( c - e ) Measured relative fraction of m + 2 labeling of TCA cycle intermediates after feeding [ U - 13C ] - glucose ( red ; mean s . d . of n = 3 ) , deconvoluted signal ( green ) , expected labeling dynamics considering loss in synchronization ( black ; representing TCA cycle oxidation of glucose - derived acetyl - CoA ) .

O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O B-SMALL_MOLECULE O O O O O O O

(c-e) Measured relative fraction of the m+2 labeling of TCA cycle intermediates after feeding [U-13C]-glucose (red; mean and s.d. of n=3), the deconvoluted signal (green), and the expected labeling dynamics considering the loss in synchronization (black; representing TCA cycle oxidation of glucose-derived acetyl-CoA).


( f ) Oscillations in citrate m + 4 labeling after feeding [ U - 13C ] - glutamine ( mean s . d . of n = 3 ) .

O O O O O B-SMALL_MOLECULE O O O O O O O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O O O O O O O O O O

(f) Oscillations in citrate m+4 labeling after feeding [U-13C]-glutamine (mean and s.d. of n=3).


( g ) Oscillations in total citrate throughout cycle ( mean s . d . of n = 5 ) .

O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O

(g) Oscillations in the total citrate concentration throughout the cell cycle (mean and s.d. of n=5).


( h ) measured lactate secretion flux in synchronized shown in red ( mean s . d . of n = 5 ; in Equation 9 ; ) ; deconvoluted secretion flux dynamics , in case of no synchronization loss ( green ; in Equation 10 ) ; expected secretion flux on deconvoluted fluxes considering loss in synchronization , matching measured fluxes ( black ; Equation 10 ) .

O O O O B-SMALL_MOLECULE O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O

(h) The measured lactate secretion flux in synchronized cells shown in red (mean and s.d. of n=5; in Equation 9;); the deconvoluted secretion flux dynamics, in case of no synchronization loss (green; in Equation 10); and the expected secretion flux based on the deconvoluted fluxes and considering the loss in synchronization, matching the measured fluxes (black; Equation 10).


Oscillations in aspartate ( a ) concentrations throughout cycle when feeding [ U - 13C ] - glutamine ( representing oxidative TCA cycle activity ) .

O O B-SMALL_MOLECULE O O O O O O O O O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O O O O O O

Oscillations in aspartate (a)concentrations throughout the cell cycle when feeding [U-13C]-glutamine (representing oxidative TCA cycle activity).


Oscillations in malate ( b ) concentrations throughout cycle when feeding [ U - 13C ] - glutamine ( representing oxidative TCA cycle activity ) .

Oscillations in malate (b) concentrations throughout the cell cycle when feeding [U-13C]-glutamine (representing oxidative TCA cycle activity).


( c ) Uniform malate m + 4 labeling throughout cycle ( combined with increase in malate in S phase representing increased oxidative TCA cycle flux in S phase ) .

O O O O B-SMALL_MOLECULE O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O

(c) Uniform malate m+4 labeling throughout the cell cycle (combined with the increase in malate concentration in S phase representing increased oxidative TCA cycle flux in S phase).


( d ) Oscillations in pyrimidines m + 3 labeling throughout cycle when feeding [ U - 13C ] - glutamine ( representing de novo pyrimidine biosynthesis ) .

O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O O O O O O

(d) Oscillations in pyrimidines m+3 labeling throughout the cell cycle when feeding [U-13C]-glutamine (representing de novo pyrimidine biosynthesis).


( e ) Oscillations in lactate m + 3 labeling throughout cycle when feeding [ U - 13C ] - glutamine ( representing malic enzyme activity ) .

O O O O O B-SMALL_MOLECULE O O O O O O O O O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O O O O O

(e) Oscillations in lactate m+3 labeling throughout the cell cycle when feeding [U-13C]-glutamine (representing malic enzyme activity).


( f ) Oscillations in malate m + 3 throughout cycle when feeding [ U - 13C ] - glucose ( representing pyruvate carboxylase activity ) .

O O O O O B-SMALL_MOLECULE O O O O O O O O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O O O O O

(f) Oscillations in malate m+3 throughout the cell cycle when feeding [U-13C]-glucose (representing pyruvate carboxylase activity).


( g ) Oscillations in citrate m + 5 when feeding [ U - 13C ] - glutamine throughout cycle ( representing reductive IDH flux ) .

O O O O O B-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O O O O O O O

(g) Oscillations in citrate m+5 when feeding [U-13C]-glutamine throughout the cell cycle (representing reductive IDH flux).


( h ) Oscillations in acetyl - CoA m + 2 when feeding [ U - 13C ] - glucose , representing oxidative glucose metabolism .

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

(h) Oscillations in acetyl-CoA m+2 when feeding [U-13C]-glucose, representing oxidative glucose metabolism.


Oscillations in acetyl - CoA m + 2 when feeding [ U - 13C ] - glutamine , representing reductive glutamine metabolism ( i ) . For measurements of metabolite concentrations ( a - b ) , red marks represent mean s . d . of n = 5 . For measurements of fractional isotopic labeling ( c - i ) , red marks represent mean s . d . of n = 3 .

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

Oscillations in acetyl-CoA m+2 when feeding [U-13C]-glutamine, representing reductive glutamine metabolism (i). For measurements of metabolite concentrations (a-b), red marks represent mean and s.d. of n=5. For measurements of fractional isotopic labeling (c-i), red marks represent mean and s.d. of n=3.


Figure 5 : Complementary oscillations of glucose versus glutamine - derived fluxes in TCA cycle . ( a - i ) Oscillations in metabolic flux throughout cycle ( in mM / h ) , computed on metabolic modelling of measured oscillations in metabolite concentrations isotopic labeling ( red green marks represent optimal estimates of transient flux with 95 % c . i . ) . Blue lines represent average fluxes inferred in a non - synchronized population . As shown , glucose - derived flux into TCA cycle peak in late G1 phase , while oxidative reductive glutamine metabolism dominates S phase .

O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-EXP_ASSAY O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O

Figure 5: Complementary oscillations of glucose versus glutamine-derived fluxes in TCA cycle. (a-i) Oscillations in metabolic flux throughout the cell cycle (in mM/h), computed based on metabolic modelling of measured oscillations in metabolite concentrations and isotopic labeling (red and green marks represent optimal estimates of transient flux with 95% c.i.). Blue lines represent average fluxes inferred in a non-synchronized cell population. As shown, glucose-derived flux into TCA cycle peak in late G1 phase, while oxidative and reductive glutamine metabolism dominates S phase.


( a - b ) One hour treatment of synchronized with DCA inhibits oscillations in citrate m + 2 ( a ) from isotopic glucose ( representing glycolytic flux into TCA cycle ) .

O O O O O O O O O B-EXP_ASSAY O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O

(a-b) One hour treatment of synchronized cells with DCA inhibits the oscillations in citrate m+2 (a) from isotopic glucose (representing glycolytic flux into TCA cycle).


( a - b ) One hour treatment of synchronized with DCA inhibits oscillations in acetyl - CoA m + 2 ( b ) from isotopic glucose ( representing glycolytic flux into TCA cycle ) .

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

(a-b) One hour treatment of synchronized cells with DCA inhibits the oscillations in acetyl-CoA m+2 (b) from isotopic glucose (representing glycolytic flux into TCA cycle).


It further inhibits oscillations in citrate m + 5 / m + 4 ( c ) from isotopic glutamine ( representing oxidative versus reductive TCA cycle flux ) .

O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O

It further inhibits oscillations in citrate m+5/m+4 ratio (c) from isotopic glutamine (representing oxidative versus reductive TCA cycle flux).


It further inhibits oscillations in acetyl - CoA m + 2 ( d ) from isotopic glutamine ( representing oxidative versus reductive TCA cycle flux ) .

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

It further inhibits oscillations in acetyl-CoA m+2 (d) from isotopic glutamine (representing oxidative versus reductive TCA cycle flux).


( e ) fraction of in G1 , S , G2 / M phases in synchronized HeLa after 3 hour treatment with DCA ( normalized by measurements in untreated control ) .

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

(e) The fraction of cells in G1, S, and G2/M phases in synchronized HeLa cells after 3 hour treatment with DCA (normalized by measurements in untreated control cells).


( f ) fraction of in G1 , S , G2 / M phases in non - synchronized HeLa HCT - 116 after 24 hour treatment with DCA ( normalized by measurements in untreated control ) . As shown , DCA significantly increases fraction of in S phase , inhibiting progression into G2 phase ; showing mean s . d . of n = 3 for isotopic labeling forms FACS measurements ( a - f ) . Statistical significance of changes in fraction of in each cycle phase following DCA treatment is calculated on two - , unequal variance t - ( e - f ) .

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

(f) The fraction of cells in G1, S, and G2/M phases in non-synchronized HeLa and HCT-116 cells after 24 hour treatment with DCA (normalized by measurements in untreated control cells). As shown, DCA significantly increases the fraction of cells in S phase, inhibiting cellular progression into G2 phase; showing mean and s.d. of n=3 for all isotopic labeling forms and FACS measurements (a-f). Statistical significance of changes in the fraction of cells in each cell cycle phase following DCA treatment is calculated based on two-tailed, unequal variance t-test (e-f).


B - D BSP amyloid aggregation in vitro is pH - dependent in 10 mM EDTA ( B ) , - dependent in 10 mM EDTA at pH 4 . 5 ( C ) strongly inhibited by millimolar Ca2 + at various concentrations ( D ) .

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

B-D BSP amyloid aggregation in vitro is pH-dependent in 10 mM EDTA (B), protein concentration-dependent in 10 mM EDTA and at pH 4.5 (C) and strongly inhibited by millimolar Ca2+ at various protein concentrations (D).


E - H showing fibril of BSP12 ( E ) , BSP123 ( F ) , BSP3 ( G ) , BSP45 ( H ) .

O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O

E-H TEM images showing fibril morphology of BSP12 (E), BSP123 (F), BSP3 (G), BSP45 (H).


I - J Amyloid aggregation by BSP subconstructs . BSP12 exhibits more prominent aggregation propensity than BSP3 BSP45 in 10 mM EDTA at pH 4 . 5 ( I ) . conjunction of module 3 with modules 1 2 promotes aggregation ( J ) .

O O O B-SMALL_MOLECULE B-EXP_ASSAY O O O O O O O O B-EXP_ASSAY O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O

I-J Amyloid aggregation by BSP subconstructs. BSP12 exhibits more prominent aggregation propensity than BSP3 and BSP45 in 10 mM EDTA and at pH 4.5 (I). The conjunction of module 3 with modules 1 and 2 promotes aggregation (J).


B Representative microscopy of BSP its subconstructs ( 20 μM ) at varying pHs . PEG 8K is 2 % . Scale bar , 20 μm .

O O B-EXP_ASSAY O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O

B Representative fluorescence microscopy images of BSP and its subconstructs (20 μM) at varying pHs. PEG 8K concentration is 2 %. Scale bar, 20 μm.


E of droplet is shown for each under various Ca2 + conditions . Each sample is quantified by total ( left ) mean ( right ) . Each experiment was repeated three times , data shown as mean ± SD .

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

E Quantification of droplet area is shown for each protein under various Ca2+ concentration conditions. Each sample is quantified by total area (left) and mean area (right). Each experiment was repeated three times, data shown as mean ± SD.


( A ) Identified acetylated histone peptides plotted against their log2 SILAC ( 1h after 16 J / m2 UV / mock ) , ranked by SILAC

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

(A) Identified acetylated histone peptides plotted against their log2 SILAC ratio (1h after 16 J/m2 UV/mock), ranked by SILAC ratio


( C ) Western blot of acid extracted histones WCE from HeLa , treated with HATi ( CTK7A , 100 μM CPTH2 , 50 μM ) or HDACi ( TSA , 1 μM ) for 4h or mock treated as

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

(C) Western blot of acid extracted histones and WCE from HeLa cells, treated with HATi (CTK7A, 100 μM and CPTH2, 50 μM) or HDACi (TSA, 1 μM) for 4h or mock treated as indicated


( D ) A representative western blot of histone acetylation of HeLa lysed at indicated time points after UV - irradiation ( 16 J / m2 ) . Blots were stained with α - acetyl - lysine ( top panels , high low exposure ) , α - histone H4 ( middle panel ) α - tubulin ( bottom panel )

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

(D) A representative western blot of histone acetylation levels of HeLa cells lysed at indicated time points after UV-irradiation (16 J/m2). Blots were stained with α-acetyl-lysine (top panels, high and low exposure), α-histone H4 (middle panel) and α-tubulin (bottom panel)


( F ) Representative western blots of HeLa WCE from different time points after UV - C irradiation ( 16J / m2 ) using indicated histone modification specific antibodies . Histone H4 tubulin were used as loading

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

(F) Representative western blots of HeLa WCE from different time points after UV-C irradiation (16J/m2) using the indicated histone modification specific antibodies. Histone H4 and tubulin were used as loading controls


( A ) Representative western blots of histone acetylation of HeLa lysed at indicated time points after UV irradiation ( 16 J / m2 , left panel ) , α - amanitin ( second panel , 100 µg / ml ) , THZ1 ( third panel , 1 μM ) or flavopiridol ( right panel , 1 μM ) treatment . Blots were stained with α - acetyl - lysine ( top panel ) , α - histone H4 ( middle panel ) α - tubulin ( bottom panel )

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

(A) Representative western blots of histone acetylation levels of HeLa cells lysed at indicated time points after UV irradiation (16 J/m2, left panel), α-amanitin (second panel, 100 µg/ml), THZ1 (third panel, 1 μM) or flavopiridol (right panel, 1 μM) treatment. Blots were stained with α-acetyl-lysine (top panel), α-histone H4 (middle panel) and α-tubulin (bottom panel)


( C ) Representative western blots of histone acetylation of HeLa pre - treated with transcription inhibitors ( mock ( left ) , THZ1 ( middle , 1 μM ) or flavopiridol ( right , 1 μM ) an hour before UV irradiation lysed at indicated time points after UV ( 16J / m2 ) . Blots were stained with indicated

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

(C) Representative western blots of histone acetylation levels of HeLa cells pre-treated with transcription inhibitors (mock (left), THZ1 (middle, 1 μM) or flavopiridol (right, 1 μM) an hour before UV irradiation and lysed at the indicated time points after UV (16J/m2). Blots were stained with the indicated antibodies


( A ) Representative western blots of histone acetylation of HeLa obtained at indicated time points after UV irradiation ( 16 J / m2 ) , HU / AraC treatment ( 100 mM / 10 μM ) or MMC treatment ( 10 µg / ml ) . Blots were stained with α - acetyl - lysine ( top panel ) , α - histone H4 ( middle panel ) α - tubulin ( bottom panel )

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

(A) Representative western blots of histone acetylation levels of HeLa cells obtained at the indicated time points after UV irradiation (16 J/m2), HU/AraC treatment (100 mM/10 μM) or MMC treatment (10 µg/ml). Blots were stained with α-acetyl-lysine (top panel), α-histone H4 (middle panel) and α-tubulin (bottom panel)


( C ) Representative Western blots , stained with indicated antibodies of HeLa pre - treated o / n with an S - phase inhibitor ( PHA 767491 hydrochloride , 10 μM ) lysed at indicated time points after UV ( 16 J / m2 )

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

(C) Representative Western blots, stained with the indicated antibodies of HeLa cells pre-treated o/n with an S-phase inhibitor (PHA 767491 hydrochloride, 10 μM) and lysed at indicated time points after UV (16 J/m2)


A Treatment scheme of experimental timeline Immunofluorescence of telogen backskin hair follicles ( HF ) for Integrin α6 ( Itgα6 , red ) , active Caspase 3 ( aCas3 , green ) DAPI ( blue ) in control ( Ctr ) Bcl - 2 inhibitor ( ABT - 199 ) treated mice . B : bulge , CH : club hair , DP : dermal papilla , HF : hair follicle , HG : secondary hair germ . Scale bar , 50 µm B of backskin HF with aCas3 + in bulge , hair germ dermal papilla of Ctr ABT - 199 treated mice ( n = 4 mice biological replicates ; mean ± standard deviation ( SD ) ; * * * p < 0 . 001 , ns = not significant , two - way ANOVA ) . nd = not detected .

O O O O O O B-EXP_ASSAY O O B-TISSUE I-TISSUE I-TISSUE O B-TISSUE O O B-GENEPROD I-GENEPROD O B-GENEPROD O O O O O B-GENEPROD I-GENEPROD O O O O O B-SMALL_MOLECULE O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O B-ORGANISM O O O B-TISSUE O B-TISSUE O B-TISSUE I-TISSUE O B-TISSUE O B-TISSUE I-TISSUE O B-TISSUE O B-TISSUE I-TISSUE O B-TISSUE O B-TISSUE I-TISSUE I-TISSUE O O O O O O O O B-TISSUE I-TISSUE O O O O B-TISSUE O B-TISSUE I-TISSUE I-TISSUE I-TISSUE O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-ORGANISM O O O O B-ORGANISM O O O O O O O O O O O O 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 Treatment scheme of experimental timeline and Immunofluorescence staining of telogen backskin hair follicles (HF) for Integrin α6 (Itgα6, red), active Caspase 3 (aCas3, green) and DAPI (blue) in control (Ctr) and Bcl-2 inhibitor (ABT-199) treated mice. B: bulge, CH: club hair, DP: dermal papilla, HF: hair follicle, HG: secondary hair germ. Scale bar, 50 µm B Quantification of backskin HF with aCas3+ cells in bulge, hair germ and dermal papilla of Ctr and ABT-199 treated mice (n=4 mice biological replicates; mean ± standard deviation (SD); *** p<0.001, ns=not significant, two-way ANOVA test). nd=not detected.


C , D Treatment scheme , FACS plot ( C ) ( D ) of keratinocytes stained for CD34 Itgα6 after 6 days of treatment with vehicle solution ( Ctr ) or ABT - 199 ( n = 6 biological replicates ; mean ± standard deviation ( SD ) ; * * p < 0 . 01 , ns = not significant , two - unpaired Student ' s t - ) . suprabasal ( sbBSC , CD34 + / Itgα6low ) , basal ( bBSC , CD34 + / Itgα6high ) BSCs n - B ( non - bulge epidermal ) . * * p < 0 . 005 .

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

C,D Treatment scheme, FACS plot (C) and quantification (D) of keratinocytes stained for CD34 and Itgα6 after 6 days of treatment with vehicle solution (Ctr) or ABT-199 (n=6 biological replicates; mean ± standard deviation (SD); ** p<0.01, ns=not significant, two-tailed unpaired Student's t-test). suprabasal (sbBSC, CD34+/Itgα6low), basal (bBSC, CD34+/Itgα6high) BSCs and n-B (non-bulge epidermal cells). ** p<0.005.


B Treatment scheme of experimental timeline histology of backskin sections of control ( Ctr ) ABT - 199 treated mice . Scale bar , 200 µm .

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

B Treatment scheme of experimental timeline and histology of backskin sections of control (Ctr) and ABT-199 treated mice. Scale bar, 200 µm.


D qRT - PCR for mRNA expression of transcription factors Lef1 Runx1 in epidermis of ABT - 199 treated control ( Ctr ) mice at P77 ( n = 3 biological replicates ; mean ± standard error of mean ( ) ; * * p < 0 . 01 , ns = not significant , two - unpaired Student ' s t - ) .

O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD I-GENEPROD O B-TISSUE O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O 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 qRT-PCR for mRNA expression of the transcription factors Lef1 and Runx1 in epidermis of ABT-199 treated and control (Ctr) mice at P77 (n=3 biological replicates; mean ± standard error of mean (SEM); ** p<0.01, ns=not significant, two-tailed unpaired Student's t-test).


C qRT - PCR for mRNA expression of anoikis - related markers Bim Bmf in sorted n - B ( non - bulge ) , bBSC ( basal BSCs ) sbBSC ( suprabasal BSCs ) ( n = 3 biological replicates ; mean ± standard error of mean ( ) ; * p < 0 . 05 , ns = not significant , two - way ANOVA ) .

O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY O O O O O B-GENEPROD I-GENEPROD O O O O O O O O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O 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 qRT-PCR for mRNA expression of the anoikis-related markers Bim and Bmf in sorted n-B (non-bulge), bBSC (basal BSCs) and sbBSC (suprabasal BSCs) (n=3 biological replicates; mean ± standard error of mean (SEM); * p<0.05, ns=not significant, two-way ANOVA test).


C FACS plot of keratinocytes stained for CD34 Itgα6 in 8 week old control ( Ctr ) Bcl - 2EOE mice ( n = 3 biological replicates ; mean ± standard deviation ( SD ) ; ns = not significant , two - unpaired Student ' s t - ) . suprabasal ( sbBSC , CD34 + / Itgα6low ) , basal ( bBSC , CD34 + / Itgα6high ) BSCs .

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C FACS plot and quantification of keratinocytes stained for CD34 and Itgα6 in 8 week old control (Ctr) and Bcl-2EOE mice (n=3 biological replicates; mean ± standard deviation (SD); ns=not significant, two-tailed unpaired Student's t-test). suprabasal (sbBSC, CD34+/Itgα6low), basal (bBSC, CD34+/Itgα6high) BSCs.


D Immunofluorescence of telogen backskin HF for BrdU incorporation ( 1 h , green ) DAPI ( blue ) in 8 week old control ( Ctr ) Bcl - 2EOE mice . Scale bar , 50 µm .

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D Immunofluorescence staining of telogen backskin HF for BrdU incorporation (1 h pulse, green) and DAPI (blue) in 8 week old control (Ctr) and Bcl-2EOE mice. Scale bar, 50 µm.


A Histology of P19 catagen backskin HF of control ( Ctr ) Bcl - 2EOE mice . RS : retracting strand , Scale bar , 50 µm . B of width of retracting strands of control ( Ctr ) Bcl - 2EOE mice ( n = 4 ; multiple measurements / biological replicate ; whiskers : min max ; boxes : median ± first third quartile ; * * * * p < 0 . 0001 , two - unpaired Student ' s t - ) .

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A Histology of P19 catagen backskin HF of control (Ctr) and Bcl-2EOE mice. RS: retracting strand, Scale bar, 50 µm. B Analysis of length and width of retracting strands of control (Ctr) and Bcl-2EOE mice (n=4; multiple measurements/biological replicate; whiskers: min and max; boxes: median ± first and third quartile; **** p<0.0001, two-tailed unpaired Student's t-test).


C Itgα6 aCas3 Immunofluorescence of catagen tailskin HF of control ( Ctr ) Bcl - 2EOE mice ( n = 4 biological replicates ) . Dashed lines : retracting strands . Scale bar , 100 µm . D of aCas3 + in RS of P19 Ctr Bcl - 2EOE mice ( n = 4 biological replicates ; mean ± standard deviation ( SD ) ; * * * * p < 0 . 0001 , two - unpaired Student ' s t - ) . HF : hair follicle .

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C Itgα6 and aCas3 Immunofluorescence staining of catagen tailskin HF of control (Ctr) and Bcl-2EOE mice (n=4 biological replicates). Dashed lines: retracting strands. Scale bar, 100 µm. D Quantification of aCas3+ cells in the RS of P19 Ctr and Bcl-2EOE mice (n=4 biological replicates; mean ± standard deviation (SD); **** p<0.0001, two-tailed unpaired Student's t-test). HF: hair follicle.


F , G qRT - PCR for mRNA expression of quiescence - related markers Bmp6 ( F ) Fgf18 ( G ) in epidermis of control ( Ctr ) Bcl - 2EOE mice ( P27 ) , ( n = 4 biological replicates ; mean ± standard error of mean ( ) ; * p < 0 . 05 , * * * p < 0 . 001 , two - unpaired Student ' s t - ) .

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F,G qRT-PCR for mRNA expression of the quiescence-related markers Bmp6 (F) and Fgf18 (G) in epidermis of control (Ctr) and Bcl-2EOE mice (P27), (n=4 biological replicates; mean ± standard error of mean (SEM); * p<0.05, *** p<0.001, two-tailed unpaired Student's t-test).


B Immunofluorescence of epidermal tail whole mounts of control ( Ctr ) , K15ΔNLef1 K15ΔNLef1 ; Bcl - 2EOE mice , stained for keratin 15 ( Krt15 , green ) , activated Caspase 3 ( aCas3 , red ) DAPI ( blue ) . Note increased number of aCas + in bulge of K15ΔNLef1 mice ( arrows ) . B : bulge , HG : secondary hair germ , SG : sebaceous . Scale bar , 100µm .

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B Immunofluorescence staining of epidermal tail whole mounts of control (Ctr), K15ΔNLef1 and K15ΔNLef1;Bcl-2EOE mice, stained for keratin 15 (Krt15, green), activated Caspase 3 (aCas3, red) and DAPI (blue). Note increased number of aCas+ cells in the bulge of K15ΔNLef1 mice (arrows). B: bulge, HG: secondary hair germ, SG: sebaceous gland. Scale bar, 100µm.


D Immunofluorescence of epidermal tail whole mounts of control ( Ctr ) , K15ΔNLef1 K15ΔNLef1 ; Bcl - 2EOE mice , stained for keratin 15 ( Krt15 , green ) , BrdU ( 1 h , red ) DAPI ( blue ) . Note increased number of BrdU + in bulge of K15ΔNLef1 mice ( arrows ) . B : bulge , HG : secondary hair germ , SG : sebaceous . Scale bar ,

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D Immunofluorescence staining of epidermal tail whole mounts of control (Ctr), K15ΔNLef1 and K15ΔNLef1;Bcl-2EOE mice, stained for keratin 15 (Krt15, green), BrdU (1 h pulse, red) and DAPI (blue). Note increased number of BrdU+ cells in the bulge of K15ΔNLef1 mice (arrows). B: bulge, HG: secondary hair germ, SG: sebaceous gland. Scale bar, 100µm


B , C Tumour incidence ( B ) frequency ( C ) in Ctr , Bcl - 2EOE , K15ΔNLef1 K15ΔNLef1 ; Bcl - 2EOE mice ( n = 10 biological replicates ; mean ± standard error of mean ( ) ) .

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B,C Tumour incidence (B) and frequency (C) in Ctr, Bcl-2EOE, K15ΔNLef1 and K15ΔNLef1;Bcl-2EOE mice (n=10 biological replicates; mean ± standard error of mean (SEM)).


D Histology of sebaceous tumours of K15ΔNLef1 K15ΔNLef1 ; Bcl - 2EOE mice . Scale bar , 100µm .

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D Histology of sebaceous tumours of K15ΔNLef1 and K15ΔNLef1;Bcl-2EOE mice. Scale bar, 100µm.


A ) Hek293T expressing Lamplight - 1D4 ( anti - 1D4 = green , DAPI = blue , Top ) schematic of BRET , measuring interaction of liberated Gβγ - Venus with GRK3 fragment - Nanoluciferase ( Bottom ) . Scale bar = 20µm . B ) Lamplight - driven response to increasing 1s 405nm C ) 405nm irradiance response curve D ) Response to 1s 405nm ( blue ) 525nm ( green ) E ) Response to 405nm ( blue ) followed by 525nm of different intensities ( green )

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

A) Hek293T cells expressing Lamplight-1D4 (anti-1D4 = green, DAPI = blue, Top) and schematic of BRET assay, measuring interaction of liberated Gβγ-Venus with GRK3 fragment-Nanoluciferase (Bottom). Scale bar = 20µm. B) Lamplight-driven response to increasing intensity 1s 405nm light C) 405nm irradiance response curve D) Response to 1s 405nm (blue line) and 525nm light (green line) E) Response to 405nm (blue line) followed by 525nm light of different intensities (green line)


A ) Hek293T expressing Lamplight - 1D4 ( anti - 1D4 = green , DAPI = blue , Top ) schematic of BRET , measuring interaction of liberated Gβγ - Venus with GRK3 fragment - Nanoluciferase ( Bottom ) . Scale bar = 20µm .

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A) Hek293T cells expressing Lamplight-1D4 (anti-1D4 = green, DAPI = blue, Top) and schematic of BRET assay, measuring interaction of liberated Gβγ-Venus with GRK3 fragment-Nanoluciferase (Bottom). Scale bar = 20µm.


G ) Response to 405nm alone or with 525nm

G) Response to 405nm alone or with 525nm light


H - I ) Representative time courses of Lamplight - driven responses to 8s mixed stimuli ( grey bar ) with different 405nm : 525nm ratios from 1 ( 100 % 405nm ) to 0 ( 100 % 525nm ) from ( H ) or ( I ) after 8s 405nm ( blue bar ) J ) Sustained Lamplight - driven activity ( mean BRET 20 - 80s after stimulus ) after exposure to mixed stimuli from ( ON ) or 405nm - adapted state ( OFF ) . Lamplight data fit with linear trendline . Best fitting parameters for ON , slope = 0 . 311 , Y - intercept = 0 . 04 , R2 = 0 . 85 . For OFF , slope = 0 . 30 , Y - intercept = 0 . 07 , R2 = 0 . 69 ) .

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H-I) Representative time courses of Lamplight-driven responses to 8s mixed stimuli (grey bar) with different 405nm: 525nm ratios from 1 (100% 405nm) to 0 (100% 525nm) from (H) dark or (I) after 8s 405nm light (blue bar) J) Sustained Lamplight-driven activity (mean BRET ratio 20-80s after stimulus) after exposure to mixed stimuli from dark (ON) or 405nm light-adapted state (OFF). Lamplight data fit with linear trendline. Best fitting parameters for ON, slope = 0.311, Y-intercept = 0.04, R2 = 0.85. For OFF, slope = 0.30, Y-intercept = 0.07, R2 = 0.69).


H - I ) Representative time courses of Lamplight - driven responses to 8s mixed stimuli ( grey bar ) with different 405nm : 525nm ratios from 1 ( 100 % 405nm ) to 0 ( 100 % 525nm ) from ( H ) or ( I ) after 8s 405nm ( blue bar )

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H-I) Representative time courses of Lamplight-driven responses to 8s mixed stimuli (grey bar) with different 405nm: 525nm ratios from 1 (100% 405nm) to 0 (100% 525nm) from (H) dark or (I) after 8s 405nm light (blue bar)


B ) Representative trace of hyperpolarisation to 405nm in a Lamplight - expressing SCN neuron C ) Hyperpolarisation to 405nm was observed over a range of intensities . Recordings from 3 different across 2 SCN slices

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B) Representative trace of hyperpolarisation to 405nm light in a Lamplight-expressing SCN neuron C) Hyperpolarisation to 405nm was observed over a range of intensities. Recordings from 3 different cells across 2 SCN slices


B ) Representative trace of hyperpolarisation to 405nm in a Lamplight - expressing SCN

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B) Representative trace of hyperpolarisation to 405nm light in a Lamplight-expressing SCN neuron


D ) Representative trace showing hyperpolarisation was sustained during after 405nm ( 5s ) reversed by 525nm ( 15s )

D) Representative trace showing hyperpolarisation was sustained during dark after 405nm light (5s) and reversed by 525nm light (15s)


E ) Hyperpolarisation depolarisation of a Lamplight - expressing neuron by 405nm 525nm , respectively , across multiple stimulus presentations

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E) Hyperpolarisation and depolarisation of a Lamplight-expressing neuron by 405nm and 525nm light, respectively, across multiple stimulus presentations


E ) Hyperpolarisation depolarisation of a Lamplight - expressing neuron by 405nm 525nm , respectively , across multiple stimulus

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F ) Three example traces from a single exposed to 405nm followed by 525nm . traces were fit with exponential decay ( 405nm , blue ) or association curves ( 525nm , green ) for 6 trials across 2

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F) Three example traces from a single cell exposed to 405nm followed by 525nm light. Individual traces were fit with exponential decay (405nm, blue) or association curves (525nm, green) for 6 trials across 2 cells


G , H ) Parameters from best - fit curves used to calculate ( G ) latency ( ms ) ( H ) half - life ( ms ) .

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G, H) Parameters from best-fit curves used to calculate (G) latency (ms) and (H) half-life (ms).


I ) Simultaneous 525nm 405nm prevents spike silencing J ) Constant 405nm ( 12 . 9 log photons ) presented with 525nm of decreasing from 14 . 9 - 12 . 9log photons in 6 steps of 10s ( left ) or 20s ( right ) . 525nm antagonised 405nm - driven hyperpolarisation for intensities except 12 . 9log photons

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I) Simultaneous 525nm and 405nm light prevents spike silencing J) Constant 405nm light (12.9 log photons) presented with 525nm light of decreasing intensity from 14.9 - 12.9log photons in 6 steps of 10s (left) or 20s (right). 525nm light antagonised the 405nm-driven hyperpolarisation for all intensities except 12.9log photons


I ) Simultaneous 525nm 405nm prevents spike silencing

I) Simultaneous 525nm and 405nm light prevents spike silencing


A ) L7Cre rd1 retina expressing Lamplight - T2A - mCherry AAV . Anti - mCherry ( ) was widespread ( Left , scale bar = 500µm ) . bodies found in different focal planes consistent with expression in ganglion ( Top Right , scale bar = 50µm ) bipolar ( Bottom Right , scale bar = 50µm ) .

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A) L7Cre rd1 retina expressing Lamplight-T2A-mCherry AAV. Anti-mCherry staining (white) was widespread (Left, scale bar = 500µm). Fluorescent cell bodies found in different focal planes consistent with expression in ganglion (Top Right, scale bar = 50µm) and bipolar cells (Bottom Right, scale bar = 50µm).


B ) Representative single unit showing characteristic ipRGC response .

B) Representative single unit showing characteristic ipRGC response.


C ) Three Lamplight response categories - transient excitation ( N = 35 biological replicates ) , transient inhibition ( N = 6 biological replicates ) , sustained inhibition ( N = 8 biological replicates ) . Left panel shows representative single unit response to 2s 405nm or 525nm . Mean baseline normalised firing ( grey shading shows ) is shown in centre left ( for entire recording , 500ms bins ) , centre right far right panels ( during 405nm 525nm stimuli , respectively 100ms bins , N as above , except N = 5 biological replicates for sustained response to 525nm ) .

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C) Three Lamplight response categories - transient excitation (N = 35 biological replicates), transient inhibition (N = 6 biological replicates), and sustained inhibition (N = 8 biological replicates). Left panel shows representative single unit response to 2s 405nm or 525nm light. Mean baseline normalised firing (grey shading shows SEM) is shown in centre left (for entire recording, 500ms bins), centre right and far right panels (during 405nm and 525nm stimuli, respectively 100ms bins, N as above, except N = 5 biological replicates for sustained response to 525nm).


D ) Representative single unit showing responses over 20 stimulus repeats .

D) Representative single unit showing light responses over 20 stimulus repeats.


E ) Distribution of response latencies for 3 Lamplight - driven responses to 405nm , as well as reversal caused by 525nm in sustained inhibition units . Two outliers with slow latency ( > 2 . 5s ) in transient excitation group are likely responding to stimulus offset ( N = 35 for Transient Excitation , N = 6 for Transient Inhibition , N = 7 for Sustained Inhibition N = 5 for Sustained Excitation , biological replicates ) .

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E) Distribution of response latencies for 3 Lamplight-driven responses to 405nm, as well as reversal caused by 525nm in sustained inhibition units. Two outliers with slow latency (>2.5s) in transient excitation group are likely responding to stimulus offset (N = 35 for Transient Excitation, N = 6 for Transient Inhibition, N = 7 for Sustained Inhibition and N = 5 for Sustained Excitation, all biological replicates).


F ) Inhibition responses were smaller less sustained for mixed stimuli ( 50 % 405nm , 50 % 525nm ) compared to 405nm only ( N = 14 biological replicates ) . G ) Excitation responses were attenuated for mixed stimuli compared to 405nm only . Left panel shows representative retinal single unit ( N = 35 biological replicates ) . Data information : For ( F - G ) Left panel shows representative single unit . Right panel shows distribution of difference in firing rate for 405nm vs mixed stimulus . representative single units show perievent rasters ( first trial at top ) firing rate histograms ( Bin = 500ms ) . Timing of stimuli shown by shaded vertical bars . stimuli are 16 log total Lamplight effective photons / cm2 / s . Error bars show . * p < 0 . 05 , * * * p < 0 . 001 ( One - sample t - compared to zero ) .

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F) Inhibition responses were smaller and less sustained for mixed stimuli (50% 405nm, 50% 525nm) compared to 405nm only (N = 14 biological replicates). G) Excitation responses were attenuated for mixed stimuli compared to 405nm only. Left panel shows representative retinal single unit (N = 35 biological replicates). Data information: For (F-G) Left panel shows representative single unit. Right panel shows distribution of difference in firing rate for 405nm vs mixed stimulus. All representative single units show perievent rasters (first trial at top) and firing rate histograms (Bin size = 500ms). Timing of light stimuli shown by shaded vertical bars. All light stimuli are 16 log total Lamplight effective photons/cm2/s. Error bars show SEM. * p<0.05, *** p < 0.001 (One-sample t-test compared to zero).


F ) Inhibition responses were smaller less sustained for mixed stimuli ( 50 % 405nm , 50 % 525nm ) compared to 405nm only ( N = 14 biological replicates ) .

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F) Inhibition responses were smaller and less sustained for mixed stimuli (50% 405nm, 50% 525nm) compared to 405nm only (N = 14 biological replicates).


( E - G ) cortical neurons from E15 cerebral cortices were transfected with indicated plasmids , incubated for two days in vitro treated with Tf - 555 for 30 minutes before fixation . were immunostained with indicated antibodies . are obtained with high - resolution microscopy ( Nikon ) . Blue alone channels are shown in black . Lower panels in ( E ) ( F ) are high magnification indicated by or blue rectangles in upper panels . graph in ( G ) shows Pearson ' s correlation coefficient of Tf - 555 Rab11 or APPL1 . Each score represents mean with points . Control : n = 23 , Rab21 - sh115 : n = 18 , Rab5 - sh232 ( Tf - Rab11 ) : n = 23 , Rab5 - sh232 ( Tf - APPL1 ) : n = 22 .

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(E-G) Primary cortical neurons from E15 cerebral cortices were transfected with the indicated plasmids, incubated for two days in vitro and treated with Tf-555 for 30 minutes before fixation. Cells were immunostained with the indicated antibodies. The images are obtained with high-resolution microscopy (Nikon). Blue alone channels are shown in black and white images. Lower panels in (E) and (F) are high magnification images indicated by white or blue rectangles in upper panels. The graph in (G) shows the Pearson's correlation coefficient of Tf-555 and Rab11 or APPL1. Each score represents the mean with the individual points. Control: n = 23 cells, Rab21-sh115: n = 18 cells, Rab5-sh232 (Tf - Rab11): n = 23 cells, Rab5-sh232 (Tf - APPL1): n = 22 cells.


Cerebral cortices at E17 , electroporated with indicated plasmids plus pCAG - EGFP at E14 . ( C ) of locomoting neurons with more than three neurites in IZ . Each score represents mean with points . Control : n = 4 brains ( 60 ) , Rab21 - sh115 : n = 3 brains ( 65 ) , Rab5 - sh232 : n = 4 brains ( 111 ) .

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Cerebral cortices at E17, electroporated with the indicated plasmids plus pCAG-EGFP at E14. (C) The ratio of locomoting neurons with more than three primary neurites in the IZ. Each score represents the mean with the individual points. Control: n = 4 brains (60 cells), Rab21-sh115: n = 3 brains (65 cells), Rab5-sh232: n = 4 brains (111 cells).


( E ) Time - lapse imaging of control Rab21 - sh115 - electroporated in cortical slices from E16 cerebral cortices , electroporated with indicated plasmids at E14 . After formation of leading process , control neurons rapidly eliminated their immature neurites ( yellow arrows ) , whereas Rab21 - knockdown neurons retained immature neurites ( arrows ) for long periods . Time interval of each frame is 30 min .

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(E) Time-lapse imaging of control and Rab21-sh115-electroporated cells in cortical slices from E16 cerebral cortices, electroporated with the indicated plasmids at E14. After formation of the leading process, control neurons rapidly eliminated their immature neurites (yellow arrows), whereas the Rab21-knockdown neurons retained the immature neurites (white arrows) for long periods. Time interval of each frame is 30 min.


cortical neurons from E15 cerebral cortices were transfected with pCAG - PM - mAG1 ( green ) incubated for two days in vitro . were immunostained with indicated antibodies ( red blue / ) . transfected PM - mAG1 is a marker for plasma . are obtained with high - resolution microscopy ( Nikon ) lower panels are high magnification near plasma . Blue alone channels are shown in black . Each score represents mean of ratios with points . Rab5 : n = 18 ( middle right ) , Rab21 : n = 31 ( left middle ) or 20 ( right ) , Caveolin - 1 : n = 43 ( left ) .

B-CELL_TYPE I-CELL_TYPE O O B-TISSUE I-TISSUE O O O O O B-SUBCELLULAR O B-GENEPROD O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O B-SUBCELLULAR O B-GENEPROD O O O O B-SUBCELLULAR O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O

Primary cortical neurons from E15 cerebral cortices were transfected with pCAG-PM-mAG1 (green) and incubated for two days in vitro. Cells were immunostained with the indicated antibodies (red and blue/white). The transfected PM-mAG1 is a marker for the plasma membrane. The images are obtained with high-resolution microscopy (Nikon) and the lower panels are high magnification images near the plasma membrane. Blue alone channels are shown in black and white images. Each score represents the mean of ratios with the individual points. Rab5: n = 18 cells (middle and right), Rab21: n = 31 cells (left and middle) or 20 cells (right), Caveolin-1: n = 43 cells (left).


( H - I ) HeLa were immunostained with indicated antibodies . are obtained with high - resolution microscopy ( Nikon ) . graph in ( I ) shows Pearson ' s correlation coefficient between caveolin - 1 Rab5 or Rab21 . Each score represents mean of ratios with points . Rab5 : n = 80 , Rab21 : n = 62 .

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

(H-I) HeLa cells were immunostained with the indicated antibodies. The images are obtained with high-resolution microscopy (Nikon). The graph in (I) shows the Pearson's correlation coefficient between caveolin-1 and Rab5 or Rab21. Each score represents the mean of ratios with the individual points. Rab5: n = 80 cells, Rab21: n = 62 cells.


cortical neurons from E15 cerebral cortices were transfected with indicated plasmids plus pCAG - EGFP , incubated for two days in vitro treated with BODIPY - LacCer ( LacCer ) ( green ) ( A - B ) for 30 minutes before fixation . arrows in ( A ) indicate perinuclear accumulation of LacCer . Blue alone channels are shown in black . graphs in ( B ) show of with perinuclear accumulation of LacCer ( B ) which was quantified in a blinded counting . Each score represents mean with points . B : n = 5 biological replicates ( Control : 115 , Cav1 - sh490 : 127 , Rab21 - sh115 : 136 , Rab5 - sh232 : 216 , Rab21 - sh115 + wt - Rab21 : 132 )

B-CELL_TYPE I-CELL_TYPE O O B-TISSUE I-TISSUE O O O O O O O O B-GENEPROD O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O B-GENEPROD O O O O O B-GENEPROD O O O O O B-GENEPROD O O O O O B-GENEPROD O O O

Primary cortical neurons from E15 cerebral cortices were transfected with the indicated plasmids plus pCAG-EGFP , incubated for two days in vitro and treated with BODIPY-LacCer (LacCer) (green) (A-B) for 30 minutes before fixation. White arrows in (A) indicate the perinuclear accumulation of LacCer. Blue alone channels are shown in black and white images. The graphs in (B) show the ratio of cells with perinuclear accumulation of LacCer (B) which was quantified in a blinded counting. Each score represents the mean with the individual points. B: n = 5 biological replicates (Control: 115 cells, Cav1-sh490: 127 cells, Rab21-sh115: 136 cells, Rab5-sh232: 216 cells, Rab21-sh115 + wt-Rab21: 132 cells)


cortical neurons from E15 cerebral cortices were transfected with pCAG - PM - mAG1 , incubated for two days in vitro stained with indicated antibodies . Blue alone channels are shown in black . Arrowheads indicate accumulation of N - cadherin at plasma . Lower panels in ( D ) are high magnification indicated by or blue rectangles . are obtained with high - resolution microscopy ( Nikon ) .

B-CELL_TYPE I-CELL_TYPE O O B-TISSUE I-TISSUE O O O O O B-SUBCELLULAR O B-GENEPROD O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O

Primary cortical neurons from E15 cerebral cortices were transfected with pCAG-PM-mAG1, incubated for two days in vitro and stained with the indicated antibodies. Blue alone channels are shown in black and white images. Arrowheads indicate accumulation of N-cadherin at the plasma membrane. Lower panels in (D) are high magnification images indicated by white or blue rectangles. The images are obtained with high-resolution microscopy (Nikon).


Immature neurons in IZ of cerebral cortices at E17 , electroporated with indicated plasmids plus pCAG - PM - mAG1 pCAG - HA - N - cadherin at E14 . Frozen sections were immunostained with anti - mAG1 anti - HA antibodies . Arrow arrowheads indicate accumulation of HA - N - cadherin at perinuclear regions or plasma , respectively . Control : n = 77 from 3 biological replicates , Rab21 - sh115 : n = 108 from 3 biological replicates .

B-CELL_TYPE I-CELL_TYPE O B-TISSUE O B-TISSUE I-TISSUE O O O O O O O O O O B-SUBCELLULAR O B-GENEPROD O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-EXP_ASSAY O O O B-GENEPROD O O B-GENEPROD O O O O O O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-SUBCELLULAR O O B-SUBCELLULAR O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O

Immature neurons in the IZ of the cerebral cortices at E17, electroporated with the indicated plasmids plus pCAG-PM-mAG1 and pCAG-HA-N-cadherin at E14. Frozen sections were immunostained with anti-mAG1 and anti-HA antibodies. Arrow and arrowheads indicate the accumulation of HA-N-cadherin at the perinuclear regions or the plasma membrane, respectively. Control: n = 77 cells from 3 biological replicates, Rab21-sh115: n = 108 cells from 3 biological replicates.


( A - B ) cortical neurons from E15 cerebral cortices were transfected with indicated plasmids plus pCAG - PM - mAG1 ( green ) incubated for two days in vitro . Immunocytochemical analyses with anti - caveolin - 1 ( red ) antibody were performed . are obtained with high - resolution microscopy ( Nikon ) . graph in ( B ) indicates of caveolin - 1 signals in plasma to total intensities of caveolin - 1 in each immature neuron . Each score represents mean of ratios with points . Control : n = 20 , Rab21 - sh115 : n = 22 , Rab21 - sh115 + wt - Rab21 : n = 23 . Overexpression of wt - Rab21 resulted in neuronal , which making it difficult to determine accurate of wt - Rab21 in rescue experiments .

O O O O O B-CELL_TYPE I-CELL_TYPE O O B-TISSUE I-TISSUE O O O O O O O O B-SUBCELLULAR O B-GENEPROD O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-SUBCELLULAR O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-CELL_TYPE I-CELL_TYPE O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O B-GENEPROD O O O O O B-GENEPROD O O O O O O O O O B-GENEPROD O O B-CELL_TYPE O O O O O O O O O O O B-GENEPROD O O O O

(A-B) Primary cortical neurons from E15 cerebral cortices were transfected with the indicated plasmids plus pCAG-PM-mAG1 (green) and incubated for two days in vitro. Immunocytochemical analyses with anti-caveolin-1 (red) antibody were performed. The images are obtained with high-resolution microscopy (Nikon). The graph in (B) indicates the ratio of the caveolin-1 staining signals in the plasma membrane to total fluorescence intensities of caveolin-1 in each immature neuron. Each score represents the mean of ratios with the individual points. Control: n = 20 cells, Rab21-sh115: n = 22 cells, Rab21-sh115 + wt-Rab21: n = 23 cells. Overexpression of wt-Rab21 resulted in neuronal cell death, which making it difficult to determine the accurate DNA concentration of wt-Rab21 in the rescue experiments.


( H - I ) cortical neurons from E15 cerebral cortices were transfected with indicated plasmids , incubated for two days in vitro , treated with Bafilomycin A1 ( Baf A1 ) for 6 h stained with indicated antibodies . are obtained with high - resolution microscopy ( Nikon ) lower panels are high magnification of lysosomes , indicated by or blue rectangles . Blue alone channels are shown in black . graph in ( I ) shows Pearson ' s correlation coefficient between caveolin - 1 Lamp1 , a lysosomal marker , in control Rab21 - sh115 - transfected neurons . Each score represents mean of ratios with points . Control : n = 31 , Rab21 - sh115 : n = 22 .

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

(H-I) Primary cortical neurons from E15 cerebral cortices were transfected with the indicated plasmids, incubated for two days in vitro, treated with Bafilomycin A1 (Baf A1) for 6 h and stained with the indicated antibodies. The images are obtained with high-resolution microscopy (Nikon) and the lower panels are high magnification images of the lysosomes, indicated by white or blue rectangles. Blue alone channels are shown in black and white images. The graph in (I) shows the Pearson's correlation coefficient between caveolin-1 and Lamp1, a lysosomal marker, in control and Rab21-sh115-transfected neurons. Each score represents the mean of ratios with the individual points. Control: n = 31 cells, Rab21-sh115: n = 22 cells.


( A ) Locomoting neurons in upper IZ of cerebral cortices at E17 , electroporated with indicated plasmids plus pCAG - EGFP at E14 . ( B ) of locomoting neurons with more than three neurites . Each score represents mean of ratios with points . Control : n = 4 brains , Rab21 - sh115 : n = 5 brains , Rab21 - sh115 + Caveolin - 1 : n = 6 brains . ( C ) box - - whisker plot shows average leading process of locomoting neurons in IZ . Control : n = 115 , Rab21 - sh115 : n = 188 , Rab21 - sh115 + Caveolin - 1 : n = 185 . (

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(A) Locomoting neurons in the upper IZ of the cerebral cortices at E17, electroporated with the indicated plasmids plus pCAG-EGFP at E14. (B) The ratio of locomoting neurons with more than three primary neurites. Each score represents the mean of ratios with the individual points. Control: n = 4 brains, Rab21-sh115: n = 5 brains, Rab21-sh115+Caveolin-1: n = 6 brains. (C) The box-and-whisker plot shows average leading process length of the locomoting neurons in the IZ. Control: n = 115 cells, Rab21-sh115: n = 188 cells, Rab21-sh115+Caveolin-1: n = 185 cells. (


( D - E ) Cerebral cortices at P0 , electroporated with indicated plasmids plus pCAG - EGFP at E14 . lower graphs in ( D ) graph in ( E ) show estimation of migration , as measured by EGFP intensities in distinct regions of cerebral cortices using Leica SP5 software . Each bar in graph in ( E ) represents mean of relative intensities . Rab21 - sh115 : n = 4 brains , Rab21 - sh115 + CAG - wt - caveolin - 1 : n = 6 brains . II - IV : layers II - IV of cortical plate , V - VI : layers V - VI of cortical plate , IZ : intermediate zone , WM : matter , SVZ / VZ : subventricular zone / ventricular zone .

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

(D-E) Cerebral cortices at P0, electroporated with the indicated plasmids plus pCAG-EGFP at E14. The lower graphs in (D) and the graph in (E) show the estimation of cell migration, as measured by EGFP fluorescence intensities in distinct regions of the cerebral cortices using Leica SP5 software. Each bar in the graph in (E) represents the mean percentage of relative intensities. Rab21-sh115: n = 4 brains, Rab21-sh115+CAG-wt-caveolin-1: n = 6 brains. II-IV: layers II-IV of the cortical plate, V-VI: layers V-VI of the cortical plate, IZ: intermediate zone, WM: white matter, SVZ/VZ: subventricular zone/ventricular zone.


B . Analyses of Hog1 phosphorylation by immunoblotting with anti - phospho - p38 ( Hog1 - P ) anti - Hog1 ( total Hog1 ) antibodies . of indicated genotypes were stimulated with indicated concentrations of NaCl for indicated time . Strains used are TM257 , KT207 KY594 - 1 .

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

B. Analyses of Hog1 phosphorylation by immunoblotting with anti-phospho-p38 (Hog1-P) and anti-Hog1 (total Hog1) antibodies. Cells of the indicated genotypes were stimulated with the indicated concentrations of NaCl for the indicated time. Strains used are TM257, KT207 and KY594-1.


C . Analyses of Hog1 phosphorylation by Phos - tag band - shift . Yeast KY594 - 1 was stimulated with indicated concentrations of NaCl for 5 min . percentages of phosphorylated Hog1 [ Hog1 - P ( % ) ] were calculated as explained in Materials Methods , are shown beneath panel . Data information : Representative results from three independent experiments .

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

C. Analyses of Hog1 phosphorylation by Phos-tag band-shift assay. Yeast strain KY594-1 was stimulated with the indicated concentrations of NaCl for 5 min. The percentages of phosphorylated Hog1 [Hog1-P (%)] were calculated as explained in Materials and Methods, and are shown beneath the panel. Data information: Representative results from three independent experiments.


D . Analyses of Hog1 phosphorylation by immunoblotting with anti - phospho - p38 ( Hog1 - P ) anti - Hog1 ( total Hog1 ) antibodies . Yeast KT219 was transformed with indicated STE11 mutant gene carried by a single - copy plasmid that is expressed from STE11 promoter : vec , vector ; WT , wild - type ; DDD , S281D / S285D / T286D . were incubated with ( + ) or without ( - ) 1 M NaCl for 5 min .

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

D. Analyses of Hog1 phosphorylation by immunoblotting with anti-phospho-p38 (Hog1-P) and anti-Hog1 (total Hog1) antibodies. Yeast strain KT219 was transformed with the indicated STE11 mutant gene carried by a single-copy plasmid that is expressed from the STE11 promoter: vec, vector; WT, wild-type; DDD, S281D/S285D/T286D. Cells were incubated with (+) or without (-) 1 M NaCl for 5 min.


E - H . Analyses of Hog1 phosphorylation by Phos - tag band - shift . Yeast strains ( E ) KY603 - 3 ; ( F ) TM142 ; ( G ) TM257 ; ( H ) FP54 were stimulated with indicated concentrations of NaCl for 5 min . Data information : Representative results from three independent experiments .

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

E-H. Analyses of Hog1 phosphorylation by Phos-tag band-shift assay. Yeast strains (E) KY603-3; (F) TM142; (G) TM257; and (H) FP54 were stimulated with the indicated concentrations of NaCl for 5 min. Data information: Representative results from three independent experiments.


I . Comparison of NaCl dose - responses of Hog1 activation by various strains . Phos - tag band - shift assays were independently repeated three times , average values were plotted . Data information : ( I ) Error bars are ( n = 3 ) .

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

I. Comparison of the NaCl dose-responses of Hog1 activation by various strains. Phos-tag band-shift assays were independently repeated three times, and average values were plotted. Data information: (I) Error bars are SEM (n=3).


A - C . Immunoblot analyses of Hog1 phosphorylation . Yeast strains ( A ) KT235 ; ( B ) KT209 ; ( C ) KT234 were stimulated with indicated concentrations of NaCl for 10 min , phosphorylated Hog1 ( Hog1 - P ) total Hog1 in lysates were detected by immunoblotting . relevant genotypes of strains are indicated in top row of each panel , are schematically shown in diagrams at left . second row from top indicates genes carried by a single - copy plasmid that are expressed from their own promoters . vec , vector ; WT , wild - type .

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

A-C. Immunoblot analyses of Hog1 phosphorylation. Yeast strains (A) KT235; (B) KT209; and (C) KT234 were stimulated with the indicated concentrations of NaCl for 10 min, and phosphorylated Hog1 (Hog1-P) and total Hog1 in cell lysates were detected by immunoblotting. The relevant genotypes of the strains are indicated in the top row of each panel, and are schematically shown in the diagrams at left. The second row from the top indicates the genes carried by a single-copy plasmid that are expressed from their own promoters. vec, vector; WT, wild-type.


D . Phos - tag band - shift analyses of Hog1 phosphorylation . yeast KT234 carrying single - copy expression plasmid YCplac22I ' - Pbs2 S514D / T518D was stimulated with indicated concentrations of NaCl for 5 min .

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D. Phos-tag band-shift analyses of Hog1 phosphorylation. The yeast strain KT234 carrying the single-copy expression plasmid YCplac22I'-Pbs2 S514D/T518D was stimulated with the indicated concentrations of NaCl for 5 min.


E . Comparison of NaCl dose - responses of Hog1 activation by constitutively - active Ste11 - Q301P constitutively - active Pbs2 - DD . Phos - tag band - shift assays were independently repeated three times , average values were plotted . Data information : ( E ) Error bars are ( n = 3 ) .

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

E. Comparison of the NaCl dose-responses of Hog1 activation by constitutively-active Ste11-Q301P and constitutively-active Pbs2-DD. Phos-tag band-shift assays were independently repeated three times, and the average values were plotted. Data information: (E) Error bars are SEM (n=3).


C . Immunoblot analyses of Hog1 phosphorylation . yeast KT235 was transformed with pRS416 - FLAG - Hog1 ( WT ) or its indicated deletion derivatives . FLAG - Hog1 was immunoprecipitated ( IP ) , immunoblotted ( IB ) with anti - phospho - p38 ( for Hog1 - P ; upper panel ) or anti - FLAG ( for total FLAG - Hog1 ; lower panel )

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C. Immunoblot analyses of Hog1 phosphorylation. The yeast strain KT235 was transformed with pRS416-FLAG-Hog1 (WT) or its indicated deletion derivatives. FLAG-Hog1 was immunoprecipitated (IP), and immunoblotted (IB) with anti-phospho-p38 (for Hog1-P; upper panel) or anti-FLAG (for total FLAG-Hog1; lower panel)


D - G . Phos - tag band - shift of Hog1 phosphorylation . Yeast ( D - E ) KT235 or ( F - G ) KT290 carrying single - copy expression plasmid YCplac22I ' - Pbs2 S514D / T518D was transformed with either pRS416 - Hog1 ( WT ) or its indicated mutant derivatives , was treated with indicated concentrations of NaCl for 5 min . ( D ) ( F ) show typical results , ( E ) ( G ) summarizes averages of three independent experiments . Data information : ( E , G Error bars are ( n = 3 ) .

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

D-G. Phos-tag band-shift assay of Hog1 phosphorylation. Yeast strain (D-E) KT235 or (F-G) KT290 carrying the single-copy expression plasmid YCplac22I'-Pbs2 S514D/T518D was transformed with either pRS416-Hog1 (WT) or its indicated mutant derivatives, and was treated with the indicated concentrations of NaCl for 5 min. (D) and (F) show typical results, and (E) and (G) summarizes the averages of three independent experiments. Data information: (E, G Error bars are SEM (n=3).


H . Phos - tag band - shift of Hog1 phosphorylation . yeast FP4 was transformed with single - copy expression plasmid pRS416 - Hog1 ( WT ) or pRS416 - Hog1 - ΔL16 , was treated with indicated concentrations of NaCl for 5 min . averages of three independent experiments are shown . Data information : Error bars are ( n = 3 ) .

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H. Phos-tag band-shift assay of Hog1 phosphorylation. The yeast strain FP4 was transformed with the single-copy expression plasmid pRS416-Hog1 (WT) or pRS416-Hog1-ΔL16, and was treated with the indicated concentrations of NaCl for 5 min. The averages of three independent experiments are shown. Data information: Error bars are SEM (n=3).


A . Phos - tag band - shift analyses of osmostress - induced Hog1 phosphorylation . yeast FP4 ( hog1Δ ) was transformed with single - copy expression plasmid pRS416 - Hog1 - ΔL16 , was stimulated with indicated concentrations of NaCl for 5 min . B . Averages of three independent experiments from A were plotted . Results for Hog1 - WT ( from Fig 1I ) are included for comparison . Data information : Representative results from three independent experiments . Error bars are ( n = 3 ) .

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

A. Phos-tag band-shift analyses of osmostress-induced Hog1 phosphorylation. The yeast strain FP4 (hog1Δ) was transformed with the single-copy expression plasmid pRS416-Hog1-ΔL16, and was stimulated with the indicated concentrations of NaCl for 5 min. B. Averages of three independent experiments from A were plotted. Results for Hog1-WT (from Fig 1I) are included for comparison. Data information: Representative results from three independent experiments. Error bars are SEM (n=3).


C - D . yeast KT259 ( ste11Δ hog1Δ ) was used . Data information : Representative results from three independent experiments . Error bars are ( n = 3 ) .

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C-D. the yeast strain KT259 (ste11Δ hog1Δ) was used. Data information: Representative results from three independent experiments. Error bars are SEM (n=3).


E - F . yeast KY523 ( ssk2 / 22Δ hog1Δ ) was used . Data information : Representative results from three independent experiments . Error bars are ( n = 3 ) .

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E-F. the yeast strain KY523 (ssk2/22Δ hog1Δ) was used. Data information: Representative results from three independent experiments. Error bars are SEM (n=3).


Detection of Pbs2 phosphorylation at S514 yeast strains KT003 ( pbs2Δ ) , KT005 ( ste11Δ pbs2Δ ) , TM280 ( ssk2 / 22Δ pbs2Δ ) KT043 ( ste11Δ ssk2 / 22Δ pbs2Δ ) were transformed with YCplac22I ' - Pbs2 - HA , were treated with indicated concentrations of NaCl for 5 min . Pbs2 - HA was immunoprecipitated from extract , phosphorylated Pbs2 was analyzed by ( A ) Phos - tag band - shift In ( A ) , positions of Pbs2 - HA phosphorylated ( S514 - P ) unphosphorylated ( S514 - OH ) at S514 are indicated .

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Detection of Pbs2 phosphorylation at S514 The yeast strains KT003 (pbs2Δ), KT005 (ste11Δ pbs2Δ), TM280 (ssk2/22Δ pbs2Δ) and KT043 (ste11Δ ssk2/22Δ pbs2Δ) were transformed with YCplac22I'-Pbs2-HA, and were treated with the indicated concentrations of NaCl for 5 min. Pbs2-HA was immunoprecipitated from cell extract, and phosphorylated Pbs2 was analyzed by (A) Phos-tag band-shift assay In (A), the positions of Pbs2-HA phosphorylated (S514-P) and unphosphorylated (S514-OH) at S514 are indicated.


Detection of Pbs2 phosphorylation at T518 . yeast strains KT003 ( pbs2Δ ) , KT005 ( ste11Δ pbs2Δ ) , TM280 ( ssk2 / 22Δ pbs2Δ ) KT043 ( ste11Δ ssk2 / 22Δ pbs2Δ ) were transformed with YCplac22I ' - Pbs2 - HA , were treated with indicated concentrations of NaCl for 5 min . Pbs2 - HA was immunoprecipitated from extract , phosphorylated Pbs2 was analyzed by ( B ) anti - phospho - T518 immunoblotting .

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Detection of Pbs2 phosphorylation at T518. The yeast strains KT003 (pbs2Δ), KT005 (ste11Δ pbs2Δ), TM280 (ssk2/22Δ pbs2Δ) and KT043 (ste11Δ ssk2/22Δ pbs2Δ) were transformed with YCplac22I'-Pbs2-HA, and were treated with the indicated concentrations of NaCl for 5 min. Pbs2-HA was immunoprecipitated from cell extract, and phosphorylated Pbs2 was analyzed by (B) anti-phospho-T518 immunoblotting.


C - F . NaCl dose - response analyses of Pbs2 phosphorylation . ( C - D ) KT005 ( ste11Δ pbs2Δ ) ( E - F ) TM280 ( ssk2 / 22Δ pbs2Δ ) were transformed with YCplac22I ' - Pbs2 - HA , were treated with indicated concentrations of NaCl for 5 min . ( C E ) S514 phosphorylation was analyzed using Phos - tag band - shift . PC ; Positive Control . ( D F ) T518 phosphorylation was analyzed using anti - phospho - T518 immunoblotting . G - H . Average values of three independent experiments from ( C - D ) ( E - F ) , respectively , were plotted . AU , arbitrary unit . Data information : ( C - F ) Representative results from three independent experiments . ( G - H ) Error bars are ( n = 3 ) .

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

C-F. NaCl dose-response analyses of Pbs2 phosphorylation. (C-D) KT005 (ste11Δ pbs2Δ) and (E-F) TM280 (ssk2/22Δ pbs2Δ) were transformed with YCplac22I'-Pbs2-HA, and were treated with the indicated concentrations of NaCl for 5 min. (C and E) S514 phosphorylation was analyzed using the Phos-tag band-shift assay. PC; Positive Control. (D and F) T518 phosphorylation was analyzed using anti-phospho-T518 immunoblotting. G-H. Average values of three independent experiments from (C-D) and (E-F), respectively, were plotted. AU, arbitrary unit. Data information: (C-F) Representative results from three independent experiments. (G-H) Error bars are SEM (n=3).


I . Detection of di - phosphorylated Pbs2 . KT005 ( ste11Δ pbs2Δ ) transformed with YCplac22I ' - Pbs2 - HA was treated with indicated concentrations of NaCl for 5 min . Pbs2 - HA was immunoprecipitated from extracts , subjected to Phos - tag SDS - PAGE . Blots of these gels were probed with ( upper panel ) anti - phospho - T518 or ( lower panel ) anti - HA . J . Same as in ( I ) , except that yeast KT003 ( pbs2Δ ) was used .

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

I. Detection of di-phosphorylated Pbs2. KT005 (ste11Δ pbs2Δ) transformed with YCplac22I'-Pbs2-HA was treated with the indicated concentrations of NaCl for 5 min. Pbs2-HA was immunoprecipitated from cell extracts, and subjected to Phos-tag SDS-PAGE. Blots of these gels were probed with (upper panel) anti-phospho-T518 or (lower panel) anti-HA. J. Same as in (I), except that the yeast strain KT003 (pbs2Δ) was used.


B . Phos - tag band - shift analyses of osmostress - induced Hog1 phosphorylation . KT005 ( ste11Δ pbs2Δ ) was transformed with YCplac22I ' - Pbs2 - HA ( WT ) or its indicated mutant derivatives . were stimulated with ( + ) or without ( - ) 0 . 6 M NaCl for 5 min .

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

B. Phos-tag band-shift analyses of osmostress-induced Hog1 phosphorylation. KT005 (ste11Δ pbs2Δ) was transformed with YCplac22I'-Pbs2 -HA (WT) or its indicated mutant derivatives. Cells were stimulated with (+) or without (-) 0.6 M NaCl for 5 min.


C - F . Phos - tag band - shift analyses of Hog1 phosphorylation . KT291 ( ste11Δ pbs2Δ hog1Δ ) was transformed with ( C D ) pRS416 - Hog1 or ( E F ) pRS416 - Hog1 - ΔL16 , together with ( C E ) YCplac22I ' - Pbs2 - S514A - HA or ( D F ) YCplac22I ' - Pbs2 - T518A - HA . were stimulated with indicated concentrations of NaCl for 5 min . G - H . Average values of three independent experiments from ( C E ) ( D F ) , respectively , are plotted . Data information : ( C - F ) Representative results from three independent experiments . ( G - H ) Error bars are ( n = 3 ) .

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

C-F. Phos-tag band-shift analyses of Hog1 phosphorylation. KT291 (ste11Δ pbs2Δ hog1Δ) was transformed with (C and D) pRS416-Hog1 or (E and F) pRS416-Hog1-ΔL16, together with (C and E) YCplac22I'-Pbs2-S514A-HA or (D and F) YCplac22I'-Pbs2-T518A-HA. Cells were stimulated with the indicated concentrations of NaCl for 5 min. G-H. Average values of three independent experiments from (C and E) and (D and F), respectively, are plotted. Data information: (C-F) Representative results from three independent experiments. (G-H) Error bars are SEM (n=3).


A Phos - tag band - shift of Hog1 phosphorylation . KT235 ( ΔS / O / H / M ssk2 / 22Δ hog1Δ STE11 - Q301P ) was transformed with pRS416 - Hog1 ( WT ) or its indicated mutant derivatives . extracts were prepared from fresh cultures without applying osmostress . For each HOG1 mutant plasmid , three independent cultures were assayed . Data information : Error bars are ( n = 3 ) .

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

A Phos-tag band-shift assay of Hog1 phosphorylation. KT235 (ΔS/O/H/M ssk2/22Δ hog1Δ STE11-Q301P) was transformed with pRS416-Hog1 (WT) or its indicated mutant derivatives. Cell extracts were prepared from fresh cultures without applying osmostress. For each HOG1 mutant plasmid, three independent cultures were assayed. Data information: Error bars are SEM (n=3).


B . yeast strains KT260 ( ssk2 / 22Δ ste11Δ hog1Δ ) , KT235 ( ΔS / O / H / M ssk2 / 22Δ hog1Δ STE11 - Q301P ) , KT248 ( ΔS / O / H / M ssk2 / 22Δ hog1Δ pbs2Δ ) carrying YCplac22I ' - Pbs2 - DD , were used . Data information : Error bars are ( n = 3 ) .

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

B. the yeast strains KT260 (ssk2/22Δ ste11Δ hog1Δ), KT235 (ΔS/O/H/M ssk2/22Δ hog1Δ STE11-Q301P), and KT248 (ΔS/O/H/M ssk2/22Δ hog1Δ pbs2Δ) carrying YCplac22I'-Pbs2-DD, were used. Data information: Error bars are SEM (n=3).


C . KT250 ( ΔS / O / H / M ssk2 / 22Δ hog1Δ STE11 - WT ) KT235 ( ΔS / O / H / M ssk2 / 22Δ hog1Δ STE11 - Q301P ) were transformed with pRS416 - Hog1 ( WT ) or pRS416 - Hog1 - N149H D162G ( N149H D162G ) together with pRS414 - 8xCRE - lacZ . Expression of Hog1 reporter gene 8xCRE - lacZ in absence of osmostress was assayed . Data information : ( C ) Error bars are ( n = 4 ) .

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C. KT250 (ΔS/O/H/M ssk2/22Δ hog1Δ STE11-WT) and KT235 (ΔS/O/H/M ssk2/22Δ hog1Δ STE11-Q301P) were transformed with pRS416-Hog1 (WT) or pRS416-Hog1-N149H D162G (N149H D162G) together with pRS414-8xCRE-lacZ. Expression of the Hog1 reporter gene 8xCRE-lacZ in the absence of osmostress was assayed. Data information: (C) Error bars are SEM (n=4).


D . KT248 ( ΔS / O / H / M ssk2 / 22Δ hog1Δ pbs2Δ ) was transformed with pRS416 - Hog1 ( WT ) or its indicated mutant derivatives together with pRS414 - 8xCRE - lacZ . Expression of Hog1 reporter gene 8xCRE - lacZ in absence of osmostress was assayed . Data information : Error bars are ( n = 3 ) .

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

D. KT248 (ΔS/O/H/M ssk2/22Δ hog1Δ pbs2Δ) was transformed with pRS416-Hog1 (WT) or its indicated mutant derivatives together with pRS414-8xCRE-lacZ. Expression of the Hog1 reporter gene 8xCRE-lacZ in the absence of osmostress was assayed. Data information: Error bars are SEM (n=3).


E - F . Phos - tag band - shift of Hog1 phosphorylation . Yeast strains of indicated genotypes ( shown below graph ) were transformed with pRS416 - Hog1 ( WT ) or its indicated mutant derivatives ( shown inside graph ) . used : ( E ) FP4 , KT259 , KT260 , KT292 , KY523 ; ( F ) KY523 , KT293 , KT294 , KT295 . N / H , N149H ; D / G , D162G . Data information : Error bars are ( n = 3 ) .

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E-F. Phos-tag band-shift assay of Hog1 phosphorylation. Yeast strains of the indicated genotypes (shown below the graph) were transformed with pRS416-Hog1 (WT) or its indicated mutant derivatives (shown inside the graph). Strain used: (E) FP4, KT259, KT260, KT292, and KY523; and (F) KY523, KT293, KT294, and KT295. N/H, N149H; D/G, D162G. Data information: Error bars are SEM (n=3).


A . KT299 ( MATa hkr1Δ msb2Δ ssk2 / 22Δ ) was exposed to indicated concentrations of α - factor for 15 min in absence of osmostress . Phosphorylation of Fus3 Kss1 was detected by immunoblotting .

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A. KT299 (MATa hkr1Δ msb2Δ ssk2/22Δ) was exposed to the indicated concentrations of α-factor for 15 min in the absence of osmostress. Phosphorylation of Fus3 and Kss1 was detected by immunoblotting.


B . KT299 ( MATa ssk2 / 22Δ hkr1Δ msb2Δ ) was exposed to indicated concentrations of α - factor for 15 min in presence or absence of 0 . 8 M NaCl , Hog1 phosphorylation was determined using Phos - tag band - shift . Data information : ( B ) Representative results from three independent experiments .

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

B. KT299 (MATa ssk2/22Δ hkr1Δ msb2Δ) was exposed to the indicated concentrations of α-factor for 15 min in the presence or absence of 0.8 M NaCl, and Hog1 phosphorylation was determined using the Phos-tag band-shift assay. Data information: (B) Representative results from three independent experiments.


C . Average values of four independent experiments were plotted . Data information : Error bars are ( C ) n =

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C. Average values of four independent experiments were plotted. Data information: Error bars are SEM (C) n=4


D . HM06 - 1 ( MATa ΔS / O / H / M ssk2 / 22Δ ) was exposed to indicated concentrations ( log scale ) of α - factor for 15 min in presence or absence of 1 . 0 M NaCl , Hog1 phosphorylation was determined using Phos - tag band - shift . Average values of three or more independent experiments were plotted . Data information : Error bars are : ( D ) , n = 3 or

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D. HM06-1 (MATa ΔS/O/H/M ssk2/22Δ) was exposed to the indicated concentrations (log scale) of α-factor for 15 min in the presence or absence of 1.0 M NaCl, and Hog1 phosphorylation was determined using the Phos-tag band-shift assay. Average values of three or more independent experiments were plotted. Data information: Error bars are SEM: (D), n=3 or more


E . KT306 ( MATa hkr1Δ msb2Δ ssk2 / 22Δ hog1Δ ) was transformed with pRS416 - Hog1 ( WT ) or pRS416 - Hog1 - N149H D162G ( N / H D / G ) , was exposed to 10 μM α - factor for indicated time in absence of osmostress , Hog1 phosphorylation was determined using Phos - tag band - shift . Average of three independent experiments are plotted . Data information : Error bars are ( E ) n = 3 .

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E. KT306 (MATa hkr1Δ msb2Δ ssk2/22Δ hog1Δ) was transformed with pRS416-Hog1 (WT) or pRS416-Hog1-N149H D162G (N/H D/G), and was exposed to 10 μM α-factor for the indicated time in the absence of osmostress, and Hog1 phosphorylation was determined using the Phos-tag band-shift assay. Average of three independent experiments are plotted. Data information: Error bars are SEM (E) n=3.


G . Typical FRET ( YFP / CFP ) showing p38 activation . HeLa carrying p38 reporter PerKy - p38 ( Tomida et al . , 2015 ) were stably transfected with an expression vector for indicated p38α mutant . FRET was performed as described in Materials Methods . H . Distribution of p38 activity in from sets ( a ) - ( d ) in ( G ) . Data information : ( G ) Scale bars : 20 μm . ( H ) Statistics , Student ' s two - t - .

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

G. Typical FRET (YFP/CFP ratio) images showing p38 activation. HeLa cells carrying the p38 reporter PerKy-p38 (Tomida et al., 2015) were stably transfected with an expression vector for the indicated p38α mutant proteins. FRET analysis was performed as described in Materials and Methods. H. Distribution of p38 activity in individual cells from sets (a) - (d) in (G). Data information: (G) Scale bars: 20 μm. (H) Statistics, Student's two-tailed t-test.


A . An example of time - course experiments for osmostress - induced Hog1 phosphorylation . yeast KY603 - 3 ( ΔS / O / H / M ssk2 / 22Δ STE11 - Q301P ) was stimulated with 1 . 0 M NaCl for indicated times , of phosphorylated Hog1 ( Hog1 - P ) was determined using a Phos - tag band - shift .

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

A. An example of the time-course experiments for the osmostress-induced Hog1 phosphorylation. The yeast strain KY603-3 (ΔS/O/H/M ssk2/22Δ STE11-Q301P) was stimulated with 1.0 M NaCl for the indicated times, and the percentage of phosphorylated Hog1 (Hog1-P) was determined using a Phos-tag band-shift assay.


Compilations of time - courses of osmostress - induced Hog1 activation in ( B ) KY603 - 3 ( ΔS / O / H / M ssk2 / 22Δ STE11 - Q301P ) , For clarity , time - course curves are shown in two panels for lower higher ranges of NaCl concentrations . Data information : For each data point , n = 1 or more .

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

Compilations of the time-courses of osmostress-induced Hog1 activation in (B) KY603-3 (ΔS/O/H/M ssk2/22Δ STE11-Q301P), For clarity, time-course curves are shown in two panels for lower and higher ranges of NaCl concentrations. Data information: For each data point, n=1 or more.


Compilations of time - courses of osmostress - induced Hog1 activation in ( C ) TM257 ( ssk2 / 22Δ ) , ( D ) TM142 ( wild - type ) . For clarity , time - course curves are shown in two panels for lower higher ranges of NaCl concentrations . color chart below ( D ) indicates concentrations of NaCl used . Data information : For each data point , n = 1 or more .

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

Compilations of the time-courses of osmostress-induced Hog1 activation in (C) TM257 (ssk2/22Δ), and (D) TM142 (wild-type). For clarity, time-course curves are shown in two panels for lower and higher ranges of NaCl concentrations. The color chart below (D) indicates the concentrations of NaCl used. Data information: For each data point, n=1 or more.


E . Effects of Hog1 kinase activity osmostress - induced glycerol accumulation on time - courses of Hog1 activation . were stimulated with 0 . 4 M NaCl ( left panel ) or 1 . 6 M NaCl ( right panel ) for indicated times , of phosphorylated Hog1 was determined using Phos - tag band - shift . Strains used were : TM142 ( WT ) , KT254 ( gpd1Δ gpd2Δ stl1Δ ) , TM232 ( hog1Δ ) carrying pRS416 - HOG1 - K52S K53N . K / S K / N , K52S K53N ; gpd1 / 2 , gpd1Δ gpd2Δ . Data information : ( E ) Error bars are ( n = 3 or more ) .

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

E. Effects of Hog1 kinase activity and osmostress-induced glycerol accumulation on the time-courses of Hog1 activation. Cells were stimulated with 0.4 M NaCl (left panel) or 1.6 M NaCl (right panel) for the indicated times, and the percentage of phosphorylated Hog1 was determined using Phos-tag band-shift assay. Strains used were: TM142 (WT), KT254 (gpd1Δ gpd2Δ stl1Δ), and TM232 (hog1Δ) carrying pRS416-HOG1-K52S K53N. K/S K/N, K52S K53N; gpd1/2, gpd1Δ gpd2Δ. Data information: (E) Error bars are SEM (n=3 or more).