( A ) Phenotypes of wild type ( Col ) , kup9 mutants ( kup9 - 1 and kup9 - 2 ) and the kup9 - 1 / ProKUP9 : KUP9 complementation lines ( COM1 and COM2 ) . Seeds were germinated and grown on low - K + ( LK , 50 μM ) or high - K + ( HK , 5 mM ) medium for 7 d . Scale bar , 1 cm .

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(A) Phenotypes of wild type (Col), kup9 mutants (kup9-1 and kup9-2) and the kup9-1/ProKUP9:KUP9 complementation lines (COM1 and COM2). Seeds were germinated and grown on low-K+ (LK, 50 μM) or high-K+ (HK, 5 mM) medium for 7 d. Scale bar, 1 cm.


( B ) Primary root length of the plants tested Data are means ± SE ( n = 25 , individual plants ) . Student ' s t - test ( * * P < 0 . 01 ) was used to analyze statistical significance , and " # " represents the control .

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(B) Primary root length of the plants tested Data are means ± SE (n = 25, individual plants). Student's t-test (**P < 0.01) was used to analyze statistical significance, and "#" represents the control.


( D ) RT - qPCR analysis of KUP9 transcript levels in various materials . Data are means ± SE ( n = 3 , biological replicates ; each replicate contains 120 individual plants ) .

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(D) RT-qPCR analysis of KUP9 transcript levels in various materials. Data are means ± SE (n = 3, biological replicates; each replicate contains 120 individual plants).


( E ) and ( F ) K + content of the various plant materials tested in ( A ) . Data are means ± SE ( n = 3 , biological replicates ; each replicate contains 120 - 150 individual plants ) .

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(E) and (F) K+ content of the various plant materials tested in (A). Data are means ± SE (n = 3, biological replicates; each replicate contains 120-150 individual plants).


( A ) Root meristem zones of wild type ( Col ) , kup9 mutants ( kup9 - 1 and kup9 - 2 ) and the kup9 - 1 / ProKUP9 : KUP9 complementation lines ( COM1 and COM2 ) . Seeds were germinated and grown on LK or HK medium for 7 d . The meristem zone lengths are marked with red lines . Scale bars , 50 μm .

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(A) Root meristem zones of wild type (Col), kup9 mutants (kup9-1 and kup9-2) and the kup9-1/ProKUP9:KUP9 complementation lines (COM1 and COM2). Seeds were germinated and grown on LK or HK medium for 7 d. The meristem zone lengths are marked with red lines. Scale bars, 50 μm.


( B ) , ( C ) and ( D ) represent different zone lengths , different zone cell numbers , and cell length of the indicated seedlings germinated and grown on LK or HK medium for 7 d . MEZ , meristem zone ; EZ , elongation zone ; MAZ , maturation zone . Data are means ± SE ( n = 10 - 15 , individual plants ) . Student ' s t - test ( * * P < 0 . 01 ) was used to analyze statistical significance , and " # " represents the control .

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(B), (C) and (D) represent different zone lengths, different zone cell numbers, and cell length of the indicated seedlings germinated and grown on LK or HK medium for 7 d. MEZ, meristem zone; EZ, elongation zone; MAZ, maturation zone. Data are means ± SE (n = 10-15, individual plants). Student's t-test (**P < 0.01) was used to analyze statistical significance, and "#" represents the control.


( A ) to ( F ) Expression of KUP9 as determined in the ProKUP9 : GUS line . GUS staining of 5 - day - old seedling ( A ) , root maturation zone ( B ) , transverse section of maturation zone ( C ) and ( D ) , primary root tip ( E ) , and root apex ( F ) . Ep , epidermis ; Co , cortex ; En , endodermis ; Pe , pericyle ; Xy , xylem ; Ph , phloem . Scale bars , 5 mm in ( A ) , 1 mm in ( B ) , 10 μm in ( C ) , 5 μm in ( D ) , 1 mm in ( E ) , and 20 μm in ( F ) .

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(A) to (F) Expression of KUP9 as determined in the ProKUP9:GUS line. GUS staining of 5-day-old seedling (A), root maturation zone (B), transverse section of maturation zone (C) and (D), primary root tip (E), and root apex (F). Ep, epidermis; Co, cortex; En, endodermis; Pe, pericyle; Xy, xylem; Ph, phloem. Scale bars, 5 mm in (A), 1 mm in (B), 10 μm in (C), 5 μm in (D), 1 mm in (E), and 20 μm in (F).


( G ) and ( H ) Expression of KUP9 protein as determined in the kup9 - 1 / ProKUP9 : KUP9 - GFP transgenic plants . Green fluorescence shows the subcellular localization of KUP9 - GFP . PI ( propidium iodide ) and ER tracker were used to indicate the positions of the plasma membrane ( PM ) and endoplasmic reticulum ( ER ) and are shown as red and cyan fluorescence , respectively . Scale bars for root tip , 10 μm in ( G ) and 5 μm in ( H ) . Scale bars for EZ and MAZ , 20 μm .

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(G) and (H) Expression of KUP9 protein as determined in the kup9-1/ProKUP9:KUP9-GFP transgenic plants. Green fluorescence shows the subcellular localization of KUP9-GFP. PI (propidium iodide) and ER tracker were used to indicate the positions of the plasma membrane (PM) and endoplasmic reticulum (ER) and are shown as red and cyan fluorescence, respectively. Scale bars for root tip, 10 μm in (G) and 5 μm in (H). Scale bars for EZ and MAZ, 20 μm.


( I ) Co - localization analyses of KUP9 - GFP with ER marker NIP1 ; 1 - mCherry in their crossing line . Scale bars , 20 μm .

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(I) Co-localization analyses of KUP9-GFP with ER marker NIP1;1-mCherry in their crossing line. Scale bars, 20 μm.


( J ) Co - localization analyses of KUP9 - GFP with the ER marker BIP2 using immunological staining . Scale bars , 5 μm .

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(J) Co-localization analyses of KUP9-GFP with the ER marker BIP2 using immunological staining. Scale bars, 5 μm.


( A ) QC cell observation in wild type and the kup9 mutants using the QC marker lines ProWOX5 : GFP and ProQC25 : GUS . Seeds were germinated on HK medium for 4 d , then the seedlings were transferred to LK or HK medium for the indicated times . Scale bars , 10 μm .

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(A) QC cell observation in wild type and the kup9 mutants using the QC marker lines ProWOX5:GFP and ProQC25:GUS. Seeds were germinated on HK medium for 4 d, then the seedlings were transferred to LK or HK medium for the indicated times. Scale bars, 10 μm.


( B ) QC division rates of wild type and the kup9 mutants after LK treatment for 96 h . The statistical method is described in Materials and Methods . Data are means ± SE ( n = 150 , individual plants ) . Student ' s t - test ( * * P < 0 . 01 ) was used to analyze statistical significance , and " # " represents control .

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(B) QC division rates of wild type and the kup9 mutants after LK treatment for 96 h. The statistical method is described in Materials and Methods. Data are means ± SE (n = 150, individual plants). Student's t-test (**P < 0.01) was used to analyze statistical significance, and "#" represents control.


( A ) Auxin levels in root tips , as indicated by the ProDR5 : GFP reporter line . Seeds were germinated on HK medium for 4 d , then the seedlings were transferred to LK or HK medium for the indicated times . Scale bar , 10 μm . ( B ) Quantification of GFP fluorescence in the QC cells shown in ( A ) . Data are means ± SE ( n = 20 , individual plants ) . Student ' s t - test ( * * P < 0 . 01 ) was used to analyze statistical significance .

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(A) Auxin levels in root tips, as indicated by the ProDR5:GFP reporter line. Seeds were germinated on HK medium for 4 d, then the seedlings were transferred to LK or HK medium for the indicated times. Scale bar, 10 μm. (B) Quantification of GFP fluorescence in the QC cells shown in (A). Data are means ± SE (n = 20, individual plants). Student's t-test (**P < 0.01) was used to analyze statistical significance.


( A ) Phenotypes of various plants . Seeds were germinated and grown on LK or HK medium with or without NAA for 7 d . Scale bar , 1 cm .

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(A) Phenotypes of various plants. Seeds were germinated and grown on LK or HK medium with or without NAA for 7 d. Scale bar, 1 cm.


( C ) QC cell observation using the QC marker line ProWOX5 : GFP . Seeds were germinated on HK medium for 4 d , then the seedlings were transferred to LK or HK medium with or without NAA ( 100 nM ) for 96 h . Scale bar , 10 μm .

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(C) QC cell observation using the QC marker line ProWOX5:GFP. Seeds were germinated on HK medium for 4 d, then the seedlings were transferred to LK or HK medium with or without NAA (100 nM) for 96 h. Scale bar, 10 μm.


( D ) QC division rates of the plants tested The statistical method is described in Materials and Methods . Data are means ± SE ( n = 50 - 60 , individual plants ) . Student ' s t - test ( * * P < 0 . 01 ) was used to analyze statistical significance , and " # " represents control .

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(D) QC division rates of the plants tested The statistical method is described in Materials and Methods. Data are means ± SE (n = 50-60, individual plants). Student's t-test (**P < 0.01) was used to analyze statistical significance, and "#" represents control.


Phenotypes of the kup9 - 1 / ProWOX5 : AMI1 ( A ) transgenic lines . Seeds were germinated and grown on LK or HK medium with or without IAM for 7 d . Scale bars , 1 cm .

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Phenotypes of the kup9-1/ProWOX5:AMI1 (A) transgenic lines. Seeds were germinated and grown on LK or HK medium with or without IAM for 7 d. Scale bars, 1 cm.


Primary root length of the plants tested Data are means ± SE ( n = 25 , individual plants ) . Student ' s t - test ( * * P < 0 . 01 ) was used to analyze statistical significance , and " # " represents control .

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Primary root length of the plants tested Data are means ± SE (n = 25, individual plants). Student's t-test (**P < 0.01) was used to analyze statistical significance, and "#" represents control.


Phenotypes of the kup9 - 1 / ProKUP9 : AMI1 ( C ) transgenic lines . Seeds were germinated and grown on LK or HK medium with or without IAM for 7 d . Scale bars , 1 cm .

Phenotypes of the kup9-1/ProKUP9:AMI1 (C) transgenic lines. Seeds were germinated and grown on LK or HK medium with or without IAM for 7 d. Scale bars, 1 cm.


( A ) Expression of KUP9 - GFP in BY - 2 tobacco cells . HDEL - mCherry was used as an ER marker . Scale bar , 10 μm .

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(A) Expression of KUP9-GFP in BY-2 tobacco cells. HDEL-mCherry was used as an ER marker. Scale bar, 10 μm.


( B ) 3H - IAA accumulation in the BY - 2 cells expressing the indicated proteins . PIN4 - GFP and KUP10 - GFP were used as positive control and negative control , respectively . Data are means ± SE ( n = 3 , biological replicates ; each replicate contains 3 - 4 mL suspension cells ) . Student ' s t - test ( * P < 0 . 05 , * * P < 0 . 01 ) was used to analyze statistical significance , and " # " represent control .

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(B) 3H-IAA accumulation in the BY-2 cells expressing the indicated proteins. PIN4-GFP and KUP10-GFP were used as positive control and negative control, respectively. Data are means ± SE (n = 3, biological replicates; each replicate contains 3-4 mL suspension cells). Student's t-test (*P < 0.05, **P < 0.01) was used to analyze statistical significance, and "#" represent control.


( C ) Expression of KUP9 - GFP and OST4 - GFP - KUP9 in Xenopus oocytes . Scale bar , 100 μm .

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(C) Expression of KUP9-GFP and OST4-GFP-KUP9 in Xenopus oocytes. Scale bar, 100 μm.


( D ) 3H - IAA accumulation in the oocytes expressing KUP9 - GFP and PIN4 at the indicated times . PIN4 was used as a positive control . The oocytes were incubated in the bath solution containing 3H - IAA for the indicated times . Data are means ± SE ( n = 6 , biological replicates ; each replicate contains 6 oocytes ) .

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(D) 3H-IAA accumulation in the oocytes expressing KUP9-GFP and PIN4 at the indicated times. PIN4 was used as a positive control. The oocytes were incubated in the bath solution containing 3H-IAA for the indicated times. Data are means ± SE (n = 6, biological replicates; each replicate contains 6 oocytes).


( E ) IAA content in the oocytes expressing the indicated proteins . The oocytes were injected with the same amount of IAA and incubated in IAA - free bath solution for 6 h . IAA content was measured using UPLC - MS . Data are means ± SE ( n = 3 , biological replicates ; each replicate contains 6 oocytes ) .

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(E) IAA content in the oocytes expressing the indicated proteins. The oocytes were injected with the same amount of IAA and incubated in IAA-free bath solution for 6 h. IAA content was measured using UPLC-MS. Data are means ± SE (n = 3, biological replicates; each replicate contains 6 oocytes).


( F ) K + efflux activity of the oocytes expressing KUP9 and NRT1 . 5 . The oocytes were not injected with IAA . The oocytes were incubated in K + - free bath solution for 6 h . Data are means ± SE ( n = 3 , biological replicates ; each replicate contains 6 oocytes ) .

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(F) K+ efflux activity of the oocytes expressing KUP9 and NRT1.5. The oocytes were not injected with IAA. The oocytes were incubated in K+-free bath solution for 6 h. Data are means ± SE (n = 3, biological replicates; each replicate contains 6 oocytes).


( G ) K + efflux activity of the oocytes expressing the indicated proteins . The oocytes were injected with H2O , IAA , or BA . Data are means ± SE ( n = 3 , biological replicates ; each replicate contains 6 oocytes ) .

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(G) K+ efflux activity of the oocytes expressing the indicated proteins. The oocytes were injected with H2O, IAA, or BA. Data are means ± SE (n = 3, biological replicates; each replicate contains 6 oocytes).


( H ) K + efflux activity of the oocytes expressing the indicated proteins . IAA was injected into the oocytes . Then the oocytes were incubated in K + - free bath solutions at different pH for 6 h . Data are means ± SE ( n = 3 - 6 , biological replicates ; each replicate contains 6 oocytes ) .

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(H) K+ efflux activity of the oocytes expressing the indicated proteins. IAA was injected into the oocytes. Then the oocytes were incubated in K+-free bath solutions at different pH for 6 h. Data are means ± SE (n = 3-6, biological replicates; each replicate contains 6 oocytes).


( I ) IAA efflux activity of oocytes expressing the indicated proteins under different external K + conditions ( - K , 0 mM ; + K , 50 mM ) . The oocytes were injected with the same amount of IAA , and then incubated in IAA - free bath solution for 6 h . IAA content was measured using UPLC - MS . Data are means ± SE ( n = 3 , biological replicates ; each replicate contains 6 oocytes ) . Student ' s t - test ( * P < 0 . 05 , * * P < 0 . 01 ) was used to analyze statistical significance , and " # " represent control .

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(I) IAA efflux activity of oocytes expressing the indicated proteins under different external K+ conditions (-K, 0 mM; +K, 50 mM). The oocytes were injected with the same amount of IAA, and then incubated in IAA-free bath solution for 6 h. IAA content was measured using UPLC-MS. Data are means ± SE (n = 3, biological replicates; each replicate contains 6 oocytes). Student's t-test (*P < 0.05, **P < 0.01) was used to analyze statistical significance, and "#" represent control.


( A ) K + levels of various materials using the fluorescent dye Asante Potassium Green - 2 ( AM ) . Scale bar , 50 μm . ( B ) Quantification of K + concentration under LK and HK conditions . Data are means ± SE ( n = 25 , individual plants ) . Student ' s t - test ( * * P < 0 . 01 ) was used to analyze statistical significance , and " # " represents control .

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(A) K+ levels of various materials using the fluorescent dye Asante Potassium Green-2 (AM). Scale bar, 50 μm. (B) Quantification of K+ concentration under LK and HK conditions. Data are means ± SE (n = 25, individual plants). Student's t-test (**P < 0.01) was used to analyze statistical significance, and "#" represents control.


( C ) to ( E ) The contents of free IAA ( C ) , IA - Asp ( D ) , and IA - Glu ( E ) in the root tips of wild type and the kup9 - 1 mutant . Data are means ± SE ( n = 3 , biological replicates ; each replicate contains 1000 - 1200 individual plants ) . Student ' s t - test ( * P < 0 . 05 , * * P < 0 . 01 ) was used to analyze statistical significance , and " # " represents control .

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(C) to (E) The contents of free IAA (C), IA-Asp (D), and IA-Glu (E) in the root tips of wild type and the kup9-1 mutant. Data are means ± SE (n = 3, biological replicates; each replicate contains 1000-1200 individual plants). Student's t-test (*P < 0.05, **P < 0.01) was used to analyze statistical significance, and "#" represents control.


( F ) Phenotype of the kup9 - 1 / ProKUP9 : PIN8 transgenic lines . Seeds were germinated and grown on LK or HK medium for 7 d . Scale bar , 1 cm .

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(F) Phenotype of the kup9-1/ProKUP9:PIN8 transgenic lines. Seeds were germinated and grown on LK or HK medium for 7 d. Scale bar, 1 cm.


( G ) Primary root length of the plants tested Data are means ± SE ( n = 25 , individual plants ) . Student ' s t - test ( * * P < 0 . 01 ) was used to analyze statistical significance .

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(G) Primary root length of the plants tested Data are means ± SE (n = 25, individual plants). Student's t-test (**P < 0.01) was used to analyze statistical significance.


( H ) QC division rates of the indicated plants . The statistical method is described in Materials and Methods . Data are means ± SE ( n = 50 - 60 , individual plants ) . Student ' s t - test ( * * P < 0 . 01 ) was used to analyze statistical

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(H) QC division rates of the indicated plants. The statistical method is described in Materials and Methods. Data are means ± SE (n = 50-60, individual plants). Student's t-test (**P < 0.01) was used to analyze statistical significance


a , TEM analysis of P7 wt retinae ( N = 4 eyes ) showing a ( photoreceptor - Multivesicular Body ) PR - MVB in close proximity to ( Muller glia ) MG ( red dashed box ) . PR - photoreceptor cell , MG ( blue dashed lines ) - muller glia cell , MVB ( yellow and yellow arrow ) - multivesicular body , m ( green ) - mitochondria , cc ( pink ) - connecting cilium , RPE ( purple and purple dashed line ) - retina pigment epithelium , RPE melanin - black arrows . Scale bar = 2µm left , 1µm right ;

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a, TEM analysis of P7 wt retinae (N = 4 eyes) showing a (photoreceptor- Multivesicular Body) PR-MVB in close proximity to (Muller glia) MG (red dashed box). PR-photoreceptor cell, MG (blue dashed lines) -muller glia cell, MVB (yellow and yellow arrow)- multivesicular body, m (green)-mitochondria, cc (pink)-connecting cilium, RPE (purple and purple dashed line) -retina pigment epithelium, RPE melanin-black arrows. Scale bar = 2µm left, 1µm right;


f , Representative Dot Blot of 100K EV pellet ( each dot represents 3 pooled EV isolations , derived from 60 * 106 cells ; N = 8 experiments ) vs cell lysate ( CL ) from P8 Nrl . Gfp + / + photoreceptors . EV markers = LAMP1 , CD8 , CD9 ; Golgi marker = GM130 ; phototransduction markers = Recoverin , Rhodopsin , Rod α - Transducin ;

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f, Representative Dot Blot of 100K EV pellet (each dot represents 3 pooled EV isolations, derived from 60*106 cells; N = 8 experiments) vs cell lysate (CL) from P8 Nrl.Gfp+/+ photoreceptors. EV markers = LAMP1, CD8, CD9; Golgi marker = GM130; phototransduction markers = Recoverin, Rhodopsin, Rod α-Transducin;


g , RTqPCR analysis of 100K P8 photoreceptor pellets for Gnat1 , Rec , Rho , Crx , Cre and Gfp , relative to β - actin , comparing EVs ( N = 8 ) against the appropriate ( Nrl . Gfp + / + or Nrl . Cre + / - ) photoreceptor cell lysate ( N = 3 ) . Gnat1 , Cre and Gfp mRNA were present in all relevant samples ;

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g, RTqPCR analysis of 100K P8 photoreceptor pellets for Gnat1, Rec, Rho, Crx, Cre and Gfp, relative to β-actin, comparing EVs (N = 8) against the appropriate (Nrl.Gfp+/+ or Nrl.Cre+/-) photoreceptor cell lysate (N = 3). Gnat1, Cre and Gfp mRNA were present in all relevant samples;


i , representative flow cytometry analysis of Nrl . Cre + / - & mTmGfloxed co - cultures . Samples were analyzed for expression of myrGFP ( recombination ) and CD73 ( photoreceptor identity ) versus CD73 - ve fraction ( other retinal cells ) . N > 3 independent cultures for each condition with technical triplicate for each sample ; One way ANOVA , non - parametric , Kruskal - Wallis with Dunns ' multiple comparisons

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i, representative flow cytometry analysis of Nrl.Cre+/- & mTmGfloxed co-cultures. Samples were analyzed for expression of myrGFP (recombination) and CD73 (photoreceptor identity) versus CD73-ve fraction (other retinal cells). N > 3 independent cultures for each condition with technical triplicate for each sample; One way ANOVA, non-parametric, Kruskal-Wallis with Dunns' multiple comparisons test


g , Quantification of no . of TdTomato + ve host photoreceptor cells seen following transplantation of live ( 224 ± 181 ) versus UV - treated ( dead ) ( 3 ± 4 ) Nrl . Cre + / - P8 photoreceptors ( N = 4 eyes per condition ) . Graph shows mean ± S . D .

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g, Quantification of no. of TdTomato+ve host photoreceptor cells seen following transplantation of live (224 ± 181) versus UV-treated (dead) (3 ± 4) Nrl.Cre+/- P8 photoreceptors (N = 4 eyes per condition). Graph shows mean ± S.D.


i & j , Representative images of depth colour coding ( ImageJ ) of x , y , z live imaging of Nrl . Gfp + / + photoreceptor cultures showing ( i ) neurites ( blue arrows ) growing along the substrate ( asterisks denote secondary branching of long GFP + neurites ) , and in ( j ) , a free - floating PhNT ( magenta arrow ) . Scale bar = 10 µm ;

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i & j, Representative images of depth colour coding (ImageJ) of x,y,z live imaging of Nrl.Gfp+/+ photoreceptor cultures showing (i) neurites (blue arrows) growing along the substrate (asterisks denote secondary branching of long GFP+ neurites), and in (j), a free-floating PhNT (magenta arrow). Scale bar = 10 µm;


k & l , Quantification of the localization of Nrl . Gfp + / + processes in the z - axis ( depth ) ( N = 4 independent cultures , n = 1096 cells , nneurites = 440 , nnanotubes = 31 where ( k ) neurites . P values = * * * * p < 0 . 0001 ( bottom vs top ) ; * * p = 0 . 002 ( bottom vs middle ) ; * * p = 0 . 003 ( middle vs top ) and ( l ) PhNTs ; P - values = * * * * p < 0 . 0001 ( bottom vs middle & middle vs top ) . One way ANOVA , non - parametric , Kruskal - Wallis with Dunns ' multiple comparisons test shows means vary significantly .

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k & l, Quantification of the localization of Nrl.Gfp+/+ processes in the z-axis (depth) (N = 4 independent cultures, n = 1096 cells, nneurites = 440, nnanotubes = 31 where (k) neurites. P values = **** p < 0.0001 (bottom vs top); **p = 0.002 (bottom vs middle); **p = 0.003 (middle vs top) and (l) PhNTs; P-values = **** p < 0.0001 (bottom vs middle & middle vs top) . One way ANOVA, non-parametric, Kruskal-Wallis with Dunns' multiple comparisons test shows means vary significantly.


d , representative time lapse images of cGFP mobility between two PhNT - connected Nrl . Gfp + / + photoreceptors ( dashed ellipse = bleached area ) versus an isolated cell ( solid ellipse = bleached area ) and clustered cells ( dot ellipse = bleached area ) . Images show pre - bleach ( t = - 5 ' ' ) , bleach ( t = 0 ' ' ) , and post - bleach ( t = 35 ' ' and 215 ' ' ) . Unbleached reference cells also indicated ( arrows ) ; Green = cytoplasm ; Scale bar = 5µm .

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d, representative time lapse images of cGFP mobility between two PhNT-connected Nrl.Gfp+/+ photoreceptors (dashed ellipse = bleached area) versus an isolated cell (solid ellipse = bleached area) and clustered cells (dot ellipse = bleached area). Images show pre-bleach (t = -5''), bleach (t = 0''), and post-bleach (t = 35'' and 215''). Unbleached reference cells also indicated (arrows); Green = cytoplasm; Scale bar = 5µm.


d , Lysosomes can be exchanged , rarely , between PhNT - connected cells . Lysosomes were labelled with SiR - Lyso and their movements were analysed by TrackMate software . Images show deconvolution of 3D reconstructions of lysosomes ( surface ; red ) and cGFP ( volume ; green ) from time - lapse live imaging series of Nrl . Gfp + / + photoreceptor cultures ( green ) ; the position of a transferred lysosome is marked as1 ( t = 0 ) , 2 ( t = 3 ' ) , 3 ( t = 6 ' ) , 4 ( t = 9 ' ) ; N . B . Deconvolution shows a segment - like process extending from cell " A " and a PhNT connecting cells " A " and " B " ; Scale bar = 2 µm ;

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d, Lysosomes can be exchanged, rarely, between PhNT-connected cells. Lysosomes were labelled with SiR-Lyso and their movements were analysed by TrackMate software. Images show deconvolution of 3D reconstructions of lysosomes (surface; red) and cGFP (volume; green) from time-lapse live imaging series of Nrl.Gfp+/+ photoreceptor cultures (green); the position of a transferred lysosome is marked as1 (t = 0), 2 (t = 3'), 3 (t = 6'), 4 (t = 9'); N.B. Deconvolution shows a segment-like process extending from cell "A" and a PhNT connecting cells "A" and "B"; Scale bar = 2 µm;


e , Mitochondria can be exchanged , rarely , between PhNT - connected cells . Mitochondria movements determined by TrackMate software . Images show deconvolution of 3D reconstructions of mitochondria ( surface ; red ) and cGFP ( volume ; green ) from time - lapse live imaging series ; position of transferred mitochondrion is marked as 1 ( t = 0 ) , 2 ( t = 6 ' ) , 3 ( t = 12 ' ) , 4 ( t = 18 ' ) ; Mitochondria labelled with mito - Tracker - Orange ; Scale bar = 5 µm .

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e, Mitochondria can be exchanged, rarely, between PhNT-connected cells. Mitochondria movements determined by TrackMate software. Images show deconvolution of 3D reconstructions of mitochondria (surface; red) and cGFP(volume; green) from time-lapse live imaging series; position of transferred mitochondrion is marked as 1 (t = 0), 2 (t = 6'), 3 (t = 12'), 4 (t = 18'); Mitochondria labelled with mito-Tracker-Orange; Scale bar = 5 µm.


a , MIP image of wildtype ( wt ) retina transplanted with P8 Nrl . Gfp + / + CD73 + MACS - enriched photoreceptors ; dashed box indicates ROI subjected to 3D deconvolution and volume reconstruction ( right ) and shows PhNT - like connection between donor and host photoreceptor at the level of host inner segment . Additional images show 3D segmentation of z = 0 and z = 38 focal planes , and reveal the PhNT ; green = GFP , blue = nuclei ; Scale bar = 10 µm in MIP and 5 µm volume reconstruction ; b & c , assessment of properties of PhNT - like processes between donor and host photoreceptors ( n = 19 PhNT - like processes ; N = 10 eyes ) showing ( b ) diameter and ( c ) length ;

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a, MIP image of wildtype (wt) retina transplanted with P8 Nrl.Gfp+/+ CD73+ MACS-enriched photoreceptors; dashed box indicates ROI subjected to 3D deconvolution and volume reconstruction (right) and shows PhNT-like connection between donor and host photoreceptor at the level of host inner segment. Additional images show 3D segmentation of z = 0 and z = 38 focal planes, and reveal the PhNT; green = GFP, blue = nuclei; Scale bar = 10 µm in MIP and 5 µm volume reconstruction; b & c, assessment of properties of PhNT-like processes between donor and host photoreceptors (n = 19 PhNT-like processes; N = 10 eyes) showing (b) diameter and (c) length;


d , MIP image of wt retina transplanted with P8 Nrl . Gfp + / + x myrRFP / + photoreceptors ; showing wt host ONL photoreceptor labeled with cGFP ( green ) but not with myrRFP ( red ) ; Scale bar = 50µm ; e , quantification and statistical analysis of material transfer events following subretinal transplantation of live ( cGFP = 3775 ± 804 , myrRFP = 436 ± 145 ) or UV - treated ( cGFP = 1 . 3 ± 2 . 5 , myrRFP = 4 . 3 ± 5 . 7 ) Nrl . Gfp + / + x myr - Rfp + / + photoreceptors into wt hosts ( N = 8 and N = 4 retinae , respectively ) ; One - way ANOVA , parametric , two tail , Tukey ' s multiple comparisons ; Graph shows mean ± S . D .

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 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, MIP image of wt retina transplanted with P8 Nrl.Gfp+/+ x myrRFP/+ photoreceptors; showing wt host ONL photoreceptor labeled with cGFP (green) but not with myrRFP (red); Scale bar = 50µm; e, quantification and statistical analysis of material transfer events following subretinal transplantation of live (cGFP = 3775 ± 804, myrRFP = 436 ± 145) or UV-treated (cGFP = 1.3 ± 2.5, myrRFP = 4.3 ± 5.7) Nrl.Gfp+/+x myr-Rfp+/+ photoreceptors into wt hosts (N = 8 and N = 4 retinae, respectively); One-way ANOVA, parametric, two tail, Tukey's multiple comparisons; Graph shows mean ± S.D.


f - h , Assessment of potential for transfer of donor Gfp and Gnat1 mRNA during transplantation using RNAscopeTM . f , Representative MIP images of Gnat1 - / - eyes transplanted with Nrl . Gfp + / + donor cells and processed in situ for Gfp mRNA ( red ) and Gnat1 mRNA ( magenta ) ; blue = nuclei ; ROIs indicated in ( f ) show GFP + ve donor cell mass ( CM ) and GFP + ve cells within Gnat1 - / - host outer nuclear layer ( ONL ) ; g & h , quantification of Gfp and Gnat1 mRNA ( mean fluorescence intensity per cell ) , respectively ( N = 3 retinae ) ; One - way ANOVA , non - parametric , two tailed analysis showed no significant differences in signal intensity for either Gnat1 or Gfp mRNA between GFP + ve and GFP - ve host photoreceptors ;

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

f-h, Assessment of potential for transfer of donor Gfp and Gnat1 mRNA during transplantation using RNAscopeTM. f, Representative MIP images of Gnat1-/- eyes transplanted with Nrl.Gfp+/+ donor cells and processed in situ for Gfp mRNA (red) and Gnat1 mRNA (magenta); blue = nuclei; ROIs indicated in (f) show GFP+ve donor cell mass (CM) and GFP+ve cells within Gnat1-/- host outer nuclear layer (ONL); g & h, quantification of Gfp and Gnat1 mRNA (mean fluorescence intensity per cell), respectively (N = 3 retinae); One-way ANOVA, non-parametric, two tailed analysis showed no significant differences in signal intensity for either Gnat1 or Gfp mRNA between GFP+ve and GFP-ve host photoreceptors;


i , Manipulation of actin signalling alters material transfer in vivo : ( far left ) MIP image showing GFP + ve host photoreceptor ( green ) expressing Rod α - transducin ( red ) after transplantation of retinal cells transduced with lenti - CAG - P2A . Gfp ( transduction control ) into Gnat1 - / - recipient ; Scale bar = 10 µm ; adjacent images show Gnat1 - / - eyes transplanted with P2 retinal cells transduced with ( left ) plenti - CAG - P2A . GFP ( green ) , or ( middle ) plenti - CAG - RhoA - P2A . GFP ( green ) , or ( right ) plenti - CAG - ΔΝRac1 - P2A . GFP ( green ) ; green = GFP , blue = nuclei ; Scale bar = 20 µm ; j , quantification of the number of GFP + cells in Gnat1 - / - host ONL , ( N = 5 retinae per condition ) ; plenti - CAG - P2A . GFP ( 2328 ± 234 ) , plenti - CAG - RhoA - P2A . GFP ( 668 ± 481 ) , plenti - CAG - ΔΝRac1 - P2A . GFP ( 547 ± 306 ) ; One - way ANOVA , non - parametric , Dunn ' s multiple comparisons test . Graph shows mean ± SD .

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i, Manipulation of actin signalling alters material transfer in vivo: (far left) MIP image showing GFP+ve host photoreceptor (green) expressing Rod α-transducin (red) after transplantation of retinal cells transduced with lenti-CAG-P2A.Gfp (transduction control) into Gnat1-/- recipient; Scale bar = 10 µm; adjacent images show Gnat1-/- eyes transplanted with P2 retinal cells transduced with (left) plenti-CAG-P2A.GFP (green), or (middle) plenti-CAG-RhoA-P2A.GFP (green), or (right) plenti-CAG-ΔΝRac1-P2A.GFP (green); green = GFP, blue = nuclei; Scale bar = 20 µm; j, quantification of the number of GFP+ cells in Gnat1-/- host ONL, (N = 5 retinae per condition); plenti-CAG-P2A.GFP (2328 ± 234), plenti-CAG-RhoA-P2A.GFP (668 ± 481), plenti-CAG-ΔΝRac1-P2A.GFP (547 ± 306); One-way ANOVA, non-parametric, Dunn's multiple comparisons test. Graph shows mean ± SD.


b , Representative tile scan of un - injected Nrl . Cre + / - / TdTomato + / - chimera showing endogenous recombination ( white ) ; ROIs , as indicated with dashed or dot boxes ; Scale bars = 50 µm ;

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b, Representative tile scan of un-injected Nrl.Cre +/-/TdTomato+/- chimera showing endogenous recombination (white); ROIs, as indicated with dashed or dot boxes; Scale bars = 50 µm;


c & d , ROI from un - injected Nrl . Cre + / - / TdTomato + / - chimera showing spontaneous recombination presenting in mosaic stripe patterning ; Scale bar = 50 µm in c and 10 µm in d ;

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c & d, ROI from un-injected Nrl.Cre +/-/TdTomato+/- chimera showing spontaneous recombination presenting in mosaic stripe patterning; Scale bar = 50 µm in c and 10 µm in d;


e & f , Representative images of Nrl . Cre + / - / TdTomato + / - chimera after receiving a subretinal injection of AAV - Nrl . Cre . The injected eyes show widespread recombination , which also shows the anticipated mosaic stripe patterning ; Scale bar = 50 µm in ( e ) and 10 µm in ( f ) ;

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e & f, Representative images of Nrl.Cre+/-/TdTomato+/- chimera after receiving a subretinal injection of AAV-Nrl.Cre. The injected eyes show widespread recombination, which also shows the anticipated mosaic stripe patterning; Scale bar = 50 µm in (e) and 10 µm in (f);


A , B . Quantification of oryzalin root growth assays in single ( A ) and double ( B ) mutants . DAG = days after germination . Graphs show mean ± SD of three biological replicates with at least 10 plants per genotype per replicate . Asterisks indicate a significant difference in root length in a two - way ANOVA followed by Tukey ' s multiple comparisons test ( * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 and * * * * P < 0 . 0001 ) .

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A, B. Quantification of oryzalin root growth assays in single (A) and double (B) mutants. DAG = days after germination. Graphs show mean ± SD of three biological replicates with at least 10 plants per genotype per replicate. Asterisks indicate a significant difference in root length in a two-way ANOVA followed by Tukey's multiple comparisons test (* P < 0.05, ** P < 0.01, *** P < 0.001 and **** P < 0.0001).


E . Silique pictures of cycb1 double mutant combinations . White arrowheads indicate aborted ovules and seeds . Scale bars : 500 μm . F , G . Quantification of aborted seeds in single ( F ) and double ( G ) mutants . Graphs represents the average seed abortion rate per plant ± SD of three biological replicates , n = 550 - 1029 seeds analyzed per genotype . Asterisks indicate significant differences in seed abortion rate in an ordinary one - way ANOVA test , followed by a Dunnett ' s multiple comparisons test ( * * * * P < 0 . 0001 ) .

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E. Silique pictures of cycb1 double mutant combinations. White arrowheads indicate aborted ovules and seeds. Scale bars: 500 μm. F, G. Quantification of aborted seeds in single (F) and double (G) mutants. Graphs represents the average seed abortion rate per plant ± SD of three biological replicates, n = 550-1029 seeds analyzed per genotype. Asterisks indicate significant differences in seed abortion rate in an ordinary one-way ANOVA test, followed by a Dunnett's multiple comparisons test (**** P < 0.0001).


C , D . Quantification of endosperm nuclei in cycb1 single ( C ) and double ( D ) mutants . Boxes and whiskers represent min to max values with the median indicated as a central horizontal line , n = 26 - 30 seeds per genotype . Asterisks show significant differences in the number of endosperm nuclei per seed in a Kruskal - Wallis test followed by Dunn ' s multiple comparisons test ( * * * * P < 0 . 0001 ) .

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C, D. Quantification of endosperm nuclei in cycb1 single (C) and double (D) mutants. Boxes and whiskers represent min to max values with the median indicated as a central horizontal line, n = 26-30 seeds per genotype. Asterisks show significant differences in the number of endosperm nuclei per seed in a Kruskal-Wallis test followed by Dunn's multiple comparisons test (**** P < 0.0001).


A . DIC images of abnormal embryo sacs in cycb1 mutant combinations . Red arrowheads indicate the visible nuclei in Col - 0 embryo sacs ( central and egg cells ) and the corresponding structures in the quadruple cycb1 ; 1 - / - cycb1 ; 2 + / - cycb1 ; 3 + / - cyb1 ; 4 - mutant . Scale bars : 20 μm . B . Quantification of the different abnormal embryo sac structures in cycb1 mutant combinations ( n = 202 - 459 embryo sacs per genotype ) . Different letters indicate significant differences in the proportion of abnormal embryo sacs in a Chi - squared test followed by the Marascuilo procedure to identify significant pairwise comparisons . WT = wildtype .

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A. DIC images of abnormal embryo sacs in cycb1 mutant combinations. Red arrowheads indicate the visible nuclei in Col-0 embryo sacs (central and egg cells) and the corresponding structures in the quadruple cycb1;1-/- cycb1;2+/- cycb1;3+/- cyb1;4- mutant. Scale bars: 20 μm. B. Quantification of the different abnormal embryo sac structures in cycb1 mutant combinations (n = 202-459 embryo sacs per genotype). Different letters indicate significant differences in the proportion of abnormal embryo sacs in a Chi-squared test followed by the Marascuilo procedure to identify significant pairwise comparisons. WT = wildtype.


C . DIC images of embryo sacs 3 DAP with wildtype pollen ( female x male ) . Red arrowheads indicate the visible embryo sac nuclei in the crosses with the quadruple cycb1 ; 1 - / - cycb1 ; 2 + / - cycb1 ; 3 + / - cyb1 ; 4 - mutant as a female donor , while the control Col - 0 x Col - 0 cross exhibits a developing embryo . Scale bars : 20 μm . D . Quantification of seed abortion in different cycb1 mutant combinations . Graph represents the average seed abortion rate per plant ± SD of three biological replicates , n = 463 - 579 seeds analyzed per genotype . Asterisks indicate significant differences in seed abortion rate in an ordinary one - way ANOVA test , followed by a Dunnett ' s multiple comparisons test ( * * * * P < 0 . 0001 ) .

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

C. DIC images of embryo sacs 3 DAP with wildtype pollen (female x male). Red arrowheads indicate the visible embryo sac nuclei in the crosses with the quadruple cycb1;1-/- cycb1;2+/- cycb1;3+/- cyb1;4- mutant as a female donor, while the control Col-0 x Col-0 cross exhibits a developing embryo. Scale bars: 20 μm. D. Quantification of seed abortion in different cycb1 mutant combinations. Graph represents the average seed abortion rate per plant ± SD of three biological replicates, n = 463-579 seeds analyzed per genotype. Asterisks indicate significant differences in seed abortion rate in an ordinary one-way ANOVA test, followed by a Dunnett's multiple comparisons test (**** P < 0.0001).


A , B . DAPI staining of pollen in cycb1 mutants , including pollen configurations found in cycb1 ; 1 - / - cycb1 ; 2 + / - cycb1 ; 3 + / - cycb1 ; 4 - / - mutants ( B ) . Scale bars : 5 μm . C . Quantification of DAPI - stained pollen configurations in different cycb1 mutant combinations , n = 420 - 616 pollen grains per genotype . Different letters indicate significant differences in the proportion of abnormal pollen ( uni - and bicellular ) in a Chi - squared test followed by the Marascuilo procedure to identify significant pairwise comparisons .

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A, B. DAPI staining of pollen in cycb1 mutants, including pollen configurations found in cycb1;1-/- cycb1;2+/- cycb1;3+/- cycb1;4-/- mutants (B). Scale bars: 5 μm. C. Quantification of DAPI-stained pollen configurations in different cycb1 mutant combinations, n = 420-616 pollen grains per genotype. Different letters indicate significant differences in the proportion of abnormal pollen (uni- and bicellular) in a Chi-squared test followed by the Marascuilo procedure to identify significant pairwise comparisons.


D . Alexander staining of mature pollen indicating pollen viability . Scale bars : 5 μm . E . Quantification of Alexander - stained pollen viability , n = 403 - 498 pollen grains per genotype . Different letters indicate significant differences in the proportion of dead pollen in a Chi - squared test followed by the Marascuilo procedure to identify significant pairwise comparisons .

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D. Alexander staining of mature pollen indicating pollen viability. Scale bars: 5 μm. E. Quantification of Alexander-stained pollen viability, n = 403-498 pollen grains per genotype. Different letters indicate significant differences in the proportion of dead pollen in a Chi-squared test followed by the Marascuilo procedure to identify significant pairwise comparisons.


A , B , C . Co - immunolocalization against tubulin ( magenta ) and KNOLLE ( green ) in root meristematic cells . Nuclei were counterstained with DAPI for the DNA ( cyan ) . White arrowheads indicate laggards in the metaphase stage . Scale bars : 5 μm .

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A, B, C. Co-immunolocalization against tubulin (magenta) and KNOLLE (green) in root meristematic cells. Nuclei were counterstained with DAPI for the DNA (cyan). White arrowheads indicate laggards in the metaphase stage. Scale bars: 5 μm.


D . Quantification of wildtype ( WT ) , double and misplaced PPBs . Different letters indicate significant differences in the proportions of the different arrays per category in a Chi - squared test followed by the Marascuilo procedure to identify significant pairwise comparisons . Ten roots were analyzed per genotype . E . Quantification of PPBs with prominent perinuclear microtubules ( MTs ) . Boxes and whiskers represent min to max values with the median indicated as a central horizontal line , n = 10 roots per genotype . Asterisks show significant differences in the percentage of PPBs with prominent perinuclear microtubules per root in an ANOVA test followed by Dunnett ' s multiple comparisons test ( * * P < 0 . 01 and * * * * P < 0 . 0001 ) . F . Quantification of spindles with lagging chromosomes . Boxes and whiskers represent min to max values with the median indicated as a central horizontal line , n = 10 roots per genotype . Asterisks show significant differences in the percentage of spindles with lagging chromosomes per root in a Kruskal - Wallis test followed by Dunn ' s multiple comparisons test ( * * P < 0 . 01 and ns = non - significant ) .

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D. Quantification of wildtype (WT), double and misplaced PPBs. Different letters indicate significant differences in the proportions of the different arrays per category in a Chi-squared test followed by the Marascuilo procedure to identify significant pairwise comparisons. Ten roots were analyzed per genotype. E. Quantification of PPBs with prominent perinuclear microtubules (MTs). Boxes and whiskers represent min to max values with the median indicated as a central horizontal line, n = 10 roots per genotype. Asterisks show significant differences in the percentage of PPBs with prominent perinuclear microtubules per root in an ANOVA test followed by Dunnett's multiple comparisons test (** P < 0.01 and **** P < 0.0001). F. Quantification of spindles with lagging chromosomes. Boxes and whiskers represent min to max values with the median indicated as a central horizontal line, n = 10 roots per genotype. Asterisks show significant differences in the percentage of spindles with lagging chromosomes per root in a Kruskal-Wallis test followed by Dunn's multiple comparisons test (** P < 0.01 and ns = non-significant).


A . Co - immunolocalization against tubulin ( magenta ) and KNOLLE ( green ) in root meristematic cells . Nuclei were counterstained with DAPI for the DNA ( cyan ) . Scale bars : 5 μm . B . Quantification of wildtype ( WT ) and abnormal phragmoplasts . Different letters indicate significant differences in the proportions of the different arrays per category in a Chi - squared test followed by the Marascuilo procedure to identify significant pairwise comparisons . Ten roots were analyzed per genotype . C . Quantification of the different mitotic stages in roots of the different genotypes . Different letters indicate significant differences in the proportions of the different arrays per category in a Chi - squared test followed by the Marascuilo procedure to identify significant pairwise comparisons . Ten roots were analyzed per genotype .

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A. Co-immunolocalization against tubulin (magenta) and KNOLLE (green) in root meristematic cells. Nuclei were counterstained with DAPI for the DNA (cyan). Scale bars: 5 μm. B. Quantification of wildtype (WT) and abnormal phragmoplasts. Different letters indicate significant differences in the proportions of the different arrays per category in a Chi-squared test followed by the Marascuilo procedure to identify significant pairwise comparisons. Ten roots were analyzed per genotype. C. Quantification of the different mitotic stages in roots of the different genotypes. Different letters indicate significant differences in the proportions of the different arrays per category in a Chi-squared test followed by the Marascuilo procedure to identify significant pairwise comparisons. Ten roots were analyzed per genotype.


A . Kinase assays against Histone H1 . Top and middle panels indicate shorter and longer exposures respectively of the same kinase assays . Bottom panel is a CBB staining of Histone H1 showing equal loading of the protein . A : CDKA ; 1 , B1 : CDKB1 ; 1 , B2 : CDKB2 ; 2 . B . Kinase assays against GIP1 . Top and and middle panels indicate shorter and longer exposures respectively of the same kinase assays . Bottom panel is a CBB staining of GIP1 showing equal loading of the protein . A : CDKA ; 1 , B1 : CDKB1 ; 1 , B2 : CDKB2 ; 2 . C . Western blot against StrepIII - tagged proteins to show loaded amounts of the CDKs . A : CDKA ; 1 , B1 : CDKB1 ; 1 , B2 : CDKB2 ; 2 . Double CDKB2 ; 2 bands are likely due to a truncation of the expressed protein .

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A. Kinase assays against Histone H1. Top and middle panels indicate shorter and longer exposures respectively of the same kinase assays. Bottom panel is a CBB staining of Histone H1 showing equal loading of the protein. A: CDKA;1, B1: CDKB1;1, B2: CDKB2;2. B. Kinase assays against GIP1. Top and and middle panels indicate shorter and longer exposures respectively of the same kinase assays. Bottom panel is a CBB staining of GIP1 showing equal loading of the protein. A: CDKA;1, B1: CDKB1;1, B2: CDKB2;2. C. Western blot against StrepIII-tagged proteins to show loaded amounts of the CDKs. A: CDKA;1, B1: CDKB1;1, B2: CDKB2;2. Double CDKB2;2 bands are likely due to a truncation of the expressed protein.


D . Time - lapse of confocal microscope pictures of root meristematic cells tagged with GFP - GIP1 in Col - 0 ( top panel ) and cycb1 ; 1 cycb1 ; 2 ( bottom panel ) . GIP1 localizes at the nuclear polar caps , followed by co - localization with microtubules at the spindle and phragmoplast arrays . In cycb1 ; 1 cycb1 ; 2 double mutants , GIP1 exhibited an abnormal localization , being found at the spindle ( magenta arrowheads ) and phragmoplast ( white arrowheads ) midzones , which are normally devoid of the protein . Scale bars : 5 μm .

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D. Time-lapse of confocal microscope pictures of root meristematic cells tagged with GFP-GIP1 in Col-0 (top panel) and cycb1;1 cycb1;2 (bottom panel). GIP1 localizes at the nuclear polar caps, followed by co-localization with microtubules at the spindle and phragmoplast arrays. In cycb1;1 cycb1;2 double mutants, GIP1 exhibited an abnormal localization, being found at the spindle (magenta arrowheads) and phragmoplast (white arrowheads) midzones, which are normally devoid of the protein. Scale bars: 5 μm.


C Wild - type and tel1 ∆ strains expressing a fully functional Top1 - HA tagged protein were arrested in G1 with α - factor ( αf ) and released into YEPD supplemented with CPT ( 50 μM ) . Western blot with anti - HA antibodies ( top ) and Coomassie staining ( bottom ) of protein extracts prepared at the indicated time points

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C Wild-type and tel1∆ strains expressing a fully functional Top1-HA tagged protein were arrested in G1 with α-factor (αf) and released into YEPD supplemented with CPT (50 μM). Western blot with anti-HA antibodies (top) and Coomassie staining (bottom) of protein extracts prepared at the indicated time points


A - C Exponentially growing cell cultures ( exp ) were arrested in G1 with α - factor ( αf ) and released into YEPD with or without CPT ( 50 μM ) . Cell samples were harvested at the indicated time points to evaluat Rad53 phosphorylation by western blot with anti - Rad53 antibodies ( C )

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A-C Exponentially growing cell cultures (exp) were arrested in G1 with α-factor (αf) and released into YEPD with or without CPT (50 μM). Cell samples were harvested at the indicated time points to evaluat Rad53 phosphorylation by western blot with anti-Rad53 antibodies (C)


E , F G1 - arrested cell cultures ( αf ) were released in YEPD supplemented with CPT ( 50 μM ) ( E ) or in YEPD supplemented with CPT ( 10 μM ) with ( + noc ) or without ( - noc ) nocodazole ( F ) . Western blot analysis with anti - Rad53 antibodies

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E, F G1-arrested cell cultures (αf) were released in YEPD supplemented with CPT (50 μM) (E) or in YEPD supplemented with CPT (10 μM) with (+noc) or without (-noc) nocodazole (F). Western blot analysis with anti-Rad53 antibodies


A - C Exponentially growing cell cultures were arrested in G1 with α - factor ( αf ) and released in YEPD supplemented with CPT ( 50 μM ) . ( A ) Western blot analysis with anti - Rad53 antibodies

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A-C Exponentially growing cell cultures were arrested in G1 with α-factor (αf) and released in YEPD supplemented with CPT (50 μM). (A) Western blot analysis with anti-Rad53 antibodies


( B - E ) G1 - arrested cell cultures ( αf ) were released in YEPD supplemented with CPT ( 50 μM ) . Sample collected at the indicated time points were subjected to western blot analysis with anti - Rad53 antibodie

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(B-E) G1-arrested cell cultures (αf) were released in YEPD supplemented with CPT (50 μM). Sample collected at the indicated time points were subjected to western blot analysis with anti-Rad53 antibodie


( B - E ) G1 - arrested cell cultures ( αf ) were released in YEPD supplemented with CPT ( 50 μM ) . Sample collected at the indicated time points were subjected to western blot analysis with anti - Rad53 antibodies ( B , D

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(B-E) G1-arrested cell cultures (αf) were released in YEPD supplemented with CPT (50 μM). Sample collected at the indicated time points were subjected to western blot analysis with anti-Rad53 antibodies (B, D


F , G G1 - arrested cell cultures ( αf ) were released into YEPD supplemented with CPT ( 50 μM ) . Western blot analysis with anti - Rad53 antibodies

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F, G G1-arrested cell cultures (αf) were released into YEPD supplemented with CPT (50 μM). Western blot analysis with anti-Rad53 antibodies


A Agarose gel image illustrating PCR amplicons of ltaS ( LBA0447 ) , slpB , and slpX deletions in NCK2187 .

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A Agarose gel image illustrating PCR amplicons of ltaS (LBA0447), slpB, and slpX deletions in NCK2187.


B SDS - PAGE gel of 5 M LiCl ‐ purified S ‐ layer fractions from the parental strains , NCK56 and NCK1909 , NCK2030 ( LTA + , SlpA + , SlpB − , SlpX − ) , and NCK2187 ( LTA − , SlpA + , SlpB − , SlpX − ) .

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B SDS-PAGE gel of 5 M LiCl‐purified S‐layer fractions from the parental strains, NCK56 and NCK1909, NCK2030 (LTA+, SlpA+, SlpB−, SlpX−), and NCK2187 (LTA−, SlpA+, SlpB−, SlpX−).


C B6 mice were orally gavaged with 109 CFU erythromycin ‐ resistant NCK56 or NCK2187 . Fecal pellets were collected daily and tested for the presence of erythromycin ‐ resistant strains . n = 3 mice / group . Data are representative of five independent experiments and are shown as mean ± SEM .

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C B6 mice were orally gavaged with 109 CFU erythromycin‐resistant NCK56 or NCK2187. Fecal pellets were collected daily and tested for the presence of erythromycin‐resistant strains. n = 3 mice/group. Data are representative of five independent experiments and are shown as mean ± SEM.


D ColonicLPL cells were co ‐ cultured with NCK56 or NCK2187 ( 1 : 1 ) , and secreted cytokines were measured in the supernatants . Data are shown as mean ± SEM and are representative of two experiments performed in triplicate . * P 0 . 05 , * * P 0 . 01 , * * * P 0 . 001 .

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D ColonicLPL cells were co‐cultured with NCK56 or NCK2187 (1:1), and secreted cytokines were measured in the supernatants. Data are shown as mean ± SEM and are representative of two experiments performed in triplicate. *P 0.05, **P 0.01, ***P 0.001.


A B6 mice were orally gavaged with 109CFU NCK56 ( blue ) or NCK2187 ( green ) on days 0 , 3 , 6 , and 9 , or left untreated , and immune responses in the colon analyzed at day 14 by flow cytometry .

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A B6 mice were orally gavaged with 109CFU NCK56 (blue) or NCK2187 (green) on days 0, 3, 6, and 9, or left untreated, and immune responses in the colon analyzed at day 14 by flow cytometry.


B , C B6 FoxP3 ‐ GFP mice were treated and evaluated as in ( A ) . Regulatory cytokine production in FoxP3 ‐ GFP + ( green ‐ dotted bars ) versus FoxP3 ‐ GFP − ( white bars ) cells was measured by intracellular staining and FACS analyses ( C ) .

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B, C B6 FoxP3‐GFP mice were treated and evaluated as in (A). Regulatory cytokine production in FoxP3‐GFP+ (green‐dotted bars) versus FoxP3‐GFP− (white bars) cells was measured by intracellular staining and FACS analyses (C).


A B6 Rag1 − / − mice were injected with 106CD4 + CD45RBhiT cells and then orally gavaged with NCK56 ( red ) , NCK2187 ( green ) , or SlpA ( blue ) , 1 and 3 days after transfer , and subsequently once a week for four consecutive weeks , or left untreated ( magenta ) . A group of mice was co ‐ transferred with CD4 + CD25 + T cells as a positive control for protection ( Tregs ; gray ) . Colitis severity was determined in part by weight loss , diarrhea scores , and FOB . See Supplementary Tables S1 , S2 and S3 for statistical analyses results .

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A B6 Rag1−/−mice were injected with 106CD4+CD45RBhiT cells and then orally gavaged with NCK56 (red), NCK2187 (green), or SlpA (blue), 1 and 3 days after transfer, and subsequently once a week for four consecutive weeks, or left untreated (magenta). A group of mice was co‐transferred with CD4+CD25+T cells as a positive control for protection (Tregs; gray). Colitis severity was determined in part by weight loss, diarrhea scores, and FOB. See Supplementary Tables S1, S2 and S3 for statistical analyses results.


B , C Colitis scores based on histopathology and gross morphology of the colons of mice treated as in ( A ) were also used as measures of disease . Scale bar = 50 μm .

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B, C Colitis scores based on histopathology and gross morphology of the colons of mice treated as in (A) were also used as measures of disease. Scale bar = 50 μm.


D Circulating levels of proinflammatory cytokines were measured in the sera of the mice transferred and treated as in ( A ) , or sham adoptive transferred ( white bars ) .

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D Circulating levels of proinflammatory cytokines were measured in the sera of the mice transferred and treated as in (A), or sham adoptive transferred (white bars).


Colitis was induced in B6 Rag1 − / − mice as described in Fig . A , B Colonic expression of tight junction ‐ associated genes Cldn3 and Ocln , determined by RT - PCR relative to 18S rRNA ( A ) , were used as measures of epithelial barrier integrity . Sham adoptive transferred B6 Rag1 − / − mice ( white bars ) were used as baseline controls in some cases . n = 5 mice / group . Data represent three individual experiments and are shown as mean ± SEM . * P 0 . 05 , * * P 0 . 01 . Black asterisks compare NCK2187 to PBS ‐ treated adoptively transferred mice , and red asterisks to NCK56 ‐ treated mice .

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Colitis was induced in B6 Rag1−/−mice as described in Fig .A, B Colonic expression of tight junction‐associated genes Cldn3 and Ocln, determined by RT-PCR relative to 18S rRNA (A), were used as measures of epithelial barrier integrity. Sham adoptive transferred B6 Rag1−/−mice (white bars) were used as baseline controls in some cases. n = 5 mice/group. Data represent three individual experiments and are shown as mean ± SEM. *P 0.05, **P 0.01. Black asterisks compare NCK2187 to PBS‐treated adoptively transferred mice, and red asterisks to NCK56‐treated mice.


Colitis was induced in B6 Rag1 − / − mice as described in Fig . A , B Passive transepithelial absorption of FITC ‐ dextran ( B ) , was used as measures of epithelial barrier integrity . Sham adoptive transferred B6 Rag1 − / − mice ( white bars ) were used as baseline controls in some cases . n = 5 mice / group . Data represent three individual experiments and are shown as mean ± SEM . * P 0 . 05 , * * P 0 . 01 . Black asterisks compare NCK2187 to PBS ‐ treated adoptively transferred mice , and red asterisks to NCK56 ‐ treated mice .

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Colitis was induced in B6 Rag1−/−mice as described in Fig .A, B Passive transepithelial absorption of FITC‐dextran (B), was used as measures of epithelial barrier integrity. Sham adoptive transferred B6 Rag1−/−mice (white bars) were used as baseline controls in some cases. n = 5 mice/group. Data represent three individual experiments and are shown as mean ± SEM. *P 0.05, **P 0.01. Black asterisks compare NCK2187 to PBS‐treated adoptively transferred mice, and red asterisks to NCK56‐treated mice.


A B6 mice were orally gavaged with 109CFU NCK56 or NCK2187 , and the colonic gene expression of C ‐ type lectin receptors were measured by RT - PCR . Each box represents an individual mouse ; n = 4 . Data represent three individual experiments .

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A B6 mice were orally gavaged with 109CFU NCK56 or NCK2187, and the colonic gene expression of C‐type lectin receptors were measured by RT-PCR. Each box represents an individual mouse; n = 4. Data represent three individual experiments.


B Binding of SlpA to various hFc fusion proteins was analyzed by flow cytometry . Gray tinted line = SlpA ‐ coated beads only ; orange = SlpA ‐ coated beads + secondary antibody ; green = SlpA ‐ coated beads + control fusion protein ; blue = SlpA ‐ coated beads + SIGNR1 ‐ hFc ; red = SlpA ‐ coated beads + SIGNR3 ‐ hFc . Binding assay results were confirmed five independent times .

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B Binding of SlpA to various hFc fusion proteins was analyzed by flow cytometry. Gray tinted line = SlpA‐coated beads only; orange = SlpA‐coated beads + secondary antibody; green = SlpA‐coated beads + control fusion protein; blue = SlpA‐coated beads + SIGNR1‐hFc; red = SlpA‐coated beads + SIGNR3‐hFc. Binding assay results were confirmed five independent times.


C Binding of SlpA to SIGNR3 , but not SIGNR1 expressed by CHO ‐ S cells . Gray tinted line = untransfected CHO ‐ S cells ; blue = untransfected CHO ‐ S cells + labeled SlpA ; green = SIGNR1 ‐ transfected CHO ‐ S cells + labeled SlpA ; red = SIGNR3 ‐ transfected CHO ‐ S cells + labeled SlpA . Binding assays in CHO ‐ S cells were performed three individual times .

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C Binding of SlpA to SIGNR3, but not SIGNR1 expressed by CHO‐S cells. Gray tinted line = untransfected CHO‐S cells; blue = untransfected CHO‐S cells + labeled SlpA; green = SIGNR1‐transfected CHO‐S cells + labeled SlpA; red = SIGNR3‐transfected CHO‐S cells + labeled SlpA. Binding assays in CHO‐S cells were performed three individual times.


D Binding of SlpA to recombinant human DC ‐ SIGN / CD209 ‐ Fc chimera was analyzed by flow cytometry . Gray tinted line = SlpA ‐ coated beads only ; orange = SlpA ‐ coated beads + secondary antibody ; green = SlpA ‐ coated beads + control fusion protein ; red = SlpA ‐ coated beads + DC ‐ SIGN ‐ Fc . Binding assays were performed and confirmed three times .

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D Binding of SlpA to recombinant human DC‐SIGN/CD209‐Fc chimera was analyzed by flow cytometry. Gray tinted line = SlpA‐coated beads only; orange = SlpA‐coated beads + secondary antibody; green = SlpA‐coated beads + control fusion protein; red = SlpA‐coated beads + DC‐SIGN‐Fc. Binding assays were performed and confirmed three times.


E Binding of SlpA to DC ‐ SIGN expressed by CHO ‐ S cells . Gray tinted line = untransfected CHO ‐ S cells ; blue = untransfected CHO ‐ S cells + labeled SlpA ; red = DC ‐ SIGN ‐ transfected CHO ‐ S cells + labeled SlpA . Binding assays in CHO ‐ S cells were performed three individual times .

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E Binding of SlpA to DC‐SIGN expressed by CHO‐S cells. Gray tinted line = untransfected CHO‐S cells; blue = untransfected CHO‐S cells + labeled SlpA; red = DC‐SIGN‐transfected CHO‐S cells + labeled SlpA. Binding assays in CHO‐S cells were performed three individual times.


F , G IL ‐ 1β production by colonic DCs of naïve WT B6 GF or B6 Signr3 − / − ( KO ) mice treated with NCK56 ( blue ) or NCK2187 ( green ) four times , or left untreated ( black ) , as determined by flow cytometry . n = 5 mice / group . Data represent four individual experiments and are shown as mean ± SEM . * P 0 . 05 . Black asterisks compare NCK2187 to untreated ( PBS ) mice , and red asterisks to NCK56 ‐ treated mice .

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F, G IL‐1β production by colonic DCs of naïve WT B6 GF or B6 Signr3−/− (KO) mice treated with NCK56 (blue) or NCK2187 (green) four times, or left untreated (black), as determined by flow cytometry. n = 5 mice/group. Data represent four individual experiments and are shown as mean ± SEM. *P 0.05. Black asterisks compare NCK2187 to untreated (PBS) mice, and red asterisks to NCK56‐treated mice.


H Frequency of colonic FoxP3 + Tregs in KO mice treated with NCK56 or NCK2187 was measured by flow cytometry . n = 5 mice / group . Data represent four individual experiments and are shown as mean ± SEM .

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H Frequency of colonic FoxP3+ Tregs in KO mice treated with NCK56 or NCK2187 was measured by flow cytometry. n = 5 mice/group. Data represent four individual experiments and are shown as mean ± SEM.


WT B6 or B6 Signr3 − / − ( KO ) mice were orally gavaged with NCK56 , NCK2187 , or SlpA on days − 3 and − 1 , and 3 % DSS was given in the drinking water . Mice were gavaged with bacteria or purified SlpA every other day for an additional three times and monitored for disease progression . A Colitis severity was determined in part by weight loss . n = 5 mice / group .

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WT B6 or B6 Signr3−/− (KO) mice were orally gavaged with NCK56, NCK2187, or SlpA on days −3 and −1, and 3% DSS was given in the drinking water. Mice were gavaged with bacteria or purified SlpA every other day for an additional three times and monitored for disease progression.A Colitis severity was determined in part by weight loss. n = 5 mice/group.


WT B6 or B6 Signr3 − / − ( KO ) mice were orally gavaged with NCK56 , NCK2187 , or SlpA on days − 3 and − 1 , and 3 % DSS was given in the drinking water . Mice were gavaged with bacteria or purified SlpA every other day for an additional three times and monitored for disease progression . B , C Colitis scores based on histopathology and gross morphology of the colons were also used as measures of disease . Scale bar = 50 μm . n = 5 mice / group . Empty bars = WT ; lined bars = KO ; white bars = untreated ; purple bars = DSS ; red bars = DSS + NCK56 ; green bars = DSS + NCK2187 ; blue bars = DSS + SlpA .

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WT B6 or B6 Signr3−/− (KO) mice were orally gavaged with NCK56, NCK2187, or SlpA on days −3 and −1, and 3% DSS was given in the drinking water. Mice were gavaged with bacteria or purified SlpA every other day for an additional three times and monitored for disease progression.B, C Colitis scores based on histopathology and gross morphology of the colons were also used as measures of disease. Scale bar = 50 μm. n = 5 mice/group. Empty bars = WT; lined bars = KO; white bars = untreated; purple bars = DSS; red bars = DSS + NCK56; green bars = DSS + NCK2187; blue bars = DSS + SlpA.


WT B6 or B6 Signr3 − / − ( KO ) mice were orally gavaged with NCK56 , NCK2187 , or SlpA on days − 3 and − 1 , and 3 % DSS was given in the drinking water . Mice were gavaged with bacteria or purified SlpA every other day for an additional three times and monitored for disease progression . D Colonoscopies were performed in the different groups with a Multi ‐ Purpose Rigid ™ Telescope attached to a TELE PACK X on day 10 .

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WT B6 or B6 Signr3−/− (KO) mice were orally gavaged with NCK56, NCK2187, or SlpA on days −3 and −1, and 3% DSS was given in the drinking water. Mice were gavaged with bacteria or purified SlpA every other day for an additional three times and monitored for disease progression.D Colonoscopies were performed in the different groups with a Multi‐Purpose Rigid™ Telescope attached to a TELE PACK X on day 10.


WT B6 or B6 Signr3 − / − ( KO ) mice were orally gavaged with NCK56 , NCK2187 , or SlpA on days − 3 and − 1 , and 3 % DSS was given in the drinking water . Mice were gavaged with bacteria or purified SlpA every other day for an additional three times and monitored for disease progression . E Mean relative colonic expression of tight junction ‐ associated genes in WT mice . n = 5 mice / group .

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WT B6 or B6 Signr3−/− (KO) mice were orally gavaged with NCK56, NCK2187, or SlpA on days −3 and −1, and 3% DSS was given in the drinking water. Mice were gavaged with bacteria or purified SlpA every other day for an additional three times and monitored for disease progression.E Mean relative colonic expression of tight junction‐associated genes in WT mice. n = 5 mice/group.


WT B6 or B6 Signr3 − / − ( KO ) mice were orally gavaged with NCK56 , NCK2187 , or SlpA on days − 3 and − 1 , and 3 % DSS was given in the drinking water . Mice were gavaged with bacteria or purified SlpA every other day for an additional three times and monitored for disease progression . F Fecalalbumin levels in WT mice as a measure of intestinal permeability . n = 5 mice / group .

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WT B6 or B6 Signr3−/− (KO) mice were orally gavaged with NCK56, NCK2187, or SlpA on days −3 and −1, and 3% DSS was given in the drinking water. Mice were gavaged with bacteria or purified SlpA every other day for an additional three times and monitored for disease progression.F Fecalalbumin levels in WT mice as a measure of intestinal permeability. n = 5 mice/group.


Signr3 + / + ( WT ) or Signr3 − / − ( KO ) mice were orally gavaged with NCK56 , NCK2187 , or SlpA on days − 1 and − 3 , and 3 % DSS was given in the drinking water . Mice were gavaged with bacteria or purified SlpA every other day for an additional three times , and immunity was analyzed by flow cytometry at day 10 . A Representative plots indicate the frequency of neutrophils in the colons of untreated or DSS ‐ treated WT ( left ) and KO mice ( right ) . Empty bars = WT ; lined bars = KO ; white bars = untreated ; purple bars = DSS ; red bars = DSS + NCK56 ; green bars = DSS + NCK2187 ; blue bars = DSS + SlpA .

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Signr3+/+ (WT) or Signr3−/− (KO) mice were orally gavaged with NCK56, NCK2187, or SlpA on days −1 and −3, and 3% DSS was given in the drinking water. Mice were gavaged with bacteria or purified SlpA every other day for an additional three times, and immunity was analyzed by flow cytometry at day 10.A Representative plots indicate the frequency of neutrophils in the colons of untreated or DSS‐treated WT (left) and KO mice (right). Empty bars = WT; lined bars = KO; white bars = untreated; purple bars = DSS; red bars = DSS + NCK56; green bars = DSS + NCK2187; blue bars = DSS + SlpA.


Signr3 + / + ( WT ) or Signr3 − / − ( KO ) mice were orally gavaged with NCK56 , NCK2187 , or SlpA on days − 1 and − 3 , and 3 % DSS was given in the drinking water . Mice were gavaged with bacteria or purified SlpA every other day for an additional three times , and immunity was analyzed by flow cytometry at day 10 . B , C Colonic DCs and macrophages ( MΦs ) were analyzed by flow cytometry for the production of IL ‐ 1β ( B ) , and colonic FoxP3 + Tregs were evaluated for co ‐ expression of RORγt + ( C ) . n = 5 mice / group . Gray tinted line = isotype control ; black = untreated ; purple = DSS ; red = DSS + NCK56 ; green = DSS + NCK2187 ; blue = DSS + SlpA .

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Signr3+/+ (WT) or Signr3−/− (KO) mice were orally gavaged with NCK56, NCK2187, or SlpA on days −1 and −3, and 3% DSS was given in the drinking water. Mice were gavaged with bacteria or purified SlpA every other day for an additional three times, and immunity was analyzed by flow cytometry at day 10.B, C Colonic DCs and macrophages (MΦs) were analyzed by flow cytometry for the production of IL‐1β (B), and colonic FoxP3+ Tregs were evaluated for co‐expression of RORγt+ (C). n = 5 mice/group. Gray tinted line = isotype control; black = untreated; purple = DSS; red = DSS + NCK56; green = DSS + NCK2187; blue = DSS + SlpA.


CRTH2 subcellular localization in GFP - fused CRTH2 expressing plasmid - transfected cells . Red arrows indicate CRTH2 localized in the ER and white arrows indicate CRTH2 localized in the plasma membrane . Green , GFP - CRTH2 ; blue , DAPI ; red , Calnexin ; Scale bar , 20 μm .

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CRTH2 subcellular localization in GFP-fused CRTH2 expressing plasmid-transfected cells. Red arrows indicate CRTH2 localized in the ER and white arrows indicate CRTH2 localized in the plasma membrane. Green, GFP-CRTH2; blue, DAPI; red, Calnexin; Scale bar, 20 μm.


Western blot analysis of CRTH2 expression in cell membrane and ER in NIH 3T3 and HEK293T cells . Myc - tagged CRTH2 expressing plasmid was transfected into NIH 3T3 and HEK293T cells . Whole cell lysate , plasma , and ER fractions were obtained 48 h after transfection . Calnexin and ATP1B were used as markers for ER and cell membrane , respectively .

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Western blot analysis of CRTH2 expression in cell membrane and ER in NIH 3T3 and HEK293T cells. Myc-tagged CRTH2 expressing plasmid was transfected into NIH 3T3 and HEK293T cells. Whole cell lysate, plasma, and ER fractions were obtained 48 h after transfection. Calnexin and ATP1B were used as markers for ER and cell membrane, respectively.


Representative images of CRTH2 subcellular localization in NIH 3T3 cells in response to TGF - β1 ( 10 ng / mL ) treatment . Red arrows indicate CRTH2 localized in the ER and white arrows indicate CRTH2 localized in the plasma membrane . Green , GFP - CRTH2 ; blue , DAPI ; red , Calnexin ; Scale bar , 20 μm .

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Representative images of CRTH2 subcellular localization in NIH 3T3 cells in response to TGF-β1 (10 ng/mL) treatment. Red arrows indicate CRTH2 localized in the ER and white arrows indicate CRTH2 localized in the plasma membrane. Green, GFP-CRTH2; blue, DAPI; red, Calnexin; Scale bar, 20 μm.


Co - immunoprecipitation analysis of association of CRTH2 with LARP6 . HEK293T cells were co - transfected with Myc - CRTH2 and Flag - LARP6 expressing plasmids . Whole cell lysates from HEK293T cells were immunoprecipitated and then immunoblotted with the indicated antibodies .

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Co-immunoprecipitation analysis of association of CRTH2 with LARP6. HEK293T cells were co-transfected with Myc-CRTH2 and Flag-LARP6 expressing plasmids. Whole cell lysates from HEK293T cells were immunoprecipitated and then immunoblotted with the indicated antibodies.


Co - IP analysis of association of CRTH2 with GPR78 . HEK293T cells were co - transfected with Myc - GPR78 expressing plasmids .

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Co-IP analysis of association of CRTH2 with GPR78. HEK293T cells were co-transfected with Myc-GPR78 expressing plasmids.


Effect of LARP6 knockdown on Col1a1 , Col1a2 and Col3a1 mRNA stability in WT and CRTH2 - / - fibroblasts . * P < 0 . 05 vs WT , ( two - way ANOVA ) ; n = 8 for all groups .

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Effect of LARP6 knockdown on Col1a1, Col1a2 and Col3a1 mRNA stability in WT and CRTH2-/- fibroblasts. *P < 0.05 vs WT, (two-way ANOVA); n = 8 for all groups.


Effect of LARP6 knockdown on Collagen I and III protein expression in WT and CRTH2 - / - fibroblasts .

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Effect of LARP6 knockdown on Collagen I and III protein expression in WT and CRTH2-/- fibroblasts.


, Representative images of Masson ′ s trichrome staining of lung sections from CRTH2flox / floxCol1a2 - CreERT ( F - CRTH2 KO ) and CRTH2flox / flox ( Control ) mice 14 days after bleomycin challenge . Blue indicates the collagen deposition regions . Scale bar , 100 μm . Quantification of collagen content in mouse lung sections after bleomycin challenge . * P < 0 . 05 vs control , # P < 0 . 05 vs saline group ( two - way ANOVA ) ; saline groups , n = 4 each ; bleomycin groups , n = 8 each .

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, Representative images of Masson′s trichrome staining of lung sections from CRTH2flox/floxCol1a2-CreERT (F-CRTH2 KO) and CRTH2flox/flox (Control) mice 14 days after bleomycin challenge. Blue indicates the collagen deposition regions. Scale bar, 100 μm. Quantification of collagen content in mouse lung sections after bleomycin challenge. *P < 0.05 vs control, #P < 0.05 vs saline group (two-way ANOVA); saline groups, n = 4 each; bleomycin groups, n = 8 each.


Representative images of Masson ′ s trichrome staining of liver sections from control and F - CRTH2 KO mice 4 weeks after CCl4 challenge ; Scale bar , 100 μm .

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Representative images of Masson′s trichrome staining of liver sections from control and F-CRTH2 KO mice 4 weeks after CCl4 challenge; Scale bar, 100 μm.


Representative images of Masson ′ s trichrome staining of heart sections from control and F - CRTH2 KO mice 21 days after ISO challenge ; Scale bar , 50 μm . Quantification of collagen content in mouse heart sections after ISO challenge . * P < 0 . 05 vs control , # P < 0 . 05 vs saline group ( two - way ANOVA ) ; PBS groups , n = 3 each , ISO groups , n = 7 - 8 .

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Representative images of Masson′s trichrome staining of heart sections from control and F-CRTH2 KO mice 21 days after ISO challenge; Scale bar, 50 μm. Quantification of collagen content in mouse heart sections after ISO challenge. *P < 0.05 vs control, #P < 0.05 vs saline group (two-way ANOVA); PBS groups, n = 3 each, ISO groups, n = 7-8.


Representative images of Masson ' s trichrome staining of lung sections from F - LARP6 KO and F - LARP6 / F - CRTH2 double KO ( F - DKO ) mice 14 days after bleomycin challenge ; Scale bar , 100 μm . , Quantification of collagen content in mouse lung sections after bleomycin challenge . # P < 0 . 05 vs saline group ( two - way ANOVA ) ; saline groups , n = 4 each , bleomycin groups , n = 7 - 8 . Hydroxyproline content in lungs from F - LARP6 KO and F - DKO mice treated with bleomycin . # P < 0 . 05 vs saline group ( two - way ANOVA ) ; saline groups , n = 4 each , bleomycin groups , n = 8 each .

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Representative images of Masson's trichrome staining of lung sections from F-LARP6 KO and F-LARP6/F-CRTH2 double KO (F-DKO) mice 14 days after bleomycin challenge; Scale bar, 100 μm. , Quantification of collagen content in mouse lung sections after bleomycin challenge. #P < 0.05 vs saline group (two-way ANOVA); saline groups, n = 4 each, bleomycin groups, n = 7-8. Hydroxyproline content in lungs from F-LARP6 KO and F-DKO mice treated with bleomycin. #P < 0.05 vs saline group (two-way ANOVA); saline groups, n = 4 each, bleomycin groups, n = 8 each.


Representative images of Masson ' s trichrome staining of liver sections from F - LARP6 KO and F - DKO mice 4 weeks after CCl4 challenge ; Scale bar , 100 μm . Quantification of collagen content in mouse liver sections after CCl4 challenge . # P < 0 . 05 vs mineral oil group ( two - way ANOVA ) ; mineral oil groups , n = 4 each , CCl4 groups , n = 8 each . Hydroxyproline content in livers from F - LARP6 KO and F - DKO mice treated with CCl4 . # P < 0 . 05 vs mineral oil group ( two - way ANOVA ) ; saline groups , n = 4 each , CCl4 groups , n = 8 each .

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Representative images of Masson's trichrome staining of liver sections from F-LARP6 KO and F-DKO mice 4 weeks after CCl4 challenge; Scale bar, 100 μm. Quantification of collagen content in mouse liver sections after CCl4 challenge. #P < 0.05 vs mineral oil group (two-way ANOVA); mineral oil groups, n = 4 each, CCl4 groups, n = 8 each. Hydroxyproline content in livers from F-LARP6 KO and F-DKO mice treated with CCl4. #P < 0.05 vs mineral oil group (two-way ANOVA); saline groups, n = 4 each, CCl4 groups, n = 8 each.


Representative images of Masson ' s trichrome staining of heart sections from F - LARP6 KO and F - DKO mice 21 days after ISO challenge ; Scale bar , 50 μm . Quantification of collagen content in mouse heart sections after ISO challenge . # P < 0 . 05 vs saline group ( two - way ANOVA ) ; saline groups , n = 3 each , ISO groups , n = 7 each . , Hydroxyproline content in hearts from F - LARP6 KO and F - DKO mice treated with ISO . # P < 0 . 05 vs saline group ( two - way ANOVA ) ; saline groups , n = 4 each , ISO groups , n = 8 each .

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Representative images of Masson's trichrome staining of heart sections from F-LARP6 KO and F-DKO mice 21 days after ISO challenge; Scale bar, 50 μm. Quantification of collagen content in mouse heart sections after ISO challenge. #P < 0.05 vs saline group (two-way ANOVA); saline groups, n = 3 each, ISO groups, n = 7 each. , Hydroxyproline content in hearts from F-LARP6 KO and F-DKO mice treated with ISO. #P < 0.05 vs saline group (two-way ANOVA); saline groups, n = 4 each, ISO groups, n = 8 each.


Microscale thermophoresis ( MST ) analysis of the binding affinity of Bumetanide with MBP -

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Microscale thermophoresis (MST) analysis of the binding affinity of Bumetanide with MBP-LARP6