( D ) Super - resolution of immunofluorescence for Tom20 ( blue ) , NCOA4 ( red ) FTMT ( green ) in Huh7 after DFP ( 1 mM ) treatment , using single - molecule microscope . arrows indicate colocalization of these .

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(D) Super-resolution images of immunofluorescence staining for Tom20 (blue), NCOA4 (red) and FTMT (green) in Huh7 cells after DFP (1 mM) treatment, using the single-molecule fluorescence microscope. The white arrows indicate colocalization of these proteins.


( E ) Immunofluorescence for Tom20 ( blue ) , FTMT - GFP ( green ) , recombinant FTMTR82A - GFP ( green ) , NCOA4 - mCherry ( red ) NCOA4I489A / W497A - mCherry ( red ) in Huh7 . Boxed areas are enlarged on right . pointed by arrows in overlaid picture indicate colocalization of Tom20 , FTMT - GFP NCOA4 - mCherry . yellow shown by arrows in overlaid picture indicate colocalization of FTMT - GFP NCOA4 - mCherry . Scale bar : 10 μm 5 μm in enlarged .

O O O B-EXP_ASSAY O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O O O B-GENEPROD O O O O B-CELL_LINE O O O O O O O O O O O O O B-EXP_ASSAY O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD O B-GENEPROD O O O O O O O B-EXP_ASSAY O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD I-GENEPROD O B-GENEPROD O O O O O O O O O O O

(E) Immunofluorescence staining for Tom20 (blue), FTMT-GFP (green), recombinant FTMTR82A-GFP (green), NCOA4-mCherry (red) and NCOA4I489A/W497A-mCherry (red) in Huh7 cells. Boxed areas are enlarged on the right. The white puncta pointed by white arrows in the overlaid picture indicate the colocalization of Tom20, FTMT-GFP and NCOA4-mCherry. The yellow puncta shown by white arrows in the overlaid picture indicate the colocalization of FTMT-GFP and NCOA4-mCherry. Scale bar: 10 μm and 5 μm in enlarged images.


( F ) Extracted from Huh7 expressing recombinant as indicated were mixed with anti - GFP - coupled magnetic beads immunoblotted with anti - mCherry anti - GFP antibodies . Co - immunoprecipitation revealed a specific interaction of NCOA4 - mCherry with FTMT - GFP or FTMTD57A - GFP , which has a substitution of alanine ( A ) for aspartic acid ( D ) at position 57 in FTMT mitochondrial leader ( lanes 1 3 ) .

O O O O O B-CELL_LINE O O O O O O O O O B-GENEPROD O O O O B-EXP_ASSAY O O O B-GENEPROD O O B-GENEPROD O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O B-GENEPROD B-SUBCELLULAR O O O O O O O

(F) Extracted proteins from Huh7 cells expressing the recombinant proteins as indicated were mixed with anti-GFP-coupled magnetic beads and immunoblotted with anti-mCherry and anti-GFP antibodies. Co-immunoprecipitation revealed a specific interaction of NCOA4-mCherry with FTMT-GFP or FTMTD57A-GFP, which has a substitution of alanine (A) for aspartic acid (D) at position 57 in the FTMT mitochondrial leader sequence (lanes 1 and 3).


( G ) Following DFP ( 1 . 0 mM ) or ammonium iron sulfate ( FAS ) treatment as indicated , extracted from Huh7 expressing FTMT - GFP NCOA4 - mCherry were mixed with anti - GFP - coupled magnetic beads immunoblotted with anti - mCherry anti - GFP antibodies .

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(G) Following DFP (1.0 mM) or ammonium iron sulfate (FAS) treatment as indicated, extracted proteins from Huh7 cells expressing FTMT-GFP and NCOA4-mCherry were mixed with anti-GFP-coupled magnetic beads and immunoblotted with anti-mCherry and anti-GFP antibodies.


( A ) Flowcytometric of mitochondrial potential of isolated mitochondria . mitochondria isolated from Huh7 with CCCP treatment with or without DFP treatment were incubated with JC - 1 for 1 h at 30 ° C subjected to flow cytometric ( n = 4 , biological replicates ) by excitation with a blue ( 488 nm ) with simultaneous emission assessment using a 585 / 42 detector for JC - 1 red a 530 / 30 for JC - 1 green . trapezoid areas show population of depolarized mitochondria . of proportion of depolarized mitochondria in Huh7 with or without DFP treatment is shown in lower panel . central horizontal bar error bards indicate mean ± SD . Two sample t was used for statistical . * * : P < 0 . 01 .

O O O B-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY O O B-SUBCELLULAR O B-SUBCELLULAR O O B-CELL_LINE O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O 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-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O B-SUBCELLULAR O O O O O B-SUBCELLULAR O B-CELL_LINE O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) Flowcytometric analysis of the mitochondrial membrane potential of the isolated mitochondria. The mitochondria isolated from Huh7 cells with CCCP treatment and with or without DFP treatment were incubated with JC-1 for 1 h at 30°C and subjected to flow cytometric analysis (n=4, biological replicates) by excitation with a blue laser (488 nm) with simultaneous emission assessment using a 585/42 detector for JC-1 red and a 530/30 for JC-1 green. The trapezoid areas show the population of depolarized mitochondria. Quantification of the proportion of depolarized mitochondria in Huh7 cells with or without DFP treatment is shown in the lower panel. The central horizontal bar and the error bards indicate mean ± SD. Two sample t test was used for statistical analysis. **: P<0.01.


( B ) oxygen consumption rate was measured using a Seahorse XF24 Extracellular Flux Analyzer ( n = 4 , technical replicates ) . Huh7 were incubated with 1 mM of DFP for different lengths of time ( 0 , 3 , 6 , 18 , 24 h ) . Oxygen consumption was measured , respiration rate was analyzed by injecting of 1μM oligomycin , 1 μM carbonyl cyanide 4 - ( trifluoromethoxy ) phenylhydrazone ( FCCP ) 10 μM rotenone / antimycin A . OCR : oxygen consumption rate . central point error bards indicate mean ± SD .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O B-CELL_LINE O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE O B-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O

(B) The oxygen consumption rate was measured using a Seahorse XF24 Extracellular Flux Analyzer (n=4, technical replicates). Huh7 cells were incubated with 1 mM of DFP for different lengths of time (0, 3, 6, 18, and 24 h). Oxygen consumption was measured, and the respiration rate was analyzed by injecting of 1μM oligomycin, 1 μM carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP) and 10 μM rotenone/antimycin A. OCR: oxygen consumption rate. The central point and the error bards indicate mean ± SD.


( C ) Immunofluorescence for FTMT - GFP ( green ) LC3 ( red ) in Huh7 transfected with FTMT - GFP . proportion of colocalization was quantified for four randomly selected areas ( technical replicates ) using ImageJ software version 1 . 46 ( NIH , Bethesda , MD ) . central horizontal bar error bards indicate mean ± SD . Tukey ' s honestly significant was used for statistical . * * : P < 0 . 01 .

O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O O O O B-CELL_LINE O O B-GENEPROD O B-GENEPROD I-GENEPROD O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C) Immunofluorescence staining for FTMT-GFP (green) and LC3 (red) in Huh7 cells transfected with FTMT-GFP. The proportion of colocalization was quantified for four randomly selected areas (technical replicates) using ImageJ software version 1.46 (NIH, Bethesda, MD). The central horizontal bar and the error bards indicate mean ± SD. The Tukey's honestly significant test was used for statistical analysis. **: P<0.01.


( D ) Immunofluorescence for Ferritin - GFP heavy chain ( FTH1 ) ( green ) LC3 ( red ) in Huh7 transfected with FTH1 - GFP . proportion of colocalization was quantified as described in ( C ) . * : P < 0 . 05 , * * : P < 0 . 01 .

O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O O O O B-GENEPROD O O O O B-CELL_LINE O O B-GENEPROD O B-GENEPROD I-GENEPROD O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O

(D) Immunofluorescence staining for Ferritin-GFP heavy chain (FTH1) (green) and LC3 (red) in Huh7 cells transfected with FTH1-GFP. The proportion of colocalization was quantified as described in (C). *: P<0.05, **: P<0.01.


( E ) Immunoblots for precursor form processed form of FTMT - GFP , endogenous FTMT , NCOA4 , LC3 , Tom20 using mitochondrial fractions of Huh7 expressing FTMT - GFP with or without digestion with proteinase K in presence or absence of DFP . mtHSP70 was used as loading control .

O O O B-EXP_ASSAY O O O O O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-SUBCELLULAR O O B-CELL_LINE O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD I-GENEPROD O O O O O B-SMALL_MOLECULE O B-GENEPROD O O O O O O

(E) Immunoblots for the precursor form and processed form of FTMT-GFP, endogenous FTMT, NCOA4, LC3, and Tom20 using the mitochondrial fractions of Huh7 cells expressing FTMT-GFP with or without digestion with proteinase K in the presence or absence of DFP. mtHSP70 was used as the loading control.


( F ) Immunoblots for FTMT , VDAC , mtHSP70 using mitochondrial from Huh7 with or without DFP treatment . mitochondrial was fractionated from Huh7 by means of sodium carbonate treatment . mtHSP70 was used as non - - integral control VDAC as - integral control . mtHSP70 at right - side in third lane indicates positive control obtained from mitochondrial fraction of Huh7 without DFP treatment .

O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-SUBCELLULAR O B-CELL_LINE O O O B-SMALL_MOLECULE O O B-SUBCELLULAR O O O B-CELL_LINE O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O B-GENEPROD O O O O O O O O B-GENEPROD O O O O O B-GENEPROD O O O O O O O O O O O O B-SUBCELLULAR O O B-CELL_LINE O B-SMALL_MOLECULE O O

(F) Immunoblots for FTMT, VDAC, and mtHSP70 using the mitochondrial membrane from Huh7 cells with or without DFP treatment. The mitochondrial membrane was fractionated from Huh7 cells by means of sodium carbonate treatment. mtHSP70 was used as the non-membrane-integral protein control and VDAC as the membrane-integral protein control. mtHSP70 at the right-hand side in the third lane indicates the positive control obtained from the mitochondrial fraction of Huh7 cells without DFP treatment.


( A ) Immunofluorescence for polarized mitochondria ( red ) , Tom20 ( blue ) , LC3 ( green ) in Huh7 untreated or treated with DFP ( 1 mM ) for 24 h , or with CCCP ( 30 μM ) for 6 h . Boxed areas are enlarged on right . Scale bar : 10 μm , 5 μm in enlarged .

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(A) Immunofluorescence staining for the polarized mitochondria (red), Tom20 (blue), and LC3 (green) in Huh7 cells untreated or treated with DFP (1 mM) for 24 h, or with CCCP (30 μM) for 6 h. Boxed areas are enlarged on the right. Scale bar: 10 μm, and 5 μm in enlarged images.


( B ) Immunofluorescence for polarized mitochondria ( red ) , Tom20 ( blue ) , LC3 ( blue ) , FTMTD57A ( green ) using Huh7 expressing FTMTD57A . Boxed areas are enlarged on right . in overlaid pictures indicate colocalization of polarized mitochondria , Tom20 FTMTD57A or colocalization of polarized mitochondria , FTMTD57A , LC3 . Scale bar : 10 μm , 5 μm in enlarged .

O O O B-EXP_ASSAY O O B-SUBCELLULAR O O O O B-GENEPROD O O O O B-GENEPROD O O O O B-GENEPROD O O O O B-CELL_LINE O B-GENEPROD O O O O O O O O O O B-EXP_ASSAY O B-EXP_ASSAY O O B-SUBCELLULAR O B-GENEPROD I-GENEPROD O B-EXP_ASSAY O O B-SUBCELLULAR O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O

(B) Immunofluorescence staining for the polarized mitochondria (red), Tom20 (blue), LC3 (blue), and FTMTD57A (green) using Huh7 cells expressing FTMTD57A. Boxed areas are enlarged on the right. The white puncta in overlaid pictures indicate the colocalization of the polarized mitochondria, Tom20 and FTMTD57A or the colocalization of the polarized mitochondria, FTMTD57A, and LC3. Scale bar: 10 μm, and 5 μm in enlarged images.


( C ) STAM mice were fed a high - fat diet orally administered 0 . 0375 mg / g of DFP , 0 . 075 mg / g of DFP or distilled water from 4 of to 16 of ( n = 6 ) . Arrows indicate liver tumors . Scale bar : 1 . 0 cm . number maximum of liver tumors were compared among three groups . central horizontal bar error bards indicate mean ± SD . Tukey ' s honestly significant was used for statistical . * : P < 0 . 05 , * * : P < 0 . 01 .

O O O O B-ORGANISM O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O B-DISEASE I-DISEASE O O O O O O O O O O O O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C) STAM mice were fed a high-fat diet and orally administered 0.0375 mg/g body weight of DFP, 0.075 mg/g body weight of DFP or distilled water from 4 weeks of age to 16 weeks of age (n=6). Arrows indicate liver tumors. Scale bar: 1.0 cm. The number and maximum size of liver tumors were compared among the three groups. The central horizontal bar and the error bards indicate mean ± SD. The Tukey's honestly significant test was used for statistical analysis. *: P<0.05, **: P<0.01.


( D ) Histology of nontumorous liver ( H & E Sirius red stain , original magnification x40 ) superoxide anion production in livers of STAM mice fed a high - fat diet with or without DFP ( 0 . 075 mg / g ) administration . Frozen liver sections of mice were stained with dihydroethidium for assessment of superoxide anion . Scale bar : 100 μm .

O O O B-EXP_ASSAY O O B-TISSUE O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE O O O B-TISSUE O O B-ORGANISM O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O B-TISSUE O O B-ORGANISM O B-EXP_ASSAY O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O O O O O O

(D) Histology of the nontumorous liver (H&E and Sirius red stain, original magnification x40) and superoxide anion production in the livers of STAM mice fed a high-fat diet with or without DFP (0.075 mg/g) administration. Frozen liver sections of mice were stained with dihydroethidium for assessment of superoxide anion. Scale bar: 100 μm.


( E ) Histology of hepatocellular carcinoma using H & E in STAM mice fed a high - fat diet . boxed is enlarged on right ( x40 in left panel x400 in right panel ) . has increased density , an increased of nucleus to cytoplasm , thickened trabeculae Scale bar : 100 μm .

O O O B-EXP_ASSAY O B-DISEASE I-DISEASE O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-ORGANISM O 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-EXP_ASSAY O O O O B-SUBCELLULAR O B-SUBCELLULAR O O O O O O O O O

(E) Histology of the hepatocellular carcinoma using H&E staining in STAM mice fed a high-fat diet. The boxed area is enlarged on the right (x40 in left panel and x400 in right panel). The tumor has increased cell density, an increased ratio of nucleus to cytoplasm, and thickened trabeculae Scale bar: 100 μm.


( F ) Electron microscopic of livers in control ( C57BL / 6 ) DMBA + HFD mice that were orally administered 0 . 075 mg / g of DFP or distilled water . Scale bars : 2 . 0 μm . boxed areas ( x 1 , 500 ) are enlarged below ( x6 , 000 on left x10 , 000 on right ) . arrow indicates mitophagosome - like structures . mean number of mitophagosome - like structures / 100 μm2 was compared for four randomly selected areas ( technical replicates ) among four groups ( control , DMBA + HFD mice treated with 0 , 0 . 0375 or 0 . 075 mg / g of DFP ) ( n = 6 ) . central horizontal bar error bards indicate mean ± SD . Tukey ' s honestly significant was used for statistical . * : P < 0 . 05 .

O O O B-EXP_ASSAY I-EXP_ASSAY O B-TISSUE O O O O O O O B-SMALL_MOLECULE O O B-ORGANISM O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O 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-SUBCELLULAR O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O B-ORGANISM O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(F) Electron microscopic images of livers in control (C57BL/6) and DMBA + HFD mice that were orally administered 0.075 mg/g of DFP or distilled water. Scale bars: 2.0 μm. The boxed areas (x 1,500) are enlarged below (x6,000 on left and x10,000 on right). The white arrow indicates mitophagosome-like structures. The mean number of mitophagosome-like structures/100 μm2 was compared for four randomly selected areas (technical replicates) among the four groups (control, DMBA + HFD mice treated with 0, 0.0375 or 0.075 mg/g of DFP) (n=6). The central horizontal bar and the error bards indicate mean ± SD. The Tukey's honestly significant test was used for statistical analysis. *: P<0.05.


( A ) Immunoblots for FTMT , frataxin , mitoferrin , HSP60 , FTH1 TfR using hepatic mitochondrial fraction lysate whole liver lysate in control ( C57BL / 6J ) mice STAM mice treated with DFP ( 0 , 0 . 0375 , or 0 . 075 mg / g ) ( n = 6 ) . expression of FTMT , frataxin , mitoferrin , FTH1 , TfR were normalized to HSP60 or β - actin , respectively . central horizontal bar error bards indicate mean ± SD . Tukey ' s honestly significant was used for statistical . * : P < 0 . 05 , * * : P < 0 . 01 . ( B ) mean number of mitophagosome - like structures / 100 μm2 from four randomly selected areas in livers of DFP ( 0 . 075 mg / g ) - administered mice were compared between control FTMT siRNA - treated groups for STAM mice ( n = 6 ) DMBA + HFD mice ( n = 6 ) . central horizontal bar error bards indicate mean ± SD . Two sample t was used for statistical . NC : negative control siRNA , * : P < 0 . 05 .

O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD O B-TISSUE B-SUBCELLULAR O O O B-TISSUE O O O O O O O O B-ORGANISM O B-ORGANISM O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O 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-SUBCELLULAR O O O O O O O O O O O O B-TISSUE O B-SMALL_MOLECULE O O O O O O O O O O B-ORGANISM O O O O B-GENEPROD O O O O O O B-ORGANISM O O O O O B-SMALL_MOLECULE O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) Immunoblots for FTMT, frataxin, mitoferrin, HSP60, FTH1 and TfR using the hepatic mitochondrial fraction lysate and whole liver lysate in control (C57BL/6J) mice and STAM mice treated with DFP (0, 0.0375, or 0.075 mg/g body weight) (n=6). The expression of FTMT, frataxin, mitoferrin, FTH1, and TfR were normalized to HSP60 or β-actin, respectively. The central horizontal bar and the error bards indicate mean ± SD. The Tukey's honestly significant test was used for statistical analysis. *: P<0.05, **: P<0.01. (B) The mean number of mitophagosome-like structures/100 μm2 from four randomly selected areas in the livers of DFP (0.075 mg/g body weight)-administered mice were compared between control and FTMT siRNA-treated groups for STAM mice (n=6) and DMBA + HFD mice (n=6). The central horizontal bar and the error bards indicate mean ± SD. Two sample t test was used for statistical analysis. NC: negative control siRNA, *: P<0.05.


( C ) Frozen liver sections of DFP ( 0 . 075 mg / g ) - administered mice ( STAM mice DMBA + HFD ) with or without FTMT knockdown were stained with dihydroethidium . Red indicates ROS production . Scale bar : 100 μm . siNC : negative control siRNA .

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(C) Frozen liver sections of DFP (0.075 mg/g body weight)-administered mice (STAM mice and DMBA + HFD) with or without FTMT knockdown were stained with dihydroethidium. Red fluorescence indicates the ROS production. Scale bar: 100 μm. siNC: negative control siRNA.


( D , E ) STAM mice ( D ) were fed a high - fat diet without DFP treatment or orally administered with 0 . 075 mg / g of DFP from of 4 , injected with FTMT siRNA ( 10 μl / g ) or negative control siRNA through tail vein every 2 for 8 prior to sacrifice , followed until 16 of ( n = 6 ) . DMBA + HFD mice ( E ) were fed a high - fat diet , treated with DFP siRNA in same manner with STAM mice , followed until 30 of ( n = 6 ) . number maximum of liver tumors were compared among three groups ( control without DFP , negative control siRNA , FTMT siRNA with DFP ) . central horizontal bar error bards indicate mean ± SD . Tukey ' s honestly significant was used for statistical . * : P < 0 . 05 , * * : P < 0 . 01 . Representative liver are shown in upper panel . Scale bar : 1 cm .

O O O O O O B-ORGANISM O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O B-GENEPROD O O O O O O O O O O O O B-TISSUE I-TISSUE O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O B-ORGANISM O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O B-ORGANISM O O O O O O O O O O O O O O B-DISEASE I-DISEASE O O O O O O O O B-SMALL_MOLECULE O O O O O B-GENEPROD O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O 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-TISSUE O O O O O O O O O O O O

(D, E) STAM mice (D) were fed a high-fat diet without DFP treatment or orally administered with 0.075 mg/g body weight of DFP from the age of 4 weeks, injected with FTMT siRNA (10 μl/g body weight) or negative control siRNA through the tail vein every 2 weeks for 8 weeks prior to sacrifice, and followed until 16 weeks of age (n=6). DMBA + HFD mice (E) were fed a high-fat diet, treated with DFP and siRNA in the same manner with STAM mice, and followed until 30 weeks of age (n=6). The number and maximum size of liver tumors were compared among the three groups (control without DFP, negative control siRNA, and FTMT siRNA with DFP). The central horizontal bar and the error bards indicate mean ± SD. The Tukey's honestly significant test was used for statistical analysis. *: P<0.05, **: P<0.01. Representative liver images are shown in the upper panel. Scale bar: 1 cm.


A . Coomassie Blue stained SDS - PAGE gel of yeast Elongator isolated by FLAG affinity purification glycerol gradient ultracentrifugation from a encoding C - terminally FLAG - tagged Elp1 .

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A. Coomassie Blue stained SDS-PAGE gel of yeast Elongator isolated by FLAG affinity purification and glycerol gradient ultracentrifugation from a strain encoding C-terminally FLAG-tagged Elp1.


B . Representative segment of an electron micrograph of negatively stained Elongator after stabilization by gradient fixation ( Lane 6 of Fig 1A ) . Circled particles correspond to intact Elongator . scale bar corresponds to 50 nm .

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B. Representative segment of an electron micrograph of negatively stained Elongator after stabilization by gradient fixation (Lane 6 of Fig 1A). Circled particles correspond to intact Elongator. White scale bar corresponds to 50 nm.


C . Representative 2D class averages of full Elongator from a total of 17 , 074 particles . class averages shown in this figure have side width of 500 Å .

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C. Representative 2D class averages of the full Elongator complex from a total of 17,074 particles. All class averages shown in this figure have side width of 500 Å.


D . Representative 2D class averages of Elp123 subcomplex prepared through a high salt dissociation procedure .

D. Representative 2D class averages of the Elp123 subcomplex prepared through a high salt dissociation procedure.


E . Six evenly - sized densities are arranged in one Elongator lobe . A projection of Elp456 hexamer X - ray structure ( PDB code : 4A8J ) is shown for comparison .

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E. Six evenly-sized densities are arranged in one Elongator lobe. A projection of the Elp456 hexamer X-ray structure (PDB code: 4A8J) is shown for comparison.


F . of Elp2 localization using a C - terminal MBP tag . Class averages of untagged Elongator is shown for comparison . arrowheads point towards additional densities in tagged .

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F. Analysis of Elp2 localization using a C-terminal MBP tag. Class averages of untagged Elongator is shown for comparison. White arrowheads point towards additional densities in the tagged complex.


G . Elp3 localization using a C - terminal MBP tag , denoted by arrowhead .

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G. Elp3 localization analysis using a C-terminal MBP tag, denoted by the white arrowhead.


H . Mapping of Elp1 by incubating purified Elongator containing an Elp1 C - terminal FLAG tag with α - FLAG antibody . additional antibody density is denoted by arrowhead .

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H. Mapping of Elp1 by incubating purified Elongator containing an Elp1 C-terminal FLAG tag with α-FLAG antibody. The additional antibody density is denoted by the white arrowhead.


I . Three dimensional reconstruction of intact Elongator at 25Å .

I. Three dimensional reconstruction of the intact Elongator complex at 25Å.


J . Schematic of approximate subunit locations on 2D EM .

J. Schematic of the approximate subunit locations based on 2D EM analysis.


K . Inset showing cavity formed through centre of putative Elp456 density . Black scale bars in this figure correspond to 50 Å .

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K. Inset showing the cavity formed through the centre of the putative Elp456 density. Black scale bars in this figure correspond to 50 Å.


A . Number of peptides detected of each Elongator subunit purified through Elp4 - FLAG pulldown from yeast lacking ELP1 , ELP2 , or ELP3 as a fraction of number of Elp4 peptides . data points are represented as circles , while averages are represented as bars .

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A. Number of peptides detected of each Elongator subunit purified through Elp4-FLAG pulldown from yeast lacking ELP1, ELP2, or ELP3 as a fraction of the number of Elp4 peptides. Individual data points are represented as circles, while the averages are represented as bars.


B . Purified Elongator was incubated with amine crosslinker disuccinimidyl suberate ( DSS ) analyzed by mass spectrometry . Schematic representation of major crosslinking connectivity of Elp123 is shown .

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B. Purified Elongator was incubated with the primary amine crosslinker disuccinimidyl suberate (DSS) and analyzed by mass spectrometry. Schematic representation of the major crosslinking connectivity of Elp123 is shown.


C . Major crosslinking connectivity of Elp456 .

C. Major crosslinking connectivity of the Elp456 complex.


B . Plasmids containing ELP2 four loop mutants were transformed into elp2Δ yeast . strains were then plated onto - Ura media containing caffeine . pRS416 vector is used for these experiments .

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B. Plasmids containing ELP2 and the four loop mutants were transformed into elp2Δ yeast. The strains were then plated onto -Ura media containing caffeine. The pRS416 vector is used for these experiments.


C . Growth curve of loop mutants in - Ura media with without K . lactis toxin zymocin . Shaded regions correspond to at each time point . N = 3 .

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C. Growth curve analysis of loop mutants in -Ura media with and without K. lactis toxin zymocin. Shaded regions correspond to the SEM at each time point. N=3.


D . Lysates of Elp4 - FLAG - containing yeast expressing Elp2 loop mutants were incubated with α - FLAG resin eluted with 3xFLAG peptides then analyzed by Western Blot using α - HA α - FLAG

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D. Lysates of Elp4-FLAG-containing yeast expressing the Elp2 loop mutants were incubated with α-FLAG resin and eluted with 3xFLAG peptides then analyzed by Western Blot using α-HA and α-FLAG antibody


A . Fitting of Elp123 structures into 3D reconstruction of Elongator . homology structure of Elp1 N - terminal double WD40 - domains , homology structure of Elp3 on X - ray structure of D . mccartyi , X - ray structure of Elp1 C - terminal dimer ( 5CQS ) , X - ray structure of Elp2 ( 4XFV ) . Scale bar corresponds to 50 Å , applies to panels A - C .

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A. Fitting of Elp123 structures into the 3D reconstruction of Elongator. The homology structure of the Elp1 N-terminal double WD40-domains, the homology structure of Elp3 based on the X-ray structure of D. mccartyi, the X-ray structure of Elp1 C-terminal dimer (5CQS), and the X-ray structure of Elp2 (4XFV). Scale bar corresponds to 50 Å, and applies to panels A-C.


B . Fitting of Elp456 heterohexameric ATPase ring ( 4A8J ) into six - membered ring from larger Elongator lobe .

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B. Fitting of the Elp456 heterohexameric ATPase ring (4A8J) into the six-membered ring from the larger Elongator lobe.


C . comparison of a representative tRNA molecule ( 1VTQ ) cavity observed through middle of Elp456 hexamer .

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C. Size comparison of a representative tRNA molecule (1VTQ) and the cavity observed through the middle of the Elp456 hexamer.


D . Alternate views of Elongator model showing subunit crosslinking patterns .

D. Alternate views of the Elongator model showing the subunit crosslinking patterns.


A . CXMS EM data , combined with X - ray structures of Elp1 C - terminus ( 5CQS ) , Elp2 ( 4XFV ) , Elp456 ( 4A8J ) , homology models of Elp1 tandem WD - 40 domains Elp3 , were used to estimate relative positions of each subunit to generate a multi - scale model of Elongator subunit organization .

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A. CXMS and EM data, combined with the X-ray structures of Elp1 C-terminus (5CQS), Elp2 (4XFV), Elp456 (4A8J), and homology models of the Elp1 tandem WD-40 domains and Elp3, were used to estimate the relative positions of each subunit to generate a multi-scale model of Elongator subunit organization.


B . Side view of Elongator architectural model highlighting cavity ( red outline ) spanning through centre of Elp456 hexamer .

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B. Side view of the Elongator architectural model highlighting the cavity (red outline) spanning through the centre of the Elp456 hexamer.


C . Comparison of Elp123 conformations of intact Elongator ( gold ) Elp123 subcomplex ( cyan ) . Elp456 - unloaded lobe of Elongator one lobe of Elp123 were aligned .

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C. Comparison of the Elp123 conformations of the intact Elongator complex (gold) and the Elp123 subcomplex (cyan). The Elp456-unloaded lobe of Elongator and one lobe of Elp123 were aligned.


A . Silver - stained SDS - PAGE of Elongator glycerol gradient fractions purified using low salt ( 75 mM NaCl ) buffer . fraction containing most Elp4 , Elp5 , Elp6 is designated by an asterisk ( * ) corresponding fraction from a glutaraldehyde - containing glycerol gradient is used for EM .

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A. Silver-stained SDS-PAGE of Elongator glycerol gradient fractions purified using low salt (75 mM NaCl) buffer. The fraction containing the most Elp4, Elp5, and Elp6 is designated by an asterisk (*) and the corresponding fraction from a glutaraldehyde-containing glycerol gradient is used for EM analysis.


B . Comparison of class averages of Elongator purified in buffer containing regular or low salt concentrations ( 150 mM 75 mM NaCl , respectively ) . Densities resembling a second copy of Elp456 is indicated by triangles .

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B. Comparison of class averages of Elongator purified in buffer containing regular or low salt concentrations (150 mM and 75 mM NaCl, respectively). Densities resembling a second copy of Elp456 is indicated by white triangles.


D . Coomassie - stained SDS - PAGE of rElp456 glycerol gradient fractions .

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D. Coomassie-stained SDS-PAGE of rElp456 glycerol gradient fractions.


E . Coomassie - stained SDS - PAGE of Elongator mixed with rElp456 glycerol gradient fractions . fraction containing most intact complexes is indicated with an asterisk ( * ) , corresponding fraction from a glutaraldehyde - containing glycerol is used for EM .

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E. Coomassie-stained SDS-PAGE of the Elongator mixed with rElp456 glycerol gradient fractions. The fraction containing the most intact complexes is indicated with an asterisk (*), and the corresponding fraction from a glutaraldehyde-containing glycerol is used for EM analysis.


F . Comparison of class averages of natively purified Elongator with without rElp456 incubation . Densities resembling a second copy of Elp456 are indicated by triangles .

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F. Comparison of class averages of natively purified Elongator with and without rElp456 incubation. Densities resembling a second copy of Elp456 are indicated by white triangles.


( B ) Plaque formation was monitored by automated scanning Levin - Reisman et al . , 2010 ( ) on a lawn of infected PY79 ( WT ) ( 10 - 6 PFU / ml ) spread over an MB agar plate . Shown are captured at indicated time points ( hrs ) . Scale bar 2 mm . Corresponds to Movie EV2 . ( C - D ) dynamic of SPP1 ( C ) Phi29 ( D ) plaque formation following PY79 ( WT ) infection was monitored as described in ( B ) . Shown is diameter of plaques for each phage ( n ≥ 12 ) , with average highlighted in red .

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(B) Plaque formation was monitored by automated scanning Levin-Reisman et al., 2010() on a lawn of infected PY79 (WT) (10-6 PFU/ml) cells spread over an MB agar plate. Shown are images captured at the indicated time points (hrs). Scale bar 2 mm. Corresponds to Movie EV2. (C-D) The dynamic of SPP1 (C) and Phi29 (D) plaque formation following PY79 (WT) infection was monitored as described in (B). Shown is the diameter of individual plaques for each phage (n ≥ 12), with the average highlighted in red.


( A ) indicated strains were infected with SPP1 ( 10 - 6 PFU / ml ) , spread over MB plates , plaque diameter was monitored after 20 hrs of incubation . Shown is average plaque diameter SD for each ( n ≥ 54 ) .

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(A) The indicated strains were infected with SPP1 (10-6 PFU/ml), spread over MB plates, and plaque diameter was monitored after 20 hrs of incubation. Shown is average plaque diameter and SD for each strain (n ≥ 54).


( D ) PY79 ( WT ) ET19 ( ∆ sigX ) were infected with SPP1 at either high ( phages : 1 : 1 ) or low ( 1 : 20 ) MOI , OD600nm was followed at 2 min intervals . Shown is a representative experiment out of 6 biological repeats , average values SD of 8 technical repeats .

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(D) PY79 (WT) and ET19 (∆sigX) cells were infected with SPP1 at either high (phages:bacteria 1:1) or low (1:20) MOI, and OD600nm was followed at 2 min intervals. Shown is a representative experiment out of 6 biological repeats, and the average values and SD of 8 technical repeats.


( E ) BDR2637 ( Pveg - mCherry ) ( WT , purple ) ET191 ( PrrnE - gfp , ∆ sigX ) ( ∆ sigX , cyan ) were mixed , infected with low concentrations ( 10 - 8 PFU / ml ) of SPP1 , placed on an agarose pad plaque formation was followed by time lapse confocal microscopy . Shown are overlay from mCherry ( purple ) GFP ( cyan ) signals of lawn captured at indicated time points ( hrs ) . plaque is seen as a hole formed on lawn . Scale bar 100 µm . Corresponds to Movie EV4 .

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(E) BDR2637 (Pveg-mCherry) (WT, purple) and ET191 (PrrnE-gfp, ∆sigX) (∆sigX, cyan) cells were mixed, infected with low concentrations (10-8 PFU/ml) of SPP1, placed on an agarose pad and plaque formation was followed by time lapse confocal microscopy. Shown are overlay images from mCherry (purple) and GFP (cyan) signals of the bacterial lawn captured at the indicated time points (hrs). The plaque is seen as a hole formed on the bacterial lawn. Scale bar 100 µm. Corresponds to Movie EV4.


( F ) ET27 ( PsigX - gfp ) were infected with low concentrations ( 10 - 8 PFU / ml ) of SPP1 , placed on an agarose pad , plaque formation was followed by time lapse confocal microscopy . Shown are from GFP ( upper panels ) corresponding phase contrast ( lower panels ) , captured at indicated time points ( hrs ) . plaque is seen as a hole formed on lawn . Scale bar 100 µm .

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(F) ET27 (PsigX-gfp) cells were infected with low concentrations (10-8 PFU/ml) of SPP1, placed on an agarose pad, and plaque formation was followed by time lapse confocal microscopy. Shown are fluorescence from GFP (upper panels) and corresponding phase contrast images (lower panels), captured at the indicated time points (hrs). The plaque is seen as a hole formed on the bacterial lawn. Scale bar 100 µm.


( C ) ET26 ( PsigX - sigX - gfp ) were visualized by microscopy . Shown are signal from SigX - GFP ( cyan ) ( left panel ) , an overlay image of phase contrast ( grey ) signal from SigX - GFP ( cyan ) ( right panel ) . Scale bar 1 μm .

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(C) ET26 (PsigX-sigX-gfp) cells were visualized by fluorescence microscopy. Shown are signal from SigX-GFP (cyan) (left panel), and an overlay image of phase contrast (grey) and signal from SigX-GFP (cyan) (right panel). Scale bar 1 μm.


( D ) BDR2637 ( Pveg - mCherry ) ( WT , purple ) ET261 ( ∆ yueB , PsigX - sigX - gfp ) ( cyan ) were mixed , infected with SPP1 at 5 : 1 ( phages : ) MOI , placed on an agarose pad followed by time lapse microscopy . Shown are overlay from mCherry ( purple ) , SigX - GFP ( cyan ) , phase contrast ( grey ) , captured at indicated time points post infection . Scale bar 1 μm .

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(D) BDR2637 (Pveg-mCherry) (WT, purple) and ET261 (∆yueB, PsigX-sigX-gfp) (cyan) cells were mixed, infected with SPP1 at 5:1 (phages:bacteria) MOI, placed on an agarose pad and followed by time lapse fluorescence microscopy. Shown are overlay images from mCherry (purple), SigX-GFP (cyan), and phase contrast (grey), captured at the indicated time points post infection. Scale bar 1 μm.


( B ) ET29 ( Pveg - mCherry , PIPTG - sigX ) ( purple ) were grown in presence of IPTG mixed with PY79 ( WT ) . mixture was infected with SPP1 - lysin - yfp 5 : 1 ( phages : ) MOI , placed on an IPTG - containing agarose pad , followed by time lapse microscopy . Shown are overlay of phase contrast ( grey ) , signal from mCherry labeled ( purple ) , signal from Lysin - SPP1 - YFP ( cyan ) , captured at indicated time points post infection ( upper panels ) . Corresponding signal from Lysin - SPP1 - YFP ( cyan ) is shown separately ( lower panels ) . Arrows highlight delayed infection of ET29 . Scale bar 1 μm .

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(B) ET29 (Pveg-mCherry, PIPTG-sigX) (purple) cells were grown in the presence of IPTG and mixed with PY79 (WT) cells. The mixture was infected with SPP1-lysin-yfp 5:1 (phages:bacteria) MOI, placed on an IPTG-containing agarose pad, and followed by time lapse fluorescence microscopy. Shown are overlay images of phase contrast (grey), signal from mCherry labeled cells (purple), and signal from Lysin-SPP1-YFP (cyan), captured at the indicated time points post infection (upper panels). Corresponding signal from Lysin-SPP1-YFP (cyan) is shown separately (lower panels). Arrows highlight the delayed infection of ET29 cells. Scale bar 1 μm.


( E ) OF83 ( Ppen - lacIΔ11 - cfp ) ( WT ) ET40 ( Ppen - lacIΔ11 - cfp , PIPTG - sigX ) , grown in presence or absence of IPTG , were infected with SPP1 - delX110lacO64 at 5 : 1 ( phages : ) MOI . formation of LacI - CFP foci on phage was monitored 10 min post infection . Non - infected OF83 were used for comparison . Shown are overlay of phase contrast ( grey ) signal from LacI - CFP ( cyan ) . Scale bar 1 µm . ( F ) of experiment described in ( E ) . Shown is of LacI - CFP foci 10 min post infection of OF83 ET40 by SPP1 , with average values SD ( n ≥ 850 for each population ) .

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(E) OF83 (Ppen-lacIΔ11-cfp) (WT) and ET40 (Ppen-lacIΔ11-cfp, PIPTG-sigX) cells, grown in the presence or absence of IPTG, were infected with SPP1-delX110lacO64 at 5:1 (phages:bacteria) MOI. The formation of LacI-CFP foci on phage DNA was monitored 10 min post infection. Non-infected OF83 cells were used for comparison. Shown are overlay images of phase contrast (grey) and signal from LacI-CFP (cyan). Scale bar 1 µm. (F) Quantification of the experiment described in (E). Shown is the percentage of LacI-CFP foci 10 min post infection of OF83 and ET40 cells by SPP1, with average values and SD (n ≥ 850 cells for each population).


( A ) ET28 ( PIPTG - sigX ) ET42 ( ∆ dltA , PIPTG - sigX ) , grown in presence or absence of IPTG , were infected with SPP1 at 1 : 20 ( phages : ) MOI , OD600nm was followed at 2 min intervals . Shown is a representative experiment out of 6 biological repeats , with average values SD of 8 technical repeats .

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(A) ET28 (PIPTG-sigX) and ET42 (∆dltA, PIPTG-sigX) cells, grown in the presence or absence of IPTG, were infected with SPP1 at 1:20 (phages:bacteria) MOI, and OD600nm was followed at 2 min intervals. Shown is a representative experiment out of 6 biological repeats, with the average values and SD of 8 technical repeats.


( D ) PY79 ( WT ) ET41 ( ΔdltA ) were infected with SPP1 ( 10 - 6 PFU / ml ) , spred over an MB agar plate , plaque diameter was monitored after 20 hrs of incubation . Shown is plaque diameter distributions for each ( n ≥ 40 ) .

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(D) PY79 (WT) and ET41 (ΔdltA) cells were infected with SPP1 (10-6 PFU/ml), spred over an MB agar plate, and plaque diameter was monitored after 20 hrs of incubation. Shown is plaque diameter distributions for each strain (n ≥ 40).


( F ) AR16 ( PrrnE - gfp ) ( WT , cyan ) ET411 ( Pveg - mCherry , ∆ dltA ) ( ∆ dltA , purple ) were mixed , infected with low concentrations ( 10 - 8 PFU / ml ) of SPP1 , placed on an agarose pad , plaque formation was followed by time lapse confocal microscopy . Shown are overlay from GFP ( cyan ) mCherry ( purple ) signals of lawn captured at indicated time points ( hrs ) . plaque is seen as a hole formed on lawn . Scale bar 150 µm .

O O O B-ORGANISM O O O B-GENEPROD O O O O O O B-ORGANISM O O O B-GENEPROD O O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O O O B-EXP_ASSAY O O O O B-ORGANISM O O O O B-SMALL_MOLECULE O O B-EXP_ASSAY I-EXP_ASSAY O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(F) AR16 (PrrnE-gfp) (WT, cyan) and ET411 (Pveg-mCherry, ∆dltA) (∆dltA, purple) cells were mixed, infected with low concentrations (10-8 PFU/ml) of SPP1, placed on an agarose pad, and plaque formation was followed by time lapse confocal microscopy. Shown are overlay images from GFP (cyan) and mCherry (purple) signals of the bacterial lawn captured at the indicated time points (hrs). The plaque is seen as a hole formed on the bacterial lawn. Scale bar 150 µm.


( G ) ET43 ( dltA - yfp ) were infected with low concentrations ( 10 - 8 PFU / ml ) of SPP1 , placed on an agarose pad , plaque formation was followed by time lapse confocal microscopy . Shown are fluoresce from DltA - YFP signal ( upper panels ) corresponding phase contrast ( lower panels ) , captured at indicated time points ( hrs ) . plaque is seen as a hole formed on lawn . Scale bar 100 µm .

O O O B-ORGANISM O B-GENEPROD O B-GENEPROD O O O O O O O O O O B-EXP_ASSAY O O O O B-ORGANISM O O O O B-SMALL_MOLECULE O O B-EXP_ASSAY I-EXP_ASSAY O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O B-GENEPROD O B-GENEPROD O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(G) ET43 (dltA-yfp) cells were infected with low concentrations (10-8 PFU/ml) of SPP1, placed on an agarose pad, and plaque formation was followed by time lapse confocal microscopy. Shown are fluoresce from DltA-YFP signal (upper panels) and corresponding phase contrast images (lower panels), captured at the indicated time points (hrs). The plaque is seen as a hole formed on the bacterial lawn. Scale bar 100 µm.


A Normal human fibroblasts HGPS fibroblasts were serum - starved for 48 h stained for progerin ( red ) , acetylated tubulin ( green ) ( blue ) . representative show that with high progerin ( inset 2 ) , but not low progerin ( inset 1 ) , displays defective cilia formation . Scale bars , 20 μm or 2 μm ( in magnified ) .

O O B-ORGANISM B-CELL_TYPE B-DISEASE B-CELL_TYPE O B-TISSUE O O O O O B-EXP_ASSAY O B-GENEPROD O O O O B-EXP_ASSAY B-GENEPROD O O O O O O O O O O O O B-GENEPROD O O O O O O O O B-GENEPROD O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O

A Normal human fibroblasts and HGPS fibroblasts were serum-starved for 48 h and stained for progerin (red), acetylated tubulin (green) and DNA (blue). The representative images show that the cell with high progerin (inset 2), but not low progerin (inset 1), displays defective cilia formation. Scale bars, 20 μm or 2 μm (in the magnified images).


B progerin of was measured by Zeiss ZEN2 software ( n ≥ 148 ) . dashed indicates background level of progerin . A . U . , arbitrary units .

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B The progerin fluorescence intensity of the cells was measured by the Zeiss ZEN2 software (n ≥ 148). The dashed line indicates the background level of progerin. A.U., arbitrary units.


were grown in growth medium stained for progerin ( red ) F - actin with phalloidin ( green ) . with high or low progerin expression was defined by progerin above or below 3 . 5 x 106 ( A . U . ) , respectively . spreading was measured according to phalloidin ( n ≥ 30 ) . Scale bars , 50 m .

O O O O O B-EXP_ASSAY O B-GENEPROD O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O B-SMALL_MOLECULE O O O O O O O O B-GENEPROD B-EXP_ASSAY O O O B-GENEPROD O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O B-SMALL_MOLECULE O O O O O O O O O O O O

The cells were grown in the growth medium and stained for progerin (red) and F-actin with phalloidin (green). The cell with high or low progerin expression was defined by the progerin fluorescence intensity above or below 3.5 x 106 (A.U.), respectively. The cell spreading area was measured according to the phalloidin staining (n ≥ 30). Scale bars, 50 m.


D were serum - starved for 32 h to allow cilia formation then treated with ( + ) or without ( - ) 250 nM cytochalasin D ( Cyto D ) for another 16 h before fixation . were stained for progerin ( red ) , acetylated tubulin ( green ) , F - actin ( ) , ( blue ) . numbers of actin filaments within 60 μm2 around cilia ( as illustrated by circle ) from under serum - starvation without Cyto D treatment were measured ( n ≥ 55 ) . Scale bars , 20 μm or 2 μm ( in magnified insets ) .

O O B-TISSUE O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O B-EXP_ASSAY O B-GENEPROD O O O O B-EXP_ASSAY B-GENEPROD O O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O B-SUBCELLULAR O O O O O O O O B-TISSUE O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O

D The cells were serum-starved for 32 h to allow cilia formation and then treated with (+) or without (-) 250 nM cytochalasin D (Cyto D) for another 16 h before fixation. The cells were stained for progerin (red), acetylated tubulin (green), F-actin (white), and DNA (blue). The numbers of actin filaments within 60 μm2 around the cilia (as illustrated by the circle) from the cells under serum-starvation without Cyto D treatment were measured (n ≥ 55). Scale bars, 20 μm or 2 μm (in the magnified insets).


E , F were subjected to immunofluorescence stain as in ( D ) . of with cilia in total counted ( E , n ≥ 50 ) cilia ( F , n ≥ 50 ) were measured .

O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O B-SUBCELLULAR O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O

E,F The cells were subjected to immunofluorescence stain as in (D). The percentage of the cells with cilia in the total counted cells (E, n ≥ 50) and the cilia length (F, n ≥ 50) were measured.


A skeletal ( tabialis anterior ) from control ( Lmna + / + Lmna + / - ) Lmna - / - mice were stained for acetylated tubulin ( green ) nucleus ( blue ) . representative with low high magnification are shown . insets are enlarged for cilia .

O B-TISSUE O B-TISSUE I-TISSUE O O O O B-GENEPROD O O O B-GENEPROD O O O O B-GENEPROD O O O B-ORGANISM O B-EXP_ASSAY O B-EXP_ASSAY B-GENEPROD O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O B-SUBCELLULAR O

A The skeletal (tabialis anterior) muscles from control (Lmna+/+ and Lmna+/-) and Lmna-/- mice were stained for acetylated tubulin (green) and nucleus (blue). The representative images with low and high magnification are shown. The insets are enlarged images for cilia.


B kidneys from control Lmna - / - mice were stained for acetylated tubulin ( green ) AQ1 ( a marker for proximal tubules , red ) . insets show cilia of proximal renal tubule .

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B The kidneys from control and Lmna-/- mice were stained for acetylated tubulin (green) and AQ1 (a marker for the proximal tubules, red). The insets show the cilia of proximal renal tubule cells.


C uterus from control Lmna - / - mice was stained for acetylated tubulin ( green ) keratin 8 ( red ) . insets show cilia at stroma of uterus .

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C The uterus from control and Lmna-/- mice was stained for acetylated tubulin (green) and keratin 8 (red). The insets show the cilia at the stroma of the uterus.


D ovary from control Lmna - / - mice were stained for acetylated tubulin ( green ) keratin 8 ( red ) . insets show cilia of granulosa in follicles of ovary .

O B-TISSUE O O B-GENEPROD O O O B-ORGANISM O B-EXP_ASSAY O B-EXP_ASSAY B-GENEPROD O O O B-GENEPROD I-GENEPROD O O O O O O B-SUBCELLULAR O B-CELL_TYPE O B-CELL_TYPE O B-TISSUE O

D The ovary from control and Lmna-/- mice were stained for acetylated tubulin (green) and keratin 8 (red). The insets show the cilia of the granulosa cells in the follicles of the ovary.


E , F cilia ( E , n = 150 - 180 ) cilia positive ( F , n ≥ 1197 ) in each organ from 4 - week - old Lmna - / - mice ( n = 3 ) their littermate controls ( n = 3 ) were measured .

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E, F The cilia length (E, n=150-180) and cilia positive cells (F, n ≥ 1197) in each organ from 4-week-old Lmna-/- mice (n=3) and their littermate controls (n=3) were measured.


A RPE were infected with lentiviruses encoding shRNAs specific to lamin A / C ( shLamin A / C ) or luciferase ( shLuc ) . Exogenous lamin A was re - expressed in lamin A / C - depleted ( shLamin A / C - Lamin A ) . An equal amount of whole lysates was analyzed by immunoblotting with antibodies as indicated .

O B-CELL_TYPE O O O B-ORGANISM O O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD O O O O O B-GENEPROD I-GENEPROD O O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD O O O O O O O O O O O B-EXP_ASSAY O O O O O

A RPE cells were infected with lentiviruses encoding shRNAs specific to lamin A/C (shLamin A/C) or luciferase (shLuc). Exogenous lamin A was re-expressed in the lamin A/C-depleted cells (shLamin A/C-Lamin A). An equal amount of whole cell lysates was analyzed by immunoblotting with antibodies as indicated.


B were serum - starved for 48 h stained for acetylated tubulin ( green ) , γ - tubulin ( red ) ( blue ) . depletion of lamin A / C ( shLamin A / C ) led to impaired ciliogenesis , as manifested by no cilia ( inset 1 ) or short cilia ( inset 2 ) . Scale bars , 20 μm or 2 μm ( insets ) .

O O B-TISSUE O O O O O B-EXP_ASSAY O B-EXP_ASSAY B-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-EXP_ASSAY O O O O O B-SUBCELLULAR O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O

B The cells were serum-starved for 48 h and stained for acetylated tubulin (green), γ-tubulin (red) and DNA (blue). The depletion of lamin A/C (shLamin A/C) led to impaired ciliogenesis, as manifested by no cilia (inset 1) or short cilia (inset 2). Scale bars, 20 μm or 2 μm (insets).


C of with cilia in total counted ( n ≥ 626 ) was measured .

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C The percentage of the cells with cilia in the total counted cells (n ≥ 626) was measured.


D of cilia was measured ( n ≥ 87 in each group ) .

D The length of cilia was measured (n ≥ 87 in each group).


E were stained for acetylated tubulin ( green ) γ - tubulin ( red ) after various intervals of serum starvation . of cilia - positive at each time point ( n ≥ 372 ) was measured .

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E Cells were stained for acetylated tubulin (green) and γ-tubulin (red) after various intervals of serum starvation. The percentage of cilia-positive cells at each time point (n ≥ 372) was measured.


A - C RPE as described in Fig 3 were stained for paxillin ( as a marker for focal adhesion ; green ) , F - actin ( red ) ( blue ) . representative are shown ( A ) . Scale bars , 20 μm or 5 μm ( insets ) . spreading of ( B , n ≥ 108 ) number of focal adhesions per ( C , n ≥ 30 ) were measured .

O O O B-CELL_TYPE O O O O O O B-EXP_ASSAY O B-GENEPROD O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O 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-EXP_ASSAY O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O

A-C The RPE cells as described in Fig 3 were stained for paxillin (as a marker for focal adhesion; green), F-actin (red) and DNA (blue). The representative images are shown (A). Scale bars, 20 μm or 5 μm (insets). The cell spreading area of the cells (B, n ≥ 108) and the number of focal adhesions per cell (C, n ≥ 30) were measured.


D - G were serum - starved for 32 h then treated with ( + ) or without ( - ) Cyto D ( 250 nM ) for another 16 h . were stained for acetylated tubulin ( ) , pericentrin ( green ) , F - actin ( red ) ( blue ) . representative are shown ( D ) . left insets show merged signals of acetylated tubulin pericentrin . Scale bars , 20 μm or 2 μm ( magnified ) . numbers of actin filaments within 60 μm2 around basal ( as illustrated by circle ) from under serum - starvation without Cyto D treatment were measured ( E , n ≥ 132 ) . of with cilia in total counted ( F , n ≥ 357 ) of cilia ( G , n ≥ 126 ) were measured .

O O O O B-TISSUE O O O O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O B-EXP_ASSAY O B-EXP_ASSAY B-GENEPROD O O O B-GENEPROD O O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY B-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O B-SUBCELLULAR O O O O O O O O B-TISSUE O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O

D-G The cells were serum-starved for 32 h and then treated with (+) or without (-) Cyto D (250 nM) for another 16 h. The cells were stained for acetylated tubulin (white), pericentrin (green), F-actin (red) and DNA (blue). The representative images are shown (D). The left insets show the merged signals of acetylated tubulin and pericentrin. Scale bars, 20 μm or 2 μm (magnified images). The numbers of actin filaments within 60 μm2 around the basal body (as illustrated by the circle) from the cells under serum-starvation without Cyto D treatment were measured (E, n ≥ 132). The percentage of the cells with cilia in the total counted cells (F, n ≥ 357) and the length of cilia (G, n ≥ 126) were measured.


A An equal amount of whole lysates from RPE or those expressing shLamin A / C or luciferase ( shLuc ) was analyzed by immunoblotting with antibodies as indicated .

O O O O O O O O B-CELL_TYPE O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O O O O O B-EXP_ASSAY O O O O O

A An equal amount of whole cell lysates from RPE cells or those expressing shLamin A/C or luciferase (shLuc) was analyzed by the immunoblotting with antibodies as indicated.


RPE were infected with lentiviruses expressing shRNAs to nesprin 2 ( shNesprine 2 , clone # 1 # 2 ) or luciferase ( shLuc ) as a control . An equal amount of whole lysates was analyzed by immunoblotting with antibodies as indicated ( B ) .

B-CELL_TYPE O O O B-ORGANISM O O O B-GENEPROD I-GENEPROD O O B-GENEPROD O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O

RPE cells were infected with lentiviruses expressing shRNAs to nesprin 2 (shNesprine 2, clone #1 and #2) or luciferase (shLuc) as a control. An equal amount of whole cell lysates was analyzed by immunoblotting with antibodies as indicated (B).


were serum - starved for 48 h then stained for acetylated tubulin ( ) , pericentrin ( green ) , F - actin ( red ) ( blue ) . representative are shown ( C ) left insets show merged signals of acetylated tubulin pericentrin . Scale bars , 20 μm or 2 μm ( magnified ) . numbers of F - actin stress within 60 μm2 around basal bodies ( as illustrated by circle ) were measured ( D , n ≥ 53 ) .

O B-TISSUE O O O O O O B-EXP_ASSAY O B-EXP_ASSAY B-GENEPROD O O O B-GENEPROD O O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY B-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O O O O O

The cells were serum-starved for 48 h and then stained for acetylated tubulin (white), pericentrin (green), F-actin (red) and DNA (blue). The representative images are shown (C) The left insets show the merged signals of acetylated tubulin and pericentrin. Scale bars, 20 μm or 2 μm (magnified images). The numbers of F-actin stress fibers within 60 μm2 around the basal bodies (as illustrated by the circle) were measured (D, n ≥ 53).


E , F RPE expressing shNesprine 2 or shLuc were serum - starved for 32 h then treated with ( + ) or without ( - ) Cyto D for another 16 h . of with cilia in total counted ( E , n ≥ 197 ) of cilia ( F , n ≥ 52 ) were measured .

O O O B-CELL_TYPE O O B-GENEPROD O O O B-TISSUE O O O O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O B-SUBCELLULAR O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O

E,F RPE cells expressing shNesprine 2 or shLuc were serum-starved for 32 h and then treated with (+) or without (-) Cyto D for another 16 h. The percentage of the cells with cilia in the total counted cells (E, n ≥ 197) and the length of cilia (F, n ≥ 52) were measured.


A , B RPE expressing shRNAs specific to lamin A / C or nesprin 2 ( shNesprin 2 , clones # 1 # 2 ) were stained for G - actin with DNase I ( green ) . Representative are shown ( A ) . Scale bars , 20 μm . proportion of G - actin to whole was measured ( B , n ≥ 67 ) .

O O O B-CELL_TYPE O O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD O O B-GENEPROD O O O O O O O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O

A,B RPE cells expressing shRNAs specific to lamin A/C or nesprin 2 (shNesprin 2, clones #1 and #2) were stained for G-actin with DNase I (green). Representative images are shown (A). Scale bars, 20 μm. The proportion of nuclear G-actin fluorescence intensity to the whole cell was measured (B, n ≥ 67).


C An equal amount of whole lysates from RPE ( control ) those expressing shRNAs to importin 9 ( shImportin 9 , clones # 1 # 2 ) was analyzed by immunoblotting with antibodies as indicated .

O O O O O O O O B-CELL_TYPE O O O O O O O B-GENEPROD I-GENEPROD O O B-GENEPROD O O O O O O O O O O B-EXP_ASSAY O O O O O

C An equal amount of whole cell lysates from RPE cells (control) and those expressing shRNAs to importin 9 (shImportin 9, clones #1 and #2) was analyzed by immunoblotting with antibodies as indicated.


D - F expressing shRNAs to importin 9 were stained for G - actin ( green ) F - actin ( red ) . representative are shown ( D ) . Scale bars , 20 μm or 10 μm ( insets ) . proportion of G - actin to whole ( E , n > 152 ) spreading ( F , n ≥ 139 ) were measured .

O O O O O O B-GENEPROD I-GENEPROD O B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O 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-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O

D-F The cells expressing shRNAs to importin 9 were stained for G-actin (green) and F-actin (red). The representative images are shown (D). Scale bars, 20 μm or 10 μm (insets). The proportion of nuclear G-actin intensity to the whole cell (E, n > 152) and the cell spreading area (F, n ≥ 139) were measured.


G - I RPE those expressing shRNAs to importin 9 were serum - starved for 32 h then treated with ( + ) or without ( - ) Cyto D for another 16 h then stained for acetylated tubulin ( ) , pericentrin ( green ) F - actin ( red ) . Representative are shown ( G ) . left insets show merged signals of acetylated tubulin pericentrin . Scale bars , 20 μm or 2 μm ( magnified ) . of with cilia ( H , n ≥ 385 ) of cilia ( I , n ≥ 111 ) were measured .

O O O B-CELL_TYPE O O O O B-GENEPROD I-GENEPROD O B-TISSUE O O O O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O B-EXP_ASSAY O B-EXP_ASSAY B-GENEPROD O O O B-GENEPROD O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O O O O O O B-EXP_ASSAY B-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O B-SUBCELLULAR O O O O O O O O O O

G-I RPE cells and those expressing shRNAs to importin 9 were serum-starved for 32 h and then treated with (+) or without (-) Cyto D for another 16 h and then stained for acetylated tubulin (white), pericentrin (green) and F-actin (red). Representative images are shown (G). The left insets show the merged signals of acetylated tubulin and pericentrin. Scale bars, 20 μm or 2 μm (magnified images). The percentage of the cells with cilia (H, n ≥ 385) and the length of cilia (I, n ≥ 111) were measured.


A RPE expressing shRNAs specific to luciferase ( shLuc . ) , lamin A / C , or nesprin 2 were grown in growth medium ( + serum ) or serum - starved for 1 h ( - serum ) then stained for Arp2 ( red ) , CEP164 ( a marker for mother centriole , green ) acetylated tubulin ( ) . representative are shown . Note that Arp2 was found to localize exclusively at mother centriole ( upper panels ) or at both mother daughter centrioles ( lower panels ) . Scale bars , 0 . 5 μm . of with Arp2 localized exclusively at mother centriole in total counted was measured ( n = 131 - 240 in each group ) .

O B-CELL_TYPE O O O O B-GENEPROD O O O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD I-GENEPROD O O O O O O O B-TISSUE O O B-TISSUE O O O O O O O B-TISSUE O O B-EXP_ASSAY O B-GENEPROD O O O O B-GENEPROD O O O O O B-SUBCELLULAR O O O B-EXP_ASSAY B-GENEPROD O O O O O O O O O B-GENEPROD O O O B-EXP_ASSAY O O O B-SUBCELLULAR O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O B-GENEPROD B-EXP_ASSAY O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O

A RPE cells expressing shRNAs specific to luciferase (shLuc.), lamin A/C, or nesprin 2 were grown in the growth medium (+ serum) or serum-starved for 1 h (- serum) and then stained for Arp2 (red), CEP164 (a marker for the mother centriole, green) and acetylated tubulin (white). The representative images are shown. Note that Arp2 was found to localize exclusively at the mother centriole (upper panels) or at the both mother and daughter centrioles (lower panels). Scale bars, 0.5 μm. The percentage of cells with Arp2 localized exclusively at the mother centriole in the total counted cells was measured (n=131-240 cells in each group).


B were serum - starved for 48 h then stained for acetylated tubulin ( green ) , Arp2 ( red ) , γ - tubulin ( ) . representative are shown . Scale bars , 1 μm ( upper panels ) 0 . 5 μm ( lower panels ) . Note that with Arp2 at both mother daughter centrioles fails to form cilium .

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B The cells were serum-starved for 48 h and then stained for acetylated tubulin (green), Arp2 (red), and γ-tubulin (white). The representative images are shown. Scale bars, 1 μm (upper panels) and 0.5 μm (lower panels). Note that the cell with Arp2 at the both mother and daughter centrioles fails to form cilium.


C were serum - starved for 48 h then stained for myosin Va ( red ) , acetylated tubulin ( green ) γ - tubulin ( ) . Scale bars , 0 . 5 μm . cartoon illustrates that myosin Va - associated vesicles ( CV + PCV ) specifically accumulate at mother centriole in most control ( as in " one centriole " image ) . CV , ciliary vesicle . PCV , periciliary vesicle . of with myosin Va exclusively at mother centriole in total counted was measured ( n = 160 - 200 in each group ) .

O O B-TISSUE O O O O O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD O O O O B-EXP_ASSAY B-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD O O B-SUBCELLULAR O B-SUBCELLULAR O B-SUBCELLULAR O O O O O B-SUBCELLULAR O O O O O O O O B-SUBCELLULAR O O O O B-SUBCELLULAR O B-SUBCELLULAR I-SUBCELLULAR O B-SUBCELLULAR O B-SUBCELLULAR I-SUBCELLULAR O O O B-GENEPROD I-GENEPROD O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O

C The cells were serum-starved for 48 h and then stained for myosin Va (red), acetylated tubulin (green) and γ-tubulin (white). Scale bars, 0.5 μm. The cartoon illustrates that myosin Va-associated vesicles (CV+PCV) specifically accumulate at the mother centriole in most control cells (as in the "one centriole" image). CV, ciliary vesicle. PCV, periciliary vesicle. The percentage of cells with myosin Va exclusively at the mother centriole in the total counted cells was measured (n=160-200 cells in each group).


A RPE expressing shRNAs specific to luciferase ( shLuc ) or lamin A / C ( shLamin A / C ) were serum - starved for 48 h then stained for acetylated tubulin ( Ac - tubulin , green ) CEP164 ( red ) . representative show that of CEP164 at mother centriole of cilia - negative ( left column ) cilia - positive ( right column ) basal bodies are similar . graphs shown are of CEP164 at cilia - negative basal bodies cilia - positive basal bodies .

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A RPE cells expressing shRNAs specific to luciferase (shLuc) or lamin A/C (shLamin A/C) were serum-starved for 48 h and then stained for acetylated tubulin (Ac-tubulin, green) and CEP164 (red). The representative images show that the fluorescence intensity of CEP164 at the mother centriole of cilia-negative (left column) and cilia-positive (right column) basal bodies are similar. The graphs shown are the quantification of CEP164 at cilia-negative basal bodies and cilia-positive basal bodies.


B were serum - starved for 32 h then treated with ( + ) or without ( - ) Cyto D for another 16 h . were stained for acetylated tubulin ( Ac - tubulin , green ) TTBK2 ( red ) . graphs shown are quantifications of TTBK2 at cilia - negative basal bodies cilia - positive basal bodies .

O O B-TISSUE O O O O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O B-EXP_ASSAY O B-EXP_ASSAY B-GENEPROD O B-EXP_ASSAY O B-GENEPROD O O O B-GENEPROD O O O O O O O O O B-GENEPROD O B-SUBCELLULAR O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O B-SUBCELLULAR I-SUBCELLULAR O

B The cells were serum-starved for 32 h and then treated with (+) or without (-) Cyto D for another 16 h. The cells were stained for acetylated tubulin (Ac-tubulin, green) and TTBK2 (red). The graphs shown are the quantifications of TTBK2 intensity at cilia-negative basal bodies and cilia-positive basal bodies.


C as in ( B ) were stained for acetylated tubulin ( Ac - tubulin , green ) CP110 ( red ) . representative show that CP110 is not removed from mother centriole at some of cilia - negative basal bodies ( left column ) , whereas it is removed from mother centriole at cilia - positive basal bodies ( right column ) . graphs shown are quantifications of CP110 - removal at cilia - negative basal bodies cilia - positive basal bodies .

O O O O O O O B-EXP_ASSAY O B-EXP_ASSAY B-GENEPROD O B-EXP_ASSAY O B-GENEPROD O O O B-GENEPROD O O O O O O O B-GENEPROD O O O O O B-SUBCELLULAR O O O B-SUBCELLULAR O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O B-SUBCELLULAR O B-SUBCELLULAR O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O B-GENEPROD O O O B-SUBCELLULAR O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O B-SUBCELLULAR I-SUBCELLULAR O

C The cells as in (B) were stained for acetylated tubulin (Ac-tubulin, green) and CP110 (red). The representative images show that CP110 is not removed from the mother centriole at some of cilia-negative basal bodies (left column), whereas it is removed from the mother centriole at cilia-positive basal bodies (right column). The graphs shown are the quantifications of CP110-removal at cilia-negative basal bodies and cilia-positive basal bodies.


B t - SNE plots showing integrated of Prrx1 - Cre - ( 8 - week - old mice , n = 3 ) Lepr - Cre - traced ( 8 - week - old mice , n = 4 ) . were colored by clusters ( left ) or samples ( right ) . Top 20 PCs were chosen for clustering .

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B t-SNE plots showing integrated analysis of Prrx1-Cre- (8-week-old mice, n=3 males) and Lepr-Cre-traced (8-week-old mice, n=4 males) cells. Cells were colored by clusters (left) or samples (right). Top 20 PCs were chosen for the clustering.


D Violin plots showing expression of feature genes for each cluster .

D Violin plots showing the expression of feature genes for each cluster.


E In situ hybridization of Col3a1 on femur sections . Col3a1 was expressed in perichondral ( a ) periosteal ( b ) regions , but not in bone marrow . B : Bone ; BM : Bone marrow ; Peri : Periosteum ; Endo : Endosteum . Scale bars were 100 µm . Arrowheads indicated periosteum .

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E In situ hybridization of Col3a1 on femur sections. Col3a1 was expressed in the perichondral (a) and periosteal (b) regions, but not in the bone marrow. B: Bone; BM: Bone marrow; Peri: Periosteum; Endo: Endosteum. Scale bars were 100 µm. Arrowheads indicated the periosteum.


E Violin plots showing expression of feature genes for each cluster .

E Violin plots showing the expression of feature genes for each cluster.


F Immunofluorescent of distal femur in 8 - week - old Lepr - Cre ; tdTomato mice . Arrowheads indicated tdTomato + periosteal . Two representative periosteal regions were magnified ( a b ) . GP : Growth plate . B : Bone . BM : Bone marrow . Peri : Periosteum . Scale bars were 100 µm . G Immunofluorescent of distal femur in 8 - week - old Lepr - Cre ; tdTomato mice 7 days after fracture . Aggrecan ( green ) , tdTomato ( red ) , DAPI ( blue ) . Arrowheads indicated tdTomato + periosteal . Two representative regions were magnified ( a b ) . Scale bars were 100 µm .

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F Immunofluorescent images of the distal femur in 8-week-old Lepr-Cre; tdTomato mice. Arrowheads indicated tdTomato+ periosteal cells. Two representative periosteal regions were magnified (a and b). GP: Growth plate. B: Bone. BM: Bone marrow. Peri: Periosteum. Scale bars were 100 µm. G Immunofluorescent images of the distal femur in 8-week-old Lepr-Cre; tdTomato mice 7 days after fracture. Aggrecan (green), tdTomato (red), DAPI (blue). Arrowheads indicated tdTomato+ periosteal cells. Two representative regions were magnified (a and b). Scale bars were 100 µm.


B Dot plots showing gene expression patterns of selected genes in each cluster .

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B Dot plots showing the gene expression patterns of selected genes in each cluster.


D Flow cytometry of BrdU incorporation in uncultured Lepr - Cre + Notch3 + / - BMSCs ( n = 4 independent experiments ) . Eight - week - old Lepr - Cre ; tdTomato mice were given a single intraperitoneal injection of BrdU ( 100 mg / kg mass ) maintained on 0 . 5 mg / ml of BrdU in drinking water for 14 days .

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D Flow cytometry analysis of BrdU incorporation in uncultured Lepr-Cre+Notch3+/- BMSCs (n=4 independent experiments). Eight-week-old Lepr-Cre; tdTomato mice were given a single intraperitoneal injection of BrdU (100 mg/kg body mass) and maintained on 0.5 mg/ml of BrdU in the drinking water for 14 days.


E Immunofluorescent of distal femur in 8 - week - old Lepr - Cre ; tdTomato mice . Notch3 ( green ) , tdTomato ( red ) , CD31 ( gray ) , DAPI ( blue ) . Sinusoid ( a ) , arteriole ( b ) , artery ( c ) avascular bone marrow ( BM , d ) regions were shown . Arrowheads indicated Notch3 + tdTomato + adventitial closely associated with ECs . Arrows indicated Notch3 - tdTomato + reticular in bone marrow that were not associated with ECs . Scale bars were 10 µm . F Localization of Lepr - Cre + Notch3 + relative to bone marrow ECs ( n = 5 biological replicates from three independent experiments ) . within 5 µm diameter of ECs were considered adjacent .

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E Immunofluorescent images of the distal femur in 8-week-old Lepr-Cre; tdTomato mice. Notch3 (green), tdTomato (red), CD31 (gray), DAPI (blue). Sinusoid (a), arteriole (b), artery (c) and avascular bone marrow (BM, d) regions were shown. Arrowheads indicated Notch3+ tdTomato+ adventitial cells closely associated with ECs. Arrows indicated Notch3- tdTomato+ reticular cells in the bone marrow that were not associated with ECs. Scale bars were 10 µm. F Localization quantification of Lepr-Cre+Notch3+ cells relative to bone marrow ECs (n=5 biological replicates from three independent experiments). Cells within 5 µm diameter of ECs were considered adjacent. G


H Volcano plot showing DEGs in bulk - seq ( n = 3 biological replicates ) . Green dots represent down - regulated genes . Red dots represent up - regulated genes .

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H Volcano plot showing DEGs in the bulk RNA-seq analysis (n=3 biological replicates). Green dots represent down-regulated genes. Red dots represent up-regulated genes.


O showing average expression ( column - wide Z score ) of secreted factors with clustering .

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O Heatmap showing the average expression levels (column-wide Z score) of secreted factors with clustering.


C showing pseudotime - dependent gene expression . Bars on top were colored as in ( A ) . Representative genes were highlighted on both sides .

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C Heatmap showing pseudotime-dependent gene expression. Bars on the top were colored as in (A). Representative genes were highlighted on both sides.


F Representative of adipogenic , osteogenic chondrogenic differentiation following shRNA knockdown in Lepr - Cre + BMSCs . Scale bars were 100 µm .

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F Representative images of adipogenic, osteogenic and chondrogenic differentiation following shRNA knockdown in Lepr-Cre+ BMSCs. Scale bars were 100 µm.


G qPCR of expression of Pparg , Bglap Acan in control or knockdown group ( n = 3 independent experiments ) . statistical significance of differences was analyzed by one - way ANOVAs with Tukey ' s multiple comparison . Data represented Mean ± SD . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

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G qPCR analysis of the expression levels of Pparg, Bglap and Acan in control or knockdown group (n=3 independent experiments). The statistical significance of differences was analyzed by one-way ANOVAs with Tukey's multiple comparison tests. Data represented Mean ± SD. *p<0.05, **p<0.01, ***p<0.001.


B Violin plots showing expression of feature genes for each cluster .

B Violin plots showing the expression of feature genes for each cluster.


F Immunostaining of Sca - 1 on femur sections of 8 - week - old Lepr - Cre ; tdTomato mice . Arrows indicated Sca - 1 + tdTomato + inside cortical bones of femur diaphysis . Arrowheads indicated Sca - 1 + tdTomato + in femur periosteum . B : Bone . BM : Bone marrow . Peri : Periosteum . Scale bars were 100 µm .

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F Immunostaining of Sca-1 on femur sections of 8-week-old Lepr-Cre; tdTomato mice. Arrows indicated Sca-1+ tdTomato+ cells inside cortical bones of the femur diaphysis. Arrowheads indicated Sca-1+ tdTomato+ cells in the femur periosteum. B: Bone. BM: Bone marrow. Peri: Periosteum. Scale bars were 100 µm.


G Representative flow cytometry plots of long bone periosteum in 8 - week - old Lepr - Cre ; tdTomato mice . of DAPI - CD45 - Ter119 - Tie2 - tdTomato + Sca - 1 + / - were shown .

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G Representative flow cytometry plots of long bone periosteum in 8-week-old Lepr-Cre; tdTomato mice. The percentage of DAPI- CD45- Ter119- Tie2- tdTomato+ Sca-1+/- cells were shown.


I , J average number of CFU - F colonies formed ( I ) average number per colony ( J ) were shown ( n = 4 independent experiments ) . statistical significance of differences was assessed using one - way ANOVAs with Tukey ' s multiple comparison . data represented Mean ± SD . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

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I, J The average number of CFU-F colonies formed (I) and the average cell number per colony (J) were shown (n=4 independent experiments). The statistical significance of differences was assessed using one-way ANOVAs with Tukey's multiple comparison tests. All data represented Mean ± SD. *p<0.05, **p<0.01, ***p<0.001.


A , B Representative oil red after 7 days of adipogenic differentiation in Lepr - Cre + subsets ( A ) qPCR of Adipoq Pparg expression ( B ) ( n = 3 independent experiments ) . Scale bars were 100 µm .

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A, B Representative oil red staining after 7 days of adipogenic differentiation in Lepr-Cre+ subsets (A) and qPCR analysis of Adipoq and Pparg expression (B) (n=3 independent experiments). Scale bars were 100 µm.


C , D In vitro chondrogenic differentiation of sorted periosteal . Representative alcian blue after chondrogenic differentiation for 21 days cryosection ( C ) . Scale bars were 100 µm . Chondrogenic efficiencies were quantified by qPCR of Acan Col2a1 expression ( D ) ( n = 3 independent experiments ) .

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C, D In vitro chondrogenic differentiation of sorted periosteal cells. Representative alcian blue staining after chondrogenic differentiation for 21 days and cryosection (C). Scale bars were 100 µm. Chondrogenic efficiencies were quantified by qPCR analysis of Acan and Col2a1 expression (D) (n=3 independent experiments).


E , F In vitro osteogenic differentiation of sorted periosteal . Representative alizarin red after osteogenic differentiation for 14 days . Scale bars were 200 µm . Osteogenic efficiencies were quantified by qPCR of Sp7 Bglap expression ( F ) ( n = 3 independent experiments ) .

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E, F In vitro osteogenic differentiation of sorted periosteal cells. Representative alizarin red staining after osteogenic differentiation for 14 days. Scale bars were 200 µm. Osteogenic efficiencies were quantified by qPCR analysis of Sp7 and Bglap expression (F) (n=3 independent experiments).


( H ) Immunoblot representing mTOR - dependent targets with defined 5 ' TOP motifs 3 hours 2 days after TAC . n = 3 biological replicates per condition .

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(H) Immunoblot representing levels mTOR-dependent targets with defined 5'TOP motifs 3 hours and 2 days after TAC. n=3 biological replicates per condition.


( J ) Immunoblots quantifications of expression of mTOR targets two days after TAC surgery after mTOR inhibition with Torin 1 . * p ≤ 0 . 05 vs sham surgery , # p ≤ 0 . 05 vs TAC 2d surgery . t - , n = 5 biological replicates per group . Data information : Error bars indicate means ±

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(J) Immunoblots and quantifications of protein expression of mTOR targets two days after TAC surgery and after mTOR inhibition with Torin 1.* p≤ 0.05 vs sham surgery, # p≤ 0.05 vs TAC 2d surgery. t-test, n=5 biological replicates per group. Data information: Error bars indicate means ± SEM


( K ) RT - qPCR for indicated mRNAs 2 days after TAC for indicated groups , n = 5 biological replicates per group . Data information : Error bars indicate means ±

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(K) RT-qPCR for indicated mRNAs 2 days after TAC for indicated groups, n=5 biological replicates per group. Data information: Error bars indicate means ± SEM


( C ) expression of Cand2 in NRCMs treated with PE for 3 24 h , 24h of Torin 1 treatment . mTORC1 induction was monitored by downstream targets , p4EBP1 pS6K . rpS5 - example of mTOR - dependent mRNA used as a positive control . Csq - calsequestrin used as a houskeeping .

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(C) Protein expression of Cand2 in NRCMs treated with PE for 3 and 24 h, and 24h of Torin 1 treatment. mTORC1 induction was monitored by downstream targets, p4EBP1 and pS6K proteins. rpS5 - example of mTOR-dependent mRNA used as a positive control. Csq - calsequestrin used as a houskeeping protein.


( F , G ) Ribo - seq - seq expression data of Cand2 in sham TAC operated ( CPM - per million ) . One - way ANOVA , * * - P ≤ 0 . 01 , n = 2 - 4 biological replicates per group . Data information : Error bars indicate means ±

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(F, G) Ribo-seq and RNA-seq expression data of Cand2 in sham and TAC operated animals (CPM - count per million). One-way ANOVA, ** - P ≤ 0.01, n=2-4 biological replicates per group. Data information: Error bars indicate means ± SEM


( J ) Schematic representation of reporter consisting of human Cand2 5 ' UTR downstream Renilla luciferase coding . A potential TOP - like motif is highlighted in a grey box . Black arrows indicate transcription start sites in adult heart according to Database of Transcriptional Start Sites ( Release 10 . 1 ) . effect of Cand2 5 ' UTR on translation measured by luciferase activity in normal conditions after mTORC1 block with Torin 1 . eEF2 - positive control of reporter with defined 5 ' TOP motif non - TOP 5 ' UTR of β - actin used as a negative control . Total content in lysates used for luciferase . t - , n = 4 - 5 biological replicates per group , * * - P ≤ 0 . 01 , * * * - P ≤ 0 . 001 .

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(J) Schematic representation of reporter consisting of human Cand2 5'UTR and downstream Renilla luciferase coding sequence. A potential TOP-like motif is highlighted in a grey box. Black arrows indicate transcription start sites in the adult heart according to the Database of Transcriptional Start Sites (Release 10.1). The effect of Cand2 5'UTR on translation measured by luciferase activity in normal conditions and after mTORC1 block with Torin 1. eEF2 - positive control of reporter with defined 5'TOP motif and non-TOP 5'UTR of β-actin used as a negative control. Total protein content in lysates used for luciferase assay. t-test, n=4-5 biological replicates per group, ** - P ≤ 0.01, *** - P ≤ 0.001.


( A ) Representative immunoblot of Cand2 in mouse organs .

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(A) Representative immunoblot of Cand2 protein levels in mouse organs.


( C ) Immunofluorescent of paraffin sections from hearts from WT Cand2 KO mice . CAND2 ( stained in red ) , ACTIN ( stained in green ) nuclei ( stained with DAPI , blue ) in . Scale bar

O O O B-EXP_ASSAY O O O O B-TISSUE O O B-GENEPROD O B-ORGANISM O B-GENEPROD O B-EXP_ASSAY O O O O B-GENEPROD O B-EXP_ASSAY O O O B-SUBCELLULAR O B-EXP_ASSAY O B-SMALL_MOLECULE O O O O O O O

(C) Immunofluorescent staining of paraffin sections from hearts from WT and Cand2 KO mice. CAND2 (stained in red), ACTIN (stained in green) and nuclei (stained with DAPI, blue) in. Scale bar 20μm


( F ) Immunoblot of subcellular fractionation of left ventricle ( LV ) . Gapdh Histone 3D ( H3D ) used as markers , respectively . WCL - whole lysate , cyt - fraction , nuc - fraction .

O O O B-EXP_ASSAY O O O O B-TISSUE I-TISSUE O B-TISSUE O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O O O O O O O O O O O B-SUBCELLULAR O O O B-SUBCELLULAR O O O

(F) Immunoblot of subcellular fractionation of left ventricle (LV). Gapdh and Histone 3D (H3D) used as cytoplasmic and nuclear markers, respectively. WCL - whole cell lysate, cyt - cytoplasmic fraction, nuc - nuclear fraction.


( E ) NRCMs measurement after Cand2 OE 24h PE stimulation . Analyzed by One - way ANOVA . n > 150 from n = 3 independent experiments . Violin plot : middle horizontal lane - median , upper lane - 3rd quartile , lower lane - 1st quartile . Data information : Error bars indicate means ± ; * - P ≤ 0 . 05 , * * - P ≤ 0 . 01 , * * * - P ≤ 0 . 001 , * * * * - P ≤ 0 .

O O O B-CELL_TYPE O O B-GENEPROD O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(E) NRCMs size measurement after Cand2 OE and 24h PE stimulation. Analyzed by One-way ANOVA. n>150 cells from n=3 independent experiments. Violin plot: the middle horizontal lane - median, upper lane - 3rd quartile, lower lane - 1st quartile. Data information: Error bars indicate means ± SEM; * - P ≤ 0.05, ** - P ≤ 0.01, *** - P ≤ 0.001, **** - P ≤ 0.0001


( F ) Nppa mRNA in vitro after Cand2 OE PE treatment measured by RT - qPCR . Analysed by One - way ANOVA . n = 5 - 9 biological replicates per conditions . Data information : Error bars indicate means ± ; * - P ≤ 0 . 05 , * * - P ≤ 0 . 01 , * * * - P ≤ 0 . 001 , * * * * - P ≤ 0 .

O O O B-GENEPROD O O O O B-GENEPROD O B-SMALL_MOLECULE O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(F) Nppa mRNA levels in vitro after Cand2 OE and PE treatment measured by RT-qPCR. Analysed by One-way ANOVA. n=5-9 biological replicates per conditions. Data information: Error bars indicate means ± SEM; * - P ≤ 0.05, ** - P ≤ 0.01, *** - P ≤ 0.001, **** - P ≤ 0.0001


( J ) Nppa Nppb mRNA 4 after TAC in WT Cand2 KO mice measured by RT - qPCR . Analyzed by One - way ANOVA . n = 6 - 13 mice per group . Data information : Error bars indicate means ± ; * - P ≤ 0 . 05 , * * - P ≤ 0 . 01 , * * * - P ≤ 0 . 001 , * * * * - P ≤ 0 . 0001

O O O B-GENEPROD I-GENEPROD O O O O O O B-GENEPROD O B-ORGANISM O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(J) Nppa and Nppb mRNA levels 4 weeks after TAC in WT and Cand2 KO mice measured by RT-qPCR. Analyzed by One-way ANOVA. n=6-13 mice per group. Data information: Error bars indicate means ± SEM; * - P ≤ 0.05, ** - P ≤ 0.01, *** - P ≤ 0.001, **** - P ≤ 0.0001


( B ) Immunoblots of Grk5 in vitro after Cand2 KD with siRNA after Cand2 OE with AAV6 . t - , n = 4 - 5 biological replicates per group . Data information : Error bars indicate means ± . * - P ≤ 0 . 05 , * * * - P ≤ 0 . 001 .

O O O B-EXP_ASSAY O B-GENEPROD O O O B-GENEPROD O O O O B-GENEPROD O O B-ORGANISM O O O O O O O O O O O O O O O O O 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) Immunoblots and quantification of Grk5 protein levels in vitro after Cand2 KD with siRNA and after Cand2 OE with AAV6. t-test, n=4-5 biological replicates per group. Data information: Error bars indicate means ± SEM. * - P ≤ 0.05, *** - P ≤ 0.001.