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 .

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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 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 .

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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 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 .

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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 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 .

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

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 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 .

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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 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 .

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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 . Body weight changes between WT and TRPV2KO mice treated with high fat diet ( HFD ) for continuous 8 weeks 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 and C . Blood glucose level ( B ) and plasma insulin level ( C ) of WT and 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 and TRPV2KO mice after 8 - week HFD treatment .

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


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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 ( 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 .

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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 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 .

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 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 .

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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 the presence or the 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 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 .

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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 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 .

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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 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 .

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 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 .

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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 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 ) .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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 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 .

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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 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 .

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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 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 .

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 cells transfected with pcDNA , BaxTBak or BaxTBakSS with Bcl - xL overexpression in the 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 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 .

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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 cells expressing Bax , Bax S184V , BakTBax or BakTBax S184V is displayed normalized to Bax activity . Data represent averages ± SEM . n ≥ 3 .

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

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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 ( ● ) and Bak ( ○ ) retrotranslocation rates vs . hydrophobicity of the MA of the constructs . R2 = 0 . 95 .

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


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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 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 .

O O O O O O O O O O O O O O O O O O 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 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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 cells expressing similar levels of mitochondrial Bax or BaxTBak analyzed by Western blot without apoptotic stimuli . Actin is used as loading control . n = 3 .

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

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 cells of Nicotiana benthamiana as indicated .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 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 .

O O O O O O O O O O O O O O O O O O O O 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 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 proteins were incubated with MBP or MBP - BZR1 for the MBP pull - down assay . Arrowhead indicates specific bands .

O O O O O O O O O O O O O O O O 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 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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 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 .

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

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 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 ) .

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

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 . 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 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 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 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 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 .

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

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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 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 ) .

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

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 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 .

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

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 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 .

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

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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 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 ) .

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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 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 .

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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 cells of Nicotiana benthamiana were co - transformed with nLUC - BIC1 or nLUC and cLUC - PIF4 or cLUC as indicated .

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

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 proteins were incubated with MBP or MBP - PIF4 for the MBP pull - down assay . Arrowhead indicates specific bands .

O O O O O O O O O O O O O O O O O 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 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 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 .

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 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 .

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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 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 ) .

O O O O O O O O O O O O O O O O O O O O O O O O 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 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 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 .

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

G,H 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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 ) .

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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 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 ) .

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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 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 .

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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 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 ) .

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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 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 .

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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 cells of Nicotiana benthamiana were co - transformed with nLUC - TaBIC1 or nLUC and 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 proteins were incubated with MBP or MBP - TaBZR1 for the MBP pull - down assay . 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 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 .

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 primary keratinocytes and HaCaT cell line . The scatter plot depicts individual 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 , 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 .

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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 . 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 .

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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 ) 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 .

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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 ) 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 .

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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 ) 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 .

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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 the two approaches , using select filters on quantification 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 the two different approaches . The stacked bar graph differentiates between glycosites identified on peptides with single and 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 ) 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 .

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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 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 .

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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 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

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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 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 .

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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 and hemidesmosome component COL17A1 .

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


( G ) Quantification of Western blot bands for COL17A1 ( full length ) 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 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 ) .

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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 and 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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

O O O O O 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

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 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 .

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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 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 .

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

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 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 .

O O O O O O O O O O O O O O O O O O O O O O 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 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 . 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 .

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

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

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

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 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 .

O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 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 ) .

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

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 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 ) .

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

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

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

O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 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 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 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 .

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 the indicated bem1 constructs tagged with 3XHA ( top panel ) . Detection of Ade13 was used as loading control ( bottom panel ) .

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

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

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


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 .

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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 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 .

O O O O O O O O O O O O O O O O O O O O O O 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 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 and subsequent colony formation of the indicated mutant cells at the indicated temperatures .

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


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 .

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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 the indicated mutant strains ( top panel ) . Detection of Ade13 was used as a loading control ( bottom panel ) .

O O O O O O O O O O O O O O O O O O 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 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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O 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 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.