F Collective assessment of USP9X XIAP expression determined by immunohistochemistry in tissue microarrays ( TMA ) derived from a cohort representing 121 evaluable human CD20 + aggressive B - lymphomas . High USP9X expression was identified in 37 cases ( 31 % ) significantly correlated with high XIAP expression ( * * , P = 0 . 004 ; Student ' s t ) .

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F Collective assessment of USP9X and XIAP expression determined by immunohistochemistry in tissue microarrays (TMA) derived from a patient cohort representing 121 evaluable human CD20+ aggressive B-cell lymphomas. High USP9X expression was identified in 37 cases (31 %) and significantly correlated with high XIAP expression levels (**, P = 0.004; Student's t test).


G Expression data derived from ( E ) was correlated with clinical follow - up . Within a cohort of 58 receiving vincristine - containing chemotherapy ( CHOP regimen : cyclophosphamide , doxorubicin , vincristine , prednisone ) without addition of anti - CD20 antibody rituximab ( left panel ) overexpression of USP9X XIAP ( n = 14 , straight ) was associated with a significantly shortened event - free survival ( EFS ) as compared to remaining ( n = 44 , dotted ) ( * , P = 0 . 050 ; log rank ) . Within a cohort of 63 receiving CHOP regimen with addition of anti - CD20 antibody rituximab overexpression of USP9X XIAP ( n = 14 , straight ) was not associated with a shortened event - free survival ( EFS ) as compared to remaining ( n = 49 , dotted ) ( P = 0 . 454 ) .

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G Expression data derived from (E) was correlated with clinical follow-up. Within a cohort of 58 patients receiving vincristine-containing chemotherapy (CHOP regimen: cyclophosphamide, doxorubicin, vincristine, prednisone) without the addition of the anti-CD20 antibody rituximab (left panel) overexpression of USP9X and XIAP (n = 14, straight line) was associated with a significantly shortened event-free survival (EFS) as compared to all remaining patients (n = 44, dotted line) (*, P = 0.050; log rank test). Within a cohort of 63 patients receiving the CHOP regimen with addition of the anti-CD20 antibody rituximab overexpression of USP9X and XIAP (n = 14, straight line) was not associated with a shortened event-free survival (EFS) as compared to all remaining patients (n = 49, dotted line) (P = 0.454).


molecular detection of nCoV - 2019 in seven during two times of sampling . information can be found in Extended Data Table 1 2 . Details on detection method can be found in material methods . BALF , bronchoalveolar lavage fluid ; OS , oral swab ; AS , anal swab .

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molecular detection of nCoV-2019 in seven patients during two times of sampling. Patient information can be found in Extended Data Table 1 and 2. Details on detection method can be found in material and methods. BALF, bronchoalveolar lavage fluid; OS, oral swab; AS, anal swab.


dynamics of nCoV - 2019 antibodies in one who showed sign of disease on 2019 . 12 . 23 ( ICU - 06 ) . For b c , cut - off was set up as 0 . 2 for IgM 0 . 3 for IgG , according to healthy controls .

dynamics of nCoV-2019 antibodies in one patient who showed sign of disease on 2019.12.23 (ICU-06). For b and c, cut-off was set up as 0.2 for IgM test and 0.3 for IgG test, according to healthy controls.


serological of nCoV - 2019 antibodies in five ( more information can be found in Extended Data Table 2 ) . Star indicates data collected from ICU - 06 on 2020 . 01 . 10 . For b c , cut - off was set up as 0 . 2 for IgM 0 . 3 for IgG , according to healthy controls .

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serological test of nCoV-2019 antibodies in five patients (more information can be found in Extended Data Table 2). Star indicates data collected from patient ICU-06 on 2020.01.10. For b and c, cut-off was set up as 0.2 for IgM test and 0.3 for IgG test, according to healthy controls.


viral particles in ultrathin sections under electron microscope at 200 kV , sample from viral infected Vero E6

viral particles in the ultrathin sections under electron microscope at 200 kV, sample from viral infected Vero E6 cells


usage . Determination of infectivity in HeLa with or without expression of ACE2 . h , human ; b , Rhinolophus sinicus bat ; c , civet ; s , swine ( pig ) ; m , mouse . ACE2 ( green ) , viral ( red ) nuclei ( blue ) was shown . Scale bar = 10 um .

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usage.Determination of virus infectivity in HeLa cells with or without the expression of ACE2. h, human; b, Rhinolophus sinicus bat; c, civet; s, swine (pig); m, mouse. ACE2 protein (green), viral protein (red) and nuclei (blue) was shown. Scale bar=10 um.


A . RT - PCR of expression of β - actin , Trpv1 , Trpv2 , Trpv3 Trpv4 using mouse differentiated brown adipocytes after 29 ( upper ) 35 ( lower ) thermal cycles with ( RT ( + ) ) without ( RT ( - ) ) reverse transcription ( RT ) . Control ( Ct . ) lanes indicate results with each plasmid as a template .

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A. RT-PCR analysis of the expression of β-actin, Trpv1, Trpv2, Trpv3 and Trpv4 using mouse differentiated brown adipocytes after 29 (upper) and 35 (lower) thermal cycles with (RT (+)) and without (RT (-)) reverse transcription (RT). Control (Ct.) lanes indicate the results with each plasmid DNA as a template.


B . Results of real - time RT - PCR of Trpv1 , Trpv2 , Trpv3 Trpv4 expression using mouse differentiated brown adipocytes . Expression of mRNA were normalized to that of ribosomal gene ( 36B4 ) , a housekeeping gene un - affected by adipogenesis . Data are presented as mean ± , n = 6 .

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B. Results of real-time RT-PCR analysis of Trpv1, Trpv2, Trpv3 and Trpv4 expression using mouse differentiated brown adipocytes. Expression levels of mRNA were normalized to that of the ribosomal protein gene (36B4), a housekeeping gene un-affected by adipogenesis. Data are presented as mean ± SEM, n = 6.


C . Results of real - time PCR of Trpv1 , Trpv2 , Trpv3 Trpv4 expression using mouse interscapular brown adipose tissue ( iBAT ) . mRNA expression were normalized to that of 36B4 . Data are presented as mean ± , n = 5 .

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C. Results of real-time PCR analysis of Trpv1, Trpv2, Trpv3 and Trpv4 expression using mouse interscapular brown adipose tissue (iBAT). mRNA expression levels were normalized to that of 36B4. Data are presented as mean ± SEM, n = 5.


D . Western blot results of TRPV2 tubulin from WT TRPV2KO brown adipocytes . Upper bands in TRPV2 blots likely indicate glycosylated forms .

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D. Western blot results of TRPV2 and tubulin from WT and TRPV2KO brown adipocytes. Upper bands in the TRPV2 blots likely indicate glycosylated forms.


E . Averaged traces of [ Ca2 + ] i changes in response to 500 µmol / L 2APB in differentiated brown adipocytes from WT ( black ) TRPV2KO ( red ) mice . One µmol / L NE was used to confirm differentiation . Five µmol / L ionomycin was used to confirm viability . values correspond to real [ Ca2 + ] i of differentiated mousebrown adipocytes . Data are presented as mean ± , n = 149 of WT , n = 112 of TRPV2KO brown adipocytes ; * * P < 0 . 01 . Unpaired Student ' s t - .

O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O 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 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. Averaged traces of [Ca2+]i changes in response to 500 µmol/L 2APB in differentiated brown adipocytes from WT (black) and TRPV2KO (red) mice. One µmol/L NE was used to confirm differentiation. Five µmol/L ionomycin was used to confirm cell viability. Ratio values correspond to the real [Ca2+]i of differentiated mousebrown adipocytes. Data are presented as mean ± SEM, n = 149 of WT cells, and n = 112 of TRPV2KO brown adipocytes; ** P < 0.01. Unpaired Student's t-test.


F . A representative trace of whole - current activated by 3 mmol / L of 2APB in presence or absence of 10 µmol / L SKF in mouse differentiated brown adipocyte . left inset indicates a voltage ramp - protocol . right inset indicates current - voltage curves of basal current ( a , black ) , current in presence of 2APB + SKF ( b , blue ) current in presence of 2APB alone ( c , red ) at time points of a , b c , respectively . G . Comparison of mean densities of basal currents ( Base , black ) , currents in presence of 2APB alone ( red ) currents in presence of 2APB + SKF ( blue ) at - 60 mV + 100 mV in mouse differentiated brown adipocytes . Data are presented as mean ± , n = 10 ; * * P < 0 . 01 vs . Base ; # # P < 0 . 01 vs . 2APB alone . One - way ANOVA followed by 2 - t - with Bonferroni correction .

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F. A representative trace of whole-cell current activated by 3 mmol/L of 2APB in the presence or absence of 10 µmol/L SKF in mouse differentiated brown adipocyte. The left inset indicates a voltage ramp-pulse protocol. The right inset indicates the current-voltage curves of basal current (a, black), current in the presence of 2APB+SKF (b, blue) and current in the presence of 2APB alone (c, red) at the time points of a, b and c, respectively.G. Comparison of the mean densities of basal currents (Base, black), currents in the presence of 2APB alone (red) and currents in the presence of 2APB+SKF (blue) at -60 mV and +100 mV in mouse differentiated brown adipocytes. Data are presented as mean ± SEM, n = 10; ** P < 0.01 vs. Base; ##P < 0.01 vs. 2APB alone. One-way ANOVA followed by 2-tailed t-test with Bonferroni correction.


A . Results of real - time RT - PCR of Trpv1 , Trpv2 , Trpv3 Trpv4 expression in mouse pre - adipocytes differentiated brown adipocytes . Expression of mRNA were normalized to those of 36B4 . Data are presented as mean ± , n = 6 . * P < 0 . 05 vs . pre - adipocytes . Unpaired Student ' s t - .

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A. Results of real-time RT-PCR analysis of Trpv1, Trpv2, Trpv3 and Trpv4 expression in mouse pre-adipocytes and differentiated brown adipocytes. Expression levels of mRNA were normalized to those of 36B4. Data are presented as mean ± SEM, n = 6. * P < 0.05 vs. pre-adipocytes. Unpaired Student's t-test.


B . number of 6 day - differentiated mousebrown adipocytes treated with TRPV2 agonists , 100 µmol / L 2APB or 10 µmol / L LPC . Mean ± , n = 6 , * P < 0 . 05 * * P < 0 . 01 vs . dimethyl sulfoxide ( DMSO ) group . One - way ANOVA followed by 2 - t - with Bonferroni correction .

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B. The number of 6 day-differentiated mousebrown adipocytes treated with TRPV2 agonists, 100 µmol/L 2APB or 10 µmol/L LPC. Mean ± SEM, n = 6, * P < 0.05 and ** P < 0.01 vs. dimethyl sulfoxide (DMSO) group. One-way ANOVA followed by 2-tailed t-test with Bonferroni correction.


C D . Comparison of numbers of 6 day - differentiated mouse brown adipocytes ( C ) triglyceride ( D ) in from WT TRPV2KO mice with different differentiation media . 1 / 10 suppl . indicates differentiation medium ten - time - diluted with DMEM . Mean ± , n = 8 , * * P < 0 . 01 vs . control group , # # P < 0 . 01 vs . WT group . One - way ANOVA followed by 2 - t - with Bonferroni correction .

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C and D. Comparison of the numbers of 6 day-differentiated mouse brown adipocytes (C) and triglyceride levels (D) in the cells from WT and TRPV2KO mice with different differentiation media. 1/10 suppl. indicates differentiation medium ten-time-diluted with DMEM. Mean ± SEM, n = 8, ** P < 0.01 vs. control group, ##P < 0.01 vs. WT group. One-way ANOVA followed by 2-tailed t-test with Bonferroni correction.


A . Basal mRNA expression of Ucp1 , peroxisome proliferator - activated receptor gamma coactivator 1 - alpha ( Pgc1α ) , peroxisome proliferator - activated receptorγ ( Pparγ ) β3 - adrenergic receptor 3 ( Adrb3 ) in differentiated brown adipocytes from WT TRPV2KO mice . Data are presented as mean ± , n = 5 ; * * P < 0 . 01 vs . WT . Unpaired Student ' s t - .

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A. Basal mRNA expression of Ucp1, peroxisome proliferator-activated receptor gamma coactivator 1-alpha (Pgc1α), peroxisome proliferator-activated receptorγ(Pparγ) and β3-adrenergic receptor 3 (Adrb3) in the differentiated brown adipocytes from WT and TRPV2KO mice. Data are presented as mean ± SEM, n = 5; ** P < 0.01 vs. WT. Unpaired Student's t-test.


B C . Changes in Ucp1 ( B ) Pgc1α ( C ) mRNA expression in differentiated brown adipocytes from WT TRPV2KO mice with without 10 µmol / L isoproterenol ( ISO ) for 4 h . Data are presented as mean ± , n = 5 ; * * P < 0 . 01 vs . DMSO group ; # P < 0 . 05 ; # # P < 0 . 01 vs . WT group . One - way ANOVA followed by 2 - t - with Bonferroni correction .

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B and C. Changes in Ucp1 (B) and Pgc1α(C) mRNA expression in the differentiated brown adipocytes from WT and TRPV2KO mice with and without 10 µmol/L isoproterenol (ISO) for 4 h. Data are presented as mean ± SEM, n = 5; ** P < 0.01 vs. DMSO group; # P < 0.05; ## P < 0.01 vs. WT group. One-way ANOVA followed by 2-tailed t-test with Bonferroni correction.


D E . Changes in Ucp1 mRNA expression ( D ) non - esterified fatty acid ( NEFA ) release ( E ) in differentiated brown adipocytes from WT TRPV2KO mice with or without 10 µmol / L forskolin for 4 h . Data are presented as mean ± , n = 6 ; * P < 0 . 05 ; * * P < 0 . 01 vs . DMSO group ; # # P < 0 . 01 vs . WT group . One - way ANOVA followed by 2 - t - with Bonferroni correction .

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D and E. Changes in Ucp1 mRNA expression (D) and non-esterified fatty acid (NEFA) release (E) in the differentiated brown adipocytes from WT and TRPV2KO mice with or without 10 µmol/L forskolin for 4 h. Data are presented as mean ± SEM, n = 6; * P < 0.05; ** P < 0.01 vs. DMSO group; ## P < 0.01 vs. WT group. One-way ANOVA followed by 2-tailed t-test with Bonferroni correction.


F G . Changes in Ucp1 mRNA ( F ) Pgc1α mRNA ( G ) in differentiated brown adipocytes treated with 10 µmol / L ISO alone or 10 µmol / L ISO plus 10 µmol / L BAPTA - AM for 4 h . Data are presented as mean ± , n = 6 ; * P < 0 . 05 ; * * P < 0 . 01 vs . DMSO group ; # # P < 0 . 01 vs . ISO group . One - way ANOVA followed by 2 - t - with Bonferroni correction .

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F and G. Changes in Ucp1 mRNA (F) and Pgc1α mRNA (G) in the differentiated brown adipocytes treated with 10 µmol/L ISO alone or 10 µmol/L ISO plus 10 µmol/L BAPTA-AM for 4 h. Data are presented as mean ± SEM, n = 6; * P < 0.05; ** P < 0.01 vs. DMSO group; ## P < 0.01 vs. ISO group. One-way ANOVA followed by 2-tailed t-test with Bonferroni correction.


A . changes from 3 - to 8 - of between two genotypes ( n = 11 ) . Data are presented as mean ± ; * P < 0 . 05 ; * * P < 0 . 01 vs . WT group . Unpaired Student ' s t - .

A. Body weight changes from 3-weeks to 8-weeks of age between the two genotypes (n = 11). Data are presented as mean ± SEM; * P < 0.05; ** P < 0.01 vs. WT group. Unpaired Student's t-test.


B C . Changes in food intake ( B ) water intake ( C ) from 3 - to 8 - of ages between WT TRPV2KO mice ( n = 6 ) .

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B and C. Changes in food intake (B) and water intake (C) from 3-weeks to 8-weeks of ages between WT and TRPV2KO mice (n = 6).


D . Weights of tissues related to energy metabolism normalized to their weights in WT TRPV2KO mice ( n = 15 ) .

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D. Weights of tissues related to energy metabolism normalized to their body weights in WT and TRPV2KO mice (n = 15).


E . expression of mRNA related to energy metabolism in iBAT from WT TRPV2KO mice . mRNA expression of Ucp1 , Pgc1a , Ppary , Adrb3 , cytochrome c oxidase subunit 4 isoform 1 ( Cox4i1 ) , PR domain containing 16 ( Prdm16 ) , Ucp2 , lipoprotein lipase ( Lpl ) cluster of differentiation 36 ( Cd36 ) were examined normalized to of 36B4 . Data are presented as mean ± , n = 15 ; * P < 0 . 05 vs . WT group ; * * P < 0 . 01 vs . WT group . Unpaired Student ' s t - .

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E. The expression levels of mRNA related to energy metabolism in iBAT from WT and TRPV2KO mice. mRNA expression levels of Ucp1, Pgc1a, Ppary, Adrb3, cytochrome c oxidase subunit 4 isoform 1 (Cox4i1), PR domain containing 16 (Prdm16), Ucp2, lipoprotein lipase (Lpl) and cluster of differentiation 36 (Cd36) were examined and normalized to the levels of 36B4. Data are presented as mean ± SEM, n = 15; * P < 0.05 vs. WT group; ** P < 0.01 vs. WT group. Unpaired Student's t-test.


A . Representative morphological results of iBAT by hematoxylin eosin from 8 - week WT TRPV2KO mice . Scale bars indicate 100 μm . B . Comparison of lipid droplet histograms from 8 - week - old WT ( black ) TRPV2KO ( red ) iBAT with hematoxylin eosin . Mean values are 9 . 5 ± 0 . 2 µm in 8 - week - old WT adipocytes , 14 . 7 ± 0 . 2 µm in 8 - week - old TRPV2KO adipocytes ( P < 0 . 01 ) . 1000 to 1400 droplets were counted . Data are presented as mean ± , n = 4 . C . Comparison of adipocyte histograms from 8 - week - old WT ( black ) TRPV2KO ( red ) iBAT with hematoxylin eosin . 800 to 1000 were counted . Data are presented as mean ± , n = 4 . D . Mean diameters of adipocytes in 8 - week - old WT mice ( black ) TRPV2KO mice ( red ) . Mean values are 15 . 8 ± 0 . 4 µm in 8 - week - old WT adipocytes , 19 . 8 ± 0 . 3 µm in 8 - week - old TRPV2KO adipocytes . Data are presented as mean ± , n = 4 ; * P < 0 . 05 vs . WT group . Unpaired Student ' s t - .

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A. Representative morphological results of iBAT by hematoxylin and eosin staining from 8-week WT and TRPV2KO mice. Scale bars indicate 100 μm.B. Comparison of lipid droplet size histograms from 8-week-old WT (black) and TRPV2KO (red) iBAT with hematoxylin and eosin staining. Mean values are 9.5 ± 0.2 µm in 8-week-old WT adipocytes, and 14.7 ± 0.2 µm in 8-week-old TRPV2KO adipocytes (P < 0.01). 1000 to 1400 droplets were counted. Data are presented as mean ± SEM, n = 4.C. Comparison of adipocyte size histograms from 8-week-old WT (black) and TRPV2KO (red) iBAT with hematoxylin and eosin staining. 800 to 1000 cells were counted. Data are presented as mean ± SEM, n = 4.D. Mean diameters of adipocytes in 8-week-old WT mice (black) and TRPV2KO mice (red). Mean values are 15.8 ± 0.4 µm in 8-week-old WT adipocytes, and 19.8 ± 0.3 µm in 8-week-old TRPV2KO adipocytes. Data are presented as mean ± SEM, n = 4; * P < 0.05 vs. WT group. Unpaired Student's t-test.


A , B , C D . Changes in mRNA expression of Ucp1 ( A ) , Pgc1α ( B ) , Pparγ ( C ) Trpv2 ( D ) in iBAT of WT TRPV2KO mice before ( 25oC ) , after 1 day 3 days cold ( 4oC ) exposure . Mean ± , n = 5 - 8 ; * P < 0 . 05 ; * * P < 0 . 01 vs . 25oC group ; # P < 0 . 05 vs . WT group . One - way ANOVA followed by 2 - t - with Bonferroni correction .

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A, B, C and D. Changes in mRNA expression of Ucp1 (A), Pgc1α (B), Pparγ (C) and Trpv2 (D) in iBAT of WT and TRPV2KO mice before (25oC), after 1 day and 3 days cold (4oC) exposure. Mean ± SEM, n = 5 - 8; * P < 0.05; ** P < 0.01 vs. 25oC group; # P < 0.05 vs. WT group. One-way ANOVA followed by 2-tailed t-test with Bonferroni correction.


E F . Western blot results of UCP1 in iBAT from WT TRPV2KO mice before ( 25oC ) after 3 days cold ( 4oC ) exposure ( E ) . Comparison of UCP1 before after cold exposure in iBAT from WT TRPV2KO mice ( F ) . Mean ± , n = 6 ; * P < 0 . 05 vs . 25oC group ; # P < 0 . 05 vs . WT group . One - way ANOVA followed by 2 - t - with Bonferroni correction .

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E and F. Western blot results of UCP1 protein in iBAT from WT and TRPV2KO mice before (25oC) and after 3 days cold (4oC) exposure (E). Comparison of UCP1 protein levels before and after cold exposure in iBAT from WT and TRPV2KO mice (F). Mean ± SEM, n = 6; * P < 0.05 vs. 25oC group; # P < 0.05 vs. WT group. One-way ANOVA followed by 2-tailed t-test with Bonferroni correction.


G H . Averages of temperatures ( G ) activities ( H ) of WT TRPV2KO mice with or without cold ( 4oC ) exposure . Data are presented as mean ± , n = 5 ; * P < 0 . 05 vs . WT group . Unpaired Student ' s t - .

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G and H. Averages of core body temperatures (G) and activities (H) of WT and TRPV2KO mice with or without cold (4oC) exposure. Data are presented as mean ± SEM, n = 5; * P < 0.05 vs. WT group. Unpaired Student's t-test.


A , B C . Changes in mRNA expression of Ucp1 ( A ) , Pgc1α ( B ) Pparγ ( C ) from WT TRPV2KO iBAT 4 h after intraperitoneal administration ( i . p . , arrows ) of saline or a selective β3 - adrenergic receptor agonist , BRL37344 ( 600 µg / kg ) . Mean ± , n = 5 - 7 ; * P < 0 . 05 vs . saline group ; # P < 0 . 01 vs . WT BRL37344 administration group . One - way ANOVA followed by 2 - t - with Bonferroni correction .

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A, B and C. Changes in mRNA expression of Ucp1 (A), Pgc1α (B) and Pparγ(C) from WT and TRPV2KO iBAT 4 h after intraperitoneal administration (i.p., arrows) of saline or a selective β3-adrenergic receptor agonist, BRL37344 (600 µg/kg body weight). Mean ± SEM, n = 5 - 7; * P < 0.05 vs. saline group; # P < 0.01 vs. WT BRL37344 administration group. One-way ANOVA followed by 2-tailed t-test with Bonferroni correction.


D E . Average traces of changes in iBAT ( D ) or rectal ( E ) from WT TRPV2KO mice after BRL37344 ( 600 µg / kg ) administration ( i . p . , arrows ) . Mean ± , n = 5 . * P < 0 . 05 vs . WT group . Unpaired Student ' s t - .

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D and E. Average traces of changes in iBAT temperature (D) or rectal body temperature (E) from WT and TRPV2KO mice after BRL37344 (600 µg/kg body weight) administration (i.p., arrows). Mean ± SEM, n = 5. * P < 0.05 vs. WT group. Unpaired Student's t-test.


F G . iBAT NE turnover rates ( F ) rate constants k ( G ) in WT TRPV2KO mice upon cold exposure ( 4oC ) for 4 h . Data are presented as mean ± , n = 8 .

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F and G. iBAT NE turnover rates (F) and rate constants k (G) in WT and TRPV2KO mice upon cold exposure (4oC) for 4 h. Data are presented as mean ± SEM, n = 8.


A . changes between WT TRPV2KO mice treated with high fat diet ( HFD ) for continuous 8 from 13 - week - old .

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A. Body weight changes between WT and TRPV2KO mice treated with high fat diet (HFD) for continuous 8 weeks from 13-week-old.


B C . Blood glucose level ( B ) plasma insulin level ( C ) of WT TRPV2KO mice after 8 - week HFD treatment .

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B and C. Blood glucose level (B) and plasma insulin level (C) of WT and TRPV2KO mice after 8-week HFD treatment.


D . Weights of tissues related to energy metabolism in WT TRPV2KO mice after 8 - week HFD treatment .

D. Weights of tissues related to energy metabolism in WT and TRPV2KO mice after 8-week HFD treatment.


E . mRNA expression of Ucp1 Pgc1α in iBAT from WT TRPV2KO mice after 8 - week HFD treatment . data are presented as mean ± , WT mice ( n = 6 ) , TRPV2KO mice ( n = 8 ) ; * P < 0 . 05 ; * * P < 0 . 01 vs . WT group . Unpaired Student ' s t - .

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E. mRNA expression of Ucp1 and Pgc1α in iBAT from WT and TRPV2KO mice after 8-week HFD treatment. All data are presented as mean ± SEM, WT mice (n = 6), TRPV2KO mice (n = 8); * P < 0.05; ** P < 0.01 vs. WT group. Unpaired Student's t-test.


B of anti - Bax anti - Bak antibody specificity using whole lysates from HCT116 wild - type , Bax KO , Bak KO Bax / Bak DKO . After detection of Bax was redecorated with anti - Bak antibodies ( Bax / Bak ) . Actin is used as loading control .

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B Analysis of anti-Bax and anti-Bak antibody specificity using whole cell lysates from HCT116 wild-type, Bax KO, Bak KO and Bax/Bak DKO. After detection of Bax the membrane was redecorated with anti-Bak antibodies (Bax/Bak). Actin is used as loading control.


C Western blot of Bak in heavy ( HM ) fraction of human heart , cerebral cortex ( CNS GM ) , kidney , brain matter ( CNS WM ) , lung , liver , skeletal muscle tissue . cytosol fraction obtained from CNS cortex ( C ) , LDH , Tom20 serve as fractionation loading controls . n = 3 .

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C Western blot analysis of Bak in the heavy membrane (HM) fraction of human heart, cerebral cortex (CNS GM), kidney, brain white matter (CNS WM), lung, liver, and skeletal muscle tissue. The cytosol fraction obtained from CNS cortex (C), LDH, and Tom20 serve as fractionation and loading controls. n = 3.


D Presence of Bak in cytosol of heart , cerebral cortex ( CNS GM ) , kidney , brain matter ( CNS WM ) , lung , liver , skeletal muscle tissue analyzed by Western blot in parallel to C . heavy heart fraction ( HM ) , LDH Tom20 serve as fractionation loading controls . n = 3 .

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D Presence of Bak in the cytosol of heart, cerebral cortex (CNS GM), kidney, brain white matter (CNS WM), lung, liver, and skeletal muscle tissue analyzed by Western blot in parallel to C. The heavy membrane heart fraction (HM), LDH and Tom20 serve as fractionation and loading controls. n = 3.


E Carbonate extraction of heavy fraction of human brain matter ( CNS WM ) lung tissue analyzed for presence of Bak by Western blot . - associated supernatant ( S ) - integral pellet ( P ) are displayed . Smac is released from mitochondrial inter space ( IMS ) during carbonate extraction CoxIV remains in pellet . n = 2 .

E Carbonate extraction of the heavy membrane protein fraction of human brain white matter (CNS WM) and lung tissue analyzed for the presence of Bak by Western blot. Membrane-associated protein supernatant (S) and membrane-integral protein pellet (P) are displayed. Smac is released from the mitochondrial inter membrane space (IMS) during carbonate extraction and CoxIV remains in the pellet. n = 2.


A Confocal of HCT116 Bax / Bak DKO transfected with GFP - Bak in absence ( left ) or presence ( right ) of Bcl - xL overexpression . Scale bar 15 µm . n ≥ 10 .

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A Confocal images of HCT116 Bax/Bak DKO cells transfected with GFP-Bak in the absence (left) or the presence (right) of Bcl-xL overexpression. Scale bar 15 µm. n ≥ 10.


B FLIP ( Loss in Photobleaching ) of mitochondrial GFP - Bak in absence ( ● , straight ) or presence ( ○ , broken ) of overexpressed Bcl - xL . of neighboring ( in Fig . 2C ) is shown as control ( , dotted ) . Data represent averages ± from 16 ( - Bcl - xL ) 40 ( + Bcl - xL ) ROI measurements .

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B FLIP (Fluorescence Loss in Photobleaching) of mitochondrial GFP-Bak in the absence (●, straight line) or the presence (○, broken line) of overexpressed Bcl-xL. Fluorescence of the neighboring cell (in Fig. 2C) is shown as control (, dotted line). Data represent averages ± SEM from 16 (-Bcl-xL) and 40 (+Bcl-xL) ROI measurements.


C FLIP of GFP - Bak in absence ( top ) or presence ( bottom ) of overexpressed Bcl - xL diminishes GFP - Bak in cytosol of targeted ( circled ) completely after 90 s GFP is detected only on mitochondria ( arrows ) . During FLIP measurements mitochondrial GFP - Bak is monitored , while cytosol is bleached repeatedly . Time points in seconds are displayed above .

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C FLIP of GFP-Bak in the absence (top) or the presence (bottom) of overexpressed Bcl-xL diminishes GFP-Bak fluorescence in the cytosol of targeted cells (circled) completely after 90 s and GFP fluorescence is detected only on the mitochondria (arrows). During FLIP measurements mitochondrial GFP-Bak fluorescence is monitored, while the cytosol is bleached repeatedly. Time points in seconds are displayed above the images.


A Western blot of GFP - Bak localization in cytosol ( C ) heavy fraction ( HM ) of HCT116 Bax / Bak DKO with or without overexpressed wild - type Bcl - xL or Bcl - xL G138A . GAPDH Tom20 serve as fractionation controls . n = 10 . Bak shuttling by Bcl - xL is independent of GFP fusion ( Fig . E2a ) . B of predominant mitochondrial localization ( grey bars ) mixed mitochondrial localization ( grey bars ) of Bak in HCT116 Bax / Bak DKO expressing GFP - Bak with or without Bcl - xL overexpression . Data represent averages ± SD from 3 independent experiments with n ≥ 100 .

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A Western blot analysis of GFP-Bak localization in cytosol (C) and heavy membrane fraction (HM) of HCT116 Bax/Bak DKO cells with or without overexpressed wild-type Bcl-xL or Bcl-xL G138A. GAPDH and Tom20 serve as fractionation controls. n = 10. Bak shuttling by Bcl-xL is independent of the GFP fusion (Fig. E2a).B Quantification of predominant mitochondrial localization (light grey bars) and mixed cytosolic and mitochondrial localization (dark grey bars) of Bak in HCT116 Bax/Bak DKO cells expressing GFP-Bak with or without Bcl-xL overexpression. Data represent averages ± SD from 3 independent experiments with n ≥ 100.


C Retrotranslocation rates measured for wild - type Bak or Bak D83R in presence or absence of Bcl - 2 , Mcl - 1 , wild - type Bcl - xL or Bcl - xL G138A . Data represent averages ± SD .

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C Retrotranslocation rates measured for wild-type Bak or Bak D83R in the presence or the absence of Bcl-2, Mcl-1, wild-type Bcl-xL or Bcl-xL G138A. Data represent averages ± SD.


D of endogenous Bak localization in cytosol ( C ) heavy fraction ( HM ) of HeLa expressing degradation - prone DD - FLAG - Bcl - xL . In absence of Shield - 1 DD - FLAG - Bcl - xL is readily degraded , resulting in unaltered Bcl - xL . Addition of 0 . 5 µM Shield - 1 instantly stabilizes DD - FLAG - Bcl - xL resulting in elevated Bcl - xL that increase of endogenous Bak ( Fig . E3C , D ) . Akt1 Tom20 serve as fractionation controls . n = 3 .

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D Analysis of endogenous Bak localization in cytosol (C) and heavy membrane fraction (HM) of HeLa cells expressing degradation-prone DD-FLAG-Bcl-xL. In the absence of Shield-1 DD-FLAG-Bcl-xL is readily degraded, resulting in unaltered Bcl-xL levels. Addition of 0.5 µM Shield-1 instantly stabilizes DD-FLAG-Bcl-xL resulting in elevated Bcl-xL levels that increase cytosolic levels of endogenous Bak (Fig. E3C, D). Akt1 and Tom20 serve as fractionation controls. n = 3.


B of HCT116 Bax / Bak DKO expressing Bax , BaxTBak , BakTBax or Bak with expressed being present largely ( bars ) , in a mixed distribution between cytosol mitochondria ( grey bars ) or largely mitochondrial ( black bars ) . Data represent averages ± from 7 independent experiments with n ≥ 100 .

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B Quantification of HCT116 Bax/Bak DKO cells expressing Bax, BaxTBak, BakTBax or Bak with the expressed protein being present largely cytosolic (white bars), in a mixed distribution between cytosol and mitochondria (grey bars) or largely mitochondrial (black bars). Data represent averages ± SEM from 7 independent experiments with n ≥ 100 cells.


C Confocal of HCT116 Bax / Bak DKO expressing Bax , BaxTBak , BakTBax or Bak . GFP / YFP of expressed variants is depicted in top panels in green in merged image on bottom . mitochondria were stained by MitoTracker far red depicted in red in merged ( bottom row ) . Scale 10 µm . n ≥ 5 .

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C Confocal images of HCT116 Bax/Bak DKO cells expressing Bax, BaxTBak, BakTBax or Bak. The GFP/YFP fluorescence of the expressed protein variants is depicted in the top panels and in green in the merged image on the bottom. The mitochondria were stained by MitoTracker far red depicted in red in the merged images (bottom row). Scale 10 µm. n ≥ 5.


D Western blot of Bax , BaxTBak , Bak BakTBax localization expressed in HCT116 Bax / Bak DKO . Cytosol ( C ) heavy fraction ( HM ) of HCT116 Bax / Bak DKO are displayed . GAPDH VDAC serve as fractionation controls . n = 3 .

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D Western blot analysis of Bax, BaxTBak, Bak and BakTBax localization expressed in HCT116 Bax/Bak DKO cells. Cytosol (C) and heavy membrane fraction (HM) of HCT116 Bax/Bak DKO cells are displayed. GAPDH and VDAC serve as fractionation controls. n = 3.


A Caspase 3 / 7 activity measured in HCT116 Bax / Bak DKO overexpressing Bax , BaxTBak , Bak or BakTBax with or without Bcl - xL overexpression in absence of apoptosis stimuli . Caspase activity is displayed in relative units ( RFU ) . pcDNA3 . 1 - transfected served as control . Data represent averages ± . n ≥ 3 . p - values according to One Way ANOVA are displayed . B Staurosporine ( STS , 1 µM ) - induced caspase 3 / 7 activity of Bax / Bak DKO overexpressing Bax , BaxTBak , Bak or BakTBax with or without Bcl - xL overexpression displayed in relative units ( RFU ) . Data represent averages ± . n ≥ 3 . p - values according to One Way ANOVA . BaxTBak activities with without Bcl - xL expression revealed no significant difference ( n . s . ) , while in presence of Bcl - xL overexpression BaxTBak activity is significantly higher than Bax , Bak or BakTBax activities ( p < 0 . 001 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Caspase 3/7 activity measured in HCT116 Bax/Bak DKO cells overexpressing Bax, BaxTBak, Bak or BakTBax with or without Bcl-xL overexpression in the absence of apoptosis stimuli. Caspase activity is displayed in relative fluorescence units (RFU). pcDNA3.1-transfected cells served as control. Data represent averages ± SEM. n ≥ 3. p-values according to One Way ANOVA are displayed.B Staurosporine (STS, 1 µM)-induced caspase 3/7 activity of Bax/Bak DKO cells overexpressing Bax, BaxTBak, Bak or BakTBax with or without Bcl-xL overexpression displayed in relative fluorescence units (RFU). Data represent averages ± SEM. n ≥ 3. p-values according to One Way ANOVA. BaxTBak activities with and without Bcl-xL expression revealed no significant difference (n.s.), while in the presence of Bcl-xL overexpression BaxTBak activity is significantly higher than Bax, Bak or BakTBax activities (p < 0.001).


C of active Bax conformation in HCT116 Bax / Bak DKO expressing wild - type Bax or BaxTBak with ( grey bars ) or without Bcl - xL overexpression ( grey bars ) by monoclonal antibody 6A7 ( Sigma ) detecting active Bax fold by imaging . were analyzed prior or after treatment with 1 µM STS in presence of pan - caspase inhibitor qVD . Data are represented as % of expressing population ± . n = 4 .

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C Analysis of the active Bax conformation in HCT116 Bax/Bak DKO cells expressing wild-type Bax or BaxTBak with (dark grey bars) or without Bcl-xL overexpression (light grey bars) by the monoclonal antibody 6A7 (Sigma) detecting the active Bax protein fold by fluorescence imaging. Cells were analyzed prior or after treatment with 1 µM STS in the presence of the pan-caspase inhibitor qVD. Data are represented as % of the expressing cell population ± SEM. n = 4.


D HCT116 Bax / Bak DKO ectopically expressing wild - type Bax or BaxTBak with ( grey bars ) or without Bcl - xL ( grey bars ) were analyzed in presence or absence of 1 µM STS qVD for retained mitochondrial cyt c . Data are represented as % of expressing population ± . n = 4 .

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D HCT116 Bax/Bak DKO cells ectopically expressing wild-type Bax or BaxTBak with (dark grey bars) or without Bcl-xL (light grey bars) were analyzed in the presence or the absence of 1 µM STS and qVD for retained mitochondrial cyt c. Data are represented as % of the expressing cell population ± SEM. n = 4.


E Flow cytometry of Annexin V - of Bax / Bak DKO expressing Bax , BaxTBak , Bak or BakTBax in absence ( red ) or presence of Bcl - xL overexpression ( black ) , following STS - treatment . of gated is displayed in color of corresponding graph . Data represent averages ± SD . n = 4 .

E Flow cytometry analysis of Annexin V-staining of Bax/Bak DKO cells expressing Bax, BaxTBak, Bak or BakTBax in the absence (red line) or the presence of Bcl-xL overexpression (black line), following STS-treatment. The percentage of gated cells is displayed in the color of the corresponding graph. Data represent averages ± SD. n = 4.


F Colony formation of Bax / Bak DKO transfected with pcDNA , Bax , BaxTBak , Bak or BakTBax with or without Bcl - xL overexpression . STS ( 1 µM ) was added for 24 hrs before were replated colonies were stained with methylene blue typically 14 days after treatment .

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F Colony formation of Bax/Bak DKO cells transfected with pcDNA, Bax, BaxTBak, Bak or BakTBax with or without Bcl-xL overexpression. STS (1 µM) was added for 24 hrs before cells were replated and colonies were stained with methylene blue typically 14 days after treatment.


G of colony formation of Bax / Bak DKO expressing Bax , BaxTBak , Bak or BakTBax with or without Bcl - xL overexpression after STS treatment ( Fig . 5F ) . Data represent averages ± . n = 4 . p - values according to One Way ANOVA . BaxTBak expressing with or without Bcl - xL expression showed no significant difference ( n . s . ) . H of colony formation of Bax / Bak DKO expressing Bax , BaxTBak , Bak or BakTBax in presence of Bcl - xL overexpression without apoptosis stimulation . Data represent averages ± . n = 5 . p - values according to One Way ANOVA .

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G Quantification of colony formation of Bax/Bak DKO cells expressing Bax, BaxTBak, Bak or BakTBax with or without Bcl-xL overexpression after STS treatment (Fig. 5F). Data represent averages ± SEM. n = 4. p-values according to One Way ANOVA. BaxTBak expressing cells with or without Bcl-xL expression showed no significant difference (n.s.).H Quantification of the colony formation of Bax/Bak DKO cells expressing Bax, BaxTBak, Bak or BakTBax in presence of Bcl-xL overexpression without apoptosis stimulation. Data represent averages ± SEM. n = 5. p-values according to One Way ANOVA.


A FLIP of BaxTBakSS without ( ○ , broken ) or with ( ● , straight ) overexpressed Bcl - xL . of a neighboring is shown as control ( , dotted ) . Data represent averages ± from 20 ( - Bcl - xL ) 16 ( + Bcl - xL ) ROI measurements .

A FLIP of BaxTBakSS without (○, broken line) or with (●, straight line) overexpressed Bcl-xL. Fluorescence of a neighboring cell is shown as control (, dotted line). Data represent averages ± SEM from 20 (-Bcl-xL) and 16 (+Bcl-xL) ROI measurements.


B Western blot of PARP cleavage in HCT116 Bax / Bak DKO overexpressing Bax , Bax S184V , BaxTBak or BaxTBak V197 / 198S . experiment was carried out in absence of apoptotic stimuli . transfected with pcDNA3 . 1 vector serve as control for PARP cleavage . Similar sample loading is controlled by actin . n = 4 .

B Western blot analysis of PARP cleavage in HCT116 Bax/Bak DKO cells overexpressing Bax, Bax S184V, BaxTBak or BaxTBak V197/198S. The experiment was carried out in the absence of apoptotic stimuli. Cells transfected with pcDNA3.1 vector serve as control for PARP cleavage. Similar sample loading is controlled by actin. n = 4.


C Colony formation of Bax / Bak DKO transfected with pcDNA , BaxTBak or BaxTBakSS with Bcl - xL overexpression in absence of apoptotic stimuli . n = 3 .

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C Colony formation of Bax/Bak DKO cells transfected with pcDNA, BaxTBak or BaxTBakSS with Bcl-xL overexpression in the absence of apoptotic stimuli. n = 3.


D Caspase 3 / 7 activity was measured in HCT116 Bax / Bak DKO overexpressing Bax , Bax S184V , BaxTBak or BaxTBak V197 / 198S . Caspase activity is displayed in relative units ( RFU ) . experiment was carried out in absence of apoptotic stimuli pcDNA3 . 1 - transfected served as control . Data represent averages ± . n = 4 . p - values according to One Way ANOVA using Holm - Sidak method are displayed .

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D Caspase 3/7 activity was measured in HCT116 Bax/Bak DKO cells overexpressing Bax, Bax S184V, BaxTBak or BaxTBak V197/198S. Caspase activity is displayed in relative fluorescence units (RFU). The experiment was carried out in the absence of apoptotic stimuli and pcDNA3.1-transfected cells served as control. Data represent averages ± SEM. n = 4. p-values according to One Way ANOVA using the Holm-Sidak method are displayed.


F STS - induced caspase 3 / 7 activity of HCT116 Bax / Bak DKO expressing Bax , Bax S184V , BakTBax or BakTBax S184V is displayed normalized to Bax activity . Data represent averages ± . n ≥ 3 .

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F STS-induced caspase 3/7 activity of HCT116 Bax/Bak DKO cells expressing Bax, Bax S184V, BakTBax or BakTBax S184V is displayed normalized to Bax activity. Data represent averages ± SEM. n ≥ 3.


G Western blot of PARP cleavage after STS treatment in HCT116 Bax / Bak DKO overexpressing Bax , Bax S184V , BakTBax or BakTBax S184V . Empty vector - transfected serve as control for PARP cleavage actin is loading control . n = 3 .

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G Western blot analysis of PARP cleavage after STS treatment in HCT116 Bax/Bak DKO cells overexpressing Bax, Bax S184V, BakTBax or BakTBax S184V. Empty vector-transfected cells serve as control for PARP cleavage and actin is the loading control. n = 3.


H Plot of Bax ( ● ) Bak ( ○ ) retrotranslocation rates vs . hydrophobicity of MA of constructs . R2 = 0 . 95 .

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H Plot of Bax (●) and Bak (○) retrotranslocation rates vs. hydrophobicity of the MA of the constructs. R2 = 0.95.


A Carbonate extraction of HM fraction of Bax / Bak DKO expressing wild - type Bcl - xL in presence or absence of Bak or Bax analyzed by Western blot . Carbonate extractable supernatant ( S ) - integral pellet ( P ) are displayed . Smac is released from IMS during carbonate extraction VDAC remains in pellet . n = 4 . B Western blot of Bcl - xLTBax carbonate extraction from heavy fraction from Bax / Bak DKO expressing Bcl - xL variant with or without Bak or Bax . Smac VDAC serve as controls for supernatant ( S ) pellet ( P ) . n = 4 . C Carbonate extraction of HM fraction of Bax / Bak DKO expressing Bcl - xL G138A with or without Bak or Bax . Smac VDAC serve as controls for supernatant ( S ) pellet ( P ) . n = 4 .

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A Carbonate extraction of the HM protein fraction of Bax/Bak DKO cells expressing wild-type Bcl-xL in the presence or the absence of Bak or Bax analyzed by Western blot. Carbonate extractable supernatant (S) and membrane-integral protein pellet (P) are displayed. Smac is released from the IMS during carbonate extraction and VDAC remains in the pellet. n = 4.B Western blot analysis of Bcl-xLTBax carbonate extraction from the heavy membrane fraction from Bax/Bak DKO cells expressing the Bcl-xL variant with or without Bak or Bax. Smac and VDAC serve as controls for supernatant (S) and pellet (P). n = 4.C Carbonate extraction of the HM protein fraction of Bax/Bak DKO cells expressing Bcl-xL G138A with or without Bak or Bax. Smac and VDAC serve as controls for supernatant (S) and pellet (P). n = 4.


D HM fraction carbonate extraction of Bax / Bak DKO expressing Bax , BaxTBak or Bax S184V . Smac VDAC serve as controls for supernatant ( S ) pellet ( P ) . n = 4 .

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D HM protein fraction and carbonate extraction of Bax/Bak DKO cells expressing Bax, BaxTBak or Bax S184V. Smac and VDAC serve as controls for supernatant (S) and pellet (P). n = 4.


E Smac release from heavy fraction ( HM ) into cytosol ( C ) in HCT116 Bax / Bak DKO overexpressing Bax BaxTBak without apoptotic stimuli analyzed by Western blot . Bax BaxTBak were expressed differentially to produce similar of mitochondrial Bax . GAPDH Tom20 serve as fractionation controls . n = 3 .

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E Smac release from the heavy membrane fraction (HM) into the cytosol (C) in HCT116 Bax/Bak DKO cells overexpressing Bax and BaxTBak without apoptotic stimuli analyzed by Western blot. Bax and BaxTBak were expressed differentially to produce similar levels of mitochondrial Bax. GAPDH and Tom20 serve as fractionation controls. n = 3.


F PARP cleavage in HCT116 Bax / Bak DKO expressing similar of mitochondrial Bax or BaxTBak analyzed by Western blot without apoptotic stimuli . Actin is used as loading control . n = 3 .

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F PARP cleavage in HCT116 Bax/Bak DKO cells expressing similar levels of mitochondrial Bax or BaxTBak analyzed by Western blot without apoptotic stimuli. Actin is used as loading control. n = 3.


A Luciferase complementation imaging ( LCI ) assays showing that BIC1 interacts with BZR1 . nLUC - BZR1 was co - transformed with cLUC - CCA1 , cLUC - TOC1 , cLUC - ELF3 , cLUC - LUX , cLUC - BIC1 , cLUC , respectively , in leaf epidermal of Nicotiana benthamiana as indicated .

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A Luciferase complementation imaging (LCI) assays showing that BIC1 interacts with BZR1. nLUC-BZR1 was co-transformed with cLUC-CCA1, cLUC-TOC1, cLUC-ELF3, cLUC-LUX, cLUC-BIC1, cLUC, respectively, in leaf epidermal cells of Nicotiana benthamiana as indicated.


B BiFC assays showing interaction of BIC1 BZR1 mainly in nucleus . Leaf epidermal of Nicotiana benthamiana were co - transformed with nYFP - BZR1 cYFP - BIC1 . BF , bright field . Scale bars represent 20 μm .

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B BiFC assays showing the interaction of BIC1 and BZR1 mainly in nucleus. Leaf epidermal cells of Nicotiana benthamiana were co-transformed with nYFP-BZR1 and cYFP-BIC1. BF, bright field. Scale bars represent 20 μm.


C In vitro pull - down assays showing BIC1 directly interacts with BZR1 . Purified GST - BIC1 were incubated with MBP or MBP - BZR1 for MBP pull - down . Arrowhead indicates specific bands .

C In vitro pull-down assays showing BIC1 directly interacts with BZR1. Purified GST-BIC1 proteins were incubated with MBP or MBP-BZR1 for the MBP pull-down assay. Arrowhead indicates specific bands.


D Co - immunoprecipitation ( Co - IP ) assays showing interaction between BIC1 BZR1 in vivo . Seedlings were grown for 6 days under long - day conditions . immunoprecipitates were detected using anti - GFP anti - MYC antibodies , respectively . * indicate specific bands . cross - reacting lower band on IP blot is heavy chain after IP .

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D Co-immunoprecipitation (Co-IP) assays showing the interaction between BIC1 and BZR1 in vivo. Seedlings were grown for 6 days under long-day conditions. The immunoprecipitates were detected using anti-GFP and anti-MYC antibodies, respectively. * indicate specific bands. The cross-reacting lower band on IP blot is the heavy chain after IP.


A , B Hypocotyl elongation of BIC1 overexpression mutant lines in response to BR treatment . Col - 0 , 35S : BIC1 - YFP , 35S : BIC1 - Flag bic1bic2 seedlings were grown for 6 days on 1 / 2 MS medium supplemented with 1 μM brassinazole ( BRZ ) plus different concentrations of epibrassinolide ( eBL ) . of representative seedlings are shown in ( A ) , hypocotyl lengths of indicated genotypes were measured are shown in ( B ) . Data are means ± SD ; n > 20 . Scale bars , 2 mm .

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A, B Hypocotyl elongation phenotypes of the BIC1 overexpression and mutant lines in response to BR treatment. Col-0, 35S:BIC1-YFP, 35S:BIC1-Flag and bic1bic2 seedlings were grown for 6 days on 1/2 MS medium supplemented with 1 μM brassinazole (BRZ) plus different concentrations of epibrassinolide (eBL). Images of the representative seedlings are shown in (A), and the hypocotyl lengths of the indicated genotypes were measured and are shown in (B). Data are means ± SD; n>20. Scale bars, 2 mm.


C RT - qPCR of BZR1 - activated gene expression in bic1bic2 double mutants . 6 - d - old Col - 0 bic1bic2 seedlings grown on 1 / 2 MS medium supplemented with 1 μM BRZ were treated with 5 μM eBL ( + eBL ) or not ( - eBL ) for 1 hour . PP2A gene served as internal control . Data are means ± SD ( n = 3 ) .

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C RT-qPCR analysis of BZR1-activated gene expression in the bic1bic2 double mutants. The 6-d-old Col-0 and bic1bic2 seedlings grown on 1/2 MS medium supplemented with 1 μM BRZ were treated with 5 μM eBL (+eBL) or not (-eBL) for 1 hour. The PP2A gene served as internal control. Data are means ± SD (n=3).


D Heat map of BIC1 - regulated genes . Col - 0 35S : BIC1 - YFP seedlings were grown on 1 / 2 MS medium for 10 days . Three biologic replicates were performed . colored bar beneath map indicates fold change ( log2 value ) .

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D Heat map of BIC1-regulated genes. The Col-0 and 35S:BIC1-YFP seedlings were grown on 1/2 MS medium for 10 days. Three biologic replicates were performed. The colored bar beneath the map indicates fold change (log2 value).


E BIC1 facilitates expression of elongation genes . RT - qPCR of genes selected in transcriptomic data . Col - 0 , 35S : BIC1 - YFP 35S : BIC1 - Flag seedlings were grown for 5 days under long - day conditions . PP2A gene served as internal control . Data are means ± SD ( n = 3 ) .

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E BIC1 facilitates the expression of cell elongation genes. RT-qPCR analysis of genes selected in transcriptomic data. The Col-0, 35S:BIC1-YFP and 35S:BIC1-Flag seedlings were grown for 5 days under long-day conditions. The PP2A gene served as internal control. Data are means ± SD (n=3).


A , B BIC1 BZR1 synergistically promote hypocotyl elongation . Col - 0 , 35S : BZR1 - MYC , 35S : BIC1 - YFP 35S : BZR1 - MYC / 35S : BIC1 - YFP were grown for 5 days under long - day conditions . of representative seedlings are shown in ( A ) , hypocotyl lengths of indicated genotypes were measured are shown in ( B ) . Data are means ± SD ( n > 20 ) . * * p < 0 . 01 , as determined by Student ' s t - . Scale bar , 2 mm .

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A, B BIC1 and BZR1 synergistically promote hypocotyl elongation. The Col-0, 35S:BZR1-MYC, 35S:BIC1-YFP and 35S:BZR1-MYC/35S:BIC1-YFP plants were grown for 5 days under long-day conditions. Images of the representative seedlings are shown in (A), and the hypocotyl lengths of the indicated genotypes were measured and are shown in (B). Data are means ± SD (n>20). **p < 0.01, as determined by Student's t-test. Scale bar, 2 mm.


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 - .

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 ) .

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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 .

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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 .

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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 .

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 ) .

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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 ) .

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 .

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 .

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

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 .

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


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

(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 .

(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 .

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 .

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