications of EdU incorporation ( I ) and NSC diameters ( J ) for genotypes in ( H ) . In ( I ) , n = 1334 , 16 BL for control ; n = 1299 , 15 BL for insc > cdk1 ; n = 1221 , 18 BL for pr - set7716 / Df and n = 1372 , 16 BL for pr - set7716 / Df with cdk1 - Mycinduced with insc - Gal4 . In ( J ) , n = 153 for control ; n = 152 for insc > cdk1 ; n = 156 for pr - set7716 / Df and n = 159 for pr - set7716 / Df with cdk1 - Myc induced with insc - Gal4 . Data information : Data were presented in quantification graphs as mean ± SD I , J ) . n numbers in I ) were total NSCs counted with total brain lobes measured , while each brain lobe was considered as one biological replicate . Statistical analyses were done comparing among different genotypes using ordinary one - way ANOVA * * * P < 0 . 001 . Scale bars , 30 μm H ) . Control : insc - Gal4 / + UAS -

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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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(H) Larval brains of control (ctrl; yw), cdk1-Myc overexpression under insc-Gal4, pr-set7716/Df(3R)ED5644 (716/Df), and pr-set7716/Df with cdk1-Myc induced with insc-Gal4 at 24h ALH were labelled with Dpn and EdU. (I, J) Quantifications of EdU incorporation (I) and NSC diameters (J) for genotypes in (H). In (I), n=1334, 16 BL for control; n=1299, 15 BL for insc>cdk1; n=1221, 18 BL for pr-set7716/Df and n=1372, 16 BL for pr-set7716/Df with cdk1-Mycinduced with insc-Gal4. In (J), n=153 for control; n=152 for insc>cdk1; n=156 for pr-set7716/Df and n=159 for pr-set7716/Df with cdk1-Myc induced with insc-Gal4.Data information: Data were presented in quantification graphs as mean ± SD I, J). n numbers in I) were total NSCs counted with total brain lobes measured, while each brain lobe was considered as one biological replicate. Statistical analyses were done comparing among different genotypes using ordinary one-way ANOVA ***P < 0.001. Scale bars, 30 μm H). Control: insc-Gal4/+ UAS-dicer2


Figure 5 ( A ) Larval NSCs of control ( ctrl ; yw ) and ebd1240 at 24h ALH were stained for Deadpan ( Dpn ) and Miranda ( Mira ) . ( B ) Quantifications of cellular extensions for genotypes in ( A ) . Control : n = 886 , 11 BL ; ebd1240 : n = 864 , 12 BL . ( C ) Larval brains of control ( ctrl ; yw ) and ebd1240 at 24h ALH were labelled with Dpn and EdU . ( D ) Quantifications of NSC without EdU incorporation for genotypes in ( C ) . Control : n = 1034 , 12 BL ; ebd1240 : n = 1139 , 16 BL . Data information : Data are presented in ( B , D as mean ± SD . n numbers in ( B , D , were total NSCs counted with total brain lobes measured , while each brain lobe was considered as one biological replicate . Statistical analyses were done comparing two different genotypes using Mann - Whitney test ( B , D ns : non - significant ; * P < 0 . 05 ; * * * P < 0 . 001 . ( H ) Larval brains of control ( ctrl ; yw ) , ebd1 - Flag overexpression under insc - Gal4 , pr - set7716 / Df ( 3R ) ED5644 ( 716 / Df ) and pr - set7716 / Df with ebd1 induced with insc - Gal4 at 24h ALH were labelled with Dpn and EdU . ( I ) Quantifications of NSCs without EdU incorporation for larval brains in ( H ) . Control : n = 1162 , 13 BL ; insc > ebd1 : n = 997 , 11 BL ; pr - set7716 / Df : n = 928 , 13 BL and pr - set7716 / Df with ebd1 induced with insc - Gal4 : n = 1284 , 16 BL . Data information : Data are presented in I ) as mean ± SD . n numbers in I ) were total NSCs counted with total brain lobes measured , while each brain lobe was considered as one biological replicate . Statistical analyses were done comparing among different genotypes using ordinary one - way ANOVA I ) . ns : non - significant ; * P < 0 . 05 ; * * * P < 0 . 001 .

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 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 O O O O O 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) Larval NSCs of control (ctrl; yw) and ebd1240 at 24h ALH were stained for Deadpan (Dpn) and Miranda (Mira). (B) Quantifications of cellular extensions for genotypes in (A). Control: n=886, 11 BL; ebd1240: n=864, 12 BL. (C) Larval brains of control (ctrl; yw) and ebd1240 at 24h ALH were labelled with Dpn and EdU. (D) Quantifications of NSC without EdU incorporation for genotypes in (C). Control: n=1034, 12 BL; ebd1240: n=1139, 16 BL. Data information: Data are presented in (B, D as mean ± SD. n numbers in (B, D, were total NSCs counted with total brain lobes measured, while each brain lobe was considered as one biological replicate. Statistical analyses were done comparing two different genotypes using Mann-Whitney test (B, D ns: non-significant; *P < 0.05; ***P < 0.001.(H) Larval brains of control (ctrl; yw), ebd1-Flag overexpression under insc-Gal4, pr-set7716/Df(3R)ED5644 (716/Df) and pr-set7716/Df with ebd1 induced with insc-Gal4 at 24h ALH were labelled with Dpn and EdU. (I) Quantifications of NSCs without EdU incorporation for larval brains in (H). Control: n=1162, 13 BL; insc>ebd1: n=997, 11 BL; pr-set7716/Df: n=928, 13 BL and pr-set7716/Df with ebd1 induced with insc-Gal4: n=1284, 16 BL. Data information: Data are presented in I) as mean ± SD. n numbers in I) were total NSCs counted with total brain lobes measured, while each brain lobe was considered as one biological replicate. Statistical analyses were done comparing among different genotypes using ordinary one-way ANOVA I). ns: non-significant; *P < 0.05; ***P < 0.001.


Figure 6 ( A ) Larval NSCs of control ( ctrl ; yw ) and cdk1B47 at 24h ALH were labelled for Earthbound1 ( Ebd1 ) , Deadpan ( Dpn ) , and Miranda ( Mira ) . ( B ) Quantifications of the corrected total cell fluorescence ( CCF ) of Ebd1 in genotypes in ( A ) . CCF = Integrated Density - ( Area of selected cell × Mean fluorescence of background readings ) . CCFs were 846 . 9 ± 394 . 2 for control ( ctrl ; yw ; n = 167 , 14 BL ) and 354 . 3 ± 245 . 3 for cdk1B47 ( n = 211 , 13 BL ) . ( C , D ) Larval NSCs of control ( UAS - dicer2 ) and UAS - cdk1 - Myc induced with insc - Gal4 at 6h ALH were labelled for Ebd1 , Dpn , and Mira . ( D ) Quantifications of the CCF of Ebd1 in genotypes in ( C ) . CCFs were 111 . 3 ± 80 . 2 for control ( n = 157 , 12 BL ) and 346 . 6 ± 221 . 1 for cdk1 - Myc ( n = 181 , 12 BL ) . Data information : Data are presented in ( B , D , as mean ± SD . n numbers in ( B , D , were total NSCs counted with total brain lobes measured , while each NSC was considered as one biological replicate . White arrows point to NSCs ( A , C , Yellow dotted boxes indicate the region of zoomed - in images ( A , C , Statistical analyses were done comparing between two different genotypes using Mann - Whitney test ( B , D ) Scale bars , 15 μm ( A , C , ( E ) qPCR analysis of cdk1 in larval brains of control ( ctrl ; yw ) and ebd1240 at 24h ALH . n = 3 as biological replicates . Statistical analyses were done comparing control ( GAPDH1 , GAPDH2 , Tbp1 and Rpl32 ) and ebd1240 , using two - tailed Student ' s t - test . The mRNA levels of control were normalized to 1 . Data information : Statistical analyses were done comparing between two different genotypes using two - tailed Student ' s t - test ( E ) . Larval NSCs of control ( ctrl ; yw ) , ebd1240 and pr - set7716 / Df ( 3R ) ED5644 ( 716 / Df ) at 24h ALH were stained for Senseless ( Sens ) , Dpn and Mira . ( F ) Data information : White arrows point to NSCs F ) . Yellow dotted boxes indicate the region of zoomed - in images F ) . Scale bars , 15 μm F ) .

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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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

Figure 6(A) Larval NSCs of control (ctrl; yw) and cdk1B47 at 24h ALH were labelled for Earthbound1 (Ebd1), Deadpan (Dpn), and Miranda (Mira). (B) Quantifications of the corrected total cell fluorescence (CCF) of Ebd1 in genotypes in (A). CCF = Integrated Density - (Area of selected cell × Mean fluorescence of background readings). CCFs were 846.9±394.2 for control (ctrl; yw; n=167, 14 BL) and 354.3±245.3 for cdk1B47 (n=211, 13 BL). (C, D) Larval NSCs of control (UAS-dicer2) and UAS-cdk1-Myc induced with insc-Gal4 at 6h ALH were labelled for Ebd1, Dpn, and Mira. (D) Quantifications of the CCF of Ebd1 in genotypes in (C). CCFs were 111.3±80.2 for control (n=157, 12 BL) and 346.6±221.1 for cdk1-Myc (n=181, 12 BL). Data information: Data are presented in (B, D, as mean ± SD. n numbers in (B, D, were total NSCs counted with total brain lobes measured, while each NSC was considered as one biological replicate. White arrows point to NSCs (A, C, Yellow dotted boxes indicate the region of zoomed-in images (A, C, Statistical analyses were done comparing between two different genotypes using Mann-Whitney test (B, D) Scale bars, 15 μm (A, C,(E) qPCR analysis of cdk1 in larval brains of control (ctrl; yw) and ebd1240 at 24h ALH. n=3 as biological replicates. Statistical analyses were done comparing control (GAPDH1, GAPDH2, Tbp1 and Rpl32) and ebd1240, using two-tailed Student's t-test. The mRNA levels of control were normalized to 1. Data information: Statistical analyses were done comparing between two different genotypes using two-tailed Student's t-test (E).Larval NSCs of control (ctrl; yw), ebd1240 and pr-set7716/Df(3R)ED5644 (716/Df) at 24h ALH were stained for Senseless (Sens), Dpn and Mira. (F) Data information: White arrows point to NSCs F). Yellow dotted boxes indicate the region of zoomed-in images F). Scale bars, 15 μm F).


Figure 1 ( B ) Quantification of embryos that expressing the ZGA reporter ( MERVL : : tdTomato ) after injection with a siRNA - repressor or mRNA - inducer . N , the total number of embryos analyzed for each condition . Error bars , mean ± S . D . ; n = 3 biological replicates per group . ( C ) Representative fluorescence image of SCNT embryos derived by different methods . The SCNT embryos produced by transfer of MERVL : : tdTomato cumulus cells ( B6D2F1 background ) into WT enucleated oocytes . n = 3 biological replicates per group . Scale bar , 100 μm . ( D ) Stacked bar plots show the fraction of embryos at blastocyst stages after injection with different mRNA , as indicated . Error bars , mean ± S . D . ; n = 3 biological replicates per group ; N , total number of embryos analyzed for each condition ; * * P < 0 . 01 , * * * P < 0 . 001 as compared with control group , by two - tailed Student ' s t - test . ( E ) Representative live - cell images of dynamic ZGA - reporter expression during SCNT embryo development . The time after initial observation is shown at the bottom of the image . n = 3 biological replicates per group . Scale bar , 50 μm . ( G ) Preimplantation development of SCNT embryos . The percentage of embryos reaching each indicated stage is shown . The D - SCNT embryos produced by transfer of dox - Dux cumulus cell ( B6D2F1 background ) into WT enucleated oocytes ( B6D2F1 background ) . Error bars , mean ± S . D . ; n = 3 biological replicates per group . Scale bar , 100 μm . ( J ) Image of full - term cloned mice derived by canonical SCNT and D - SCNT method ( left ) . The birth rate of SCNT embryos derived by different methods is indicated ( right ) . N , the total number of cloned pups obtained from each condition .

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 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 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 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

Figure 1(B) Quantification of embryos that expressing the ZGA reporter (MERVL::tdTomato) after injection with a siRNA-repressor or mRNA-inducer. N, the total number of embryos analyzed for each condition. Error bars, mean ± S.D.; n = 3 biological replicates per group.(C) Representative fluorescence image of SCNT embryos derived by different methods. The SCNT embryos produced by transfer of MERVL::tdTomato cumulus cells (B6D2F1 background) into WT enucleated oocytes. n = 3 biological replicates per group. Scale bar, 100 μm.(D) Stacked bar plots show the fraction of embryos at blastocyst stages after injection with different mRNA, as indicated. Error bars, mean ± S.D.; n = 3 biological replicates per group; N, total number of embryos analyzed for each condition; **P < 0.01, ***P < 0.001 as compared with control group, by two-tailed Student's
 t-test.(E) Representative live-cell images of dynamic ZGA-reporter expression during SCNT embryo development. The time after initial observation is shown at the bottom of the image. n = 3 biological replicates per group. Scale bar, 50 μm.(G) Preimplantation development of SCNT embryos. The percentage of embryos reaching each indicated stage is shown. The D-SCNT embryos produced by transfer of dox-Dux cumulus cell (B6D2F1 background) into WT enucleated oocytes (B6D2F1 background). Error bars, mean ± S.D.; n = 3 biological replicates per group. Scale bar, 100 μm.(J) Image of full-term cloned mice derived by canonical SCNT and D-SCNT method (left). The birth rate of SCNT embryos derived by different methods is indicated (right). N, the total number of cloned pups obtained from each condition.


Figure 2 ( B ) Durations of dox addition at different time points during chemical induction ( upper ) and assessment of AP + colonies ( bottom ) . Error bars , mean ± S . D . ; n = 3 biological replicates per condition ; * * P < 0 . 01 , * * * P < 0 . 001 as compared with condition - 1 , by two - tailed Student ' s t - test . n . s . , not significant . ( C ) Morphological changes at distinct time points during D - CiPS induction . Scale bar , 100 μm . ( D ) Representative images of the number of AP + positive colonies . n = 5 biological replicates per condition . ( E ) RT - qPCR analysis of select ZGA - related genes in dox - untreated D - CiPSCs and canonical CiPSCs at passage 1 . The value in CiPSCs was set as 1 , and data shown are mean expression values relative to GAPDH . Error bars , mean ± S . E . M . ; n = 3 biological replicates per group ; * * * P < 0 . 001 by two - tailed Student ' s t - test . ( F ) Representative immunostaining images of D - CiPSCs expressing pluripotency markers . n = 5 biological replicates . Scale bar , 50 μm . ( G ) Histology by H & E staining of teratoma tissues derived by D - CiPS cells . n = 3 biological replicates . Scale bar , 200 μm . ( I ) Karyotype analysis of D - CiPSCs and canonical CiPSCs . The majority of D - CiPSCs and CiPSCs cells possessed a normal karyotype . Scale bar , 10 μm . ( J ) Bisulfite sequencing analysis of demethylation of Oct4 and Nanog promoters in D - CiPSCs or canonical CiPSCs . Filled and empty squares represent methylated and unmethylated CpGs , respectively .

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 B-PANEL_START 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 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 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 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 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

Figure 2(B) Durations of dox addition at different time points during chemical induction (upper) and assessment of AP+ colonies (bottom). Error bars, mean ± S.D.; n = 3 biological replicates per condition; **P < 0.01, ***P < 0.001 as compared with condition-1, by two-tailed Student's
 t-test. n.s., not significant.(C) Morphological changes at distinct time points during D-CiPS induction. Scale bar, 100 μm.(D) Representative images of the number of AP+ positive colonies. n = 5 biological replicates per condition.(E) RT-qPCR analysis of select ZGA-related genes in dox-untreated D-CiPSCs and canonical CiPSCs at passage 1. The value in CiPSCs was set as 1, and data shown are mean expression values relative to GAPDH. Error bars, mean ± S.E.M.; n = 3 biological replicates per group; ***P < 0.001 by two-tailed Student's t-test.(F) Representative immunostaining images of D-CiPSCs expressing pluripotency markers. n = 5 biological replicates. Scale bar, 50 μm.(G) Histology by H&E staining of teratoma tissues derived by D-CiPS cells. n = 3 biological replicates. Scale bar, 200 μm.(I) Karyotype analysis of D-CiPSCs and canonical CiPSCs. The majority of D-CiPSCs and CiPSCs cells possessed a normal karyotype. Scale bar, 10 μm.(J) Bisulfite sequencing analysis of demethylation of Oct4 and Nanog promoters in D-CiPSCs or canonical CiPSCs. Filled and empty squares represent methylated and unmethylated CpGs, respectively.