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

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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O 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 ratio ( 1h after 16 J / m2 UV / mock ) , ranked by SILAC

O O O O 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) 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 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

O O O O O O O O O O O O O O O O O O O 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) 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 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 )

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 the indicated histone modification specific antibodies . Histone H4 and tubulin were used as loading

O O O O O O O O O O O O O O O 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) 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 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 )

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 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

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 )

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 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 )

O O O O O O O O O O O O O O O O O O O O O O O O O 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, 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 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 .

O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 ) 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 .

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

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

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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 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 ) .

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

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 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 ) .

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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 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 .

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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 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 .

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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 ) 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 ) .

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

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 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 .

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

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 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 ) .

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

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 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 .

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

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 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 ,

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

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

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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 and 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 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 )

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

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 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 .

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 B-MEASURED_VAR 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.


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 ) 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 ) .

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

H-I) 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 ) dark or ( I ) after 8s 405nm light ( 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 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

O O O O O O O O O O O O O O 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) 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 light in a Lamplight - expressing SCN

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


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

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


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

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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

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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


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

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

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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 and 405nm light prevents spike silencing

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


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 ) .

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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 ) , 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 ) .

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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 light 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 and N = 5 for Sustained Excitation , all 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 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 ) .

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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 and 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 ) 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 .

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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 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 ) .

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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 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 .

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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.


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 ) .

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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 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 .

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(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.


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 )

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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)


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 ) .

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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 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 .

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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 ) 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 .

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(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 ) 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 .

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(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 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 . (

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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 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 .

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(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 ) 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 .

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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 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 .

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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 ) 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 .

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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 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 .

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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 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 ) .

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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 ; 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 .

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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 . 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 .

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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 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 ) .

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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 . 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 )

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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 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 ) .

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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 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 ) .

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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 . 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 ) .

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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 . the yeast strain KT259 ( ste11Δ hog1Δ ) was used . Data information : Representative results from three independent experiments . Error bars are SEM ( 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 . the yeast strain KY523 ( ssk2 / 22Δ hog1Δ ) was used . Data information : Representative results from three independent experiments . Error bars are SEM ( 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 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 .

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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 . 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 .

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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 ) 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 ) .

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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 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 .

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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 . Cells were stimulated with ( + ) or without ( - ) 0 . 6 M NaCl for 5 min .

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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 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 ) .

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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 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 ) .

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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 . 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 ) .

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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 ) 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 ) .

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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 the Hog1 reporter gene 8xCRE - lacZ in the absence of osmostress was assayed . Data information : Error bars are SEM ( n = 3 ) .

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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 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 ) .

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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 the indicated concentrations of α - factor for 15 min in the absence of osmostress . Phosphorylation of Fus3 and 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 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 .

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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 SEM ( 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 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

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

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 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 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

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 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 .

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

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 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 .

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

O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 (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 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 .

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

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 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 ) .

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

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).


( A ) Representative kymographs showing tetramethylrhodamine ( TMR ) - labeled DDB movement on each type of recombinant MT lattice , as indicated . Scale bars , 5 μm and 25 sec . Pink arrowhead marks a diffusive DDB complex . See also Movie EV1 . ( B ) Quantification of DDB velocity on MT lattices lacking each of the tubulin - CTTs ( n ≥ 100 DDB complexes per condition from at least three independent experiments ) . No statistical difference was observed between WT and mutant MTs ( P ≥ 0 . 1 , unpaired t test ) ( C - D ) Quantification of the number of processive ( C ) , or diffusive ( D ) DDB complexes relative to WT MTs in the same chamber ( n ≥ 10 MTs quantified for each condition from at least three independent experiments ) . Processive ( unidirectional motion exceeding 0 . 5 μm ) and diffusive ( back - and - forth motion ) events were scored in a field of view and then divided by the lengths of MTs and time , thus normalizing to a value of number of motility events per μm MT per sec . Error bars SD .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Representative kymographs showing tetramethylrhodamine (TMR)-labeled DDB movement on each type of recombinant MT lattice, as indicated. Scale bars, 5 μm and 25 sec. Pink arrowhead marks a diffusive DDB complex. See also Movie EV1. (B) Quantification of DDB velocity on MT lattices lacking each of the tubulin-CTTs (n ≥ 100 DDB complexes per condition from at least three independent experiments). No statistical difference was observed between WT and mutant MTs (P ≥ 0.1, unpaired t test) (C-D) Quantification of the number of processive (C), or diffusive (D) DDB complexes relative to WT MTs in the same chamber (n ≥ 10 MTs quantified for each condition from at least three independent experiments). Processive (unidirectional motion exceeding 0.5 μm) and diffusive (back-and-forth motion) events were scored in a field of view and then divided by the lengths of MTs and time, thus normalizing to a value of number of motility events per μm MT per sec. Error bars SD.


( E - F ) TIRF images of 1 nM TMR - labeled GST - hDyn ( 2 mM ATP ) ( E ) , or 0 . 3 nM TMR - labeled p150 ( F ) molecules ( green ) bound to either Δα - CTT ( top ) , or Δβ - CTT MTs ( bottom ) . Pink arrows show mutant MTs in both the raw , and SD map images , highlighting differences in binding between WT and mutant MTs that are apparent in the SD map . Right , standard deviation maps from an entire time sequence reveal ensemble binding and dissociation events that lead to variations in pixel intensities ( see Methods ) . Scale bars are 5 μm . ( G ) Quantification of mean fluorescence intensity ( arbitrary units ) per μm MT for GST - hDyn ( 2 mM ATP ) or p150 bound to the indicated mutant MT relative to WT MTs in the same chamber . Image shows first frame from time series . The intensity of GST - hDyn was quantified from maximum intensity projections of the entire time sequence due to transient binding of GST - hDyn to the MT ( n ≥ 10 MTs quantified for each condition from at least three independent experiments ) . Error bars represent SD . P value ( unpaired t test ) of Δα - CTT vs . Δβ - CTT MTs = 0 . 5208 for GST - hDyn , and ≤ 0 . 0001 for p150 .

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

(E-F) TIRF images of 1 nM TMR-labeled GST-hDyn (2 mM ATP) (E), or 0.3 nM TMR-labeled p150 (F) molecules (green) bound to either Δα-CTT (top), or Δβ-CTT MTs (bottom). Pink arrows show mutant MTs in both the raw, and SD map images, highlighting differences in binding between WT and mutant MTs that are apparent in the SD map. Right, standard deviation maps from an entire time sequence reveal ensemble binding and dissociation events that lead to variations in pixel intensities (see Methods). Scale bars are 5 μm. (G) Quantification of mean fluorescence intensity (arbitrary units) per μm MT for GST-hDyn (2 mM ATP) or p150 bound to the indicated mutant MT relative to WT MTs in the same chamber. Image shows first frame from time series. The intensity of GST-hDyn was quantified from maximum intensity projections of the entire time sequence due to transient binding of GST-hDyn to the MT (n ≥ 10 MTs quantified for each condition from at least three independent experiments). Error bars represent SD. P value (unpaired t test) of Δα-CTT vs. Δβ-CTT MTs = 0.5208 for GST-hDyn, and ≤ 0.0001 for p150.


( A ) TIRF image of TMR - labeled DDB molecules ( green ) bound to either tyrosinated ( red ) or detyrosinated ( blue ) MTs . A standard deviation map of the whole time sequence is shown , revealing the preferential association of DDB with tyrosinated MTs versus detyrosinated MTs ( pink arrow ) . Representative kymographs of DDB movement are shown from each type of MT present in the same chamber . Scale bars are 5 μm and 25 sec . See also Movie EV2 . ( B - C ) Quantification of the number of processive ( B ) or diffusive ( C ) DDB complexes per μm MT per sec on detyrosinated MTs relative to tyrosinated MTs in the same chamber ( n ≥ 20 MTs quantified for each condition from three independent experiments ) . Error bars represent SD . Data from Δα - CTT MTs ( Fig . 1 ) are re - plotted for comparison . ( D ) Quantification of the frequency of processive DDB molecules as a function of the amount of tyrosinated tubulin incorporated into the lattice normalized to fully tyrosinated MTs . Data points from two independent experiments are shown .

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

(A) TIRF image of TMR-labeled DDB molecules (green) bound to either tyrosinated (red) or detyrosinated (blue) MTs. A standard deviation map of the whole time sequence is shown, revealing the preferential association of DDB with tyrosinated MTs versus detyrosinated MTs (pink arrow). Representative kymographs of DDB movement are shown from each type of MT present in the same chamber. Scale bars are 5 μm and 25 sec. See also Movie EV2. (B-C) Quantification of the number of processive (B) or diffusive (C) DDB complexes per μm MT per sec on detyrosinated MTs relative to tyrosinated MTs in the same chamber (n ≥ 20 MTs quantified for each condition from three independent experiments). Error bars represent SD. Data from Δα-CTT MTs (Fig. 1) are re-plotted for comparison. (D) Quantification of the frequency of processive DDB molecules as a function of the amount of tyrosinated tubulin incorporated into the lattice normalized to fully tyrosinated MTs. Data points from two independent experiments are shown.


( E ) TIRF images of 0 . 3 nM TMR - labeled p150 ( top ) or 1 nM TMR - labeled GST - hDyn ( bottom ) molecules ( green ) bound to either tyrosinated ( red ) or detyrosinated ( blue ) MTs . Right , standard deviation maps of protein binding over an entire time sequence and kymographs from either tyrosinated or detyrosinated MTs are shown . Scale bars are 5 μm and 25 sec . ( F ) Quantification of mean fluorescence intensity ( arbitrary units ) per μm MT for TMR - labeled GST - hDyn ( 2 mM ATP ) or p150 bound to the indicated mutant MT relative to WT MTs in the same chamber . The intensity of GST - hDyn was quantified from maximum intensity projections of the entire time sequence due to transient binding of GST - hDyn to the MT . ( n ≥ 10 MTs quantified for each condition from at least three independent experiments ) . Error bars represent SD .

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

(E) TIRF images of 0.3 nM TMR-labeled p150 (top) or 1 nM TMR-labeled GST-hDyn (bottom) molecules (green) bound to either tyrosinated (red) or detyrosinated (blue) MTs. Right, standard deviation maps of protein binding over an entire time sequence and kymographs from either tyrosinated or detyrosinated MTs are shown. Scale bars are 5 μm and 25 sec. (F) Quantification of mean fluorescence intensity (arbitrary units) per μm MT for TMR-labeled GST-hDyn (2 mM ATP) or p150 bound to the indicated mutant MT relative to WT MTs in the same chamber. The intensity of GST-hDyn was quantified from maximum intensity projections of the entire time sequence due to transient binding of GST-hDyn to the MT. (n ≥ 10 MTs quantified for each condition from at least three independent experiments). Error bars represent SD.


( A ) Immunoblot of brain tubulin with or without carboxypeptidase A ( CPA ) . Note CPA specifically removes the α - tubulin C - terminal tyrosine without affecting other PTMs .

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

(A) Immunoblot of brain tubulin with or without carboxypeptidase A (CPA). Note CPA specifically removes the α-tubulin C-terminal tyrosine without affecting other PTMs.


( B ) TIRF image of TMR - labeled DDB molecules ( green ) on either WT ( red ) or CPA - treated ( blue ) pig brain MTs . Standard deviation map of the entire time sequence shows DDB preference for WT MTs and kymographs from either WT or CPA - treated are shown . Scale bars are 5 μm and 25 sec . ( C ) Quantification of the number of processive DDB complexes per μm MT per sec on the CPA - treated MTs , relative to WT MTs in the same chamber ( n ≥ 7 MTs of each type quantified from at least three independent experiments ) . Error bars represent SD .

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 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 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

(B) TIRF image of TMR-labeled DDB molecules (green) on either WT (red) or CPA-treated (blue) pig brain MTs. Standard deviation map of the entire time sequence shows DDB preference for WT MTs and kymographs from either WT or CPA-treated are shown. Scale bars are 5 μm and 25 sec. (C) Quantification of the number of processive DDB complexes per μm MT per sec on the CPA-treated MTs, relative to WT MTs in the same chamber (n ≥ 7 MTs of each type quantified from at least three independent experiments). Error bars represent SD.


( D ) TIRF images of 0 . 3nM TMR - labeled p150 ( top ) or 1nM TMR - labeled GST - hDyn ( bottom ) molecules ( green ) bound to either WT ( red ) , or CPA - treated MTs ( blue ) . Right , standard deviation maps of protein binding over an entire time sequence and kymographs from either WT or CPA - treated are shown . Scale bars are 5 μm and 25 sec for p150 , and 5 μm and 12 sec for GST - hDyn ( E ) Quantification of mean fluorescence intensity ( arbitrary units ) per μm MT for TMR - labeled GST - hDyn ( 2mM ATP ) or p150 bound to CPA - treated MTs relative to WT MTs in the same chamber . The intensity of GST - hDyn was quantified from maximum intensity projections of the entire time sequence due to transient binding of GST - hDyn to the MT ( n ≥ 10 MTs quantified for each condition from at least three independent experiments ) . Error bars represent SD .

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

(D) TIRF images of 0.3nM TMR-labeled p150 (top) or 1nM TMR-labeled GST-hDyn (bottom) molecules (green) bound to either WT (red), or CPA-treated MTs (blue). Right, standard deviation maps of protein binding over an entire time sequence and kymographs from either WT or CPA-treated are shown. Scale bars are 5 μm and 25 sec for p150, and 5 μm and 12 sec for GST-hDyn (E) Quantification of mean fluorescence intensity (arbitrary units) per μm MT for TMR-labeled GST-hDyn (2mM ATP) or p150 bound to CPA-treated MTs relative to WT MTs in the same chamber. The intensity of GST-hDyn was quantified from maximum intensity projections of the entire time sequence due to transient binding of GST-hDyn to the MT (n ≥ 10 MTs quantified for each condition from at least three independent experiments). Error bars represent SD.


( A ) Schematic of the p150 constructs used . The p135 isoform lacks the CAP - Gly domain , but retains part of the adjacent basic domain ( red ) and coiled - coil domains ( blue ) . Right , Coomassie blue stained gel of the purified p150 and p135 proteins . Molecular weight markers are indicated .

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(A) Schematic of the p150 constructs used. The p135 isoform lacks the CAP-Gly domain, but retains part of the adjacent basic domain (red) and coiled-coil domains (blue). Right, Coomassie blue stained gel of the purified p150 and p135 proteins. Molecular weight markers are indicated.


( B ) TIRF image of either p150 ( 0 . 3 nM ) , or p135 ( 0 . 3 or 6 nM ) bound to either WT ( red ) , or CPA - treated porcine MTs ( blue ) . Note : no binding of p135 is observed at comparable concentrations to p150 indicating the lower binding affinity of this isoform . Proteins were visualized by fluorescence from the GFP tag fused to each construct .

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(B) TIRF image of either p150 (0.3 nM), or p135 (0.3 or 6 nM) bound to either WT (red), or CPA-treated porcine MTs (blue). Note: no binding of p135 is observed at comparable concentrations to p150 indicating the lower binding affinity of this isoform. Proteins were visualized by fluorescence from the GFP tag fused to each construct.


( C ) Still images from a TIRF time - series of p135 ( green ) binding to either WT ( red ) , or CPA - treated porcine MTs ( blue ) . Right , standard deviation map of p135 binding shows no preference for either type of MT , and quantification of mean fluorescence intensity ( arbitrary units ) per μm MT for p135 bound to CPA - treated MTs relative to WT MTs in the same chamber ( n ≥ 10 MTs quantified for each condition from three independent experiments ) . Scale bars are 5 μm .

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(C) Still images from a TIRF time-series of p135 (green) binding to either WT (red), or CPA-treated porcine MTs (blue). Right, standard deviation map of p135 binding shows no preference for either type of MT, and quantification of mean fluorescence intensity (arbitrary units) per μm MT for p135 bound to CPA-treated MTs relative to WT MTs in the same chamber (n ≥ 10 MTs quantified for each condition from three independent experiments). Scale bars are 5 μm.