C - E BR signaling is enhanced in 35S : BZR1 - MYC / 35S : BIC1 - YFP . Seedlings were grown for 6 days on medium supplemented with 1 μM brassinazole ( BRZ ) plus a gradient of concentrations of epibrassinolide ( eBL ) under long - day conditions . of representative seedlings when grown with 500 nM eBL ( + eBL ) or not ( - eBL ) are shown in ( C ) , hypocotyl lengths of indicated genotypes were measured are shown in ( D ) ( E ) . Data are means ± SD ( n > 20 ) . * * p < 0 . 01 , as determined by Student ' s t - . Scale bar , 2 mm .

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C-E BR signaling is enhanced in the 35S:BZR1-MYC/35S:BIC1-YFP plants. Seedlings were grown for 6 days on medium supplemented with 1 μM brassinazole (BRZ) plus a gradient of concentrations of epibrassinolide (eBL) under long-day conditions. Images of the representative seedlings when grown with 500 nM eBL (+eBL) or not (-eBL) are shown in (C), and the hypocotyl lengths of the indicated genotypes were measured and are shown in (D) and (E). Data are means ± SD (n>20). **p < 0.01, as determined by Student's t-test. Scale bar, 2 mm.


F , G BZR1 - mediated hypocotyl elongation phenotype is dependent on function of BIC1 BIC2 . Seedlings were grown for 5 days under long - day conditions . of representative seedlings are shown in ( F ) , hypocotyl lengths of indicated genotypes were measured are shown in ( G ) . Data are means ± SD ( n > 20 ) . * * p < 0 . 01 , n . s . indicates no significant difference , ( Student ' s t - ) . Scale bar , 2 mm .

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F, G BZR1-mediated hypocotyl elongation phenotype is dependent on the function of BIC1 and BIC2. Seedlings were grown for 5 days under long-day conditions. Images of the representative seedlings are shown in (F), and the hypocotyl lengths of the indicated genotypes were measured and are shown in (G). Data are means ± SD (n>20). **p < 0.01, n.s. indicates no significant difference, (Student's t-test). Scale bar, 2 mm.


H BZR1 - mediated hypocotyl elongation phenotype is dependent on function of BIC1 BIC2 . Col - 0 , 35S : BZR1 - MYC , bic1bic2 , 35S : BZR1 - MYC / bic1bic2 seedlings were grown on medium supplemented with 1 μM BRZ plus different concentrations of eBL for 6 days then hypocotyl lengths were measured . Data are means ± SD ( n > 20 ) .

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H BZR1-mediated hypocotyl elongation phenotype is dependent on the function of BIC1 and BIC2. The Col-0, 35S:BZR1-MYC, bic1bic2, 35S:BZR1-MYC/bic1bic2 seedlings were grown on medium supplemented with 1 μM BRZ plus different concentrations of eBL for 6 days and then the hypocotyl lengths were measured. Data are means ± SD (n>20).


A BR enhances stability of BIC1 . 5 - d - old 35S : BIC1 - YFP transgenic were treated with 100 μM cycloheximide ( CHX ) or co - treated with 100 μM CHX 1 μM epibrassinolide ( eBL ) . Samples were collected at indicated time points BIC1 - YFP were analyzed by western blots using anti - GFP antibody . Actin was used to verify equal loadings . of bands was measured using Adobe Photoshop CS3 Extended program . Three independent experiments were performed with similar results .

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A BR enhances the stability of BIC1 proteins. The 5-d-old 35S:BIC1-YFP transgenic plants were treated with 100 μM cycloheximide (CHX) or co-treated with 100 μM CHX and 1 μM epibrassinolide (eBL). Samples were collected at indicated time points and BIC1-YFP protein levels were analyzed by western blots using anti-GFP antibody. Actin was used to verify equal protein loadings. Intensity of the bands was measured using Adobe Photoshop CS3 Extended program. Three independent experiments were performed with similar results.


C Transactivation of BIC1 using a yeast assays . GAL4 - binding domain ( BD ) alone was used as negative control . SD - W , synthetic dextrose medium lacking Trp ; SD - WH , synthetic dextrose medium lacking both Trp His .

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C Transactivation analysis of BIC1 using a yeast assays. The GAL4 DNA-binding domain (BD) alone was used as the negative control. SD-W, synthetic dextrose medium lacking Trp; SD-WH, synthetic dextrose medium lacking both Trp and His.


E Transient transcriptional activity in Arabidopsis protoplasts illustrating transcriptional activation activity of BIC1 . relative luciferase activities were calculated by normalizing LUC values against REN . Error bars represent SD ( n = 3 ) . * * p < 0 . 01 , as determined by a Student ' s t - .

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E Transient transcriptional activity analysis in Arabidopsis protoplasts illustrating the transcriptional activation activity of BIC1. The relative luciferase activities were calculated by normalizing the LUC values against REN. Error bars represent SD (n = 3). **p < 0.01, as determined by a Student's t-test.


G Transient expression assays in Arabidopsis protoplasts showing that BIC1 BZR1 synergistically activate PRE5 promoter . ProPRE5 : LUC reporter was co - transformed with indicated effector constructs . LUC / REN represents ProPRE5 : LUC activity relative to internal control ( REN driven by 35S promoter ) . Error bars represent SD ( n = 3 ) .

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

G Transient expression assays in Arabidopsis protoplasts showing that BIC1 and BZR1 synergistically activate PRE5 promoter. The ProPRE5:LUC reporter was co-transformed with the indicated effector constructs. The LUC/REN ratio represents the ProPRE5:LUC activity relative to the internal control (REN driven by the 35S promoter). Error bars represent SD (n = 3).


H , I BIC1 - mediated hypocotyl elongation was reduced by addition of SRDX motif . Seedlings were grown for 5 days under long - day conditions . of representative seedlings are shown in ( H ) , hypocotyl lengths of indicated genotypes were measured are shown in ( I ) . # 1 , # 2 , # 3 represent different 35S : BIC1 - SRDX - Flag transgenic lines . Data are means ± SD ( n > 20 ) . * * p < 0 . 01 , as determined by Student ' s t - . Scale bar , 2 mm .

O O O B-GENEPROD O O B-TISSUE O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O B-ORGANISM O O O O O O O B-TISSUE B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

H, I BIC1-mediated hypocotyl elongation was reduced by the addition of SRDX motif. Seedlings were grown for 5 days under long-day conditions. Images of the representative seedlings are shown in (H), and the hypocotyl lengths of the indicated genotypes were measured and are shown in (I). #1, #2, #3 represent different 35S:BIC1-SRDX-Flag transgenic lines. Data are means ± SD (n>20). **p < 0.01, as determined by Student's t-test. Scale bar, 2 mm.


LCI assays showing that BIC1 interacts with PIF4 . Leaf epidermal of Nicotiana benthamiana were co - transformed with nLUC - BIC1 or nLUC cLUC - PIF4 or cLUC as indicated .

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LCI assays showing that BIC1 interacts with PIF4. Leaf epidermal cells of Nicotiana benthamiana were co-transformed with nLUC-BIC1 or nLUC and cLUC-PIF4 or cLUC as indicated.


B In vitro pull - down assays showing that BIC1 directly interacts with PIF4 . Purified GST - BIC1 were incubated with MBP or MBP - PIF4 for MBP pull - down . Arrowhead indicates specific bands .

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B In vitro pull-down assays showing that BIC1 directly interacts with PIF4. Purified GST-BIC1 proteins were incubated with MBP or MBP-PIF4 for the MBP pull-down assay. Arrowhead indicates specific bands.


C Co - IP assays showing interaction between BIC1 PIF4 in vivo . 35S : BIC1 - YFP , 35S : PIF4 - MYC 35S : PIF4 - MYC / 35S : BIC1 - YFP transgenic were grown for 6 days under long - day conditions . immunoprecipitates were detected using anti - GFP anti - MYC antibodies , respectively .

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C Co-IP assays showing the interaction between BIC1 and PIF4 in vivo. The 35S:BIC1-YFP, 35S:PIF4-MYC and 35S:PIF4-MYC/35S:BIC1-YFP transgenic plants were grown for 6 days under long-day conditions. The immunoprecipitates were detected using anti-GFP and anti-MYC antibodies, respectively.


D , E Phenotypic analyses showing that BIC1 PIF4 synergistically promote hypocotyl elongation . Col - 0 , 35S : BIC1 - YFP , 35S : PIF4 - MYC 35S : PIF4 - MYC / 35S : BIC1 - YFP were grown for 5 days under long - day conditions . of representative seedlings are shown in ( D ) , hypocotyl lengths of indicated genotypes were measured are shown in ( E ) . Data are means ± SD ( n > 20 ) . * * p < 0 . 01 , as determined by Student ' s t - . Scale bar , 2 mm .

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

D, E Phenotypic analyses showing that BIC1 and PIF4 synergistically promote hypocotyl elongation. The Col-0, 35S:BIC1-YFP, 35S:PIF4-MYC and 35S:PIF4-MYC/35S:BIC1-YFP plants were grown for 5 days under long-day conditions. Images of the representative seedlings are shown in (D), and the hypocotyl lengths of the indicated genotypes were measured and are shown in (E). Data are means ± SD (n>20). **p < 0.01, as determined by Student's t-test. Scale bar, 2 mm.


F RT - qPCR showing that BIC1 PIF4 synergistically activate PIF4 target genes . seedlings growth conditions were same as ( D ) . PP2A gene was used as an internal control . Data are means ± SD ( n = 3 ) .

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F RT-qPCR analysis showing that BIC1 and PIF4 synergistically activate PIF4 target genes. The seedlings growth conditions were same as (D). The PP2A gene was used as an internal control. Data are means ± SD (n=3).


G , H Phenotypic analyses showing that PIF4 - induced hypocotyl elongation is partially . dependent on function of BIC1 BIC2 . Col - 0 , bic1bic2 , PIF4 - MYC PIF4 - MYC / bic1bic2 were grown for 5 days under long - day conditions . of representative seedlings are shown in ( G ) hypocotyl lengths of indicated genotypes were measured are shown in ( H ) . Data are means ± SD ( n > 20 ) . * * p < 0 . 01 , as determined by Student ' s t - . Scale bar , 2 mm .

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G,H Phenotypic analyses showing that PIF4-induced hypocotyl elongation is partially. dependent on the function of BIC1 and BIC2. The Col-0, bic1bic2, PIF4-MYC and PIF4-MYC/bic1bic2 plants were grown for 5 days under long-day conditions. Images of the representative seedlings are shown in (G) and the hypocotyl lengths of indicated genotypes were measured and are shown in (H). Data are means ± SD (n>20). **p < 0.01, as determined by Student's t-test. Scale bar, 2 mm.


I RT - qPCR showing that PIF4 - induced expression of target genes is partially dependent function of BIC1 BIC2 . seedlings growth conditions were same as ( G ) . PP2A gene was analyzed as an internal control . Data are means ± SD ( n = 3 ) . * * p < 0 . 01 , as determined by Student ' s t - .

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I RT-qPCR analysis showing that PIF4-induced expression of target genes is partially dependent the function of BIC1 and BIC2. The seedlings growth conditions were same as (G). The PP2A gene was analyzed as an internal control. Data are means ± SD (n=3). **p < 0.01, as determined by Student's t-test.


J , K Phenotypic analyses showing that BIC1 - induced hypocotyl elongation is largely dependent on function of PIFs . Col - 0 , pifQ , 35S : BIC1 - Flag , 35S : BIC1 - flag / pifQ # 7 BIC1 - flag / pifQ # 8 were grown for 5 days under long - day conditions . of representative seedlings are shown in ( J ) hypocotyl lengths of indicated genotypes were measured are shown in ( K ) . Data are means ± SD ( n > 20 ) . * * p < 0 . 01 , as determined by Student ' s t - . Scale bar , 2 mm .

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J, K Phenotypic analyses showing that BIC1-induced hypocotyl elongation is largely dependent on the function of PIFs. The Col-0, pifQ, 35S:BIC1-Flag, 35S:BIC1-flag/pifQ #7 and BIC1-flag/pifQ #8 plants were grown for 5 days under long-day conditions. Images of the representative seedlings are shown in (J) and the hypocotyl lengths of indicated genotypes were measured and are shown in (K). Data are means ± SD (n>20). **p < 0.01, as determined by Student's t-test. Scale bar, 2 mm.


B ChIP - qPCR showing association of BIC1 with promoter regions of BZR1 / PIF4 target genes . 6 - d - old Col - 0 35S : BIC1 - Flag seedlings were used for ChIP assays .

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B ChIP-qPCR analysis showing the association of BIC1 with the promoter regions of BZR1/PIF4 target genes. The 6-d-old Col-0 and 35S:BIC1-Flag seedlings were used for ChIP assays.


C ChIP - qPCR showing that association of BIC1 with promoter regions of BZR1 / PIF4 target genes is dependent on PIFs . 6 - d - old 35S : BIC1 - Flag 35S : BIC1 - Flag / pifQ seedlings were used for ChIP assays .

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C ChIP-qPCR analysis showing that the association of BIC1 with the promoter regions of BZR1/PIF4 target genes is dependent on PIFs. The 6-d-old 35S:BIC1-Flag and 35S:BIC1-Flag/pifQ seedlings were used for ChIP assays.


D ChIP - qPCR showing that enrichment of BZR1 in promoter regions of its target genes is dependent on BICs . 10 - d - old 35S : BZR1 - MYC 35S : BZR1 - MYC / bic1bic2 seedlings were treated with 1 μM epibrassinolide ( eBL ) for 3 h then collected for ChIP assays .

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D ChIP-qPCR analysis showing that the enrichment of BZR1 in the promoter regions of its target genes is dependent on BICs. The 10-d-old 35S:BZR1-MYC and 35S:BZR1-MYC/bic1bic2 seedlings were treated with 1 μM epibrassinolide (eBL) for 3 h and then collected for ChIP assays.


E ChIP - qPCR showing that enrichment of PIF4 in promoter regions of its target genes is dependent on BICs . 6 - d - old 35S : PIF4 - MYC 35S : PIF4 - MYC / bic1bic2 seedlings were used for ChIP assays .

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E ChIP-qPCR analysis showing that the enrichment of PIF4 in the promoter regions of its target genes is dependent on BICs. The 6-d-old 35S:PIF4-MYC and 35S:PIF4-MYC/bic1bic2 seedlings were used for ChIP assays.


A RT - qPCR showing that BIC1 upregulates transcriptional expression of PIF4 . Col - 0 , 35S : BIC1 - YFP , 35S : BIC1 - Flag bic1bic2 were grown for 5 days under long - day conditions . PP2A gene served as internal control . Data are means ± SD ( n = 3 ) . Different letters indicate significant differences ( Fisher ' s LSD , P < 0 . 05 ) .

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A RT-qPCR analysis showing that BIC1 upregulates the transcriptional expression of PIF4. The Col-0, 35S:BIC1-YFP, 35S:BIC1-Flag and bic1bic2 plants were grown for 5 days under long-day conditions. The PP2A gene served as internal control. Data are means ± SD (n=3). Different letters indicate significant differences (Fisher's LSD, P< 0.05).


B Transcript of BIC1 PIF4 in transgenic containing a chemical - inducible construct pERGW - BIC1 . 8 - d - old pERGW - BIC1 seedlings were treated with 10 μM estradiol for indicated time points before harvest for extraction RT - qPCR . PP2A gene served as internal control . Data are means ± SD ( n = 3 ) .

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

B Transcript levels of BIC1 and PIF4 in transgenic plants containing a chemical-inducible construct pERGW-BIC1. 8-d-old pERGW-BIC1 seedlings were treated with 10 μM estradiol for indicated time points before harvest for RNA extraction and RT-qPCR analysis. The PP2A gene served as internal control. Data are means ± SD (n=3).


C ChIP - qPCR showing association of BIC1 with promoter regions of PIF4 . top panel shows diagram of promoter structure of PIF4 . Arrows indicate primer positions for ChIP - qPCR . G - box , CACGTG . 6 - d - old Col - 0 35S : BIC1 - Flag seedlings were used for ChIP assays . of each sample was immunoprecipitated ( IP ) using an anti - Flag or not ( Mock ) . Precipitated was quantified by qPCR , enrichment is displayed as between IP Mock , normalized to that of PP2A as an internal control . Data are means ± SD ( n = 3 ) . * p < 0 . 05 , as determined by Student ' s t - .

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C ChIP-qPCR analysis showing the association of BIC1 with the promoter regions of PIF4. The top panel shows the diagram of the promoter structure of PIF4. Arrows indicate the primer positions for ChIP-qPCR. G-box, CACGTG. The 6-d-old Col-0 and 35S:BIC1-Flag seedlings were used for ChIP assays. The chromatin of each sample was immunoprecipitated (IP) using an anti-Flag or not (Mock). Precipitated DNA was quantified by qPCR, and DNA enrichment is displayed as the ratio between IP and Mock, normalized to that of PP2A as an internal control. Data are means ± SD (n=3). *p < 0.05, as determined by Student's t-test.


D RT - qPCR showing transcript of endogenous PIF4 in 5 - d - old Col - 0 , 35S - PIF4 PIF4 - HA seedlings . PP2A gene served as internal control . Data are means ± SD ( n = 3 ) .

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D RT-qPCR analysis showing the transcript levels of endogenous PIF4 in the 5-d-old Col-0, 35S-PIF4 and PIF4-HA seedlings. The PP2A gene served as internal control. Data are means ± SD (n=3).


E Transient expression assays in Arabidopsis protoplasts showing that BIC1 BZR1 / PIF4 synergistically activate PIF4 promoter . ProPIF4 : LUC reporter was co - transformed with indicated effector constructs . LUC / REN ratios represent ProPIF4 : LUC activity relative to internal control ( REN driven by 35S promoter ) . Error bars represent SD ( n = 3 ) . * p < 0 . 05 , as determined by Student ' s t - .

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E Transient expression assays in Arabidopsis protoplasts showing that BIC1 and BZR1/PIF4 synergistically activate PIF4 promoter. The ProPIF4:LUC reporter was co-transformed with the indicated effector constructs. The LUC/REN ratios represent the ProPIF4:LUC activity relative to the internal control (REN driven by the 35S promoter). Error bars represent SD (n = 3). *p < 0.05, as determined by Student's t-test.


F LCI assays showing that TaBIC1 interacts with TaBZR1 . Leaf epidermal of Nicotiana benthamiana were co - transformed with nLUC - TaBIC1 or nLUC cLUC - TaBZR1 or cLUC as indicated .

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F LCI assays showing that TaBIC1 interacts with TaBZR1. Leaf epidermal cells of Nicotiana benthamiana were co-transformed with nLUC-TaBIC1 or nLUC and cLUC-TaBZR1 or cLUC as indicated.


G In vitro pull - down assays showing that TaBIC1 directly interacts with TaBZR1 . Purified GST - TaBIC1 were incubated with MBP or MBP - TaBZR1 for MBP pull - down . Arrowhead indicates specific band .

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G In vitro pull-down assays showing that TaBIC1 directly interacts with TaBZR1. Purified GST-TaBIC1 proteins were incubated with MBP or MBP-TaBZR1 for the MBP pull-down assay. Arrowhead indicates specific band.


H BiFC assays showing interaction of TaBIC1 TaBZR1 in nucleus . Leaf epidermal of Nicotiana benthamiana were co - transformed with nYFP - TaBIC1 cYFP - TaBZR1 . yellow dots represent interaction signals . BF , bright field . Scale bars , 20 μm .

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H BiFC assays showing the interaction of TaBIC1 and TaBZR1 in nucleus. Leaf epidermal cells of Nicotiana benthamiana were co-transformed with nYFP-TaBIC1 and cYFP-TaBZR1. The yellow dots represent interaction signals. BF, bright field. Scale bars, 20 μm.


( C ) Expression of GALNT isoforms in keratinocytes HaCaT . scatter plot depicts RPKM values of 2 biological replicates .

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(C) Expression of GALNT isoforms in primary keratinocytes and HaCaT cell line. The scatter plot depicts individual RPKM values of 2 biological replicates.


( D ) Expression of GalNAc - T1 , - T2 , - T3 in human skin ( upper panel ) HaCaT keratinocyte organoids ( lower panel ) . Frozen human skin or HaCaT keratinocyte organotypic skin models were stained using antibodies for GalNAc - T isoforms . Scale bar - 25 μm .

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(D) Expression of GalNAc-T1, -T2, and -T3 in human skin (upper panel) and HaCaT keratinocyte organoids (lower panel). Frozen human skin or HaCaT keratinocyte organotypic skin models were stained using antibodies for the GalNAc-T isoforms. Scale bar - 25 μm.


( E ) Phenotypic characterization of organotypic models made with HaCaT WT or GALNT1 / 2 / 3 KO keratinocytes . of tissue sections stained for differentiation marker keratin 10 ( upper panel ) or proliferation marker Ki67 ( lower panel ) . Scale bar - 50 μm . Red arrows - flattened ; red asterisks - K10 - negative region in suprabasal / granular layers ; purple asterisks - pyknotic nuclei ; green asterisks - increase in Ki67 positive .

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(E) Phenotypic characterization of organotypic models made with HaCaT WT or GALNT1/2/3 KO keratinocytes. IHC of tissue sections stained for differentiation marker keratin 10 (upper panel) or proliferation marker Ki67 (lower panel). Scale bar - 50 μm. Red arrows - flattened cells; red asterisks - K10-negative region in suprabasal/granular layers; purple asterisks - pyknotic nuclei; green asterisks - increase in Ki67 positive cells.


( F ) of epidermal thickness of skin organotypic models . Epidermal thickness was measured in 5 distinct ( 4 positions / image ) of 4 clones of GALNT isoform KO or WT ( 4 different tissues ) is presented as averages + SD . Due to high ZFN KO phenotypic inter - clonal variation to exclude off target effects , 5 clones of ZFN 3 clones of CRISPR KO ( each targeted by a different gRNA ) were used for GALNT3 KO . ANOVA followed by Dunnet ' s multiple comparison was used to compare mean epidermal thicknesses of different KOs to WT . * p ≤ 0 . 05 ; * * * * p ≤ 0 . 0001 .

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(F) Quantification of epidermal thickness of skin organotypic models. Epidermal thickness was measured in 5 distinct images (4 positions/image) of 4 clones of GALNT isoform KO or WT (4 different tissues) and is presented as averages +SD. Due to high ZFN KO phenotypic inter-clonal variation and to exclude off target effects, 5 clones of ZFN and 3 clones of CRISPR KO (each targeted by a different gRNA) were used for GALNT3 KO. ANOVA followed by Dunnet's multiple comparison test was used to compare mean epidermal thicknesses of different KOs to WT. * p ≤ 0.05; **** p ≤ 0.0001.


( G ) of keratin 10 - negative in skin organotypic tissue sections . Keratin 10 - negative was measured in 5 distinct of 4 clones of GALNT isoform KO or WT ( 4 different tissues ) is presented as averages + SD . Due to high ZFN KO phenotypic inter - clonal variation to exclude off target effects , 5 clones of ZFN 3 clones of CRISPR KO ( each targeted by a different gRNA ) were used for GALNT3 KO . ANOVA followed by Dunnet ' s multiple comparison was used to compare mean areas of different KOs to WT . * * * * p ≤ 0 . 0001 .

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(G) Quantification of keratin 10-negative area in skin organotypic tissue sections. Keratin 10-negative area was measured in 5 distinct images of 4 clones of GALNT isoform KO or WT (4 different tissues) and is presented as averages +SD. Due to high ZFN KO phenotypic inter-clonal variation and to exclude off target effects, 5 clones of ZFN and 3 clones of CRISPR KO (each targeted by a different gRNA) were used for GALNT3 KO. ANOVA followed by Dunnet's multiple comparison test was used to compare mean areas of different KOs to WT. **** p ≤ 0.0001.


( H ) Seq expression data of select differentiation markers , expressed in spinous granular layers of epidermis ( cartoon ) . Expression data is presented as log2 ( average KO / average WT ) . Significantly changed transcripts , identified in R , are marked with asterisks .

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(H) RNA Seq expression data of select differentiation markers, expressed in spinous and granular layers of the epidermis (cartoon). Expression data is presented as log2(average KO/average WT). Significantly changed transcripts, identified in R analysis, are marked with asterisks.


( A ) Numbers of quantified O - glycopeptides identified with ETD fragmentation with either dimethyl labeling or TMT labeling approach in COSMC KO or WT background , respectively . ( B ) Numbers of differentially glycosylated peptides in two approaches , using select filters on values .

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(A) Numbers of quantified O-glycopeptides identified with ETD fragmentation with either dimethyl labeling or TMT labeling approach in COSMC KO or WT background, respectively. (B) Numbers of differentially glycosylated peptides in the two approaches, using select filters on quantification values.


( C ) Numbers of unambiguous unique differential O - glycosites identified with two different approaches . stacked bar graph differentiates between glycosites identified on peptides with single multiple HexNAc / HexHexNAc modifications .

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(C) Numbers of unambiguous unique differential O-glycosites identified with the two different approaches. The stacked bar graph differentiates between glycosites identified on peptides with single and multiple HexNAc/HexHexNAc modifications.


( E ) of differential glycosites identified with TMT labeling approach using Qluqore Omics Explorer software . represents O - glycosites most significantly contributing to sample variation , using ANOVA with a q value cutoff of 0 . 1 , where each compared sample consists of three KO clones . Each horizontal depicts median of glycopeptides contributing to same glycosite . Alternative differentially glycosylated site positions are separated by semicolons on sites derived from glycopeptides with multiple HexNAc / HexHexNAc modifications .

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(E) Analysis of differential glycosites identified with the TMT labeling approach using the Qluqore Omics Explorer software. The heatmap represents O-glycosites most significantly contributing to sample variation, using ANOVA with a q value cutoff of 0.1, where each compared sample consists of three individual KO clones. Each horizontal line depicts the median quantification of glycopeptides contributing to the same glycosite. Alternative differentially glycosylated site positions are separated by semicolons on sites derived from glycopeptides with multiple HexNAc/HexHexNAc modifications.


GalNAc - T1 affects organization function of endomembrane system . HaCaT WT GALNT1 KO were stained for intracellular trafficking markers ( scale bar - 10 μm ) . ( A ) Clathrin ; ( B ) Rab5 ; ( C ) Rab11 .

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GalNAc-T1 affects the organization and function of the endomembrane system. HaCaT WT and GALNT1 KO cells were stained for intracellular trafficking markers (scale bar - 10 μm). (A) Clathrin; (B) Rab5; (C) Rab11.


GalNAc - T1 affects organization function of endomembrane system . ( D ) Steady state intracellular pH measurement in in HaCaT WT GALNT1 KO . Data is presented as average ± SD of 2 independent experiments . ANOVA followed by Dunnet ' s multiple comparison

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GalNAc-T1 affects the organization and function of the endomembrane system. (D) Steady state intracellular pH measurement in in HaCaT WT and GALNT1 KO cells. Data is presented as average ± SD of 2 independent experiments. ANOVA followed by Dunnet's multiple comparison test


( E ) GalNAc - T2 is important for to matrix adhesion . HaCaT WT GALNT2 KO were used in a to matrix adhesion using plates coated with different ECM components . Data is presented as average + SD of 3 independent experiments is normalized to WT . ANOVA followed by Dunnet ' s multiple comparison was used to compare mean relative adhesion of 2 different clones of GALNT2 KOs to WT . * p ≤ 0 . 05 ; * * p ≤ 0 . 01 .

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(E) GalNAc-T2 is important for cell to matrix adhesion. HaCaT WT and GALNT2 KO cells were used in a cell to matrix adhesion assay using plates coated with different ECM components. Data is presented as average + SD of 3 independent experiments and is normalized to WT. ANOVA followed by Dunnet's multiple comparison test was used to compare mean relative adhesion of 2 different clones of GALNT2 KOs to WT. * p ≤ 0.05; ** p ≤ 0.01.


( F ) Western blot for GalNAc - T2 specific target hemidesmosome component COL17A1 .

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(F) Western blot for GalNAc-T2 specific target and hemidesmosome component COL17A1.


( G ) of Western blot bands for COL17A1 ( full ) normalized to β actin .

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(G) Quantification of Western blot bands for COL17A1 (full length) normalized to β actin.


( I ) GalNAc - T2 may affect tight junction formation integrity . Upper panel , of WT GALNT2 KO organotypic tissue sections , where tight junctions are marked with asterisks . Lower panel , IF of WT GALNT2 KO for tight junction marker occludin ( scale bar - 50 μm ) . Inserts depict 3x zoomed in partial ( scale bar - 50 μm ) .

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(I) GalNAc-T2 may affect tight junction formation and integrity. Upper panel, TEM of WT and GALNT2 KO organotypic tissue sections, where tight junctions are marked with asterisks. Lower panel, IF staining of WT and GALNT2 KO for tight junction marker occludin (scale bar - 50 μm). Inserts depict 3x zoomed in partial images (scale bar - 50 μm).


( J ) Expression of cyclin A ( green ) in HaCaT WT GALNT3 KO organotypic tissue , visualized by IF ( scale bar - 20 μm ) .

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(J) Expression of cyclin A (green) in HaCaT WT and GALNT3 KO organotypic tissue, visualized by IF (scale bar - 20 μm).


( L - N ) Co - culture of HaCaT WT or GALNT3 KO keratinocytes with N / TERT1 keratinocytes induces differentiation . co - cultured on coverslips were stained for Notch 1 intracellular domain ( L ) , cytokeratin 10 ( M ) , or GalNAc - T3 ( N ; inserts depict 2x zoomed in partial ( scale bar - 20 μm ) ) . Green - relevant , red - N / TERT1 ( labeled with CMTMTR ) , blue - DAPI , scale bar - 20 μm . Boundaries between HaCaT N / TERT - 1 in green panel of ( L ) are marked with a dashed .

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(L-N) Co-culture of HaCaT WT or GALNT3 KO keratinocytes with N/TERT1 keratinocytes induces cellular differentiation. Cells co-cultured on coverslips were stained for cleaved Notch 1 intracellular domain (L), cytokeratin 10 (M), or GalNAc-T3 (N; inserts depict 2x zoomed in partial images (scale bar - 20 μm)). Green - relevant staining, red - N/TERT1 cells (labeled with CMTMTR), blue - DAPI, scale bar - 20 μm. Boundaries between HaCaT and N/TERT-1 cells in the green panel of (L) are marked with a dashed line.


A Representative of Bem1 - GFP PS marker LactC2 - GFP in wild type cho1 ∆ . show average projections of deconvolved z - stacks . Black pixels were inverted to improve contrast for display . B Frequency of displaying polarized Bem1 - GFP signal in wild type cho1 ∆ . Values are means ± SD , n > 100 in each of 3 independent experiments . Paired Student ' s t - were performed .

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A Representative images of Bem1-GFP and the PS marker LactC2-GFP in wild type and cho1∆ cells. Images show average intensity projections of deconvolved z-stacks. Black and white pixels were inverted to improve contrast for display. B Frequency of cells displaying polarized Bem1-GFP signal in wild type and cho1∆ cells. Values are means ± SD, n > 100 cells in each of 3 independent experiments. Paired Student's t-tests were performed.


C Imaging of PI4P probe GFP - 2xPHOsh2 in wild type cho1 ∆ . show average projections of deconvolved z - stacks in which black pixels were inverted .

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C Imaging of the PI4P probe GFP-2xPHOsh2 in wild type and cho1∆ cells. Images show average intensity projections of deconvolved z-stacks in which black and white pixels were inverted.


D Scatter dot plot showing PI4P at plasma ( see experimental procedure for details of ) in wild type cho1 ∆ . Values are means ± SD , n > 100 , observed over 3 experiments . Mann - Whitney were performed .

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D Scatter dot plot showing PI4P levels at the plasma membrane (see experimental procedure for details of the quantification) in wild type and cho1∆ cells. Values are means ± SD, n > 100 cells, observed over 3 experiments. Mann-Whitney tests were performed.


E of PI4P average in bud versus mother in cho1 ∆ mutant . Values are means ± SD , n > 100 , observed over 3 experiments . A value > 1 indicates enrichment of in bud .

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E Ratio of PI4P average fluorescence intensity in the bud versus mother cell in the cho1∆ mutant. Values are means ± SD, n > 100 cells, observed over 3 experiments. A value > 1 indicates enrichment of the fluorescence in the bud.


F of Bem1 - GFP in cho1 ∆ 9xMyc - AID - stt4 after 30 min treatment with or without 0 . 5 mM auxin . are average projections of deconvolved z - stacks .

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F Images of Bem1-GFP in cho1∆ 9xMyc-AID-stt4 cells after 30 min treatment with or without 0.5 mM auxin. Images are average intensity projections of deconvolved z-stacks.


G Frequency of with polarized Bem1 - GFP signal or Bem1 - GFP in in cho1 ∆ 9xMyc - AID - stt4 treated with or without auxin as shown in F . Values are means ± SD , n > 100 in each of 6 independent experiments . Paired Student ' s t - were performed .

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G Frequency of cells with polarized Bem1-GFP signal or Bem1-GFP in puncta in cho1∆ 9xMyc-AID-stt4 cells treated with or without auxin as shown in F. Values are means ± SD, n > 100 cells in each of 6 independent experiments. Paired Student's t-tests were performed.


H of Bem1 - GFP ( cyan ) Cdc24 - mCherry ( red ) signals in cho1 ∆ 9xMyc - AID - stt4 with or without auxin . display maximum projections of z - stacks .

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H Images of Bem1-GFP (cyan) and Cdc24-mCherry (red) signals in cho1∆ 9xMyc-AID-stt4 cells with or without auxin. Images display maximum intensity projections of z-stacks.


I Maximum projections of z - stacks displaying Bem1 - GFP in mutants indicated . Bem1 - GFP was imaged in mss4 - 102 cho1 ∆ after shifting to restrictive for 30 minutes .

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I Maximum intensity projections of z-stacks displaying Bem1-GFP in the mutants indicated. Bem1-GFP was imaged in mss4-102 cho1∆ cells after shifting to the restrictive temperature for 30 minutes.


J Frequency of with polarized Bem1 - GFP signal after incubation at time shown . Values are means ± SD , n > 200 in each of 3 independent experiments . Paired Student ' s t - were performed .

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J Frequency of cells with polarized Bem1-GFP signal after incubation at the time and temperature shown. Values are means ± SD, n > 200 cells in each of 3 independent experiments. Paired Student's t-tests were performed.


B Upper panel . SDS - PAGE stained with Coomassie blue in which Bem1 is indicated . Lower panel . liposomes were composed of 100 % phosphatidylcholine ( PC ) , 80 % PC 20 % phosphatidylethanolamine ( PE ) , 95 % PC 5 % phosphatidic acid ( PA ) , 95 % PC 5 % phosphoatidylinositol ( PI ) , 95 % PC 5 % PI ( 4 , 5 ) P2 ( PI ( 4 , 5 ) P2 ) , 95 % PC 5 % PI4P ( PI4P ) , 80 % PC 20 % phosphatidylserine ( PS ) or 75 % PC 20 % PS 5 % PI4P ( PS + PI4P ) . ( S ) supernatant , ( P ) pellet .

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B Upper panel. SDS-PAGE stained with Coomassie blue in which Bem1 is indicated. Lower panel. The liposomes were composed of 100% phosphatidylcholine (PC), 80% PC and 20% phosphatidylethanolamine (PE), 95% PC and 5% phosphatidic acid (PA), 95% PC and 5% phosphoatidylinositol (PI), 95% PC and 5% PI(4,5)P2 (PI(4,5)P2), 95% PC and 5% PI4P (PI4P), 80% PC and 20% phosphatidylserine (PS) or 75% PC 20% PS 5% PI4P (PS+PI4P). (S) supernatant, (P) pellet.


C Liposomes composed of either 100 % PC ± 40 % ergosterol or 75 % PC , 20 % PS , 5 % Bem1 ± 40 % ergosterol replacing PC were incubated with Bem1 treated as in panel ( B ) .

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C Liposomes composed of either 100% PC ± 40% ergosterol or 75% PC, 20% PS, 5% Bem1 ± 40% ergosterol replacing PC were incubated with Bem1 and treated as in panel (B).


E of indicated bem1 constructs associated with liposomes containing 75 % PC , 20 % PS 5 % PI4P .

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E Percentage of the indicated bem1 constructs associated with liposomes containing 75% PC, 20% PS and 5% PI4P.


F Lipid ordering determined by 2H solid - state NMR of liposomes containing POPC - d31 / POPS ( 4 : 1 molar ) in presence or absence of Bem1 BC motifs . Calculation of oriented - like spectra from Pake patterns ( de - Pake - ing ) simulation of 2H solid - state NMR spectra were applied to measure quadrupolar splittings for POPC - d31 determine order parameter accurately .

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F Lipid ordering determined by 2H solid-state NMR analysis of liposomes containing POPC-d31/POPS (4:1 molar ratio) in the presence or absence of Bem1 BC motifs. Calculation of oriented-like spectra from Pake patterns (de-Pake-ing) and simulation of 2H solid-state NMR spectra were applied to measure individual quadrupolar splittings for POPC-d31 and determine order parameter accurately.


Lipid ordering determined by 2H solid - state NMR of liposomes in presence or absence of Bem1 BC motifs . Calculation of oriented - like spectra from Pake patterns ( de - Pake - ing ) simulation of 2H solid - state NMR spectra were applied to measure quadrupolar splittings for POPC - d31 determine order parameter accurately . containing POPC - d31 / PI4P ( 19 : 1 molar ) .

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Lipid ordering determined by 2H solid-state NMR analysis of liposomes in the presence or absence of Bem1 BC motifs. Calculation of oriented-like spectra from Pake patterns (de-Pake-ing) and simulation of 2H solid-state NMR spectra were applied to measure individual quadrupolar splittings for POPC-d31 and determine order parameter accurately. containing POPC-d31/PI4P (19:1 molar ratio).


Lipid ordering determined by 2H solid - state NMR of liposomes in presence or absence of Bem1 BC motifs . Calculation of oriented - like spectra from Pake patterns ( de - Pake - ing ) simulation of 2H solid - state NMR spectra were applied to measure quadrupolar splittings for POPC - d31 determine order parameter accurately . containing POPC - d31 / POPC / PI4P ( 15 : 4 : 1 molar ) .

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Lipid ordering determined by 2H solid-state NMR analysis of liposomes in the presence or absence of Bem1 BC motifs. Calculation of oriented-like spectra from Pake patterns (de-Pake-ing) and simulation of 2H solid-state NMR spectra were applied to measure individual quadrupolar splittings for POPC-d31 and determine order parameter accurately. containing POPC-d31/POPC/PI4P (15:4:1 molar ratio).


Lipid ordering determined by 2H solid - state NMR of liposomes in presence or absence of Bem1 BC motifs . Calculation of oriented - like spectra from Pake patterns ( de - Pake - ing ) simulation of 2H solid - state NMR spectra were applied to measure quadrupolar splittings for POPC - d31 determine order parameter accurately . containing POPC - d31 / POPS / Yeast lipid extract / ergosterol ( 1 : 1 : 1 : 2 molar ) .

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Lipid ordering determined by 2H solid-state NMR analysis of liposomes in the presence or absence of Bem1 BC motifs. Calculation of oriented-like spectra from Pake patterns (de-Pake-ing) and simulation of 2H solid-state NMR spectra were applied to measure individual quadrupolar splittings for POPC-d31 and determine order parameter accurately. containing POPC-d31/POPS/Yeast lipid extract/ergosterol (1:1:1:2 molar ratio).


B of different full - bem1 bc mutants associated with liposomes containing 75 % PC 20 % PS 5 % PI4P .

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B Percentage of the different full-length bem1 bc mutants associated with liposomes containing 75% PC 20% PS and 5% PI4P.


C change associated with nucleotide exchange of GDP - Cdc42 for mant - GTP Cdc42 . was measured after addition of GDP - Cdc42 to reactions containing Mant - GTP ( 100 nM ) , GMP - PNP ( 100 µM ) indicated .

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C Fluorescence intensity change associated with the nucleotide exchange of GDP-Cdc42 for mant-GTP Cdc42. Fluorescence was measured after the addition of GDP-Cdc42 to reactions containing Mant-GTP (100 nM), GMP-PNP (100 µM) and the proteins indicated.


D Ten - fold serial dilutions of subsequent colony formation on indicated plates , where expression of wild type GALp - CDC42 is either induced in presence of Gal or repressed in Dex . Note how mutation of wild type Cdc42 ( KKSKK ) to MMSMM is lethal ( see blue box ) , whereas appending Bem1 BC - 1 motif to this cdc42 mutant restores viability ( red box ) .

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D Ten-fold serial dilutions of cells and subsequent colony formation on the indicated plates, where expression of wild type GALp-CDC42 is either induced in the presence of Gal or repressed in Dex. Note how mutation of the wild type Cdc42 (KKSKK) to MMSMM is lethal (see blue box), whereas appending the Bem1 BC-1 motif to this cdc42 mutant restores viability (red box).


E Representative of mEOS - cdc42 mutants signal ( cyan ) after inducing expression of GAL1p - CDC42 in presence of Gal or repressing it in presence of Dex . in blue red boxes correspond to in blue red box in panel ( D ) . are average projections in which black pixels have been inverted .

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E Representative images of the mEOS-cdc42 mutants signal (cyan) after inducing the expression of GAL1p-CDC42 in the presence of Gal or repressing it in the presence of Dex. The cells in the blue and red boxes correspond to the cells in the blue and red box in panel (D). Images are average fluorescence intensity projections in which black and white pixels have been inverted.


A Ten - fold serial dilutions of indicated mutant subsequent colony formation at temperatures indicated . Note how mutations on both BC motifs PX in bem1 compromise growth at restrictive .

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A Ten-fold serial dilutions of the indicated mutant cells and subsequent colony formation at the temperatures indicated. Note how mutations on both the BC motifs and PX in bem1 compromise growth at the restrictive temperature.


B Western blots probed with anti - HA antibody to detect indicated bem1 constructs tagged with 3XHA ( top panel ) . Detection of Ade13 was used as loading control ( bottom panel ) .

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B Western blots probed with anti-HA antibody to detect the indicated bem1 constructs tagged with 3XHA (top panel). Detection of Ade13 was used as loading control (bottom panel).


C Representative of indicated bem1 mutants tagged with GFP at BEM1 locus . inverted are average projections .

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C Representative images of the indicated bem1 mutants tagged with GFP at the BEM1 locus. The inverted fluorescence images are average intensity projections.


D Frequency of with polarized Bem1 - GFP . E Scatter dot plot showing of indicated bem1 strains at pole . measurements were made on average projections .

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D Frequency of cells with polarized Bem1-GFP. E Scatter dot plot showing the fluorescence levels of the indicated bem1 strains at the cell pole. The measurements were made on average intensity projections.


B of full - Cdc24 cdc24 ph domain mutant ( cdc24 ph ) associated with liposomes containing 75 % PC 20 % PS 5 % PI4P .

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B Percentage of full-length Cdc24 and cdc24 ph domain mutant (cdc24 ph) associated with liposomes containing 75% PC 20% PS and 5% PI4P.


C of Cdc24 cdc24 ph mutant associated with liposomes of composition shown in ( B ) in presence of indicated bem1 . Note how additive mutations in Bem1 lipid binding sequences reduce of Cdc24 associated with liposomes .

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C Percentage of Cdc24 and cdc24 ph mutant associated with liposomes of the composition shown in (B) in the presence of the indicated bem1 protein. Note how additive mutations in the Bem1 lipid binding sequences reduce the percentage of Cdc24 associated with the liposomes.


D of Cdc24 cdc24 ph associated with liposomes of composition shown in ( B ) as a function of indicated bem1 . curves denote regression fit of data to equation 2 in materials methods .

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D Percentage of Cdc24 and cdc24 ph associated with liposomes of the composition shown in (B) as a function of the indicated bem1 protein concentration. The curves denote the regression fit of the data to equation 2 in the materials and methods.


E Ten - fold serial dilutions subsequent colony formation of indicated mutant at indicated temperatures .

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E Ten-fold serial dilutions and subsequent colony formation of the indicated mutant cells at the indicated temperatures.


F DIC of indicated bem1 cdc24 mutants showing increased morphological defects ensuing from loss of lipid tethering in bem1 cdc24 mutants .

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F DIC images of the indicated bem1 and cdc24 mutants showing the increased morphological defects ensuing from loss of lipid tethering in the bem1 and cdc24 mutants.


G Western blots probed with anti - HA antibody to detect Cdc24 or cdc24 ph tagged with 3XHA in indicated mutant strains ( top panel ) . Detection of Ade13 was used as a loading control ( bottom panel ) .

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G Western blots probed with anti-HA antibody to detect Cdc24 or cdc24 ph tagged with 3XHA in the indicated mutant strains (top panel). Detection of Ade13 was used as a loading control (bottom panel).


B Global average MSD curves of mEOS - Cdc42 in strains indicated at Pole ( P ) Non - Pole ( NP ) of . Trajectories longer than 6 frames were analyzed . Number of trajectories analyzed : BEM1 ( n = 11 : Non Pole : 1814 tracks ; Pole : 706 ) ; bem1 bc - 14E px ( n = 10 : Non Pole : 1793 tracks ; Pole : 908 ) ; bem1 bc - 14E ( n = 13 : Non Pole : 1714 tracks ; Pole : 975 ) . Error bars display s . e . m . C D coefficients of mEOS - Cdc42 in strains indicated ( in box - plots displaying median ( ) , 25 - 75 percentiles ( box ) mean ( cross ) ) , which were compared using a non - parametric , two - Mann - Whitney rank sum .

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B Global average MSD curves of mEOS-Cdc42 in the strains indicated at the Pole (P) and Non-Pole (NP) of cells. Trajectories longer than 6 frames were analyzed. Number of trajectories analyzed: BEM1 (n = 11 cells: Non Pole: 1814 tracks; Pole: 706); bem1 bc-14E px (n = 10 cells: Non Pole: 1793 tracks; Pole: 908); bem1 bc-14E (n = 13 cells: Non Pole: 1714 tracks; Pole: 975). Error bars display s.e.m. C D coefficients of mEOS-Cdc42 in the strains indicated (in box-plots displaying the median (line), the 25-75 percentiles (box) and the mean (cross)), which were compared using a non-parametric, two-tailed Mann-Whitney rank sum test.


D SR - Tesseler of mEOS - Cdc42 nanocluster organization in BEM1 ( 5312 localizations shown in image ) bem1 bc - 14E px ( 3739 localizations shown ) . Insets show mEOS - Cdc42 after 491 nm widelfield excitation to identify pole . Scale bars : 2 μm . A zoom of pole region shows organization of detected nanoclusters , circled in blue , in strains indicated . Scale bar in zoom : 60 nm .

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D SR-Tesseler images of mEOS-Cdc42 nanocluster organization in BEM1 (5312 localizations shown in image) and bem1 bc-14E px cells (3739 localizations shown). Insets show mEOS-Cdc42 after 491 nm widelfield laser excitation to identify the cell pole. Scale bars: 2 μm. A zoom of the pole region shows the organization of the detected nanoclusters, circled in light blue, in the strains indicated. Scale bar in the zoom: 60 nm.


E Distribution of nanocluster at pole ( P ) non - pole ( NP ) regions of BEM1 ( diameter NP : 59 nm ± 1 nm ( s . e . m ) ; P : 74 nm ± 3 . 2 nm ) ( n = 16 . NP : 570 clusters ; P : 162 clusters ) bem1 bc - 14E px ( diameter NP : 59 nm ± 1 . 5 nm ( s . e . m ) ; P : 57 nm ± 2 . 4 nm ) ( n = 15 . NP : 421 clusters ; P : 141 clusters ) . Data are presented as scatter dot - plots displaying median as a 25 - 75 percentiles . Data were compared using non - parametric , two - Mann - Whitney rank sum .

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E Distribution of nanocluster area at the pole (P) and non-pole (NP) regions of BEM1 (diameter NP: 59 nm ± 1 nm (s.e.m); P: 74 nm ± 3.2 nm) (n = 16 cells. NP: 570 clusters; P: 162 clusters) and bem1 bc-14E px cells (diameter NP: 59 nm ± 1.5 nm (s.e.m); P: 57 nm ± 2.4 nm) (n = 15 cells. NP: 421 clusters; P: 141 clusters). Data are presented as scatter dot-plots displaying the median as a line and the 25-75 percentiles. Data were compared using non-parametric, two-tailed Mann-Whitney rank sum test.


F Active Cdc42 - GTP were quantified in strains indicated using a gic2 ( 1 - 208 ) - yeGFP probe . Values are mean ± SD for n = 30 observed over 2 experiments . Data were compared using a Mann - Whitney .

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F Active Cdc42-GTP levels were quantified in the strains indicated using a gic2(1-208)-yeGFP probe. Values are mean ± SD for n = 30 cells observed over 2 experiments. Data were compared using a Mann-Whitney test.


( c ) Substrate independent in vitro SUMOylation with SUMO2WT , SUMO2K11Ac or SUMO2K11Q was performed analyzed by immunoblotting with indicated antibodies .

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(c) Substrate independent in vitro SUMOylation assay with SUMO2WT, SUMO2K11Ac or SUMO2K11Q was performed and analyzed by immunoblotting with the indicated antibodies.


( d ) Substrate independent in vitro SUMOylation using either SUMO2WT or SUMO2K11Ac in presence of increasing amounts of ZNF451 - N . Reaction products were analyzed by immunoblotting with indicated antibodies . Note that - when compared to ( c ) assays were done at reduced concentrations of E1

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(d) Substrate independent in vitro SUMOylation assay using either SUMO2WT or SUMO2K11Ac in the presence of increasing amounts of ZNF451-N. Reaction products were analyzed by immunoblotting with the indicated antibodies. Note that - when compared to (c) assays were done at reduced concentrations of E1 and E2


( e ) In vitro SUMOylation using a fixed amount of SUMO2WT ( 220 ng ) with increasing concentrations of SUMO2K11Ac . Ratios ranged from 3 : 1 ( unmodified / K11Ac ) to 1 : 2 ( unmodified / K11Ac )

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(e) In vitro SUMOylation assay using a fixed amount of SUMO2WT (220 ng) with increasing concentrations of SUMO2K11Ac. Ratios ranged from 3:1 (unmodified/K11Ac) to 1:2 (unmodified/K11Ac)


( f ) In vitro SUMOylation , using SUMO2WT or SUMO2K11Ac , with p53 as a substrate . p53 was generated by in vitro translation / transcription in a rabbit reticulocyte lysate system . SUMO chain formation on p53 was analyzed by immunoblotting with α - p53 antibodies .

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(f) In vitro SUMOylation assay, using SUMO2WT or SUMO2K11Ac, with p53 as a substrate. p53 was generated by in vitro translation/transcription in a rabbit reticulocyte lysate system. SUMO chain formation on p53 was analyzed by immunoblotting with α-p53 antibodies.


( a ) In vitro deacetylation of recombinant SUMO2WT or SUMO2K11Ac ( 2 . 65 μM each ) with recombinant SIRT1 in presence or absence of NAD + .

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(a) In vitro deacetylation of recombinant SUMO2WT or SUMO2K11Ac (2.65 μM each) with recombinant SIRT1 in the presence or absence of NAD+.


( b ) In vitro deacetylation of recombinant SUMO2WT , SUMO2K11Ac , SUMO2K33Ac SUMO2K35Ac . Immunoblotting was performed with antibodies as indicated .

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(b) In vitro deacetylation of recombinant SUMO2WT, SUMO2K11Ac, SUMO2K33Ac and SUMO2K35Ac. Immunoblotting was performed with antibodies as indicated.


( c ) In vitro deacetylation of SUMO2K11Ac with increasing concentrations of SIRT1 or SIRT2 ( 0 . 74 µM , 1 . 5 μM , 3 µM ) , followed by immunoblotting with α - SUMO2K11Ac .

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(c) In vitro deacetylation of SUMO2K11Ac with increasing concentrations of SIRT1 or SIRT2 (0.74 µM, 1.5 μM, 3 µM), followed by immunoblotting with α-SUMO2K11Ac.


( d ) Combination of in vitro deacetylation substrate independent in vitro SUMOylation with SUMO2WT SUMO2K11Ac in presence or absence of SIRT1 ( 1 . 5 μM ) .

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(d) Combination of in vitro deacetylation and substrate independent in vitro SUMOylation with SUMO2WT and SUMO2K11Ac in the presence or absence of SIRT1 (1.5 μM).


( e ) In vitro deacetylation of SUMO2WT or SUMO2K11Ac with or without addition of SIRT1 , followed by substrate dependent in vitro SUMOylation of p53 .

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(e) In vitro deacetylation of SUMO2WT or SUMO2K11Ac with or without addition of SIRT1, followed by substrate dependent in vitro SUMOylation of p53.


( f ) Enrichment of acetylated after SIRT1 inhibition in HeLa . Western blot of SUMO2K11Ac after treatment of with SIRT1 inhibitior NAM ( 10 mM ) for 5h subsequent enrichment for acetylated via acetyl - lysine immunoprecipitation . As a control were either treated with control siRNA or siSUMO2 / 3 . samples were analyzed by immunoblotting with α - SUMO2 / 3 α - SUMO2K11Ac antibodies . α - SUMO2 / 3Ac α - SUMO2K11Ac reactive species migrating at ~ 200 kDa are marked with an asterisk .

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

(f) Enrichment of acetylated proteins after SIRT1 inhibition in HeLa cells. Western blot analysis of SUMO2K11Ac after treatment of cells with the SIRT1 inhibitior NAM (10 mM) for 5h and subsequent enrichment for acetylated proteins via acetyl-lysine immunoprecipitation. As a control cells were either treated with control siRNA or siSUMO2/3. The samples were analyzed by immunoblotting with α-SUMO2/3 and α-SUMO2K11Ac antibodies. The α-SUMO2/3Ac and α-SUMO2K11Ac reactive species migrating at ~200 kDa are marked with an asterisk.


( b ) Western blot with α - RGS - His α - HA was performed to monitor poly - SUMOylation of HA - PML . remaining samples were measured by LC - MS / MS final data was done with MaxQuant . A K11 SUMOylated SUMO2 peptide was identified is depicted with an Andromeda score of 49 . 3 . Intensities for this SUMO2 peptide for control ( gray ) arsenic ( purple ) treated samples are blotted as a bar graph . diagram shows a representative dataset derived from two replicates . corresponding peptide is displayed below graph . SUMOylation site is marked with SUMO2 ( green ) peptide fragment ions are indicated by y - ( red ) b - ( blue ) .

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

(b) Western blot analysis with α-RGS-His and α-HA was performed to monitor poly-SUMOylation of HA-PML. The remaining samples were measured by LC-MS/MS and final data analysis was done with MaxQuant. A K11 SUMOylated SUMO2 peptide was identified and is depicted with an Andromeda score of 49.3. Intensities for this SUMO2 peptide for the control (gray) and arsenic (purple) treated samples are blotted as a bar graph. The diagram shows a representative dataset derived from two replicates. The corresponding peptide is displayed below the graph. The SUMOylation site is marked with SUMO2 (green) and peptide fragment ions are indicated by y- (red) and b- (blue).


( c ) degradation of PML reactive band ( ~ 120 kDa ) was determined by immunoblotting . HeLa were either mock transfected or transfected with siRNA directed against SUMO1 / 2 / 3 . expression of RGS - His - tagged SUMO2WT or SUMO2K11Q was induced by DOX for 12 h . Prior to lysis , were treated with 1 μM As2O3 for 6 h . Western blot for wild - type SUMO2 SUMO2K11Q were blotted with α - RGS - His . Immunoblotting with α - Tubulin served as a loading control . Immunoblots for knockdown controls ( SUMO1 SUMO2 / 3 ) can be

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

(c) The degradation of the PML reactive band (~ 120 kDa) was determined by immunoblotting. HeLa cells were either mock transfected or transfected with siRNA directed against SUMO1/2/3. The expression of RGS-His-tagged SUMO2WT or SUMO2K11Q was induced by DOX for 12 h. Prior to lysis, cells were treated with 1 μM As2O3 for 6 h. Western blot analysis for wild-type SUMO2 and SUMO2K11Q were blotted with α-RGS-His. Immunoblotting with α-Tubulin served as a loading control. Immunoblots for knockdown controls (SUMO1 and SUMO2/3) can be found


( b ) Immunoblotting of samples with α - RGS - His ( SUMO variants ) α - PCNA as loading control .

O O O B-EXP_ASSAY O O O O O O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O O O O

(b) Immunoblotting of samples with α-RGS-His (SUMO variants) and α-PCNA as loading control.


( c ) Bar graph , comparing identified SUMO - SUMO2 linkages of control heat shock samples for SUMO2WT ( gray ) SUMO2K11Q ( purple ) . relative abundance ( y - axis ) of indicated SUMO - SUMO2 linkage sites ( x - axis ) are shown . measured for K11 in SUMO2WT under control conditions was set to 100 % relative abundance for residues was calculated in relation to this value . One of two independent replicates is shown .

O O O O O O O O B-GENEPROD O B-GENEPROD B-EXP_ASSAY O O O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD B-EXP_ASSAY O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(c) Bar graph, comparing the identified SUMO-SUMO2 linkages of control and heat shock samples for SUMO2WT (gray) and SUMO2K11Q (purple). The relative abundance (y-axis) of the indicated SUMO-SUMO2 linkage sites (x-axis) are shown. The measured intensity for K11 in the SUMO2WT under control conditions was set to 100% and the relative abundance for the individual residues was calculated in relation to this value. One of two independent replicates is shown.


( A ) Sirt5 deficiency leads to higher ROS in MEFs . ROS were determined in indicated MEFs as described in ' Materials Methods ' . results are average ± SD of 3 independent experiments * * p < 0 . 01 ( two - unpaired t - ) .

O O O B-GENEPROD O O O O B-SMALL_MOLECULE O B-CELL_LINE O B-SMALL_MOLECULE O O O O B-CELL_LINE O O O O O O O O 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) Sirt5 deficiency leads to higher ROS levels in MEFs. ROS levels were determined in the indicated MEFs as described in 'Materials and Methods'. The results are average ± SD of 3 independent experiments **p<0.01 (two-tailed unpaired t-test).


( B ) Sirt5 deficiency suppresses GSH production in MEFs . of [ GSH / GSSG ] was determined in extracts as described in ' Materials Methods ' . results are average ± SD of 3 independent experiments * p < 0 . 05 ( two - unpaired t - ) .

O O O B-GENEPROD O O B-SMALL_MOLECULE O O B-CELL_LINE O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(B) Sirt5 deficiency suppresses GSH production in MEFs. The ratio of [GSH/GSSG] was determined in cell extracts as described in 'Materials and Methods'. The results are average ± SD of 3 independent experiments *p<0.05 (two-tailed unpaired t-test).


( C ) Sirt5 deficiency inhibits NADPH production in MEFs . of [ NADPH / NADP + ] was determined in extracts as described in ' Materials Methods ' . results are average ± SD of 3 independent experiments * * * p < 0 . 001 ( two - unpaired t - ) .

O O O B-GENEPROD O O B-SMALL_MOLECULE O O B-CELL_LINE O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C) Sirt5 deficiency inhibits NADPH production in MEFs. The ratio of [NADPH/NADP+] was determined in cell extracts as described in 'Materials and Methods'. The results are average ± SD of 3 independent experiments ***p<0.001 (two-tailed unpaired t-test).


( D ) Sirt5 deficiency decreases growth . MEF were seeded in 6 - well plates at a density of 50 , 000 / well , growth rate was carefully monitored every 1 - 2 days by counting for 8 days . results are average ± SD of 3 independent experiments * p < 0 . 05 , * * * p < 0 . 001 ( two - unpaired t - ) .

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

(D) Sirt5 deficiency decreases cell growth. MEF cells were seeded in 6-well plates at a density of 50,000 cells/well, and cell growth rate was carefully monitored every 1-2 days by cell counting for 8 days. The results are average ± SD of 3 independent experiments *p<0.05, ***p<0.001 (two-tailed unpaired t-test).


( E ) Sirt5 deficiency leads to higher sensitivity of MEFs to Paraquat . of PARP Caspase - 3 were determined by western blot .

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

(E) Sirt5 deficiency leads to higher sensitivity of MEFs to Paraquat. The levels of cleaved PARP and Caspase-3 were determined by western blot analysis.


( F ) Sirt5 protects MEFs from ROS - induced . Wild - type Sirt5 KO MEFs were treated with Paraquat ( 500 μM ) for 24 hrs , then viability was determined by counting remaining adherent . results are average ± SD of 3 independent experiments * * p < 0 . 01 ( two - unpaired t - ) .

O O O B-GENEPROD O B-CELL_LINE O B-SMALL_MOLECULE O O O O O O B-GENEPROD O B-CELL_LINE O O O B-SMALL_MOLECULE O O O O O O O O O B-EXP_ASSAY O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O

(F) Sirt5 protects MEFs from ROS-induced cell death. Wild-type and Sirt5 KO MEFs were treated with Paraquat (500 μM) for 24 hrs, and then cell viability was determined by counting the remaining adherent cells. The results are average ± SD of 3 independent experiments **p<0.01 (two-tailed unpaired t-test).


( G - I ) Sirt5 deficiency inhibits production of NADPH GSH increases lipid peroxide in mouse brains after Paraquat injection . Sirt5 KO WT ( n = 3 ~ 5 per group ) were intraperitoneally injected with saline or paraquat ( 10 mg / kg ) once a week for 3 consecutive . ratios of [ NADPH / NADP + ] [ GSH / GSSG ] level of lipid peroxide were determined in tissue extracts . results are average ± SD . * p < 0 . 05 , n . s . : not significant ( two - unpaired t - ) .

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

(G-I) Sirt5 deficiency inhibits the production of NADPH and GSH and increases lipid peroxide in mouse brains after Paraquat injection. Female Sirt5 KO and WT littermates (n=3~5 per group) were intraperitoneally injected with saline or paraquat (10 mg/kg) once a week for 3 consecutive weeks. The ratios of [NADPH/NADP+] and [GSH/GSSG] and the level of lipid peroxide were determined in tissue extracts. The results are average ± SD. *p<0.05, n.s.: not significant (two-tailed unpaired t-test).


( J - K ) Sirt5 KO mice are more sensitive to Paraquat - induced nigrostriatal dopaminergic degeneration . Sirt5 KO WT were treated with paraquat as in ( G - I ) . brain sections were stained to detect tyrosine hydroxylase ( TH ) positive dopaminergic neurons in SNc region , as described in ' Materials Methods ' . Representative immunohistochemistry ( original magnification , 100 × ; scale bar , 100 μm ) corresponding quantifications are shown in ( J ) ( K ) , respectively . results are average ± SD . * p < 0 . 05 , n . s . : not significant ( two - unpaired t - ) .

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

(J-K) Sirt5 KO mice are more sensitive to Paraquat-induced nigrostriatal dopaminergic degeneration. Female Sirt5 KO and WT littermates were treated with paraquat as in (G-I). The brain sections were stained to detect tyrosine hydroxylase (TH) positive dopaminergic neurons in the SNc region, as described in 'Materials and Methods'. Representative immunohistochemistry images (original magnification, 100×; scale bar, 100 μm) and the corresponding quantifications are shown in (J) and (K), respectively. The results are average ± SD. *p<0.05, n.s.: not significant (two-tailed unpaired t-test).


( A ) Knockdown of SIRT5 inhibits NADPH production in HEK293T . of [ NADPH / NADP + ] was determined in extracts as described in ' Materials Methods ' . results are average ± SD of 3 independent experiments * * p < 0 . 01 , * * * p < 0 . 001 ( two - unpaired t - ) .

O O O O O B-GENEPROD O B-SMALL_MOLECULE O O B-CELL_LINE O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A)Knockdown of SIRT5 inhibits NADPH production in HEK293T cells. The ratio of [NADPH/NADP+] was determined in cell extracts as described in 'Materials and Methods'. The results are average ± SD of 3 independent experiments **p<0.01, ***p<0.001 (two-tailed unpaired t-test).


( B ) Knockdown of SIRT5 suppresses GSH production in HEK293T . of [ GSH / GSSG ] was determined in extracts as described in ' Materials Methods ' . results are average ± SD of 3 independent experiments * * p < 0 . 01 ( two - unpaired t - ) .

O O O O O B-GENEPROD O B-SMALL_MOLECULE O O B-CELL_LINE O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(B) Knockdown of SIRT5 suppresses GSH production in HEK293T cells. The ratio of [GSH/GSSG] was determined in cell extracts as described in 'Materials and Methods'. The results are average ± SD of 3 independent experiments **p<0.01 (two-tailed unpaired t-test).


( C ) Knockdown of SIRT5 leads to higher ROS in HEK293T . ROS were determined in indicated stable as described in ' Materials Methods ' . results are average ± SD of 3 independent experiments * * * p < 0 . 001 ( two - unpaired t - ) .

O O O O O B-GENEPROD O O O B-SMALL_MOLECULE O B-CELL_LINE O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C) Knockdown of SIRT5 leads to higher ROS levels in HEK293T cells. ROS levels were determined in the indicated stable cells as described in 'Materials and Methods'. The results are average ± SD of 3 independent experiments ***p<0.001 (two-tailed unpaired t-test).


( D ) Knockdown of SIRT5 leads to higher sensitivity to Paraquat . expression of PARP Caspase - 3 were determined by western blot .

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

(D) Knockdown of SIRT5 leads to higher sensitivity to Paraquat. The expression levels of PARP and Caspase-3 were determined by western blot analysis.


( E ) SIRT5 protects HEK293T from ROS - induced . indicated stable HEK293T were treated as mentioned above in ( D ) , then viability was determined by counting remaining adherent . results are average ± SD of 3 independent experiments * * p < 0 . 01 ( two - unpaired t - ) .

O O O B-GENEPROD O B-CELL_LINE O B-SMALL_MOLECULE O O O O O B-CELL_LINE O O O O O O O O O O O B-EXP_ASSAY O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O

(E) SIRT5 protects HEK293T cells from ROS-induced cell death. The indicated stable HEK293T cells were treated as mentioned above in (D), and then cell viability was determined by counting the remaining adherent cells. The results are average ± SD of 3 independent experiments **p<0.01 (two-tailed unpaired t-test).


( G ) activities of G6PD / 6PGD IDH1 / 2 are reduced in stable HEK293T with SIRT5 knockdown . Activities of indicated endogenous were determined in extracts as described in ' Materials Methods ' . results are average ± SD of 3 independent experiments * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , n . s . : not significant ( two - unpaired t - ) .

O O O O O B-GENEPROD O B-GENEPROD I-GENEPROD O O O O O O B-CELL_LINE O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) The activities of G6PD/6PGD and IDH1/2 are reduced in stable HEK293T cells with SIRT5 knockdown. Activities of the indicated endogenous proteins were determined in cell extracts as described in 'Materials and Methods'. The results are average ± SD of 3 independent experiments *p<0.05, **p<0.01, ***p<0.001, n.s.: not significant (two-tailed unpaired t-test).


( H ) G6PD IDH2 are activated by SIRT5 in vitro . Upon pre - incubation with SIRT5 in vitro , activities of indicated ectopically expressed were determined as described in ' Materials Methods ' . results are average ± SD of 3 independent experiments * * p < 0 . 01 , n . s . : not significant ( two - unpaired t - ) .

O O O B-GENEPROD I-GENEPROD O O O B-GENEPROD O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O 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) G6PD and IDH2 are activated by SIRT5 in vitro. Upon pre-incubation with SIRT5 in vitro, the activities of the indicated ectopically expressed proteins were determined as described in 'Materials and Methods'. The results are average ± SD of 3 independent experiments **p<0.01, n.s.: not significant (two-tailed unpaired t-test).


( A ) G6PD activity is not regulated by lysine succinylation . Purified Flag - tagged G6PD was incubated with or without succinyl - CoA ( 1 mM ) at 30 ° C for 15 mins , followed by measurement of G6PD enzyme activity as described in ' Materials Methods ' . results are average ± SD of 3 independent experiments . n . s . : not significant ( two - unpaired t - ) .

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

(A) G6PD activity is not regulated by lysine succinylation. Purified Flag-tagged G6PD was incubated with or without succinyl-CoA (1 mM) at 30°C for 15 mins, followed by measurement of G6PD enzyme activity as described in 'Materials and Methods'. The results are average ± SD of 3 independent experiments. n.s.: not significant (two-tailed unpaired t-test).


( B ) IDH2 activity is suppressed by lysine succinylation . Purified Flag - tagged IDH2 was incubated with or without succinyl - CoA ( 1 mM ) at 30 ° C for 15 mins , followed by measurement of IDH2 enzyme activity as described in ' Materials Methods ' . results are average ± SD of 3 independent experiments * p < 0 . 05 ( two - unpaired t - ) .

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

(B) IDH2 activity is suppressed by lysine succinylation. Purified Flag-tagged IDH2 was incubated with or without succinyl-CoA (1 mM) at 30°C for 15 mins, followed by measurement of IDH2 enzyme activity as described in 'Materials and Methods'. The results are average ± SD of 3 independent experiments *p<0.05 (two-tailed unpaired t-test).


( C ) SIRT5 knockdown increases G6PD glutarylation IDH2 succinylation , respectively , decreases both enzyme activity . Flag - G6PD or Flag - IDH2 was ectopically expressed in indicated stable HEK293T . G6PD / IDH2 were purified by immunoprecipitated with Flag beads eluted with Flag peptide , followed by determination of their lysine modifications enzyme activity . results are average ± SD of 3 independent experiments * * p < 0 . 01 , * * * p < 0 . 001 ( two - unpaired t - ) .

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

(C) SIRT5 knockdown increases G6PD glutarylation and IDH2 succinylation, respectively, and decreases both enzyme activity. Flag-G6PD or Flag-IDH2 was ectopically expressed in the indicated stable HEK293T cells. G6PD/IDH2 proteins were purified by immunoprecipitated with Flag beads and eluted with Flag peptide, followed by determination of their lysine modifications and enzyme activity. The results are average ± SD of 3 independent experiments **p<0.01, ***p<0.001 (two-tailed unpaired t-test).


( D ) SIRT5 decreases G6PD glutarylation IDH2 succinylation , increases both enzyme activity . Flag - G6PD or Flag - IDH2 was co - overexpressed with HA - SIRT5 in HEK293T . G6PD IDH2 were purified by immunoprecipitation with Flag beads eluted with Flag peptide , followed by determination of their lysine modifications enzyme activity . results are average ± SD of 3 independent experiments * p < 0 . 05 , * * p < 0 . 01 ( two - unpaired t - ) .

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

(D) SIRT5 decreases G6PD glutarylation and IDH2 succinylation, and increases both enzyme activity. Flag-G6PD or Flag-IDH2 was co-overexpressed with HA-SIRT5 in HEK293T cells. G6PD and IDH2 proteins were purified by immunoprecipitation with Flag beads and eluted with Flag peptide, followed by determination of their lysine modifications and enzyme activity. The results are average ± SD of 3 independent experiments *p<0.05, **p<0.01 (two-tailed unpaired t-test).


( E ) Cartoon representation of porcine mitochondrial IDH2 structure shows isocitrate binding pocket ( PDB ID : 1LWD ) [ 33 ] , made by Pymol ( www . pymol . org ) . is colored in green isocitrate in rainbow , with Lys 360 Lys 413 residues in IDH2 being colored in red .

O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O

(E) Cartoon representation of porcine mitochondrial IDH2 structure shows isocitrate binding pocket (PDB ID: 1LWD) [33], made by Pymol (www.pymol.org). The protein is colored in light green and isocitrate in rainbow, with Lys 360 and Lys 413 residues in IDH2 being colored in red.


( F - G ) K413 in IDH2 may be a key succinylation site that is directly targeted by SIRT5 . indicated Flag - tagged IDH2 were overexpressed in HEK293T with stable SIRT5 knockdown or SIRT5 co - overexpression . Wild - type mutant IDH2 were purified by Flag beads eluted with Flag peptide , followed by measurement of IDH2 enzyme activity . results are average ± SD of 3 independent experiments * p < 0 . 05 , n . s . : not significant ( two - unpaired t - ) .

O O O O O O O B-GENEPROD O O O O O O O O O O O B-GENEPROD O O B-GENEPROD O O B-GENEPROD O O O B-CELL_LINE O O B-GENEPROD O O B-GENEPROD O O O O O O O O B-GENEPROD O O O B-GENEPROD O O O B-GENEPROD O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(F-G) K413 in IDH2 may be a key succinylation site that is directly targeted by SIRT5. The indicated Flag-tagged IDH2 proteins were overexpressed in HEK293T cells with stable SIRT5 knockdown or SIRT5 co-overexpression. Wild-type and mutant IDH2 proteins were purified by Flag beads and eluted with Flag peptide, followed by measurement of IDH2 enzyme activity. The results are average ± SD of 3 independent experiments *p<0.05, n.s.: not significant (two-tailed unpaired t-test).


( A - B ) SIRT5 expression is not affected by chemical oxidant treatment . HEK293T were treated with Paraquat ( A ) or H2O2 ( B ) for indicated periods . mRNA expression of endogenous SIRT5 were determined by quantitative real - time PCR western blot , respectively . results are average ± SD of 3 independent experiments . n . s . : not significant ( two - unpaired t - ) .

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

(A-B) SIRT5 expression is not affected by chemical oxidant treatment. HEK293T cells were treated with Paraquat (A) or H2O2 (B) for the indicated periods. The mRNA and protein expression of endogenous SIRT5 were determined by quantitative real-time PCR and western blot analysis, respectively. The results are average ± SD of 3 independent experiments. n.s.: not significant (two-tailed unpaired t-test).


( D ) desuccinylase activity of SIRT5 is stimulated by oxidative stimuli . Flag - SIRT5 was ectopically expressed in HEK293T . transfected were treated with increased concentrations of Paraquat or H2O2 for indicated periods . Flag - SIRT5 was purified by immunoprecipitation with Flag beads , eluted with Flag peptide , then subjected to desuccinylase activity as described in ' Materials Methods ' . SIRT5 desuccinylase activity was normalized against its level . results are average ± SD of 3 independent experiments * p < 0 . 05 , * * * p < 0 . 001 ( two - unpaired t - ) .

O O O O O O B-GENEPROD O O O O O O B-GENEPROD O B-GENEPROD O O O O B-CELL_LINE O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O B-GENEPROD O B-GENEPROD O O O B-EXP_ASSAY O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O 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) The desuccinylase activity of SIRT5 is stimulated by oxidative stimuli. Flag-SIRT5 was ectopically expressed in HEK293T cells. The transfected cells were treated with increased concentrations of Paraquat or H2O2 for the indicated periods. Flag-SIRT5 was purified by immunoprecipitation with Flag beads, eluted with Flag peptide, and then subjected to the desuccinylase activity assay as described in 'Materials and Methods'. The SIRT5 desuccinylase activity was normalized against its protein level. The results are average ± SD of 3 independent experiments *p<0.05, ***p<0.001 (two-tailed unpaired t-test).


( E ) In vitro incubation with chemical oxidants does not affect desuccinylase activity of SIRT5 . Flag - tagged SIRT5 was ectopically expressed in HEK293T , then purified by immunoprecipitation with Flag - beads eluted with Flag peptide . purified Flag - SIRT5 was treated with increased concentrations of Paraquat or H2O2 at room for 1 hr , then subjected to desuccinylase activity as described in ' Materials Methods ' . SIRT5 desuccinylase activity was normalized against its level . results are average ± SD of 3 independent experiments . n . s . : not significant ( two - unpaired t - ) .

O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O O O B-CELL_LINE O O O O B-EXP_ASSAY O B-GENEPROD O O O O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(E) In vitro incubation with chemical oxidants does not affect the desuccinylase activity of SIRT5. Flag-tagged SIRT5 was ectopically expressed in HEK293T cells, and then purified by immunoprecipitation with Flag-beads and eluted with Flag peptide. The purified Flag-SIRT5 was treated with increased concentrations of Paraquat or H2O2 at room temperature for 1 hr, and then subjected to the desuccinylase activity assay as described in 'Materials and Methods'. The SIRT5 desuccinylase activity was normalized against its protein level. The results are average ± SD of 3 independent experiments. n.s.: not significant (two-tailed unpaired t-test).


( F ) Chemical oxidants enhance ability of SIRT5 to activate IDH2 . Flag - SIRT5 was ectopically expressed in HEK293T , transfected were treated with increased concentrations of Paraquat or H2O2 for indicated periods . Flag - SIRT5 was purified by immunoprecipitation with Flag beads , eluted with Flag peptide , then incubated with purified Flag - IDH2 in vitro . IDH2 enzyme activity was measured as described in ' Materials Methods ' , normalized by its level . results are average ± SD of 3 independent experiments * * p < 0 . 01 , * * * p < 0 . 001 ( two - unpaired t - ) .

O O O O O O O O B-GENEPROD O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O B-CELL_LINE O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O B-GENEPROD O B-GENEPROD O O O B-EXP_ASSAY O B-GENEPROD O O O O B-GENEPROD O O O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(F) Chemical oxidants enhance the ability of SIRT5 to activate IDH2. Flag-SIRT5 was ectopically expressed in HEK293T cells, and the transfected cells were treated with increased concentrations of Paraquat or H2O2 for the indicated periods. Flag-SIRT5 was purified by immunoprecipitation with Flag beads, eluted with Flag peptide, and then incubated with purified Flag-IDH2 in vitro. IDH2 enzyme activity was measured as described in 'Materials and Methods', normalized by its protein level. The results are average ± SD of 3 independent experiments **p<0.01, ***p<0.001 (two-tailed unpaired t-test).


( A ) Quantitative of Aβ1 - 40 in TBS ( * p = 0 . 0212 ) , Triton - X ( p = 0 . 0544 ) SDS ( * * p = 0 . 0063 ) fractions of brain homogenates from ( n = 7 ) ( n = 10 ) APP23 mice . Mean ± s . e . m . , statistical : two - unpaired t - .

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

(A) Quantitative analysis of the Aβ1-40 protein in the TBS (*p=0.0212), Triton-X (p=0.0544) and SDS (**p=0.0063) fractions of brain homogenates from male (n=7) and female (n=10) APP23 mice. Mean ± s.e.m., statistical analysis: two-tailed unpaired t-test.