( A ) Real - time quantitative PCR of the indicated genes ( M1 - like and M2 - like macrophages markers ) of A375 xenograft treated with PLX4720 , bevacizumab or COMBO . Data are presented as expression fold change ( log2 ) compared with vehicle after normalization for housekeeping gene TBP ( n = 3 tumors ) , ( * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 versus vehicle ( PLX4720 : m - Arg1 P = 5 . 65E - 06 ) ( bevacizumab : m - Ccl5 P = 0 . 025 ) ( COMBO : m - Ccl5 P = 0 . 0070 ; m - Cd40 P = 0 . 00099 ; m - Cxcl10 P = 0 . 043 ; m - Cxcl9 P = 0 . 046 ; m - Il1b P = 0 . 0367 ; m - Stat1 P = 0 . 049 ; m - Tlr2 = 0 . 00182 ; m - Arg - 1 P = 0 . 0013 ) .

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(A) Real-time quantitative PCR of the indicated genes (M1-like and M2-like macrophages markers) of A375 xenograft treated with PLX4720, bevacizumab or COMBO. Data are presented as expression fold change (log2) compared with vehicle after normalization for housekeeping gene TBP (n=3 tumors), (*P < 0.05, **P < 0.01, ***P < 0.001 versus vehicle (PLX4720: m-Arg1 P = 5.65E-06) (bevacizumab: m-Ccl5 P = 0.025) (COMBO: m-Ccl5 P = 0.0070; m-Cd40 P = 0.00099; m-Cxcl10 P = 0.043; m-Cxcl9 P = 0.046; m-Il1b P = 0.0367; m-Stat1 P = 0.049; m-Tlr2 = 0.00182; m-Arg-1 P = 0.0013).


( B ) Representative images of COMBO - treated tumor sections co - stained with an antibody against CD68 and an antibody anti - CCR7 ( M1 - like polarization ) or anti - CD206 ( M2 - like polarization ) .

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(B) Representative images of COMBO-treated tumor sections co-stained with an antibody against CD68 and an antibody anti-CCR7 (M1-like polarization) or anti-CD206 (M2-like polarization).


( C ) Graphs showing the quantification of FACS analysis of infiltrating F4 / 80 + tumor macrophages and average percentage of median fluorescence intensity ( MFI ) of TNFα + or INOS + on F4 / 80 + cells after ex vivo stimulation with PMA and ionomycin in vehicle ( n = 4 tumors ) and COMBO ( n = 6 tumors ) , * P < 0 . 05 versus vehicle ( TNFα P = 0 . 037 ; INOS = 0 . 038 ) .

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(C) Graphs showing the quantification of FACS analysis of infiltrating F4/80+ tumor macrophages and average percentage of median fluorescence intensity (MFI) of TNFα+ or INOS+ on F4/80+ cells after ex vivo stimulation with PMA and ionomycin in vehicle (n=4 tumors) and COMBO (n=6 tumors), *P < 0.05 versus vehicle (TNFα P = 0.037; INOS = 0.038).


( D ) Graphs showing the quantification of FACS analysis of infiltrating CD11b + Ly6C + tumor monocytes and average percentage of MFI of TNFα + or INOS + on CD11b + Ly6C + cells after ex vivo stimulation with PMA and ionomycin in vehicle ( n = 4 tumors ) and COMBO ( n = 6 tumors ) , * P < 0 . 05 versus vehicle ( TNFα P = 0 . 048 ; INOS = 0 . 038 ) .

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(D) Graphs showing the quantification of FACS analysis of infiltrating CD11b+Ly6C+ tumor monocytes and average percentage of MFI of TNFα+ or INOS+ on CD11b+Ly6C+ cells after ex vivo stimulation with PMA and ionomycin in vehicle (n=4 tumors) and COMBO (n=6 tumors), *P < 0.05 versus vehicle (TNFα P = 0.048; INOS = 0.038).


( E ) Graph showing the quantification of circulating CD11b + Ly6C + monocytes in peripheral blood analyzed by FACS in vehicle - ( n = 4 tumors ) and COMBO - ( n = 6 tumors ) treated tumors .

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(E) Graph showing the quantification of circulating CD11b+Ly6C+ monocytes in peripheral blood analyzed by FACS in vehicle- (n=4 tumors) and COMBO- (n=6 tumors) treated tumors.


( F ) Cell viability measured as fluorescence intensity of ZsGreen - A375 cells co - cultured with cells isolated from xenograft treated with vehicle or COMBO with after 48h . Data are representative of one experiment out of two done in triplicate * P < 0 . 05 versus vehicle ( P = 0 . 046 ) .

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(F) Cell viability measured as fluorescence intensity of ZsGreen-A375 cells co-cultured with cells isolated from xenograft treated with vehicle or COMBO with after 48h. Data are representative of one experiment out of two done in triplicate *P < 0.05 versus vehicle (P = 0.046).


( A ) Representative images of collagen deposition determined by collagen I immunofluorescent staining in A375 xenografts treated as indicated . Bar graphs indicate the collagen I + area / tumor area ( n = 5 tumors ) , * * * P < 0 . 001 versus vehicle ( P = 2 . 93E - 06 ) . ( B ) Representative images of CAF density determined by α - SMA immunofluorescence staining in A375 xenografts treated as indicated . Bar graphs indicate α - SMA + area / tumor area ( n = 5 tumors ) , * * * P < 0 . 001 versus vehicle ( P = 0 . 0006 ) . ( C ) Representative images of Lysyl Oxidase enzyme determined by LOX immunofluorescence staining in A375 xenografts treated as indicated . Bar graphs indicate the LOX + area / tumor area ( n = 3 tumors ) , * P < 0 . 05 versus vehicle ( bevacizumab P = 0 . 033 ; COMBO P = 0 . 040 ) .

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(A) Representative images of collagen deposition determined by collagen I immunofluorescent staining in A375 xenografts treated as indicated. Bar graphs indicate the collagen I+ area/tumor area (n=5 tumors), ***P < 0.001 versus vehicle (P = 2.93E-06).(B) Representative images of CAF density determined by α-SMA immunofluorescence staining in A375 xenografts treated as indicated. Bar graphs indicate α-SMA+ area/tumor area (n=5 tumors), ***P < 0.001 versus vehicle (P = 0.0006).(C) Representative images of Lysyl Oxidase enzyme determined by LOX immunofluorescence staining in A375 xenografts treated as indicated. Bar graphs indicate the LOX+ area/tumor area (n=3 tumors), *P < 0.05 versus vehicle (bevacizumab P = 0.033; COMBO P = 0.040).


( E ) Quantitative Real - time PCR of h - TGFB1 and m - Tgfb1 of A375 xenograft treated with PLX4720 , bevacizumab or COMBO . Data are presented as expression fold change ( log2 ) compared with vehicle after normalization for the housekeeping gene TBP ( n = 3 tumors ) , * P < 0 . 05 versus vehicle ( P = 0 . 043 ) .

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(E) Quantitative Real-time PCR of h-TGFB1 and m-Tgfb1 of A375 xenograft treated with PLX4720, bevacizumab or COMBO. Data are presented as expression fold change (log2) compared with vehicle after normalization for the housekeeping gene TBP (n=3 tumors), *P < 0.05 versus vehicle (P = 0.043).


( F ) Western blot for human TGFβ in total protein extract from A375 tumors treated as indicate . Vinculin was used as an internal control .

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(F) Western blot for human TGFβ in total protein extract from A375 tumors treated as indicate. Vinculin was used as an internal control.


( A ) Effect of clodronate liposomes on tumor growth inhibition induced by COMBO treatment in in mice bearing A375 tumors . Tumor growth is expressed as % change of the initial tumor . Macrophage depletion by clodronate liposome enhanced the tumor growth of tumors treated with the combination of PLX4720 and bevacizumab ( n = 5 mice / group ) , * P < 0 . 05 , * * * P < 0 . 001 versus vehicle ( vehicle Clodronate P = 0 . 049 , COMBO P = 5 . 72E - 06 ) ŦP < 0 . 05 compared to COMBO ( P = 0 . 033 ) .

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(A) Effect of clodronate liposomes on tumor growth inhibition induced by COMBO treatment in in mice bearing A375 tumors. Tumor growth is expressed as % change of the initial tumor. Macrophage depletion by clodronate liposome enhanced the tumor growth of tumors treated with the combination of PLX4720 and bevacizumab (n=5 mice/group), *P < 0.05, ***P < 0.001 versus vehicle (vehicle Clodronate P = 0.049, COMBO P = 5.72E-06) ŦP < 0.05 compared to COMBO (P = 0.033).


( B ) Representative images of macrophage infiltration determined by F4 / 80 immunofluorescence staining in A375 xenografts treated as indicated . Bar graphs indicate the F4 / 80 + area / tumor ( n = 3 tumors ) , * * * P < 0 . 001 versus vehicle ( P = 1 . 53E - 25 ) . ( C ) Representative images of collagen deposition determined by collagen I immunofluorescence staining in A375 xenografts treated as indicated . Bar graphs indicate the collagen I + area / tumor ( n = 3 tumors ) .

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(B) Representative images of macrophage infiltration determined by F4/80 immunofluorescence staining in A375 xenografts treated as indicated. Bar graphs indicate the F4/80+ area/tumor (n=3 tumors), ***P < 0.001 versus vehicle (P = 1.53E-25).(C) Representative images of collagen deposition determined by collagen I immunofluorescence staining in A375 xenografts treated as indicated. Bar graphs indicate the collagen I+ area/tumor (n=3 tumors).


( D ) Waterfall plots showing the percent change in volume ( relative to the initial tumor volume ) for the individual A375 xenografts in each treatment group ( vehicle , PLX4720 , bevacizumab and COMBO ) from week 1 to week 6 .

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(D) Waterfall plots showing the percent change in volume (relative to the initial tumor volume) for the individual A375 xenografts in each treatment group (vehicle, PLX4720, bevacizumab and COMBO) from week 1 to week 6.


( E ) Representative images of macrophage infiltration determined by F4 / 80 immunofluorescence staining in a responder and a relapsing A375 xenografts . Bar graphs indicate the F4 / 80 + area / tumor ( n = 3 mice ) , * * * P < 0 . 001 versus vehicle ( P = 1 . 25E - 06 ) . ( F ) Representative images of collagen deposition determined by collagen I immunofluorescence staining in a responder and a relapsing A375 xenografts . Bar graphs indicate the collagen I + area / tumor ( n = 3 mice ) , * * * P < 0 . 001 versus vehicle ( P = 4 . 03E - 05 ) .

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(E) Representative images of macrophage infiltration determined by F4/80 immunofluorescence staining in a responder and a relapsing A375 xenografts. Bar graphs indicate the F4/80+ area/tumor (n=3 mice), ***P < 0.001 versus vehicle (P = 1.25E-06).(F) Representative images of collagen deposition determined by collagen I immunofluorescence staining in a responder and a relapsing A375 xenografts. Bar graphs indicate the collagen I+ area/tumor (n=3 mice), ***P < 0.001 versus vehicle (P = 4.03E-05).


( G ) Real - time quantitative PCR of h - TGFB1 of A375 xenograft treated with PLX4720 , bevacizumab or COMBO . Data are presented as expression fold change ( log2 ) of relapsing tumors compared to responder tumors after normalization for housekeeping gene TBP ( n = 3 tumors ) * * * P < 0 . 001 versus vehicle ( P = 0 . 0006 ) .

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(G) Real-time quantitative PCR of h-TGFB1 of A375 xenograft treated with PLX4720, bevacizumab or COMBO. Data are presented as expression fold change (log2) of relapsing tumors compared to responder tumors after normalization for housekeeping gene TBP (n=3 tumors) ***P < 0.001 versus vehicle (P = 0.0006).


A . Sorbitol stimulates YAP phosphorylation . HEK293A cells were cultured in the presence or absence of serum , and treated with 0 . 2 M sorbitol for the indicated time points . Phos - tag gel was used to assess total YAP phosphorylation based on mobility shift . Note that YAP and TAZ are recognized by the same antibody . l . e . denotes long exposure of YAP and TAZ .

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A. Sorbitol stimulates YAP phosphorylation. HEK293A cells were cultured in the presence or absence of serum, and treated with 0.2 M sorbitol for the indicated time points. Phos-tag gel was used to assess total YAP phosphorylation based on mobility shift. Note that YAP and TAZ are recognized by the same antibody. l.e. denotes long exposure of YAP and TAZ.


B . Osmotic stress induces LATS phosphorylation . HEK293A cells were treated with 0 . 2 M sorbitol for 30 or 60 minutes in the presence or absence of serum . LATS phosphorylation at the hydrophobic motif ( HM ) was detected with the phospho - specific pLATS antibody .

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B. Osmotic stress induces LATS phosphorylation. HEK293A cells were treated with 0.2 M sorbitol for 30 or 60 minutes in the presence or absence of serum. LATS phosphorylation at the hydrophobic motif (HM) was detected with the phospho-specific pLATS antibody.


C . Osmotic stress increases LATS kinase activity . HEK293A cells were treated with 0 . 2 M sorbitol for 30 minutes . Lats1 was immunoprecipitated and an in vitro kinase assay was performed using recombinant GST - YAP as a substrate . Phosphorylation of YAP was determined by immunoblotting with phospho - YAP ( S127 ) antibody . Data are presented as mean ± SEM . * p < 0 . 05 ( two tailed student ' s t - test , n = 3 ) .

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C. Osmotic stress increases LATS kinase activity. HEK293A cells were treated with 0.2 M sorbitol for 30 minutes. Lats1 was immunoprecipitated and an in vitro kinase assay was performed using recombinant GST-YAP as a substrate. Phosphorylation of YAP was determined by immunoblotting with phospho-YAP (S127) antibody. Data are presented as mean ± SEM. *p < 0.05 (two tailed student's t-test, n = 3).


D . Osmotic stress does not lead to YAP cytoplasmic localization . HEK293A cells were treated with 0 . 2 M sorbitol for 1 hr in the presence of serum . YAP subcellular localization was determined by immunofluorescence staining ( red ) . DAPI ( blue ) was used to stain for DNA . Scale bar : 20 μm . Quantification of more nuclear ( N > C ) or more cytosolic ( N < C ) YAP signal is determined with randomly chosen fields , each with approximately 100 cells .

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D. Osmotic stress does not lead to YAP cytoplasmic localization. HEK293A cells were treated with 0.2 M sorbitol for 1 hr in the presence of serum. YAP subcellular localization was determined by immunofluorescence staining (red). DAPI (blue) was used to stain for DNA. Scale bar: 20 μm. Quantification of more nuclear (N > C) or more cytosolic (N < C) YAP signal is determined with randomly chosen fields, each with approximately 100 cells.


E . Osmotic stress does not reduce YAP target gene expression . HEK293A cells were treated with 0 . 2 M sorbitol for 4 hr . mRNA levels of CTGF and CYR61 were measured by quantitative RT - PCR and normalized to GAPDH control . Data are presented as mean ± SEM . n . s means p > 0 . 05 ( two tailed student ' s t - test , n = 3 ) .

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E. Osmotic stress does not reduce YAP target gene expression. HEK293A cells were treated with 0.2 M sorbitol for 4 hr. mRNA levels of CTGF and CYR61 were measured by quantitative RT-PCR and normalized to GAPDH control. Data are presented as mean ± SEM. n.s means p > 0.05 (two tailed student's t-test, n = 3).


F . Osmotic stress does not disrupt the interaction between YAP and TEAD1 . HEK293A cells were treated with 0 . 2 M sorbitol for 1 hr . Endogenous YAP was immunoprecipitated with YAP antibody , and TEAD1 and YAP were detected by Western blot . Cells were serum starved for 1 hr as indicated ( - serum ) .

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F. Osmotic stress does not disrupt the interaction between YAP and TEAD1. HEK293A cells were treated with 0.2 M sorbitol for 1 hr. Endogenous YAP was immunoprecipitated with YAP antibody, and TEAD1 and YAP were detected by Western blot. Cells were serum starved for 1 hr as indicated (- serum).


G . Osmotic stress does not increase the interaction between YAP and 14 - 3 - 3 . HEK293A cells were transiently transfected with Flag - YAP and Myc - 14 - 3 - 3 , then treated with 0 . 2 M sorbitol for the indicated time points . Myc - 14 - 3 - 3 was immunoprecipitated and the co - precipitated Flag - YAP was detected .

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G. Osmotic stress does not increase the interaction between YAP and 14-3-3. HEK293A cells were transiently transfected with Flag-YAP and Myc-14-3-3, then treated with 0.2 M sorbitol for the indicated time points. Myc-14-3-3 was immunoprecipitated and the co-precipitated Flag-YAP was detected.


A . Osmotic stress induces YAP nuclear translocation . HEK293A cells were serum starved for 1 hr followed by 0 . 2 M sorbitol treatment for 1 hr . YAP / TAZ ( red , upper panels ) or YAP ( red , lower panels ) were stained with two specific antibodies . DAPI ( blue ) was used to visualize cell nuclei . Scale bar : 20 μm . Quantification of more nuclear ( N > C ) or more cytosolic ( N < C ) YAP signal is determined with randomly chosen fields .

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A. Osmotic stress induces YAP nuclear translocation. HEK293A cells were serum starved for 1 hr followed by 0.2 M sorbitol treatment for 1 hr. YAP/TAZ (red, upper panels) or YAP (red, lower panels) were stained with two specific antibodies. DAPI (blue) was used to visualize cell nuclei. Scale bar: 20 μm. Quantification of more nuclear (N > C) or more cytosolic (N < C) YAP signal is determined with randomly chosen fields.


B . Osmotic stress induces TAZ nuclear translocation . YAP knockout ( KO ) HEK293A cells were serum starved for 1 hr followed by 0 . 2 M sorbitol treatment for 1 hr . TAZ localization was determined by immunofluorescence staining with the YAP / TAZ antibody ( red ) . Scale bar : 20 μm .

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B. Osmotic stress induces TAZ nuclear translocation. YAP knockout (KO) HEK293A cells were serum starved for 1 hr followed by 0.2 M sorbitol treatment for 1 hr. TAZ localization was determined by immunofluorescence staining with the YAP/TAZ antibody (red). Scale bar: 20 μm.


C . Osmotic stress induces a transient YAP nuclear translocation . HEK293A cells were serum starved for 1 hr followed by 0 . 2 M sorbitol treatment for the indicated time points .

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C. Osmotic stress induces a transient YAP nuclear translocation. HEK293A cells were serum starved for 1 hr followed by 0.2 M sorbitol treatment for the indicated time points.


D . Osmotic stress induces YAP target gene expression . Wild type ( WT ) or YAP / TAZ double knockout ( Y / T KO ) HEK293A cells were serum starved for 1 hr and treated with 0 . 2 M sorbitol for 4 hr . mRNA levels of CTGF and CYR61 were measured by quantitative RT - PCR and normalized to GAPDH control . Data are presented as mean ± SEM . * p < 0 . 05 ( two tailed student ' s t - test , n = 3 ) .

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D. Osmotic stress induces YAP target gene expression. Wild type (WT) or YAP/TAZ double knockout (Y/T KO) HEK293A cells were serum starved for 1 hr and treated with 0.2 M sorbitol for 4 hr. mRNA levels of CTGF and CYR61 were measured by quantitative RT-PCR and normalized to GAPDH control. Data are presented as mean ± SEM. *p < 0.05 (two tailed student's t-test, n = 3).


E . Osmotic stress decreases YAP and 14 - 3 - 3 interaction . HEK293A cells were transiently transfected with Flag - YAP and Myc - 14 - 3 - 3 , and were serum starved for 16 hr . Cells were then treated with 0 . 2 M sorbitol for the indicated time points . Myc - 14 - 3 - 3 was immunoprecipitated and the associated Flag - YAP was detected by Western . Data are presented as mean ± SEM . * p < 0 . 05 ( two tailed student ' s t - test , n = 3 ) .

O O O O O B-MEASURED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O B-MEASURED_VAR O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O B-MEASURED_VAR O O O O O O O O O O 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. Osmotic stress decreases YAP and 14-3-3 interaction. HEK293A cells were transiently transfected with Flag-YAP and Myc-14-3-3, and were serum starved for 16 hr. Cells were then treated with 0.2 M sorbitol for the indicated time points. Myc-14-3-3 was immunoprecipitated and the associated Flag-YAP was detected by Western. Data are presented as mean ± SEM. *p < 0.05 (two tailed student's t-test, n = 3).


A . Inhibition of p38 or JNK does not block osmotic stress - induced YAP nuclear localization . HEK293A cells were pretreated with 2 μM p38 inhibitor ( SB203580 ) or 20 μM JNK inhibitor ( SP600125 ) before treatment with 0 . 2 M sorbitol for 1 hr in the absence of serum . Quantification of YAP / TAZ subcellular staining is shown . Scale bar : 20 μm .

O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O

A. Inhibition of p38 or JNK does not block osmotic stress-induced YAP nuclear localization. HEK293A cells were pretreated with 2 μM p38 inhibitor (SB203580) or 20 μM JNK inhibitor (SP600125) before treatment with 0.2 M sorbitol for 1 hr in the absence of serum. Quantification of YAP/TAZ subcellular staining is shown. Scale bar: 20 μm.


A . Inhibition of p38 or JNK does not block osmotic stress - induced YAP nuclear localization . HEK293A cells were pretreated with 2 μM p38 inhibitor ( SB203580 ) or 20 μM JNK inhibitor ( SP600125 ) before treatment with 0 . 2 M sorbitol for 1 hr in the absence of serum . Cell lysates from identically treated samples were examined for phosphorylation of p38 substrate MK - 2 and JNK substrate c - Jun .

O O O O 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-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O

A. Inhibition of p38 or JNK does not block osmotic stress-induced YAP nuclear localization. HEK293A cells were pretreated with 2 μM p38 inhibitor (SB203580) or 20 μM JNK inhibitor (SP600125) before treatment with 0.2 M sorbitol for 1 hr in the absence of serum. Cell lysates from identically treated samples were examined for phosphorylation of p38 substrate MK-2 and JNK substrate c-Jun.


B . NLK knockout blocks osmotic stress - induced YAP nuclear localization . HEK293A cells were transiently transfected with CRISPR / Cas9 to knockout NLK . Wild type ( WT ) cells and the NLK KO cell pool were treated with 0 . 2 M sorbitol for 1 hr in the absence of serum . YAP / TAZ subcellular localization was determined by immunofluorescence staining . Scale bar : 20 μm .

O O B-CONTROLLED_VAR O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O

B. NLK knockout blocks osmotic stress-induced YAP nuclear localization. HEK293A cells were transiently transfected with CRISPR/Cas9 to knockout NLK. Wild type (WT) cells and the NLK KO cell pool were treated with 0.2 M sorbitol for 1 hr in the absence of serum. YAP/TAZ subcellular localization was determined by immunofluorescence staining. Scale bar: 20 μm.


C . NLK induces YAP nuclear translocation . HEK293A cells were co - transfected with Flag - YAP together with vector control , Myc - NLK - WT ( wild type NLK ) , or NLK - KN ( kinase negative mutant ) . Cells were serum starved for 6 hr , and YAP localization and NLK expression were determined by Flag ( green ) and Myc ( red ) antibodies , respectively . Scale bar : 20 μm .

O O B-CONTROLLED_VAR O B-MEASURED_VAR O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O B-CONTROLLED_VAR O O O O O B-MEASURED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O

C. NLK induces YAP nuclear translocation. HEK293A cells were co-transfected with Flag-YAP together with vector control, Myc-NLK-WT (wild type NLK), or NLK-KN (kinase negative mutant). Cells were serum starved for 6 hr, and YAP localization and NLK expression were determined by Flag (green) and Myc (red) antibodies, respectively. Scale bar: 20 μm.


D . NLK induces YAP phosphorylation . HA - YAP was co - transfected with NLK - WT or NLK - KN in HEK293A cells . The phos - tag gel showed NLK - WT but not NLK - KN caused a significant mobility shift of YAP .

O O B-CONTROLLED_VAR O B-MEASURED_VAR O O O O B-MEASURED_VAR O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O B-CONTROLLED_VAR O O O O B-MEASURED_VAR O O O O O O O O B-MEASURED_VAR O

D. NLK induces YAP phosphorylation. HA-YAP was co-transfected with NLK-WT or NLK-KN in HEK293A cells. The phos-tag gel showed NLK-WT but not NLK-KN caused a significant mobility shift of YAP.


E . NLK phosphorylates YAP in vitro . NLK - WT and NLK - KN were immunoprecipitated from HEK293A cells and an in vitro kinase assay was performed using recombinant GST - YAP as a substrate in the presence of ATP - γ - S . Total phosphorylation of YAP was detected by immunoblotting with thiophosphate ester antibody , which identifies the alkylated thiophosphorylation on YAP .

O O B-CONTROLLED_VAR O B-MEASURED_VAR O O O B-CONTROLLED_VAR O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O B-MEASURED_VAR O

E. NLK phosphorylates YAP in vitro. NLK-WT and NLK-KN were immunoprecipitated from HEK293A cells and an in vitro kinase assay was performed using recombinant GST-YAP as a substrate in the presence of ATP-γ-S. Total phosphorylation of YAP was detected by immunoblotting with thiophosphate ester antibody, which identifies the alkylated thiophosphorylation on YAP.


A . NLK induces YAP Ser 128 phosphorylation . Flag - YAP WT or S128A mutant was co - transfected with or without Myc - NLK . Phosphorylation was determined by Western blot using YAP S128 phosphospecific antibody .

O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O B-MEASURED_VAR O O O O

A. NLK induces YAP Ser 128 phosphorylation. Flag-YAP WT or S128A mutant was co-transfected with or without Myc-NLK. Phosphorylation was determined by Western blot using YAP S128 phosphospecific antibody.


B . NLK phosphorylates YAP Ser 128 in vitro . NLK - WT or NLK - KN was transfected into HEK293A cells and immunoprecipitated . An in vitro NLK kinase assay was performed using recombinant GST - YAP as a substrate . Phosphorylation of YAP Ser 128 was determined by immunoblotting with phospho - YAP ( S128 ) antibody .

O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O B-MEASURED_VAR O O O O O O B-MEASURED_VAR O O O O O O O O O B-MEASURED_VAR O O O O O

B. NLK phosphorylates YAP Ser 128 in vitro. NLK-WT or NLK-KN was transfected into HEK293A cells and immunoprecipitated. An in vitro NLK kinase assay was performed using recombinant GST-YAP as a substrate. Phosphorylation of YAP Ser 128 was determined by immunoblotting with phospho-YAP (S128) antibody.


C . Osmotic stress induces YAP phosphorylation at Ser 128 . In the left panel , HEK293A cells were treated with sorbitol and endogenous YAP was immunoprecipitated . In the right panel , Flag - YAP was transfected into HEK293A cells . Flag - YAP was immunoprecipitated , YAP Ser 128 phosphorylation was detected by a phosphospecific antibody . Data are presented as mean ± SEM . * p < 0 . 05 ( two tailed student ' s t - test , n = 3 ) .

O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O B-MEASURED_VAR O O O O O O O O O O B-MEASURED_VAR O O O O O O O O B-MEASURED_VAR O O O B-MEASURED_VAR O O O O O O O O O O O O O O O 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. Osmotic stress induces YAP phosphorylation at Ser 128. In the left panel, HEK293A cells were treated with sorbitol and endogenous YAP was immunoprecipitated. In the right panel, Flag-YAP was transfected into HEK293A cells. Flag-YAP was immunoprecipitated, YAP Ser 128 phosphorylation was detected by a phosphospecific antibody. Data are presented as mean ± SEM. *p < 0.05 (two tailed student's t-test, n = 3).


D . NLK deficiency reduces YAP S128 phosphorylation . Two CRISPR / CAS9 gRNA plasmids targeting different sites of NLK were transfected into HEK293A . WT cells and two pools of NLK CRISPR / CAS9 transfected cells were treated with 0 . 2 M sorbitol for the indicated time points . Data are presented as mean ± SEM . * p < 0 . 05 ( two tailed student ' s t - test , n = 4 ) .

O O B-CONTROLLED_VAR O O B-MEASURED_VAR O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O 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. NLK deficiency reduces YAP S128 phosphorylation. Two CRISPR/CAS9 gRNA plasmids targeting different sites of NLK were transfected into HEK293A. WT cells and two pools of NLK CRISPR/CAS9 transfected cells were treated with 0.2 M sorbitol for the indicated time points. Data are presented as mean ± SEM. *p < 0.05 (two tailed student's t-test, n = 4).


E . The S128D phosphomimetic mutant abolishes YAP interaction with 14 - 3 - 3 . Flag - YAP WT and mutant constructs were co - transfected with Myc - 14 - 3 - 3 into HEK293A cells . Cells were serum starved for 16 hrs . 14 - 3 - 3 was immunoprecipitated with Myc antibody and the associated YAP was detected with Flag antibody .

O O O O O O O B-MEASURED_VAR O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O B-MEASURED_VAR O O O O O O

E. The S128D phosphomimetic mutant abolishes YAP interaction with 14-3-3. Flag-YAP WT and mutant constructs were co-transfected with Myc-14-3-3 into HEK293A cells. Cells were serum starved for 16 hrs. 14-3-3 was immunoprecipitated with Myc antibody and the associated YAP was detected with Flag antibody.


F . YAP Ser 128 phosphorylation is required for disruption of YAP - 14 - 3 - 3 interaction by osmotic stress . Flag - YAP WT or Flag - YAP S128A constructs were co - transfected with Myc - 14 - 3 - 3 into HEK293A cells . Cells were serum starved for 16 hrs , and treated with 0 . 2 M sorbitol for the indicated time points or refreshed with serum containing medium for 1 hr . Refreshing medium served as a control to disrupt YAP and 14 - 3 - 3 binding . 14 - 3 - 3 was immunoprecipitated with Myc antibody , and the associated YAP was detected with Flag antibody . Data are presented as mean ± SEM . * p < 0 . 05 ( two tailed student ' s t - test , n = 3 ) .

O O B-MEASURED_VAR O O O O O O O O B-MEASURED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O O O O B-MEASURED_VAR O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O 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. YAP Ser 128 phosphorylation is required for disruption of YAP-14-3-3 interaction by osmotic stress. Flag-YAP WT or Flag-YAP S128A constructs were co-transfected with Myc-14-3-3 into HEK293A cells. Cells were serum starved for 16 hrs, and treated with 0.2 M sorbitol for the indicated time points or refreshed with serum containing medium for 1 hr. Refreshing medium served as a control to disrupt YAP and 14-3-3 binding.14-3-3 was immunoprecipitated with Myc antibody, and the associated YAP was detected with Flag antibody. Data are presented as mean ± SEM. *p < 0.05 (two tailed student's t-test, n = 3).


A . NLK does not induce nuclear translocation of the S128A mutant YAP . HEK293A cells were co - transfected with Myc - NLK and Flag - YAP WT or S128A mutant . Cells were serum starved for 6 hr , and YAP localization and NLK expression were determined by Flag ( green ) and Myc ( red ) antibodies , respectively . Scale bar : 20 μm .

O O B-CONTROLLED_VAR O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O B-CONTROLLED_VAR O O O B-MEASURED_VAR O O O O O O O O O O O O O O B-MEASURED_VAR O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O

A. NLK does not induce nuclear translocation of the S128A mutant YAP. HEK293A cells were co-transfected with Myc-NLK and Flag-YAP WT or S128A mutant. Cells were serum starved for 6 hr, and YAP localization and NLK expression were determined by Flag (green) and Myc (red) antibodies, respectively. Scale bar: 20 μm.


B . YAP Ser 128 phosphorylation is required for osmotic stress - induced YAP nuclear localization . HEK293A YAP KO cells reconstituted with Flag - YAP WT , YAP S128D , or YAP S128A mutant were treated with 0 . 2 M sorbitol for 1 hr . YAP subcellular localization was determined by Flag immunofluorescence staining ( green ) . Scale bar : 20 μm .

O O B-MEASURED_VAR O O O O O O O O O O B-MEASURED_VAR O O O O B-CONTROLLED_VAR O O O O O O B-MEASURED_VAR O O B-MEASURED_VAR O O O B-MEASURED_VAR O O O O O O O O O B-CONTROLLED_VAR O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O

B. YAP Ser 128 phosphorylation is required for osmotic stress-induced YAP nuclear localization. HEK293A YAP KO cells reconstituted with Flag-YAP WT, YAP S128D, or YAP S128A mutant were treated with 0.2 M sorbitol for 1 hr. YAP subcellular localization was determined by Flag immunofluorescence staining (green). Scale bar: 20 μm.


A . YAP S128D reconstituted cells show growth advantage under hyperosmotic conditions . YAP / TAZ dKO HEK293A cells with stable expression of YAP WT , S128A , and S128D were cultured in the absence ( left panel ) or presence of 0 . 2M sorbitol ( right panel ) for the indicated amount of time . Cell numbers were counted and normalized to day 0 . Data are presented as mean ± SEM , n = 3 .

O O B-CONTROLLED_VAR O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O 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. YAP S128D reconstituted cells show growth advantage under hyperosmotic conditions. YAP/TAZ dKO HEK293A cells with stable expression of YAP WT, S128A, and S128D were cultured in the absence (left panel) or presence of 0.2M sorbitol (right panel) for the indicated amount of time. Cell numbers were counted and normalized to day 0. Data are presented as mean ± SEM, n = 3.


B . YAP S128D reconstituted cells have lower cell death in a hyperosmotic environment . Cell cycle analyses of YAP / TAZ dKO HEK293A cells with stable expression of YAP WT , S128A , and S128D after 0 . 2 M sorbitol treatment were determined using flow cytometry . Propidium iodine ( PI ) was used for DNA staining ( Fig EV5 ) . Quantification of sub - G1 cells is shown . Data are presented as mean ± SEM . * p < 0 . 05 ( two tailed student ' s t - test , n = 3 ) .

O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O 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. YAP S128D reconstituted cells have lower cell death in a hyperosmotic environment. Cell cycle analyses of YAP/TAZ dKO HEK293A cells with stable expression of YAP WT, S128A, and S128D after 0.2 M sorbitol treatment were determined using flow cytometry. Propidium iodine (PI) was used for DNA staining (Fig EV5). Quantification of sub-G1 cells is shown. Data are presented as mean ± SEM. *p < 0.05 (two tailed student's t-test, n = 3).


C . YAP S128D reconstituted cells show reduced apoptosis under hyperosmotic conditions . Annexin V analyses of YAP / TAZ dKO HEK293A cells with stable expression of YAP WT , S128A , and S128D after 0 . 2 M sorbitol treatment were determined using flow cytometry . PE - Annexin V and 7 - AAD stained for phospholipid phosphatidylserine ( PS ) and DNA , respectively . Data are presented as mean ± SEM . * p < 0 . 05 ( two tailed student ' s t - test , n = 3 ) .

O O B-CONTROLLED_VAR O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O B-MEASURED_VAR O O O O O O O O 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. YAP S128D reconstituted cells show reduced apoptosis under hyperosmotic conditions. Annexin V analyses of YAP/TAZ dKO HEK293A cells with stable expression of YAP WT, S128A, and S128D after 0.2 M sorbitol treatment were determined using flow cytometry. PE-Annexin V and 7-AAD stained for phospholipid phosphatidylserine (PS) and DNA, respectively. Data are presented as mean ± SEM. *p < 0.05 (two tailed student's t-test, n = 3).


G , Benzidine staining of whole spleen [ top ] or marrow [ bottom ] from 3 month old mice [ left ] or 3 year old naked mole - rats [ right ] . Scale bar 250µm , arrows indicate nucleated erythroid progenitors ( NEPs ) .

O O O O O O O O O O O O O O O O O O O O O O O O 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, Benzidine staining of whole spleen [top] or marrow [bottom] from 3 month old mice [left] or 3 year old naked mole-rats [right]. Scale bar 250µm, arrows indicate nucleated erythroid progenitors (NEPs).


H , Relative counts of Benzidine - stained cytospins from whole spleen or WBM . p - value determined by Sidak ' s Two - way ANOVA comparing BM vs spleen between mouse ( n = 5 ) and naked mole - rat ( n = 8 ) . I , Relative counts of Benzidine - stained cytospins from naked mole - rat sorted spleen fractions ; p - value determined by Sidak ' s Two - way ANOVA comparing BM vs spleen ; n = 4 .

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

H, Relative counts of Benzidine-stained cytospins from whole spleen or WBM. p-value determined by Sidak's Two-way ANOVA comparing BM vs spleen between mouse (n=5) and naked mole-rat (n=8). I, Relative counts of Benzidine-stained cytospins from naked mole-rat sorted spleen fractions; p-value determined by Sidak's Two-way ANOVA comparing BM vs spleen; n=4.


A , Cross - species integration of bulk RNA - Seq datasets . Naked mole - rat BM populations CP1 - 4 were matched to human and murine counterparts for GSEA with MSigDB hallmark genesets . Primitive stem and progenitor [ left ] , hsa : LT - HSC , LIN - / CD34 + / CD38lo / CD90 + / CD45RA - ( n = 7 ) ; HSPC , LIN - / CD34 + / CD38lo ( n = 11 ) ; MPP , LIN - / CD34 + / CD38lo / CD90 - / CD45RA - ( n = 4 ) ; mmu : LT - HSC , LIN - / Sca - 1 + / Kit + / CD150 + / CD48 - ( n = 25 ) ; MPP , LIN - / Sca - 1 + / Kit + / CD150 - / CD48 + ( n = 6 ) ; hgl : LTC , LIN - / Thy1 . 1int / CD34 + ( n = 5 ) . Lymphomyeloid [ center ] , hsa : LMPP , LIN - / CD34 + / CD38lo / CD123lo / CD45RA + ( n = 9 ) ; CLP , LIN - / CD34 + / CD38hi / CD10 + / CD45RA + ( n = 6 ) ; MLP , LIN - / CD34 + / CD38lo / CD90 - / CD45RA + / CD71 - ( n = 4 ) ; CMP , LIN - / CD34 + / CD38hi / CD123lo / CD45RA - ( n = 26 ) ; GMP , LIN - / CD34 + / CD38lo / CD123lo / CD45RA + ( n = 18 ) ; mmu : LMPP , LIN - / Sca - 1 + / Kit + / Flt3hi ( n = 5 ) ; CMP , LIN - / Sca - 1 - / Kit + / CD16 / 32lo / CD34 + ( n = 8 ) ; GMP , LIN - / Sca - 1 - / Kit + / CD16 / 32hi / CD34 + ( n = 16 ) ; hgl : CP1 , LIN + / Thy1 . 1int / CD34 + ( n = 5 ) . Erythroid [ right ] , hsa : MEP , LIN - / CD34 + / CD38hi / CD123 - / CD45RA - ( n = 23 ) ; MKP , LIN - / CD41a + / CD42b + ( n = 3 ) ; ERY , LIN - / CD34lo / - / CD36 + / GYPA + / CD71 + ( n = 12 ) ; mmu : MEP , LIN - / Sca - 1 - / Kit + / CD16 / 32 - / CD34 - ( n = 15 ) ; hgl : CP3 , LIN - / Thy1 . 1lo / CD34 + ( n = 5 ) ; CP4 , LIN - / Thy1 . 1 - / CD34 + ( n = 5 ) . Within each group , cell types were pooled for each species . Shown are pathways related to proliferation and metabolism normalized enrichment score ; GeneRatio , ( signature ∩ term ) / ( signature ∩ all terms ) ; FDR , false discovery rate .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, Cross-species integration of bulk RNA-Seq datasets. Naked mole-rat BM populations CP1-4 were matched to human and murine counterparts for GSEA with MSigDB hallmark genesets. Primitive stem and progenitor [left], hsa: LT-HSC, LIN-/CD34+/CD38lo/CD90+/CD45RA- (n=7); HSPC, LIN-/CD34+/CD38lo (n=11); MPP, LIN-/CD34+/CD38lo/CD90-/CD45RA- (n=4); mmu: LT-HSC, LIN-/Sca-1+/Kit+/CD150+/CD48- (n=25); MPP, LIN-/Sca-1+/Kit+/CD150-/CD48+ (n=6); hgl: LTC, LIN-/Thy1.1int/CD34+ (n=5). Lymphomyeloid [center], hsa: LMPP, LIN-/CD34+/CD38lo/CD123lo/CD45RA+ (n=9); CLP, LIN-/CD34+/CD38hi/CD10+/CD45RA+ (n=6); MLP, LIN-/CD34+/CD38lo/CD90-/CD45RA+/CD71- (n=4); CMP, LIN-/CD34+/CD38hi/CD123lo/CD45RA- (n=26); GMP, LIN-/CD34+/CD38lo/CD123lo/CD45RA+ (n=18); mmu: LMPP, LIN-/Sca-1+/Kit+/Flt3hi (n=5); CMP, LIN-/Sca-1-/Kit+/CD16/32lo/CD34+ (n=8); GMP, LIN-/Sca-1-/Kit+/CD16/32hi/CD34+ (n=16); hgl: CP1, LIN+/Thy1.1int/CD34+ (n=5). Erythroid [right], hsa: MEP, LIN-/CD34+/CD38hi/CD123-/CD45RA- (n=23); MKP, LIN-/CD41a+/CD42b+ (n=3); ERY, LIN-/CD34lo/-/CD36+/GYPA+/CD71+ (n=12); mmu: MEP, LIN-/Sca-1-/Kit+/CD16/32-/CD34- (n=15); hgl: CP3, LIN-/Thy1.1lo/CD34+ (n=5); CP4, LIN-/Thy1.1-/CD34+ (n=5). Within each group, cell types were pooled for each species. Shown are pathways related to proliferation and metabolism normalized enrichment score; GeneRatio, (signature ∩ term) / (signature ∩ all terms); FDR, false discovery rate.


B , Seahorse assay with cell types sorted according to ( A ) ; hOPP , human oligopotent progenitors LIN - / CD34 + / CD38 + ; mOPP , LIN - / Sca - 1 - / Kit + . ECAR , extracellular acidification rate ; OCR , oxygen consumption rate . Error bars , s . e . m . ; Human cell types , n = 4 ; mouse , n = 3 , naked mole - rat , n = 3 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, Seahorse assay with cell types sorted according to (A); hOPP, human oligopotent progenitors LIN-/CD34+/CD38+; mOPP, LIN-/Sca-1-/Kit+. ECAR, extracellular acidification rate; OCR, oxygen consumption rate. Error bars, s.e.m.; Human cell types, n=4; mouse, n=3, naked mole-rat, n=3.


C , Mitotracker Red staining in mouse ( n = 4 ) , human ( n = 4 ) and naked mole - rat ( n = 5 ) BM , histogram of merged per - species data . unstained , BM from mouse [ solid ] , human [ dotted ] or NMR [ area under curve ( AUC ) ] ; yLSK , LIN - / Sca - 1 + / Kit + , 3 month old ; oLSK , 24 month ; yLTC , LIN - / Thy1 . 1int / CD34 + , 3 year old ; hHSC , human CD34 + / CD38lo ;

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, Mitotracker Red staining in mouse (n=4), human (n=4) and naked mole-rat (n=5) BM, histogram of merged per-species data. unstained, BM from mouse [solid], human [dotted] or NMR [area under curve (AUC)]; yLSK, LIN-/Sca-1+/Kit+, 3 month old; oLSK, 24 month; yLTC, LIN-/Thy1.1int/CD34+, 3 year old; hHSC, human CD34+/CD38lo;


F , Confocal fluorescence imaging of sorted mouse LT - HSCs ( LIN - / Sca - 1 + / Kit + / CD150 + / CD48 - ) and NMR LTCs across age ; Y , young ( mouse , 4 month - old ; NMR , 3 year - old ) ; O , old ( mouse , 24 month - old ) ; MA , middle - aged ( NMR , 11 year - old ) . Scale bar 10µm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, Confocal fluorescence imaging of sorted mouse LT-HSCs (LIN-/Sca-1+/Kit+/CD150+/CD48-) and NMR LTCs across age; Y, young (mouse, 4 month-old; NMR, 3 year-old); O, old (mouse, 24 month-old); MA, middle-aged (NMR, 11 year-old). Scale bar 10µm.


G , H Polarization quantification for G , mouse LT - HSC ( biological n = 3 ) ; and H , NMR LTC ( Y , n = 5 ; MA , n = 4 ) . Line at mean ; error bars , s . e . m . ; p - value determined by Sidak ' s Two - way ANOVA .

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G, H Polarization quantification for G, mouse LT-HSC (biological n=3); and H, NMR LTC (Y, n=5; MA, n=4). Line at mean; error bars, s.e.m.; p-value determined by Sidak's Two-way ANOVA.


A U2OS cells were transfected with Flag - NAT10 or control vectors . Forty - eight hours later , cells were harvested and whole cell extracts were immunoprecipitated with Flag - antibody affinity resin . The NAT10 - binding proteins were resolved by SDS - PAGE and detected by silver staining .

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A U2OS cells were transfected with Flag-NAT10 or control vectors. Forty-eight hours later, cells were harvested and whole cell extracts were immunoprecipitated with Flag-antibody affinity resin. The NAT10-binding proteins were resolved by SDS-PAGE and detected by silver staining.


B - F U2OS , HCT116 p53 + / + or HCT116 p53 - / - cell lysates were immunoprecipitated with control IgG , anti - NAT10 ( B and F ) , anti - p53 ( C ) and anti - Mdm2 ( D and E ) antibodies . The immunoprecipitates were subsequently immunoblotted with the indicated antibodies .

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B-F U2OS, HCT116 p53+/+ or HCT116 p53-/- cell lysates were immunoprecipitated with control IgG, anti-NAT10 (B and F), anti-p53 (C) and anti-Mdm2 (D and E) antibodies. The immunoprecipitates were subsequently immunoblotted with the indicated antibodies.


G Purified NAT10 was incubated with GST , GST - p53 or GST - Mdm2 proteins coupled to Glutathione Sepharose 4B . Proteins retained on the Sepharose were then analyzed by western blot using the antibodies as indicated . The amount of GST fusion proteins are shown in the lower panel .

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G Purified NAT10 was incubated with GST, GST-p53 or GST-Mdm2 proteins coupled to Glutathione Sepharose 4B. Proteins retained on the Sepharose were then analyzed by western blot using the antibodies as indicated. The amount of GST fusion proteins are shown in the lower panel.


H Full - length GST - p53 fusion protein , its deletion mutants or GST protein was used to pull - down experiments with purified NAT10 protein . The levels of the GST fusion proteins are shown in the left panel .

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H Full-length GST-p53 fusion protein, its deletion mutants or GST protein was used to pull-down experiments with purified NAT10 protein. The levels of the GST fusion proteins are shown in the left panel.


I GST pull - down assay was performed using purified GST - NAT10 deletion mutants or GST protein and overexpressed Flag - p53 or Mdm2 protein in HEK293T cells . Schematic diagram represents the constructs of GST - NAT10 deletion mutants ( right panel ) .

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I GST pull-down assay was performed using purified GST-NAT10 deletion mutants or GST protein and overexpressed Flag-p53 or Mdm2 protein in HEK293T cells. Schematic diagram represents the constructs of GST-NAT10 deletion mutants (right panel).


A Recombinant His - p53 was incubated with or without purified Flag - NAT10 as described in Experimental Procedures . The reaction products were resolved by SDS - PAGE and immunoblotted using anti - acetyl - lysine antibodies . Flag - PCAF was used as a positive control .

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A Recombinant His-p53 was incubated with or without purified Flag-NAT10 as described in Experimental Procedures. The reaction products were resolved by SDS-PAGE and immunoblotted using anti-acetyl-lysine antibodies. Flag-PCAF was used as a positive control.


B In vitro acetylation was performed and the acetylated p53 was detected as in A .

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B In vitro acetylation was performed and the acetylated p53 was detected as in A.


C In vitro acetylation assay was performed as described in A . The reaction products were resolved by SDS - PAGE and acetylated p53 was purified from the SDS - PAGE and subjected to mass spectrometry analysis .

O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O

C In vitro acetylation assay was performed as described in A. The reaction products were resolved by SDS-PAGE and acetylated p53 was purified from the SDS-PAGE and subjected to mass spectrometry analysis.


D His - p53 or His - p53 - K120R fusion protein was incubated with purified NAT10 or NAT10 GE mutant as described in Experimental Procedures . Reaction mixtures were subjected to western blot using the site specific monoclonal anti - Ac - p53 - K120 antibody .

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D His-p53 or His-p53-K120R fusion protein was incubated with purified NAT10 or NAT10 GE mutant as described in Experimental Procedures. Reaction mixtures were subjected to western blot using the site specific monoclonal anti-Ac-p53-K120 antibody.


E H1299 cells were transfected with the indicated vectors . Total proteins and the anti - Flag antibody M2 - specific immunoprecipitates were analyzed by western blot using the indicated antibodies .

O O O O O O O O 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 H1299 cells were transfected with the indicated vectors. Total proteins and the anti-Flag antibody M2-specific immunoprecipitates were analyzed by western blot using the indicated antibodies.


A U2OS cells were transfected with the indicated siRNAs . Seventy - two hours later , cell lysates were prepared and proteins from lysates were subjected to western blot using the indicated antibodies .

O O O O O O O O O O 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 U2OS cells were transfected with the indicated siRNAs. Seventy-two hours later, cell lysates were prepared and proteins from lysates were subjected to western blot using the indicated antibodies.


B U2OS cells were transfected as in A . Seventy - two hours later , total mRNAs were extracted and subjected to RT - qPCR for the indicated genes .

O O O O O O 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 U2OS cells were transfected as in A. Seventy-two hours later, total mRNAs were extracted and subjected to RT-qPCR for the indicated genes.


C U2OS cells were transfected with increasing amount of plasmid DNA expressing NAT10 . Proteins from whole cell extracts were subjected to western blot using the indicated antibodies .

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C U2OS cells were transfected with increasing amount of plasmid DNA expressing NAT10. Proteins from whole cell extracts were subjected to western blot using the indicated antibodies.


D U2OS cells were transfected with control or NAT10 siRNAs . Seventy - two hours later , cells were treated with cycloheximide ( CHX ) and harvested at the indicated time points . Proteins from cell lysates were subjected to immunoblot for the evaluation of NAT10 and p53 ( upper panels ) . Beta - actin was evaluated as a loading control ( lower panel ) . p53 half - life ( t1 / 2 ) obtained from three independent experiments was presented as mean ± SEM at the bottom .

O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O

D U2OS cells were transfected with control or NAT10 siRNAs. Seventy-two hours later, cells were treated with cycloheximide (CHX) and harvested at the indicated time points. Proteins from cell lysates were subjected to immunoblot for the evaluation of NAT10 and p53 (upper panels). Beta-actin was evaluated as a loading control (lower panel). p53 half-life (t1/2) obtained from three independent experiments was presented as mean ± SEM at the bottom.


E U2OS cells were treated with MG132 ( 10μM ) for 4 hours after transfected with the indicated siRNAs . Proteins from cell extracts were detected by western blot for the indicated proteins .

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E U2OS cells were treated with MG132 (10μM) for 4 hours after transfected with the indicated siRNAs. Proteins from cell extracts were detected by western blot for the indicated proteins.


F HCT116 p53 + / + cells were transfected with the indicated constructs and treated with MG132 for 4 hours before harvest . p53 was immunoprecipitated with anti - p53 polyclonal antibody and immunoblotted with anti - Ub antibody .

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F HCT116 p53+/+ cells were transfected with the indicated constructs and treated with MG132 for 4 hours before harvest. p53 was immunoprecipitated with anti-p53 polyclonal antibody and immunoblotted with anti-Ub antibody.


G p53 and Mdm2 double null MEF cells were transfected with the indicated constructs and treated with MG132 for 4 hours before harvest . Proteins from whole cell extracts were subjected to western blot using the indicated antibodies .

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G p53 and Mdm2 double null MEF cells were transfected with the indicated constructs and treated with MG132 for 4 hours before harvest. Proteins from whole cell extracts were subjected to western blot using the indicated antibodies.


H U2OS cells were transfected with vectors as indicated . Forty - eight hours later , cells were harvested and proteins from whole cell extracts were subjected to western blot using the indicated antibodies .

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

H U2OS cells were transfected with vectors as indicated. Forty-eight hours later, cells were harvested and proteins from whole cell extracts were subjected to western blot using the indicated antibodies.


A HCT116 cells were transfected with increasing amount of DNA expressing NAT10 . Forty - eight hours later , whole cell lysates were prepared and proteins from lysates were analyzed by western blot using the indicated antibodies .

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A HCT116 cells were transfected with increasing amount of DNA expressing NAT10. Forty-eight hours later, whole cell lysates were prepared and proteins from lysates were analyzed by western blot using the indicated antibodies.


B HCT116 cells were transfected with Flag - NAT10 and harvested at indicated time points after treated with CHX . Proteins from cell lysates were immunoblotted with the antibodies as indicated ( left panel ) . Relative Mdm2 protein levels standardized by β - actin are shown in the right . Mdm2 half - life ( t1 / 2 ) obtained from three independent experiments was presented as mean ± SEM at the bottom .

O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O

B HCT116 cells were transfected with Flag-NAT10 and harvested at indicated time points after treated with CHX. Proteins from cell lysates were immunoblotted with the antibodies as indicated (left panel). Relative Mdm2 protein levels standardized by β-actin are shown in the right. Mdm2 half-life (t1/2) obtained from three independent experiments was presented as mean ± SEM at the bottom.


C HCT116 cells transfected with the indicated constructs were harvested after treatment with MG132 for 4 hours . The Mdm2 protein was immunoprecipitated with anti - Mdm2 antibody and immunoblotted with anti - HA antibody .

O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O B-MEASURED_VAR O O O O O O O O O

C HCT116 cells transfected with the indicated constructs were harvested after treatment with MG132 for 4 hours. The Mdm2 protein was immunoprecipitated with anti-Mdm2 antibody and immunoblotted with anti-HA antibody.


D In vitro ubiquitination was performed with recombinant GST - Mdm2 ( left ) or GST - Mdm2 C464A mutant ( right ) and Flag - NAT10 or Flag - NAT10 GE in the presence of E1 , E2 and Ub in vitro as described in Experimental Procedures . The reaction products were resolved by SDS - PAGE , followed by western blot for evaluation of the indicated proteins .

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D In vitro ubiquitination was performed with recombinant GST-Mdm2 (left) or GST-Mdm2 C464A mutant (right) and Flag-NAT10 or Flag-NAT10 GE in the presence of E1, E2 and Ub in vitro as described in Experimental Procedures. The reaction products were resolved by SDS-PAGE, followed by western blot for evaluation of the indicated proteins.


E Mdm2 is ubiquitinated by NAT10 in a dose - dependent manner . In vitro ubiquitination assay were carried out with Mdm2 and different doses of purified Flag - NAT10 as described in D .

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E Mdm2 is ubiquitinated by NAT10 in a dose-dependent manner. In vitro ubiquitination assay were carried out with Mdm2 and different doses of purified Flag-NAT10 as described in D.


G U2OS cells transfected with NAT10 siRNA - 3 ( targeting 3 ' - UTR of NAT10 mRNA ) or control siRNA were co - expressed different NAT10 deletion mutants . Cell lysates were extracted and proteins from lysates were subjected to western blot using the indicated antibodies . Relative p53 protein levels standardized by β - actin are shown in the lower .

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G U2OS cells transfected with NAT10 siRNA-3 (targeting 3'-UTR of NAT10 mRNA) or control siRNA were co-expressed different NAT10 deletion mutants. Cell lysates were extracted and proteins from lysates were subjected to western blot using the indicated antibodies. Relative p53 protein levels standardized by β-actin are shown in the lower.


H In vitro ubiquitination reaction was conducted using recombinant GST - Mdm2 C464A and full - length Flag - NAT10 or Flag - NAT10 - D5 . Ubiquitinated Mdm2 was detected as in D .

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H In vitro ubiquitination reaction was conducted using recombinant GST-Mdm2 C464A and full-length Flag-NAT10 or Flag-NAT10-D5. Ubiquitinated Mdm2 was detected as in D.


I HCT116 cells were transfected with the indicated constructs . Cells were harvested after CHX treatment for the indicated times and cell lysates were prepared . Mdm2 protein levels were evaluated by western blot . Relative Mdm2 protein levels standardized by β - actin are shown in the right .

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I HCT116 cells were transfected with the indicated constructs. Cells were harvested after CHX treatment for the indicated times and cell lysates were prepared. Mdm2 protein levels were evaluated by western blot. Relative Mdm2 protein levels standardized by β-actin are shown in the right.


A U2OS cells were treated with 10nM actinomycin D and cell extracts were prepared at indicated time points . Proteins from extracts were analyzed by western blot using the indicated antibodies .

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A U2OS cells were treated with 10nM actinomycin D and cell extracts were prepared at indicated time points. Proteins from extracts were analyzed by western blot using the indicated antibodies.


B U2OS and H1299 cells were treated with actinomycin D for 12 hours . Cells were harvested and proteins from cell lysates were subjected to western blot probed with the indicated antibodies .

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B U2OS and H1299 cells were treated with actinomycin D for 12 hours. Cells were harvested and proteins from cell lysates were subjected to western blot probed with the indicated antibodies.


C U2OS cells transfected with the indicated siRNAs were treated with actinomycin D and harvested at the indicated time points . Proteins from cell lysates were subjected to western blot .

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C U2OS cells transfected with the indicated siRNAs were treated with actinomycin D and harvested at the indicated time points. Proteins from cell lysates were subjected to western blot.


D HCT116 cells were treated with 10nM of actinomycin D or 4 μM of doxorubicin ( Dox ) for 12 hours . Cells were harvested and total proteins were immunoprecipitated with anti - NAT10 antibody . Immunoprecipitates were analyzed by western blot for the indicated proteins .

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D HCT116 cells were treated with 10nM of actinomycin D or 4 μM of doxorubicin (Dox) for 12 hours. Cells were harvested and total proteins were immunoprecipitated with anti-NAT10 antibody. Immunoprecipitates were analyzed by western blot for the indicated proteins.


E HCT116 cells were treated with actinomycin D for the indicated times . Cells were fixed and immuno - stained as indicated . Immunofluorescence images were taken by confocal microscopy . Scale bar represents 10 μM .

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E HCT116 cells were treated with actinomycin D for the indicated times. Cells were fixed and immuno-stained as indicated. Immunofluorescence images were taken by confocal microscopy. Scale bar represents 10 μM.


F H1299 cells were transfected with the indicated vectors . After treated with actinomycin D , cells were harvested and the whole cell extracts were subjected to western blot using the indicated antibodies .

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F H1299 cells were transfected with the indicated vectors. After treated with actinomycin D, cells were harvested and the whole cell extracts were subjected to western blot using the indicated antibodies.


G H1299 cells were transfected with GFP - NAT10 vectors . After treated with actinomycin D for 12 hours , cells were fixed and nuclei were stained with DAPI . Bar represents 10μM .

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G H1299 cells were transfected with GFP-NAT10 vectors. After treated with actinomycin D for 12 hours, cells were fixed and nuclei were stained with DAPI. Bar represents 10μM.


H HCT116 cells transfected with the indicated constructs were treated with actinomycin D or doxorubicin . Western blot was performed with total cell extracts and Mdm2 - specific immunoprecipitates for the indicated proteins .

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H HCT116 cells transfected with the indicated constructs were treated with actinomycin D or doxorubicin. Western blot was performed with total cell extracts and Mdm2-specific immunoprecipitates for the indicated proteins.


I HCT116 cells were transfected with the indicated constructs . Cells were harvested after treatment with actinomycin D . p53 protein was immunoprecipitated with an anti - p53 polyclonal antibody and immunoblotted with p53 monoclonal antibody ( DO - 1 ) .

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I HCT116 cells were transfected with the indicated constructs. Cells were harvested after treatment with actinomycin D. p53 protein was immunoprecipitated with an anti-p53 polyclonal antibody and immunoblotted with p53 monoclonal antibody (DO-1).


A p21 promoter luciferase reporter plasmid was cotransfected with the indicated siRNAs into U2OS cells . Luciferase activity was determined as described in Experimental Procedure .

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A p21 promoter luciferase reporter plasmid was cotransfected with the indicated siRNAs into U2OS cells. Luciferase activity was determined as described in Experimental Procedure.


B U2OS cells were transfected with siRNAs as indicated . Seventy - two hours later , cells were harvested after treated with actinomycin D for 12 hours . Relative mRNA levels for the indicated genes were analyzed by RT - qPCR .

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B U2OS cells were transfected with siRNAs as indicated. Seventy-two hours later, cells were harvested after treated with actinomycin D for 12 hours. Relative mRNA levels for the indicated genes were analyzed by RT-qPCR.


C - E U2OS cells ( C and D ) and H1299 cells ( D ) transfected with the indicated siRNAs were treated with actinomycin D and harvested at the indicated time points . Cell cycle profiles were determined by FACS . Results from three independent experiments are shown as mean ± SEM . ( two - tailed Student ' s t test )

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C-E U2OS cells (C and D) and H1299 cells (D) transfected with the indicated siRNAs were treated with actinomycin D and harvested at the indicated time points. Cell cycle profiles were determined by FACS. Results from three independent experiments are shown as mean ± SEM. (two-tailed Student's t test)


F U2OS cells were transfected with the indicated siRNAs . Cells were irradiated with 50 J / m2 UV . Forty - eight hours later , apoptotic cells were determined . Quantification of apoptotic cells is presented . ( two - tailed Student ' s t test )

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F U2OS cells were transfected with the indicated siRNAs. Cells were irradiated with 50 J/m2 UV. Forty-eight hours later, apoptotic cells were determined. Quantification of apoptotic cells is presented. (two-tailed Student's t test)


G U2OS cells transfected with the indicated siRNAs were plated . Cell numbers were determined as described in Experimental Procedures at the indicated time points .

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G U2OS cells transfected with the indicated siRNAs were plated. Cell numbers were determined as described in Experimental Procedures at the indicated time points.


H Colony formation assay were performed with U2OS cells transfected with the indicated siRNAs . The right panel shows the quantification of formed colonies . * * p < 0 . 01 , * * * p < 0 . 001 ( two - tailed Student ' s t test )

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H Colony formation assay were performed with U2OS cells transfected with the indicated siRNAs. The right panel shows the quantification of formed colonies. **p<0.01, ***p<0.001 (two-tailed Student's t test)


A Twenty pairs of fresh frozen CRC tissues and corresponding normal tissues were lysed and total RNAs were extracted and subjected to RT - qPCR . ( N , Normal tissue ; T , Tumor tissue ) * * p < 0 . 01 ( non - parametric Mann - Whitney t - test )

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A Twenty pairs of fresh frozen CRC tissues and corresponding normal tissues were lysed and total RNAs were extracted and subjected to RT-qPCR. (N, Normal tissue; T, Tumor tissue) **p<0.01 (non-parametric Mann-Whitney t-test)


B Immunohistochemical staining of NAT10 in CRC tissues and normal colon tissues . NAT10 - specific immuno - complex was detected with DAB as shown as brown color . Quantification of NAT10 - positive or NAT10 - negative CRC cases is shown in the lower table .

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B Immunohistochemical staining of NAT10 in CRC tissues and normal colon tissues. NAT10-specific immuno-complex was detected with DAB as shown as brown color. Quantification of NAT10-positive or NAT10-negative CRC cases is shown in the lower table.


C Cell extracts were prepared from different human colon epithelial cell lines as indicated at the top . Proteins from the extracts were subjected to western blot for the evaluation of NAT10 and p53 .

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C Cell extracts were prepared from different human colon epithelial cell lines as indicated at the top. Proteins from the extracts were subjected to western blot for the evaluation of NAT10 and p53.


D CRISPR - Cas9 system mediated NAT10 gene editing . The genome targeting for knockdown NAT10 was performed as described in Experimental Procedures . The HCT116 NAT10 knockdown cell lines were harvested and western blot was performed on the cell lysates for the indicated proteins .

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D CRISPR-Cas9 system mediated NAT10 gene editing. The genome targeting for knockdown NAT10 was performed as described in Experimental Procedures. The HCT116 NAT10 knockdown cell lines were harvested and western blot was performed on the cell lysates for the indicated proteins.


E HCT116 Ctrl or c2 - 4 cells with NAT10 disruption were treated with actinomycin D and harvested at the indicated time points . Cell cycle profiles were determined by FACS . Results from three independent experiments are shown as mean ± SEM . ( two - tailed Student ' s t test )

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E HCT116 Ctrl or c2-4 cells with NAT10 disruption were treated with actinomycin D and harvested at the indicated time points. Cell cycle profiles were determined by FACS. Results from three independent experiments are shown as mean ± SEM. (two-tailed Student's t test)


F HCT116 Ctrl cells or c2 - 4 cells with NAT10 disruption were treated with actinomycin D Cells were harvested at the indicated time points and lysed . The total proteins were analyzed by western blot using the indicated antibodies .

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F HCT116 Ctrl cells or c2-4 cells with NAT10 disruption were treated with actinomycin D Cells were harvested at the indicated time points and lysed. The total proteins were analyzed by western blot using the indicated antibodies.


G HCT116 Ctrl or c2 - 4 cells were irradiated with 80 J / m2 UV . Forty - eight hours later , apoptotic cells were determined . Results from three independent experiments are shown as mean ± SEM . ( two - tailed Student ' s t test )

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G HCT116 Ctrl or c2-4 cells were irradiated with 80 J/m2 UV. Forty-eight hours later, apoptotic cells were determined. Results from three independent experiments are shown as mean ± SEM. (two-tailed Student's t test)


H Colony formation assay were performed with HCT116 cells ( Ctrl , c1 - 1 and c2 - 4 ) . The right panel shows the quantification of formed colonies . ( two - tailed Student ' s t test )

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H Colony formation assay were performed with HCT116 cells (Ctrl, c1-1 and c2-4). The right panel shows the quantification of formed colonies. (two-tailed Student's t test)


I HCT116 c1 - 1 and c2 - 4 cell lines were plated and cell growth curves were determined as described in Fig 6F . Error bars represent the SEM of the triplicate experiments .

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I HCT116 c1-1 and c2-4 cell lines were plated and cell growth curves were determined as described in Fig 6F. Error bars represent the SEM of the triplicate experiments.


Representative western - blots of the cortical ( Ctx ) or hippocampal ( Hip ) extracts from mice at the indicated time after seizure activity in pilocarpine ( A , B ) models . ( C , F ) Statistics of Gli1 or Shh expression levels shown in A , B or D , E respectively . n = 8 - 14 mice in C and n = 8 - 23 mice in F .

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Representative western-blots of the cortical (Ctx) or hippocampal (Hip) extracts from mice at the indicated time after seizure activity in pilocarpine (A,B) models. (C,F) Statistics of Gli1 or Shh expression levels shown in A,B or D,E respectively. n=8-14 mice in C and n=8-23 mice in F.


Representative western - blots of the cortical ( Ctx ) or hippocampal ( Hip ) extracts from mice at the indicated time after seizure activity in kindling ( D , E ) models . For D and E , samples obtained from mice evoked with a single kindling stimulation to induce seizure activity as evidenced in EEG . ( C , F ) Statistics of Gli1 or Shh expression levels shown in A , B or D , E respectively . n = 8 - 14 mice in C and n = 8 - 23 mice in F .

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Representative western-blots of the cortical (Ctx) or hippocampal (Hip) extracts from mice at the indicated time after seizure activity in kindling (D,E) models. For D and E, samples obtained from mice evoked with a single kindling stimulation to induce seizure activity as evidenced in EEG. (C,F) Statistics of Gli1 or Shh expression levels shown in A,B or D,E respectively. n=8-14 mice in C and n=8-23 mice in F.


( G ) Shh levels assayed by ELISA from mouse cortex and hippocampus at the indicated time after the initiation of status epilepticus ( SE ) induced by pilocarpine ( n = 7 - 10 ) .

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(G) Shh levels assayed by ELISA from mouse cortex and hippocampus at the indicated time after the initiation of status epilepticus (SE) induced by pilocarpine (n=7-10).


( H , I ) Shh levels assayed by ELISA in the medium of slices ( H , n = 9 ) or hippocampal neurons ( I , n = 6 ) incubated with picrotoxin ( Pic ) or Mg2 + - free ( 0Mg ) for the indicated times .

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(H,I) Shh levels assayed by ELISA in the medium of slices (H, n=9) or hippocampal neurons (I, n=6) incubated with picrotoxin (Pic) or Mg2+-free (0Mg) for the indicated times.


( J ) Representative western - blots and statistics of Gli1 expression levels from hippocampal neurons incubated with 0Mg for the indicated times ( n = 13 - 19 ) . a - tubulin ( a - Tub ) , a loading control . Data were mean + SEM . * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 vs . Control ( Ctrl ) with student ' s t test .

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(J) Representative western-blots and statistics of Gli1 expression levels from hippocampal neurons incubated with 0Mg for the indicated times (n=13-19). a-tubulin (a-Tub), a loading control. Data were mean + SEM. *p<0.05; **p<0.01; ***p<0.001 vs. Control (Ctrl) with student's t test.


( A - C ) Whole - cell recordings of spontaneous epileptiform activity from cultured hippocampal neurons . A , representative traces showing the neuronal activity under the indicated treatments . Ctrl : extracellular solution . Cyclo : cyclopamine . B , the expanded view of a single burst ( arrow ) from A ; C , statistics of the percentage of neurons showing epileptiform activity ( left , n = 6 - 7 ) and the burst frequency ( right , n = 18 - 19 ) shown in A . Cells were incubated in the presence of Cyclo in extracellular solution for 30 min , then transferred to 0Mg plus Cyclo ( 0Mg + Cyclo ) and incubated for another 30 min .

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(A-C) Whole-cell recordings of spontaneous epileptiform activity from cultured hippocampal neurons. A, representative traces showing the neuronal activity under the indicated treatments. Ctrl: extracellular solution. Cyclo: cyclopamine. B, the expanded view of a single burst (arrow) from A; C, statistics of the percentage of neurons showing epileptiform activity (left, n=6-7) and the burst frequency (right, n=18-19) shown in A. Cells were incubated in the presence of Cyclo in extracellular solution for 30 min, then transferred to 0Mg plus Cyclo (0Mg+Cyclo) and incubated for another 30 min.


( D , E ) The effects of Sant - 1 , robotnikinin ( Robot ) or 5E1 on the percentage of neurons showing epileptiform activity ( D , n = 6 - 10 ) and the burst frequency ( E , n = 15 - 22 ) . ( F - H ) Extracellular recordings of spontaneous epileptiform activity from CA1 stratum pyramidal of hippocampal slices . F , representative traces under the indicated treatments . Ctrl : artificial cerebrospinal fluid ( aCSF ) .

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(D,E) The effects of Sant-1, robotnikinin (Robot) or 5E1 on the percentage of neurons showing epileptiform activity (D, n=6-10) and the burst frequency (E, n=15-22). (F-H) Extracellular recordings of spontaneous epileptiform activity from CA1 stratum pyramidal of hippocampal slices. F, representative traces under the indicated treatments. Ctrl: artificial cerebrospinal fluid (aCSF).


( F - H ) Extracellular recordings of spontaneous epileptiform activity from CA1 stratum pyramidal of hippocampal slices . F , representative traces under the indicated treatments . Ctrl : artificial cerebrospinal fluid ( aCSF ) . G , the expanded view of a single burst ( arrow ) in F . Slices were incubated in the presence of vehicle or Cyclo in aCSF for 30 min , then transferred to picrotoxin in aCSF ( Pic ) or with Cyclo ( Pic + Cyclo ) and recorded for 30 min . H , statistics of the spontaneous burst frequency of F from 10 slices ( 7 rats ) .

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(F-H) Extracellular recordings of spontaneous epileptiform activity from CA1 stratum pyramidal of hippocampal slices. F, representative traces under the indicated treatments. Ctrl: artificial cerebrospinal fluid (aCSF). G, the expanded view of a single burst (arrow) in F. Slices were incubated in the presence of vehicle or Cyclo in aCSF for 30 min, then transferred to picrotoxin in aCSF (Pic) or with Cyclo (Pic+Cyclo) and recorded for 30 min. H, statistics of the spontaneous burst frequency of F from 10 slices (7 rats).


( I , J ) Left are representative traces showing the D - CPP - sensitive currents recorded from cultured hippocampal neurons ( I ) or slices ( J ) incubated in 0Mg or Pic , respectively , with or without Cyclo . Black lines : application of D - CPP . Right are statistics of D - CPP - sensitive current density from 18 - 26 cells ( I ) or 16 - 18 slices ( 15 rats ) ( J ) . Unless stated , 10 µM cyclopamine was used . Data were mean + SEM . * p < 0 . 05 , * * < 0 . 01 , * * * p < 0 . 001 vs . Ctrl ; # p < 0 . 05 , # # < 0 . 01 , # # # p < 0 . 001 vs . 0Mg or Pic ; & amp ; amp ; p < 0 . 05 vs . 0Mg + IgG . C - E used one - way ANOVA . H - J used student ' s t test .

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(I,J) Left are representative traces showing the D-CPP-sensitive currents recorded from cultured hippocampal neurons (I) or slices (J) incubated in 0Mg or Pic, respectively, with or without Cyclo. Black lines: application of D-CPP. Right are statistics of D-CPP-sensitive current density from 18-26 cells (I) or 16-18 slices (15 rats) (J). Unless stated, 10 µM cyclopamine was used. Data were mean + SEM. *p<0.05,**<0.01,***p<0.001 vs. Ctrl; #p<0.05,##<0.01,###p<0.001 vs. 0Mg or Pic; &amp;p<0.05 vs. 0Mg + IgG. C-E used one-way ANOVA. H-J used student's t test.


( A ) Glutamate levels assayed by HPLC in the medium of neurons incubated with indicated agents . n = 4 - 6 .

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(A) Glutamate levels assayed by HPLC in the medium of neurons incubated with indicated agents. n=4-6.


( B ) The paired - pulse ratio of field excitatory postsynaptic potentials recorded in CA1 of hippocampal slices treated with vehicle ( Ctrl ) or Shh ( 13 - 14 slices , 6 rats ) . Insets : representative traces recorded in response to paired pulse stimuli with different intervals .

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(B) The paired-pulse ratio of field excitatory postsynaptic potentials recorded in CA1 of hippocampal slices treated with vehicle (Ctrl) or Shh (13-14 slices, 6 rats). Insets: representative traces recorded in response to paired pulse stimuli with different intervals.


Statistics of 3H - glutamate uptake by neurons treated with indicated agents ( C , n = 10 )

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Statistics of 3H-glutamate uptake by neurons treated with indicated agents (C, n=10)


Statistics of 3H - glutamate uptake by neurons transfected with two lentivirus - based RNAi against EAAC1 ( R2 and R3 ) or nonsense RNAi ( Non ) in response to vehicle ( Ctrl ) or Shh ( D , n = 3 - 6 ) . No difference between R2 or R3 in Ctrl and those in Shh . Inset of ( D ) : representative western - blots for EAAC1 .

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Statistics of 3H-glutamate uptake by neurons transfected with two lentivirus-based RNAi against EAAC1 (R2 and R3) or nonsense RNAi (Non) in response to vehicle (Ctrl) or Shh (D, n=3-6). No difference between R2 or R3 in Ctrl and those in Shh. Inset of (D): representative western-blots for EAAC1.


Aspartate ( Asp ) - evoked currents at - 70 mV from hippocampal neurons transfected with RNAi or Non ( E , n = 17 - 23 ) or treated with indicated agents ( F , n = 16 - 18 ) .

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Aspartate (Asp)-evoked currents at -70 mV from hippocampal neurons transfected with RNAi or Non (E, n=17-23) or treated with indicated agents (F, n=16-18).


( G ) Upper , representative western - blots of EAAC1 and Smo from HEK293 cells transfected with empty vectors ( Ctrl ) , constitutively active form of Smoothened ( SmoA1 ) , EAAC1 , or EAAC1 plus SmoA1 . Lower , 3H - glutamate uptake by cells transfected with the indicated vectors . n = 9 .

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(G) Upper, representative western-blots of EAAC1 and Smo from HEK293 cells transfected with empty vectors (Ctrl), constitutively active form of Smoothened (SmoA1), EAAC1, or EAAC1 plus SmoA1. Lower, 3H-glutamate uptake by cells transfected with the indicated vectors. n=9.


( H , I ) Shh effects on 3H - glutamate uptake ( H , n = 6 ) or Asp - evoked currents ( I , n = 17 - 23 ) of neurons with or without pertussis toxin ( PTX ) pretreatment . For E , F , I , the uppers : representative traces . Shh : 500 ng / mL . Data were mean ± SEM . * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 vs . Ctrl or Non ( Ctrl ) with student ' s t test .

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(H,I) Shh effects on 3H-glutamate uptake (H, n=6) or Asp-evoked currents (I, n=17-23) of neurons with or without pertussis toxin (PTX) pretreatment. For E,F,I, the uppers: representative traces. Shh: 500 ng/mL. Data were mean ± SEM. *p<0.05; **p<0.01; ***p<0.001 vs. Ctrl or Non (Ctrl) with student's t test.


Effects of Cyclo ( A - C , n = 14 ) on the progression of kindling including seizure class ( A , D , G , J ) , evoked electrographic seizure duration ( ESD ) ( B , E , H , K ) and the number of stimulations required to reach equivalent seizure intensity ( C , F , I , L ) . In A - F , Ctrl are vehicle - treated mice .

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Effects of Cyclo (A-C, n=14) on the progression of kindling including seizure class (A,D,G,J), evoked electrographic seizure duration (ESD) (B,E,H,K) and the number of stimulations required to reach equivalent seizure intensity (C,F,I,L). In A-F, Ctrl are vehicle-treated mice.


Effects of 5E1 ( D - F , n = 16 - 20 ) , on the progression of kindling including seizure class ( A , D , G , J ) , evoked electrographic seizure duration ( ESD ) ( B , E , H , K ) and the number of stimulations required to reach equivalent seizure intensity ( C , F , I , L ) . In A - F , Ctrl are vehicle - treated mice .

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Effects of 5E1 (D-F, n=16-20), on the progression of kindling including seizure class (A,D,G,J), evoked electrographic seizure duration (ESD) (B,E,H,K) and the number of stimulations required to reach equivalent seizure intensity (C,F,I,L). In A-F, Ctrl are vehicle-treated mice.


Effects of ablation of Smo in CaMKIIα - positive neurons ( G - I ) , on the progression of kindling including seizure class ( A , D , G , J ) , evoked electrographic seizure duration ( ESD ) ( B , E , H , K ) and the number of stimulations required to reach equivalent seizure intensity ( C , F , I , L ) . In G - I , Ctrl : Smofl / fl induced by tamoxifen ; CaMK : Smofl / flCaMKIIα - CreERT2 induced by tamoxifen , n = 14 - 20 .

O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O

Effects of ablation of Smo in CaMKIIα-positive neurons (G-I), on the progression of kindling including seizure class (A,D,G,J), evoked electrographic seizure duration (ESD) (B,E,H,K) and the number of stimulations required to reach equivalent seizure intensity (C,F,I,L). In G-I, Ctrl: Smofl/fl induced by tamoxifen; CaMK: Smofl/flCaMKIIα-CreERT2 induced by tamoxifen, n=14-20.


Effects of ablation of Smo in Aldh1l1 - positive astrocytes ( J - L ) on the progression of kindling including seizure class ( A , D , G , J ) , evoked electrographic seizure duration ( ESD ) ( B , E , H , K ) and the number of stimulations required to reach equivalent seizure intensity ( C , F , I , L ) . In J - L , Ctrl : Smo + / fl ; Aldh1l1 : Smo + / flAldh1l1 - Cre , n = 19 - 20 .

O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O

Effects of ablation of Smo in Aldh1l1-positive astrocytes (J-L) on the progression of kindling including seizure class (A,D,G,J), evoked electrographic seizure duration (ESD) (B,E,H,K) and the number of stimulations required to reach equivalent seizure intensity (C,F,I,L). In J-L, Ctrl: Smo+/fl; Aldh1l1: Smo+/flAldh1l1-Cre, n=19-20.


( M ) Frequency of spontaneous seizures ( class 4 - 5 ) in mice administrated with Cyclo or vehicle ( Ctrl ) at 4th , 6th and 8th week after pilocarpine SE induction . n = 22 - 23 .

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(M) Frequency of spontaneous seizures (class 4-5) in mice administrated with Cyclo or vehicle (Ctrl) at 4th, 6th and 8th week after pilocarpine SE induction. n=22-23.


( N ) Percentage of mice with class 5 seizures within 8 weeks after pilocarpine SE induction . n = 22 - 23 . Cyclo : 10 mg / kg . 5E1 : 900 ng / mouse . Data were mean ± SEM from at least three independent experiments . * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 vs . Ctrl with student ' s t test .

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(N) Percentage of mice with class 5 seizures within 8 weeks after pilocarpine SE induction. n=22-23. Cyclo: 10 mg/kg. 5E1: 900 ng/mouse. Data were mean ± SEM from at least three independent experiments. *p<0.05; **p<0.01; ***p<0.001 vs. Ctrl with student's t test.


Morphological changes of vacuolar membranes and peroxisomal GFP - SKL during microautophagic degradation of peroxisomes after glucose adaptation . Double fluorescence of methanol - grown cells of P . pastoris cells at various times after the switch to glucose medium . ( a ) 0 h , ( b ) 30 min , ( c ) 2 h .

O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O

Morphological changes of vacuolar membranes and peroxisomal GFP-SKL during microautophagic degradation of peroxisomes after glucose adaptation. Double fluorescence of methanol-grown cells of P. pastoris cells at various times after the switch to glucose medium. (a) 0 h, (b) 30 min, (c) 2 h.