A . Osmotic stress induces YAP translocation . HEK293A 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 nuclei . Scale bar : 20 μm . of more ( N > C ) or more ( 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 translocation . YAP knockout ( KO ) HEK293A were serum starved for 1 hr followed by 0 . 2 M sorbitol treatment for 1 hr . TAZ localization was determined by immunofluorescence with 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 translocation . HEK293A were serum starved for 1 hr followed by 0 . 2 M sorbitol treatment for 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 were serum starved for 1 hr treated with 0 . 2 M sorbitol for 4 hr . mRNA of CTGF CYR61 were measured by quantitative RT - PCR normalized to GAPDH control . Data are presented as mean ± . * p < 0 . 05 ( two student ' s t - , 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 14 - 3 - 3 interaction . HEK293A were transiently transfected with Flag - YAP Myc - 14 - 3 - 3 , were serum starved for 16 hr . were then treated with 0 . 2 M sorbitol for indicated time points . Myc - 14 - 3 - 3 was immunoprecipitated associated Flag - YAP was detected by Western . Data are presented as mean ± . * p < 0 . 05 ( two student ' s t - , n = 3 ) .

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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 localization . HEK293A 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 absence of serum . of YAP / TAZ subcellular is shown . Scale bar : 20 μm .

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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 localization . HEK293A 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 absence of serum . lysates from identically treated samples were examined for phosphorylation of p38 substrate MK - 2 JNK substrate c - Jun .

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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 localization . HEK293A were transiently transfected with CRISPR / Cas9 to knockout NLK . Wild type ( WT ) NLK KO pool were treated with 0 . 2 M sorbitol for 1 hr in absence of serum . YAP / TAZ subcellular localization was determined by immunofluorescence . Scale bar : 20 μm .

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

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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 . phos - tag gel showed NLK - WT but not NLK - KN caused a significant mobility shift of YAP .

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

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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 .

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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 immunoprecipitated . An in vitro NLK kinase was performed using recombinant GST - YAP as a substrate . Phosphorylation of YAP Ser 128 was determined by immunoblotting with phospho - YAP ( S128 ) antibody .

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

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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 two pools of NLK CRISPR / CAS9 transfected were treated with 0 . 2 M sorbitol for indicated time points . Data are presented as mean ± . * p < 0 . 05 ( two student ' s t - , n = 4 ) .

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

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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 . were serum starved for 16 hrs , treated with 0 . 2 M sorbitol for indicated time points or refreshed with serum containing medium for 1 hr . Refreshing medium served as a control to disrupt YAP 14 - 3 - 3 binding . 14 - 3 - 3 was immunoprecipitated with Myc antibody , associated YAP was detected with Flag antibody . Data are presented as mean ± . * p < 0 . 05 ( two student ' s t - , 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 O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR 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 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

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 translocation of S128A mutant YAP . HEK293A were co - transfected with Myc - NLK Flag - YAP WT or S128A mutant . were serum starved for 6 hr , YAP localization NLK expression were determined by Flag ( green ) Myc ( red ) antibodies , respectively . Scale bar : 20 μm .

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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 localization . HEK293A YAP KO 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 ( green ) . Scale bar : 20 μm .

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

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

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

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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 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 ) .

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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 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 ) naked mole - rat ( n = 8 ) . I , Relative 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 .

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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 - Seq datasets . Naked mole - rat BM populations CP1 - 4 were matched to human murine counterparts for GSEA with MSigDB hallmark genesets . Primitive 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 , types were pooled for each species . Shown are pathways related to proliferation metabolism normalized enrichment score ; GeneRatio , ( signature ∩ term ) / ( signature ∩ 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

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 with 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 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

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 in mouse ( n = 4 ) , human ( n = 4 ) naked mole - rat ( n = 5 ) BM , histogram of merged per - species data . unstained , BM from mouse [ solid ] , human [ dotted ] or NMR [ 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

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 imaging of sorted mouse LT - HSCs ( LIN - / Sca - 1 + / Kit + / CD150 + / CD48 - ) NMR LTCs across ; 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

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

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, 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 were transfected with Flag - NAT10 or control vectors . Forty - eight hours later , were harvested whole extracts were immunoprecipitated with Flag - antibody affinity resin . NAT10 - binding were resolved by SDS - PAGE detected by silver .

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-MEASURED_VAR O O O O O O O O O O O O

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 - / - lysates were immunoprecipitated with control IgG , anti - NAT10 ( B F ) , anti - p53 ( C ) anti - Mdm2 ( D E ) antibodies . immunoprecipitates were subsequently immunoblotted with indicated antibodies .

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

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 coupled to Glutathione Sepharose 4B . retained on Sepharose were then analyzed by western blot using antibodies as indicated . amount of GST fusion are shown in lower panel .

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

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 - GST - p53 fusion , its deletion mutants or GST was used to pull - down experiments with purified NAT10 . of GST fusion are shown in left panel .

O O O O O B-CONTROLLED_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

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 was performed using purified GST - NAT10 deletion mutants or GST overexpressed Flag - p53 or Mdm2 in HEK293T . Schematic diagram represents constructs of GST - NAT10 deletion mutants ( right panel ) .

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

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 . reaction products were resolved by SDS - PAGE immunoblotted using anti - acetyl - lysine antibodies . Flag - PCAF was used as a positive control .

O O O O B-MEASURED_VAR 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

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 acetylated p53 was detected as in A .

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

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


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

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

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

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

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 were transfected with indicated vectors . Total anti - Flag antibody M2 - specific immunoprecipitates were analyzed by western blot using 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

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 were transfected with indicated siRNAs . Seventy - two hours later , lysates were prepared from lysates were subjected to western blot using 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

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 were transfected as in A . Seventy - two hours later , total mRNAs were extracted subjected to RT - qPCR for 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

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 were transfected with increasing amount of plasmid expressing NAT10 . from whole extracts were subjected to western blot using indicated antibodies .

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

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 were transfected with control or NAT10 siRNAs . Seventy - two hours later , were treated with cycloheximide ( CHX ) harvested at indicated time points . from lysates were subjected to immunoblot for evaluation of NAT10 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 ± at bottom .

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 B-MEASURED_VAR I-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

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 were treated with MG132 ( 10μM ) for 4 hours after transfected with indicated siRNAs . from extracts were detected by western blot for indicated .

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

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

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 Mdm2 double null MEF were transfected with indicated constructs treated with MG132 for 4 hours before harvest . from whole extracts were subjected to western blot using indicated antibodies .

O B-CONTROLLED_VAR I-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

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 were transfected with vectors as indicated . Forty - eight hours later , were harvested from whole extracts were subjected to western blot using 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

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 were transfected with increasing amount of expressing NAT10 . Forty - eight hours later , whole lysates were prepared from lysates were analyzed by western blot using indicated antibodies .

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

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

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 B-MEASURED_VAR 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

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 transfected with indicated constructs were harvested after treatment with MG132 for 4 hours . Mdm2 was immunoprecipitated with anti - Mdm2 antibody immunoblotted with anti - HA antibody .

O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O B-MEASURED_VAR 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 ) Flag - NAT10 or Flag - NAT10 GE in presence of E1 , E2 Ub in vitro as described in Experimental Procedures . reaction products were resolved by SDS - PAGE , followed by western blot for evaluation of indicated .

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

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 were carried out with Mdm2 different doses of purified Flag - NAT10 as described in D .

O B-MEASURED_VAR O O O B-CONTROLLED_VAR 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

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

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

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 full - Flag - NAT10 or Flag - NAT10 - D5 . Ubiquitinated Mdm2 was detected as in D .

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

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 were transfected with indicated constructs . were harvested after CHX treatment for indicated times lysates were prepared . Mdm2 were evaluated by western blot . Relative Mdm2 standardized by β - actin are shown in right .

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-MEASURED_VAR O O O O O O O O O O

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 were treated with 10nM actinomycin D extracts were prepared at indicated time points . from extracts were analyzed by western blot using indicated antibodies .

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 H1299 were treated with actinomycin D for 12 hours . were harvested from lysates were subjected to western blot probed with 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.


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

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

O O O O O 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-MEASURED_VAR O O O O O O O O O O O

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 were treated with actinomycin D for indicated times . were fixed immuno - stained as indicated . Immunofluorescence were taken by confocal microscopy . Scale bar represents 10 μM .

O O O O O O O O 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 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 were transfected with indicated vectors . After treated with actinomycin D , were harvested whole extracts were subjected to western blot using 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

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

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

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 transfected with indicated constructs were treated with actinomycin D or doxorubicin . Western blot was performed with total extracts Mdm2 - specific immunoprecipitates for indicated .

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

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 were transfected with indicated constructs . were harvested after treatment with actinomycin D . p53 was immunoprecipitated with an anti - p53 polyclonal antibody 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 indicated siRNAs into U2OS . 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 were transfected with siRNAs as indicated . Seventy - two hours later , were harvested after treated with actinomycin D for 12 hours . Relative mRNA for 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 ( C D ) H1299 ( D ) transfected with indicated siRNAs were treated with actinomycin D harvested at indicated time points . cycle profiles were determined by FACS . Results from three independent experiments are shown as mean ± . ( two - Student ' s t )

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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 were transfected with indicated siRNAs . were irradiated with 50 J / m2 UV . Forty - eight hours later , apoptotic were determined . of apoptotic is presented . ( two - Student ' s t )

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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 transfected with indicated siRNAs were plated . numbers were determined as described in Experimental Procedures at 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 were performed with U2OS transfected with indicated siRNAs . right panel shows of formed colonies . * * p < 0 . 01 , * * * p < 0 . 001 ( two - Student ' s t )

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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 corresponding normal tissues were lysed total RNAs were extracted subjected to RT - qPCR . ( N , Normal tissue ; T , tissue ) * * p < 0 . 01 ( non - parametric Mann - Whitney t - )

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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 of NAT10 in CRC tissues normal colon tissues . NAT10 - specific immuno - was detected with DAB as shown as brown color . of NAT10 - positive or NAT10 - negative CRC cases is shown in 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 extracts were prepared from different human colon epithelial lines as indicated at top . from extracts were subjected to western blot for evaluation of NAT10 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 . genome targeting for knockdown NAT10 was performed as described in Experimental Procedures . HCT116 NAT10 knockdown lines were harvested western blot was performed on lysates for indicated .

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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 with NAT10 disruption were treated with actinomycin D harvested at indicated time points . cycle profiles were determined by FACS . Results from three independent experiments are shown as mean ± . ( two - Student ' s t )

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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 or c2 - 4 with NAT10 disruption were treated with actinomycin D were harvested at indicated time points lysed . total were analyzed by western blot using 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 were irradiated with 80 J / m2 UV . Forty - eight hours later , apoptotic were determined . Results from three independent experiments are shown as mean ± . ( two - Student ' s t )

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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 were performed with HCT116 ( Ctrl , c1 - 1 c2 - 4 ) . right panel shows of formed colonies . ( two - Student ' s t )

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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 c2 - 4 lines were plated growth curves were determined as described in Fig 6F . Error bars represent of 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 cortical ( Ctx ) or hippocampal ( Hip ) extracts from mice at indicated time after seizure activity in pilocarpine ( A , B ) models . ( C , F ) Statistics of Gli1 or Shh expression shown in A , B or D , E respectively . n = 8 - 14 mice in C 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 cortical ( Ctx ) or hippocampal ( Hip ) extracts from mice at indicated time after seizure activity in kindling ( D , E ) models . For D 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 shown in A , B or D , E respectively . n = 8 - 14 mice in C 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 assayed by ELISA from mouse cortex hippocampus at indicated time after 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 assayed by ELISA in medium of slices ( H , n = 9 ) or hippocampal neurons ( I , n = 6 ) incubated with picrotoxin ( Pic ) or Mg2 + - free ( 0Mg ) for 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 statistics of Gli1 expression from hippocampal neurons incubated with 0Mg for indicated times ( n = 13 - 19 ) . a - tubulin ( a - Tub ) , a loading control . Data were mean + . * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 vs . Control ( Ctrl ) with student ' s t .

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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 - recordings of spontaneous epileptiform activity from cultured hippocampal neurons . A , representative traces showing neuronal activity under indicated treatments . Ctrl : extracellular solution . Cyclo : cyclopamine . B , expanded view of a single burst ( arrow ) from A ; C , statistics of of neurons showing epileptiform activity ( left , n = 6 - 7 ) burst frequency ( right , n = 18 - 19 ) shown in A . were incubated in presence of Cyclo in extracellular solution for 30 min , then transferred to 0Mg plus Cyclo ( 0Mg + Cyclo ) 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 ) effects of Sant - 1 , robotnikinin ( Robot ) or 5E1 on of neurons showing epileptiform activity ( D , n = 6 - 10 ) 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 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 indicated treatments . Ctrl : artificial cerebrospinal fluid ( aCSF ) . G , expanded view of a single burst ( arrow ) in F . Slices were incubated in presence of vehicle or Cyclo in aCSF for 30 min , then transferred to picrotoxin in aCSF ( Pic ) or with Cyclo ( Pic + Cyclo ) recorded for 30 min . H , statistics of 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 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 ( I ) or 16 - 18 slices ( 15 rats ) ( J ) . Unless stated , 10 µM cyclopamine was used . Data were mean + . * 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 .

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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 assayed by HPLC in 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 ) paired - 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 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 )

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 - RNAi against EAAC1 ( R2 R3 ) or nonsense RNAi ( Non ) in response to vehicle ( Ctrl ) or Shh ( D , n = 3 - 6 ) . No difference between R2 or R3 in Ctrl 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 Smo from HEK293 transfected with empty vectors ( Ctrl ) , constitutively active form of Smoothened ( SmoA1 ) , EAAC1 , or EAAC1 plus SmoA1 . Lower , 3H - glutamate uptake by transfected with 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 , uppers : representative traces . Shh : 500 ng / mL . Data were mean ± . * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 vs . Ctrl or Non ( Ctrl ) with student ' s t .

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

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

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

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

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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.


electron microscope showing various stages during microautophagic degradation of peroxisomes . three panels in each figure are as follows ( a - e , left ) FM4 - 64 ; ( center ) GFP - SKL ; ( right ) superimposed FM4 - 64 GFP - SKL signals . ( a , f ) stage 0 ; ( b , g ) early stage 1 ; ( c , h ) late stage 1 ; ( d , i ) stage 2 ; ( e ) stage 3 . ( d , arrowhead ) During capture of these , this peroxisome - containing vesicle had just pinched off from outer vacuolar . Therefore , FM4 - 64 - stained vesicle GFP - SKL could not be superimposed in this picture .

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Fluorescence and electron microscope images showing various stages during microautophagic degradation of peroxisomes. The three panels in each figure are as follows (a-e, left) FM4-64; (center) GFP-SKL; (right) superimposed FM4-64 and GFP-SKL signals. (a, f) stage 0; (b, g) early stage 1; (c, h) late stage 1; (d, i) stage 2; (e) stage 3. (d, arrowhead) During the capture of these fluorescence images, this peroxisome-containing vesicle had just pinched off from the outer vacuolar membrane. Therefore, the FM4-64-stained vesicle membrane and GFP-SKL could not be superimposed in this picture.


Kinetics of morphological changes in vacuoles peroxisomalGFP - SKL during microautophagic degradation of P . pastoris in glucose adaptation . At least 350 vacuoles or 350 clusters of peroxisomes were counted for each time point . were counted in three randomly selected fields , standard deviations are shown as vertical bars .

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Kinetics of morphological changes in vacuoles and peroxisomalGFP-SKL during microautophagic degradation of P. pastoris in glucose adaptation. At least 350 vacuoles or 350 clusters of peroxisomes were counted for each time point. Cells were counted in three randomly selected fields, and standard deviations are shown as vertical bars.


Macroautophagic degradation of peroxisomes after transfer of methanol - grown P . pastoris to ethanol medium . ( a - e ) Electron of intermediates seen during macroautophagy . ( a ) After 1 h of ethanol adaptation , peroxisomes ( P ) were often surrounded by autophagosomal membranes , higher magnifications of which are in d e . ( b ) After 1 h of ethanol adaptation , autophagosomal surrounding peroxisomes appears to have fused with vacuolar . ( c ) At 2 h a peroxisome in form of an autophagic is seen within vacuole . Magnified pictures of samples obtained at ( d ) 20 min time point showing endoplasmic reticulum in close proximity to membranes surrounding peroxisomes , at ( e ) 1 h time point demonstrating peroxisomes surrounded by multiple autophagosomal membranes . ( f ) Kinetics of morphological changes in vacuoles peroxisomal GFP - SKL . Other conditions are described in Materials Methods , are same as in Fig . 4 . ( g ) taken after 2 h of ethanol adaptation of wild - type P . pastoris .

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Macroautophagic degradation of peroxisomes after transfer of methanol-grown P. pastoris cells to ethanol medium. (a-e) Electron micrographs of intermediates seen during macroautophagy. (a) After 1 h of ethanol adaptation, peroxisomes (P) were often surrounded by autophagosomal membranes, higher magnifications of which are in d and e. (b) After 1 h of ethanol adaptation, the autophagosomal membrane surrounding the peroxisomes appears to have fused with the vacuolar membrane. (c) At 2 h a peroxisome in the form of an autophagic body is seen within the vacuole. Magnified pictures of samples obtained at the (d) 20 min time point showing the nuclear endoplasmic reticulum membrane in close proximity to the membranes surrounding the peroxisomes, and at the (e) 1 h time point demonstrating peroxisomes surrounded by multiple autophagosomal membranes. (f) Kinetics of morphological changes in vacuoles and peroxisomal GFP-SKL. Other conditions are described in Materials and Methods, and are the same as in Fig. 4. (g) Fluorescence images taken after 2 h of ethanol adaptation of wild-type P. pastoris cells.


Effect of inhibitors on transport of peroxisomal GFP - SKL to , degradation in , vacuoles during ( a ) glucose adaptation ( b ) ethanol adaptation . Wortmannin PMSF were dissolved in DMSO , added to glucose medium . ( Filled circle ) Control ( 2 % DMSO ) ; ( open circle ) 1 mM wortmannin ( with 2 % DMSO ) ; ( filled square ) 3 mM PMSF ( with 2 % DMSO ) ; ( filled triangle ) 10 mg / ml cycloheximide .

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Effect of inhibitors on transport of peroxisomal GFP-SKL to, and degradation in, vacuoles during (a) glucose adaptation and (b) ethanol adaptation. Wortmannin and PMSF were dissolved in DMSO, and added to the glucose medium. (Filled circle) Control (2% DMSO); (open circle) 1 mM wortmannin (with 2% DMSO); (filled square) 3 mM PMSF (with 2% DMSO); (filled triangle) 10 mg/ml cycloheximide.


Microautophagic degradation of peroxisomes is not observed in a vacuolar protease - negative ( STK7 ) of P . pastoris . Kinetics of morphological changes in vacuoles peroxisomal GFP - SKL during ( a ) glucose adaptation ( b ) ethanol adaptation . ( c ) taken after 2 h of glucose adaptation . Arrows show GFP - SKL in vacuolar matrix . Some showed diffusion of GFP - SKL into vacuolar matrix .

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Microautophagic degradation of peroxisomes is not observed in a vacuolar protease-negative strain (STK7) of P. pastoris. Kinetics of morphological changes in vacuoles and peroxisomal GFP-SKL during (a) glucose adaptation and (b) ethanol adaptation. (c) Fluorescence images taken after 2 h of glucose adaptation. Arrows show GFP-SKL in vacuolar matrix. Some cells showed diffusion of GFP-SKL into the vacuolar matrix.


( a ) Visualization of alcohol oxidase activity in colonies after ( a ) glucose adaptation for 8 h ethanol adaptation for 24 h in wild - type ( PPY12 ) pag mutant ( strains STK1 - STK6 , Materials Methods ) of P . pastoris . ( b ) Western - blot of samples taken from cultures shifted from methanol to glucose for indicated time . Antibodies used were AOX , alcohol oxidase ; F1 , mitochondrial F1β subunit ; G6PDH , glucose - 6 - phosphate dehydrogenase . Growth curve of wild - type PPY12 pag4 mutant ( STK4 ) after shift from methanol to glucose .

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(a) Visualization of alcohol oxidase activity in colonies after (a) glucose adaptation for 8 h and ethanol adaptation for 24 h in wild-type (strain PPY12) and pag mutant cells (strains STK1-STK6, Materials and Methods) of P. pastoris. (b) Western-blot analysis of samples taken from cultures shifted from methanol to glucose for the indicated time. Antibodies used were AOX, alcohol oxidase; F1, mitochondrial F1β subunit; G6PDH, glucose-6-phosphate dehydrogenase. Growth curve of the wild-type strain PPY12 and the pag4 mutant (strain STK4) after shift from methanol to glucose.


of ( a ) pag2 ( STK2 ) ( b ) pag3 ( STK3 ) mutants of P . pastoris after a 3 h shift to glucose . ( b ) Arrows show deeply invaginated structures of vacuolar membranes surrounding peroxisomes in pag3 mutant .

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Fluorescence images of (a) pag2 (strain STK2) and (b) pag3 (strain STK3) mutants of P. pastoris after a 3 h shift to glucose. (b) Arrows show deeply invaginated structures of vacuolar membranes surrounding peroxisomes in the pag3 mutant.


of pag4 ( STK4 ) mutant of P . pastoris after ( a ) 0 , ( b ) 3 , or ( c ) 6 h of glucose adaptation . vacuolar proliferated extensively surrounded peroxisomes during glucose adaptation .

Fluorescence images of the pag4 (strain STK4) mutant of P. pastoris after (a) 0, (b) 3, or (c) 6 h of glucose adaptation. The vacuolar membrane proliferated extensively and surrounded peroxisomes during glucose adaptation.


Microautophagy of peroxisomes in a pex1 - ts mutant ( SJH242 ) of P . pastoris expressing GFP - SKL from constitutive glyceraldehyde - 3 - phosphate dehydrogenase ( GAPDH ) promoter at restrictive for peroxisome assembly ( 34 ° C ) . ( a ) Kinetics of peroxisome degradation at 34 ° C . SJH242 was induced on methanol at 23 ° C , glucose adaptation was performed at 34 ° C . ( b ) Changes in vacuole were seen even in absence of normal peroxisomes . At 0 h , SJH242 were grown at 34 ° C when GFP - SKL was diffuse in cytosol many vacuoles showed spherical morphologies . After 2 h , ∼ 20 - 30 % vacuoles showed complicated structures representative of stage 2 .

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Microautophagy of peroxisomes in a pex1-ts mutant (strain SJH242) of P. pastoris expressing GFP-SKL from the constitutive glyceraldehyde-3-phosphate dehydrogenase (GAPDH) promoter at the restrictive temperature for peroxisome assembly (34°C). (a) Kinetics of peroxisome degradation at 34°C. Strain SJH242 was induced on methanol at 23°C, and glucose adaptation was performed at 34°C. (b) Changes in vacuole morphology were seen even in the absence of normal peroxisomes. At 0 h, SJH242 cells were grown at 34°C when GFP-SKL was diffuse in the cytosol and many vacuoles showed spherical morphologies. After 2 h, ∼20-30% vacuoles showed complicated membrane structures representative of stage 2 cells.


D . Plot of initial MT polymerization velocities at 37 ° C in presence of increasing concentrations of WT or NN . Tubulin = 5 μM . Each point represents an average of 3 independent measurements of rate of turbidity increase measured at 500 nm wavelength ( OD500 / min ) . Fitted are sigmoidal dose - response curves : Y = YB + ( ( YT - YB ) / ( 1 + 10Xi - X ) ) , where YB YT are Y values ( OD500 / min ) at bottom top plateaus , respectively , Xi is X value at inflection point . Representative MT samples from 2 : 5 μM WT : tubulin or NN : tubulin incubations are shown in E F , respectively . MT architecture distributions for WT NN are shown with pie graphs .

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D. Plot of initial MT polymerization velocities at 37 °C in the presence of increasing concentrations of WT or NN. Tubulin concentration = 5 μM. Each point represents an average of 3 independent measurements of the rate of turbidity increase measured at 500 nm wavelength (OD500/min). Fitted are sigmoidal dose-response curves: Y = YB + ((YT - YB)/(1 + 10Xi - X)), where YB and YT are Y values (OD500/min) at the bottom and top plateaus, respectively, and Xi is the X value at the inflection point. Representative MT samples from 2:5 μM WT:tubulin or NN:tubulin incubations are shown in E and F, respectively. MT architecture distributions for WT and NN are shown with pie graphs.


E , F . Representative low high magnification of MTs nucleated for 10 min at 37 ° C from 5 μM tubulin , in presence of : 2 μM wild - type DCX ( WT ) ( E ) or 2 μM NDC - NDC ( NN ) ( F ) .

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E, F. Representative low and high magnification images of MTs nucleated for 10 min at 37 °C from 5 μM tubulin, in the presence of: 2 μM wild-type DCX (WT) (E) or 2 μM NDC-NDC (NN) (F).


H . Representative kymographs with arrowheads indicating MT catastrophes .

H. Representative kymographs with arrowheads indicating MT catastrophes.


A . Representative low - pass filtered cryo - EM of MTs decorated with wild - type DCX ( WT ) or NDC - NDC construct ( NN ) after 30 sec or 1 h incubation at 37 ° C . Additional DCX were added in excess after MT deposition on EM grids to maximise MT decoration . final of WT or NN to tubulin was 50 : 5 μM , giving dense background . WT co - polymerized exclusively 13 - PF MTs , while NN both 13 - 14 - PF MTs . Rapid ( 30 sec ) MT polymerization in excess of WT produces MTs in an intermediate GTP / GDP . Pi state , whereas long - lived WT - MTs NN - MTs are in GDP state .

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A. Representative low-pass filtered cryo-EM micrographs of MTs decorated with wild-type DCX (WT) or NDC-NDC construct (NN) after 30 sec or 1 h incubation at 37 °C. Additional DCX proteins were added in excess after MT deposition on EM grids to maximise MT decoration. The final ratio of WT or NN to tubulin was 50:5 μM, giving dense protein background. WT co-polymerized exclusively 13-PF MTs, while NN both 13- and 14-PF MTs. Rapid (30 sec) MT polymerization in excess of WT produces MTs in an intermediate GTP/GDP.Pi state, whereas long-lived WT-MTs and NN-MTs are all in the GDP state.


C - F . Immunofluorescence of esg - GFP Delta ( Dl ) with midgut section from R4 region of 14 - day Drosophila ( C ) , 26 - day Drosophila ( D ) , 40 - day Drosophila ( E ) , 40 - day Drosophila in response to 0 . 5 mM ALA administration , which started at day 26 after eclosion ( F ) . esg - GFP ( green ) identifies ISCs their differentiating . Dl ( red ) was used to visualize ISCs . Data information : DAPI stained nuclei are shown in blue .

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C-F. Immunofluorescence images of esg-GFP and Delta (Dl) staining with the midgut section from the R4 region of 14-day Drosophila (C), 26-day Drosophila (D), 40-day Drosophila (E), and 40-day Drosophila in response to 0.5 mM ALA administration, which started at day 26 after eclosion (F). esg-GFP (green) identifies ISCs and their differentiating cells. Dl staining (red) was used to visualize ISCs. Data information: DAPI stained nuclei are shown in blue.


J . Western blotting results of midguts indicate a decrease of LAS in midguts of aged Drosophila . Loading controls , β - tubulin . K . of LAS band as seen in experiments ( J ) . Error bars indicate SD of three independent experiments . L Data information : Error bars represent SDs . Student ' s t - , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 , non - significant ( NS ) represents p > 0 . 05 .

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J. Western blotting results of midguts indicate a decrease of LAS in the midguts of aged Drosophila. Loading controls, β-tubulin. K. Quantification of LAS band intensity as seen in experiments (J). Error bars indicate the SD of three independent experiments. L Data information: Error bars represent SDs. Student's t-tests, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, and non-significant (NS) represents p > 0.05.


C . of content of ALA in midguts of flies with indicated genotypes using LC - ESI - MS / MS . Error bars indicate SD of three independent experiments . Data information : Error bars represent SDs . Student ' s t - , * * * * p < 0 . 0001 , non - significant ( NS ) represents p > 0 . 05 .

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C. Quantification of the content of ALA in midguts of flies with indicated genotypes using LC-ESI-MS/MS. Error bars indicate the SD of three independent experiments. Data information: Error bars represent SDs. Student's t-tests, ****p < 0.0001, and non-significant (NS) represents p > 0.05.


D - F . Immunofluorescence of midgut section from R4 region in Drosophila carrying esgts - GAL4 - driven UAS - lacZ ( D , control ) , Las RNAi ( E ) , or Las RNAi in response to ALA administration ( F ) . esg - GFP ( green ) indicates ISCs their differentiating . Dl ( red ) was used to visualize ISCs . Data information : DAPI stained nuclei are shown in blue . Scale bars represent 10

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D-F. Immunofluorescence images of the midgut section from the R4 region in Drosophila carrying esgts-GAL4-driven UAS-lacZ (D, control), Las RNAi (E), or Las RNAi in response to ALA administration (F). esg-GFP (green) indicates ISCs and their differentiating cells. Dl staining (red) was used to visualize ISCs. Data information: DAPI stained nuclei are shown in blue. Scale bars represent 10 μm


I - J . Immunofluorescence of control ( FRT40A , I ) Las RNAi ( J ) MARCM clones ( green , outlined by dotted lines ) 10 days after clone induction ( ACI ) . Pdm1 ( red ) was used to visualize ECs . K . of of Pdm1 + per clone of MARCM clones with indicated genotypes . n is indicated . Each dot corresponds to one clone . L . of MARCM clone of experiments in ( I - J ) . n is indicated . Each dot corresponds to one clone . Data information : DAPI stained nuclei are shown in blue . Scale bars represent 10 μm ( D Error bars represent SDs . Student ' s t - , * * * * p < 0 . 0001 , non - significant ( NS ) represents p > 0 . 05 .

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I-J. Immunofluorescence images of control (FRT40A, I) and Las RNAi (J) MARCM clones (green, outlined by white dotted lines) 10 days after clone induction (ACI). Pdm1 staining (red) was used to visualize ECs. K. Quantification of the ratio of Pdm1+ cells per clone of MARCM clones with indicated genotypes. n is indicated. Each dot corresponds to one clone. L. Quantification of MARCM clone size of experiments in (I-J). n is indicated. Each dot corresponds to one clone. Data information: DAPI stained nuclei are shown in blue. Scale bars represent 10 μm (D Error bars represent SDs. Student's t-tests, ****p < 0.0001, and non-significant (NS) represents p > 0.05.


A . Representative of Drosophila midguts treated with pH indicator Bromophenol Blue . Moreover , of three categories of Drosophila of indicated conditions . three categories can be divided into : homeostasis , a well - defined acidic ( yellow colored ) copper region ( CCR ) is flanked by basic ( blue colored ) anterior midgut ( AM ) posterior midgut ( PM ) . " Perturbed A " , acidic region is lost whole gut is basic ; " Perturbed B " , strongly acidic region is lost , remainder of gut also becomes less basic . numbers of quantified guts from left to right are 90 , 90 , 90 . Error bars show SD of three independent experiments .

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A. Representative images and quantification of Drosophila midguts treated with the pH indicator Bromophenol Blue. Moreover, quantification of the three categories of Drosophila of indicated conditions. The three categories can be divided into: homeostasis, a well-defined acidic (yellow colored) copper cell region (CCR) is flanked by basic (blue colored) anterior midgut (AM) and posterior midgut (PM). "Perturbed A", the acidic region is lost and the whole gut is basic; "Perturbed B", the strongly acidic region is lost, and the remainder of the gut also becomes less basic. The numbers of quantified guts from left to right are 90, 90, and 90. Error bars show the SD of three independent experiments.


C . Excretion of Drosophila treated with Bromophenol Blue . of deposits of deposit numbers are shown . Excretions are quantified in 30 fields for each group of 12 Drosophila . were repeated as three independent experiments . Data information : statistical used in other panels were student ' s t - . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 , non - significant ( NS ) represents p > 0 . 05 .

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C. Excretion of Drosophila treated with Bromophenol Blue. Images of deposits and quantification of deposit numbers are shown. Excretions are quantified in 30 fields for each group of 12 Drosophila. Tests were repeated as three independent experiments. Data information: The statistical tests used in other panels were student's t-tests. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, and non-significant (NS) represents p > 0.05.


G . Survival ( ) of W1118 Drosophila with without supplementation of ALA as indicated . numbers of quantified flies : 100 ( W1118 + 0 . 5 mM ALA ) 100 ( W1118 + 0 mM ALA ) . Three independent experiments were conducted . Data information : Error bars represent SDs . p - values for lifespan curves were calculated by log - rank . statistical used in other panels were student ' s t - . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 , non - significant ( NS ) represents p > 0 . 05 .

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G. Survival (percentage) of female W1118 Drosophila with and without supplementation of ALA as indicated. The numbers of quantified flies: 100 (W1118 + 0.5 mM ALA) and 100 (W1118 + 0 mM ALA). Three independent experiments were conducted. Data information: Error bars represent SDs. p-values for lifespan curves were calculated by the log-rank test. The statistical tests used in other panels were student's t-tests. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, and non-significant (NS) represents p > 0.05.


I . Survival ( ) of Canton - S Drosophila with without supplementation of ALA as indicated . numbers of quantified flies : 100 ( Canton - S + 0 mM ALA ) , 100 ( Canton - S + 0 . 5 mM ALA ) . Three independent experiments were conducted . J . Survival ( ) of Canton - S Drosophila with without supplementation of ALA at 26 - day as indicated . numbers of quantified flies : 100 ( Canton - S + 0 mM ALA ) 100 ( Canton - S + 0 . 5 mM ALA ) . Three independent experiments were conducted . Data information : Error bars represent SDs . p - values for lifespan curves ( were calculated by log - rank . statistical used in other panels were student ' s t - . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 , non - significant ( NS ) represents p > 0 . 05 .

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I. Survival (percentage) of female Canton-S Drosophila with and without supplementation of ALA as indicated. The numbers of quantified flies: 100 (Canton-S + 0 mM ALA), 100 (Canton-S + 0.5 mM ALA). Three independent experiments were conducted. J. Survival (percentage) of female Canton-S Drosophila with and without supplementation of ALA at 26-day as indicated. The numbers of quantified flies: 100 (Canton-S + 0 mM ALA) and 100 (Canton-S + 0.5 mM ALA). Three independent experiments were conducted. Data information: Error bars represent SDs. p-values for lifespan curves ( were calculated by the log-rank test. The statistical tests used in other panels were student's t-tests. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, and non-significant (NS) represents p > 0.05.