D , E NIH3T3 wild type ( WT ) and Rab35 knockout ( KO ) cell lines were serum - starved for 24h and stained for Arl13b , acetylated tubulin ( acetyl . tub . ) and DNA . Representative images are shown in ( D ) . Higher magnifications of regions within white boxes shown at the bottom . Scale bars ; 10 µm . ( E ) Box - and - whisker plots show quantification of ciliary Arl13b intensity in arbitrary units ( a . u . ) . Horizontal lines show 25 , 50 and 75th percentiles ; whiskers extend to minimum and maximum values . One representative experiment out of three is shown ( n > 25 cilia per experimental condition ) . Statistical significance according to Kruskal - Wallis followed by Dunn ' s post - hoc test ( * P < 0 . 05 ; P - values : NIH3T3 WT vs . Rab35 KO # 1 P = 0 . 0118 , NIH3T3 WT vs . Rab35 KO # 2 P = 0 . 0111 ) .

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

D,E NIH3T3 wild type (WT) and Rab35 knockout (KO) cell lines were serum-starved for 24h and stained for Arl13b, acetylated tubulin (acetyl. tub.) and DNA. Representative images are shown in (D). Higher magnifications of regions within white boxes shown at the bottom. Scale bars; 10 µm. (E) Box-and-whisker plots show quantification of ciliary Arl13b intensity in arbitrary units (a. u.). Horizontal lines show 25, 50 and 75th percentiles; whiskers extend to minimum and maximum values. One representative experiment out of three is shown (n > 25 cilia per experimental condition). Statistical significance according to Kruskal-Wallis followed by Dunn's post-hoc test (* P < 0.05; P-values: NIH3T3 WT vs. Rab35 KO#1 P = 0.0118, NIH3T3 WT vs. Rab35 KO#2 P = 0.0111).


F Immunoblot analysis of total ARL13B protein levels in serum - starved NIH3T3 WT and Rab35 KO cell lines . β - tubulin served as a loading control .

O O O O O 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 Immunoblot analysis of total ARL13B protein levels in serum-starved NIH3T3 WT and Rab35 KO cell lines. β-tubulin served as a loading control.


Quantification of ciliary ARL13B intensity in hTERT - RPE1 cells depleted of GEF ( G ) regulators of RAB35 . Cells transfected with indicated siRNAs were serum - starved for 48 h and stained for ARL13B , acetylated tubulin and DNA . Horizontal lines show 25 , 50 and 75th percentiles ; whiskers extend to minimum and maximum values . One representative experiment out of three is shown ( n > 30 cilia per experimental condition ) . Statistical significance according ANOVA followed by Bonferroni post - hoc test ( * * * P < 0 . 001 ; P - values : ( G ) P = 0 .

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

Quantification of ciliary ARL13B intensity in hTERT-RPE1 cells depleted of GEF (G) regulators of RAB35. Cells transfected with indicated siRNAs were serum-starved for 48 h and stained for ARL13B, acetylated tubulin and DNA. Horizontal lines show 25, 50 and 75th percentiles; whiskers extend to minimum and maximum values. One representative experiment out of three is shown (n > 30 cilia per experimental condition). Statistical significance according ANOVA followed by Bonferroni post-hoc test (*** P < 0.001; P-values: (G) P = 0.0005


Quantification of ciliary ARL13B intensity in hTERT - RPE1 cells depleted of GAP ( H ) regulators of RAB35 . Cells transfected with indicated siRNAs were serum - starved for 48 h and stained for ARL13B , acetylated tubulin and DNA . Horizontal lines show 25 , 50 and 75th percentiles ; whiskers extend to minimum and maximum values . One representative experiment out of three is shown ( n > 30 cilia per experimental condition ) . Statistical significance according ANOVA followed by Bonferroni post - hoc test ( * * * P < 0 . 001 ; P - values : ( H ) 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 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

Quantification of ciliary ARL13B intensity in hTERT-RPE1 cells depleted of GAP (H) regulators of RAB35. Cells transfected with indicated siRNAs were serum-starved for 48 h and stained for ARL13B, acetylated tubulin and DNA. Horizontal lines show 25, 50 and 75th percentiles; whiskers extend to minimum and maximum values. One representative experiment out of three is shown (n > 30 cilia per experimental condition). Statistical significance according ANOVA followed by Bonferroni post-hoc test (*** P < 0.001; P-values: (H) P < 0.0001).


I , J Ciliary ARL13B intensity quantification of hTERT - RPE1 cells transiently overexpressing GFP or GFP - RAB35 . Cells were serum - starved for 24 h and stained for ARL13B , acetylated tubulin ( acetyl . tub . ) and DNA . Representative images are shown in ( I ) . Higher magnification images of the cilia region are shown at the bottom . Scale bars ; 5 µm . Cells with ( RAB35 + cilia ) and without ( RAB35 - cilia ) GFP - RAB35 ciliary localisation are compared in ( J ) . Horizontal lines show 25 , 50 and 75th percentiles ; whiskers extend to minimum and maximum values . One representative experiment out of three is shown ( n > 25 cilia per experimental condition ) . Statistical significance according to Kruskal - Wallis followed by Dunn ' s post - hoc test ( * * * P = 0 . 0004 ) .

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

I, J Ciliary ARL13B intensity quantification of hTERT-RPE1 cells transiently overexpressing GFP or GFP-RAB35. Cells were serum-starved for 24 h and stained for ARL13B, acetylated tubulin (acetyl. tub.) and DNA. Representative images are shown in (I). Higher magnification images of the cilia region are shown at the bottom. Scale bars; 5 µm. Cells with (RAB35+ cilia) and without (RAB35- cilia) GFP-RAB35 ciliary localisation are compared in (J). Horizontal lines show 25, 50 and 75th percentiles; whiskers extend to minimum and maximum values. One representative experiment out of three is shown (n > 25 cilia per experimental condition). Statistical significance according to Kruskal-Wallis followed by Dunn's post-hoc test (*** P =0.0004).


A HEK293T cells were transiently co - transfected with ARL13B - FLAG and GFP or GFP - RAB35 . Cells were cultured either in full growth medium ( + serum ) or serum - starved for the final 8 h before harvesting . Immunoprecipitations ( IP ) were performed using anti - GFP beads . Cell lysates ( 2 . 5 % of input ) and immunoprecipitates were loaded on the same gel , subjected to immunoblot ( I ) analysis with indicated antibodies and detected with the same exposure .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 were transiently co-transfected with ARL13B-FLAG and GFP or GFP-RAB35. Cells were cultured either in full growth medium (+serum) or serum-starved for the final 8 h before harvesting. Immunoprecipitations (IP) were performed using anti-GFP beads. Cell lysates (2.5% of input) and immunoprecipitates were loaded on the same gel, subjected to immunoblot (I) analysis with indicated antibodies and detected with the same exposure.


C HEK293T cells were transiently co - transfected with mCherry - RAB35 together with GFP or indicated ARL13B - GFP wild type , mutant or truncation constructs . Cells were serum - starved for the final 8 h before harvesting . IPs were performed using anti - GFP beads and interacting proteins detected by immunoblotting ( IB ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 HEK293T cells were transiently co-transfected with mCherry-RAB35 together with GFP or indicated ARL13B-GFP wild type, mutant or truncation constructs. Cells were serum-starved for the final 8 h before harvesting. IPs were performed using anti-GFP beads and interacting proteins detected by immunoblotting (IB).


D HEK293T cells were transiently transfected with ARL13B - FLAG and serum - starved for the final 8 h before harvesting . HEK293T cell lysates expressing ARL13B - FLAG were subjected to pull - down with GFP - RAB35 bound to anti - GFP beads and preloaded with either no nucleotide , GTPγS or GDP . Bound proteins and cell lysates ( 1 % of input ) were analysed by immunoblotting . The graph shows the ratio of precipitated ARL13B and RAB35 , normalized to the no nucleotide control . Data are mean ± S . E . M . of 3 independent experiments . Statistical significance according ANOVA followed by Tukey post - hoc test ( * P = 0 . 0333 ) .

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

D HEK293T cells were transiently transfected with ARL13B-FLAG and serum-starved for the final 8 h before harvesting. HEK293T cell lysates expressing ARL13B-FLAG were subjected to pull-down with GFP-RAB35 bound to anti-GFP beads and preloaded with either no nucleotide, GTPγS or GDP. Bound proteins and cell lysates (1% of input) were analysed by immunoblotting. The graph shows the ratio of precipitated ARL13B and RAB35, normalized to the no nucleotide control. Data are mean ± S.E.M. of 3 independent experiments. Statistical significance according ANOVA followed by Tukey post-hoc test (* P =0.0333).


E HEK293T cells were transiently transfected with mCherry - RAB35 and serum - starved for the final 8 h before harvesting . HEK293T cell lysates expressing ARL13B - FLAG were subjected to pull - down with ARL13B - GFP bound to anti - GFP beads and preloaded with either no nucleotide , GTPγS or GDP . Bound proteins and cell lysates ( 1 % of input ) were analysed by immunoblotting . The graph shows the ratio of precipitated ARL13B and RAB35 , normalized to the no nucleotide control . Data are mean ± S . E . M . of 3 independent experiments . Statistical significance according ANOVA followed by Tukey post - hoc test ( * P = 0 . 0466 )

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

E HEK293T cells were transiently transfected with mCherry-RAB35 and serum-starved for the final 8 h before harvesting. HEK293T cell lysates expressing ARL13B-FLAG were subjected to pull-down with ARL13B-GFP bound to anti-GFP beads and preloaded with either no nucleotide, GTPγS or GDP. Bound proteins and cell lysates (1% of input) were analysed by immunoblotting. The graph shows the ratio of precipitated ARL13B and RAB35, normalized to the no nucleotide control. Data are mean ± S.E.M. of 3 independent experiments. Statistical significance according ANOVA followed by Tukey post-hoc test (* P =0.0466)


A , B hTERT - RPE1 cells transfected with non - targeting siRNA control ( Neg ) or RAB35 siRNA , were serum - starved for 48 h and stained for INPP5E , ARL13B , acetylated tubulin ( acetyl . tub . ) and DNA . Representative images are shown in ( A ) . Regions within white boxes shown at higher magnifications to the right . Scale bars , 10 µm . ( B ) Box - and - whisker plots show quantification of ciliary INPP5E intensity in arbitrary units ( a . u . ) . Horizontal lines show 25 , 50 and 75th percentiles ; whiskers extend to minimum and maximum values . One representative experiment out of three is shown ( n > 50 cilia per experimental condition ) . Statistical significance according to Kruskal - Wallis followed by Dunn ' s post - hoc test ( * * * P < 0 . 001 ; P - values : Neg vs . RAB35 - 1 P < 0 . 0001 , Neg vs . RAB35 - 2 P = 0 . 0002 ) .

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

A, B hTERT-RPE1 cells transfected with non-targeting siRNA control (Neg) or RAB35 siRNA, were serum-starved for 48 h and stained for INPP5E, ARL13B, acetylated tubulin (acetyl. tub.) and DNA. Representative images are shown in (A). Regions within white boxes shown at higher magnifications to the right. Scale bars, 10 µm. (B) Box-and-whisker plots show quantification of ciliary INPP5E intensity in arbitrary units (a. u.). Horizontal lines show 25, 50 and 75th percentiles; whiskers extend to minimum and maximum values. One representative experiment out of three is shown (n > 50 cilia per experimental condition). Statistical significance according to Kruskal-Wallis followed by Dunn's post-hoc test (*** P < 0.001; P-values: Neg vs. RAB35-1 P < 0.0001, Neg vs. RAB35-2 P = 0.0002).


C For co - depletion experiments , hTERT - RPE1 cells were first transfected with Neg or RAB35 siRNA . After 24h , a second transfection with Neg or ARL13B siRNA was performed and cells were serum starved for 48 h and stained as in ( A ) . Horizontal lines show 25 , 50 and 75th percentiles ; whiskers extend to minimum and maximum values . One representative experiment out of three is shown ( n > 40 cilia per experimental condition ) . Statistical significance according to Kruskal - Wallis followed by Dunn ' s post - hoc test ( * * P = 0 . 0017 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 For co-depletion experiments, hTERT-RPE1 cells were first transfected with Neg or RAB35 siRNA. After 24h, a second transfection with Neg or ARL13B siRNA was performed and cells were serum starved for 48 h and stained as in (A). Horizontal lines show 25, 50 and 75th percentiles; whiskers extend to minimum and maximum values. One representative experiment out of three is shown (n > 40 cilia per experimental condition). Statistical significance according to Kruskal-Wallis followed by Dunn's post-hoc test (** P = 0.0017).


D , E NIH3T3 wild type ( WT ) and Rab35 knockout ( KO ) cell lines were serum - starved for 24h and stained for Innpp5e , acetylated tubulin ( acetyl . tub . ) and DNA . Representative images are shown in ( D ) . Higher magnifications of regions within white boxes shown at the bottom . Scale bars ; 10 µm . ( E ) Box - and - whisker plots show quantification of ciliary Inpp5e intensity in arbitrary units ( a . u . ) . Horizontal lines show 25 , 50 and 75th percentiles ; whiskers extend to minimum and maximum values . One representative experiment out of three is shown ( n > 25 cilia per experimental condition ) . Statistical significance according to Kruskal - Wallis followed by Dunn ' s post - hoc test ( * P < 0 . 05 , * * * P < 0 . 001 ; NIH3T3 WT vs . Rab35 KO # 1 P < 0 . 0001 , NIH3T3 WT vs . Rab35 KO # 2 P = 0 . 0345 ) .

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

D, E NIH3T3 wild type (WT) and Rab35 knockout (KO) cell lines were serum-starved for 24h and stained for Innpp5e, acetylated tubulin (acetyl. tub.) and DNA. Representative images are shown in (D). Higher magnifications of regions within white boxes shown at the bottom. Scale bars; 10 µm. (E) Box-and-whisker plots show quantification of ciliary Inpp5e intensity in arbitrary units (a. u.). Horizontal lines show 25, 50 and 75th percentiles; whiskers extend to minimum and maximum values. One representative experiment out of three is shown (n > 25 cilia per experimental condition). Statistical significance according to Kruskal-Wallis followed by Dunn's post-hoc test (* P < 0.05, *** P < 0.001; NIH3T3 WT vs. Rab35 KO#1 P < 0.0001, NIH3T3 WT vs. Rab35 KO#2 P = 0.0345).


F , G Localisation of the PI ( 4 , 5 ) P2 sensor PH - PLCδ1 - GFP to cilia in live NIH3T3 WT and Rab35 KO cells . Cilia are marked with HTR6 - RFP . Representative images are shown in ( F ) . Higher magnification images of the cilia region shown to the right . Scale bars ; 10 µm . Quantification of localisation in ( G ) . Data are mean ± S . E . M . of 3 independent experiments ( n > 100 cilia per experimental condition ) . Statistical significance according to two - way ANOVA ( * P = 0 . 0242 ) .

O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Localisation of the PI(4,5)P2 sensor PH-PLCδ1-GFP to cilia in live NIH3T3 WT and Rab35 KO cells. Cilia are marked with HTR6-RFP. Representative images are shown in (F). Higher magnification images of the cilia region shown to the right. Scale bars; 10 µm. Quantification of localisation in (G). Data are mean ± S.E.M. of 3 independent experiments (n > 100 cilia per experimental condition). Statistical significance according to two-way ANOVA (* P = 0.0242).


hTERT - RPE1 cells transfected with indicated siRNAs were serum - starved for 48 h and treated with SMO agonist ( SAG ) or vehicle control ( DMSO ) for the last 24 h . Cells were stained for SMO , acetylated tubulin and DNA . Representative images are shown in ( A ) . Regions within white boxes shown at higher magnifications to the right . Scale bars , 10 µm . Graph in ( B ) shows the percentages of SMO - positive ( SMO + ) cilia . Data are mean ± S . E . M . of 5 independent experiments . Statistical significance according to ANOVA followed by Bonferroni post - hoc test ( * * * P < 0 . 001 ; P - values : Neg + SAG vs . RAB35 - 1 + SAG P < 0 . 0001 , Neg + SAG vs . RAB35 - 2 + SAG P = 0 . 0033 )

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

hTERT-RPE1 cells transfected with indicated siRNAs were serum-starved for 48 h and treated with SMO agonist (SAG) or vehicle control (DMSO) for the last 24 h. Cells were stained for SMO, acetylated tubulin and DNA. Representative images are shown in (A). Regions within white boxes shown at higher magnifications to the right. Scale bars, 10 µm. Graph in (B) shows the percentages of SMO-positive (SMO+) cilia. Data are mean ± S.E.M. of 5 independent experiments. Statistical significance according to ANOVA followed by Bonferroni post-hoc test (*** P < 0.001; P-values: Neg+SAG vs. RAB35-1+SAG P < 0.0001, Neg+SAG vs. RAB35-2+SAG P = 0.0033)


hTERT - RPE1 cells transfected with indicated siRNAs were serum - starved for 48 h and treated with SMO agonist ( SAG ) or vehicle control ( DMSO ) for the last 24 h . ( C ) Box - and - whisker plots show quantification of the average SMO intensity in the ciliary area marked by acetylated tubulin staining . All cilia : cilia identified with acetylated tubulin staining . SMO + cilia : cilia with discernible SMO localisation . Horizontal lines show 25 , 50 and 75th percentiles ; whiskers extend to minimum and maximum values . One representative experiment out of three is shown ( n ≥ 30 cilia per experimental condition ) . Statistical significance according to Kruskal - Wallis followed by Dunn ' s post - hoc test ( * P < 0 . 05 , * * * P < 0 . 001 ; P - values : Neg + SAG vs . RAB35 - 1 + SAG ( all cilia ) P < 0 . 0001 , Neg + SAG vs . RAB35 - 2 + SAG ( all cilia ) P = 0 . 0024 , Neg + SAG vs . RAB35 - 1 + SAG ( SMO + cilia ) P < 0 . 0007 , Neg + SAG vs . RAB35 - 2 + SAG ( SMO + cilia ) P = 0 . 0205 )

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

hTERT-RPE1 cells transfected with indicated siRNAs were serum-starved for 48 h and treated with SMO agonist (SAG) or vehicle control (DMSO) for the last 24 h. (C) Box-and-whisker plots show quantification of the average SMO intensity in the ciliary area marked by acetylated tubulin staining. All cilia: cilia identified with acetylated tubulin staining. SMO+ cilia: cilia with discernible SMO localisation. Horizontal lines show 25, 50 and 75th percentiles; whiskers extend to minimum and maximum values. One representative experiment out of three is shown (n ≥ 30 cilia per experimental condition). Statistical significance according to Kruskal-Wallis followed by Dunn's post-hoc test (* P < 0.05, *** P < 0.001; P-values: Neg+SAG vs. RAB35-1+SAG (all cilia) P < 0.0001, Neg+SAG vs. RAB35-2+SAG (all cilia) P = 0.0024, Neg+SAG vs. RAB35-1+SAG (SMO+ cilia) P < 0.0007, Neg+SAG vs. RAB35-2+SAG (SMO+ cilia) P = 0.0205)


For co - depletion experiments , hTERT - RPE1 cells were first transfected with Neg or RAB35 siRNA . After 24h , a second transfection with Neg or ARL13B siRNA was performed and cells were treated as in ( A ) . Representative images are shown in ( D ) . Regions within white boxes shown at higher magnifications as insets . Scale bars , 10 µm . Graph in ( E ) shows the percentages of SMO - positive cilia . Data are mean ± S . E . M . of 5 independent experiments . Statistical significance according to ANOVA followed by Bonferroni post - hoc test ( * P = 0 . 0151 , n . s . ; non - significant ) .

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

For co-depletion experiments, hTERT-RPE1 cells were first transfected with Neg or RAB35 siRNA. After 24h, a second transfection with Neg or ARL13B siRNA was performed and cells were treated as in (A). Representative images are shown in (D). Regions within white boxes shown at higher magnifications as insets. Scale bars, 10 µm. Graph in (E) shows the percentages of SMO-positive cilia. Data are mean ± S.E.M. of 5 independent experiments. Statistical significance according to ANOVA followed by Bonferroni post-hoc test (* P = 0.0151, n.s.; non-significant).


For co - depletion experiments , hTERT - RPE1 cells were first transfected with Neg or RAB35 siRNA . After 24h , a second transfection with Neg or ARL13B siRNA was performed and cells were treated ( F ) show quantification of ciliary SMO intensity . Horizontal lines show 25 , 50 and 75th percentiles ; whiskers extend to minimum and maximum values . One representative experiment out of three is shown ( n ≥ 30 cilia per experimental condition ) . Statistical significance according to Kruskal - Wallis followed by Dunn ' s post - hoc test ( * P = 0 . 0142 , n . s . ; non - significant ) .

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

For co-depletion experiments, hTERT-RPE1 cells were first transfected with Neg or RAB35 siRNA. After 24h, a second transfection with Neg or ARL13B siRNA was performed and cells were treated (F) show quantification of ciliary SMO intensity. Horizontal lines show 25, 50 and 75th percentiles; whiskers extend to minimum and maximum values. One representative experiment out of three is shown (n ≥ 30 cilia per experimental condition). Statistical significance according to Kruskal-Wallis followed by Dunn's post-hoc test (* P = 0.0142, n.s.; non-significant).


Ciliary SMO intensity quantification in hTERT - RPE1 cells transiently expressing GFP , wild type ARL13B - GFP ( WT ) or the ciliary - targeting defective mutant ARL13B - V358A . Cells were serum - starved and treated with SAG for 24 h and stained for SMO , acetylated tubulin ( acetyl . tub . ) and DNA . Representative images are shown in ( G ) .

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

Ciliary SMO intensity quantification in hTERT-RPE1 cells transiently expressing GFP, wild type ARL13B-GFP (WT) or the ciliary-targeting defective mutant ARL13B-V358A. Cells were serum-starved and treated with SAG for 24 h and stained for SMO, acetylated tubulin (acetyl. tub.) and DNA. Representative images are shown in (G).


Ciliary SMO intensity quantification in hTERT - RPE1 cells transiently expressing GFP , wild type ARL13B - GFP ( WT ) or the ciliary - targeting defective mutant ARL13B - V358A . Cells were serum - starved and treated with SAG for 24 h and stained for SMO Box - and - whisker plots in ( H ) show quantification of ciliary SMO intensity . Horizontal lines show 25 , 50 and 75th percentiles ; whiskers extend to minimum and maximum values . One representative experiment out of three is shown ( n ≥ 25 cilia per experimental condition ) . Statistical significance according to Kruskal - Wallis followed by Dunn ' s post - hoc test ( * * P = 0 . 0046 ) .

Ciliary SMO intensity quantification in hTERT-RPE1 cells transiently expressing GFP, wild type ARL13B-GFP (WT) or the ciliary-targeting defective mutant ARL13B-V358A. Cells were serum-starved and treated with SAG for 24 h and stained for SMO Box-and-whisker plots in (H) show quantification of ciliary SMO intensity. Horizontal lines show 25, 50 and 75th percentiles; whiskers extend to minimum and maximum values. One representative experiment out of three is shown (n ≥ 25 cilia per experimental condition). Statistical significance according to Kruskal-Wallis followed by Dunn's post-hoc test (** P = 0.0046).


( A ) SDS - PAGE gels stained with coomassie showing the purity of the Chlamydomonas IFT - B2 components , over - expressed in E . coli or insect cells , after the last purification step by SEC . IFT54 / 20 and IFT57 / 38 were purified as hetero - dimeric complexes whereas IFT80 , IFT172 and an N - terminal IFT172 ( 1 - 968 ) ( IFT172ΔC ) constructs were purified as single components . A cartoon representation of the predicted domain architecture of IFT - B2 components using HHPRED ( Söding et al , 2005 ) is shown at the center .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) SDS-PAGE gels stained with coomassie showing the purity of the Chlamydomonas IFT-B2 components, over-expressed in E. coli or insect cells, after the last purification step by SEC. IFT54/20 and IFT57/38 were purified as hetero-dimeric complexes whereas IFT80, IFT172 and an N-terminal IFT172(1-968) (IFT172ΔC) constructs were purified as single components. A cartoon representation of the predicted domain architecture of IFT-B2 components using HHPRED (Söding et al, 2005) is shown at the center.


( B ) Pull - down analysis for the hexameric IFT - B2 complex . A mixture of IFT172ΔC , IFT80 , and the IFT57 / 38 complex ( ' input ' ) shows only weak non - specific binding to GSH - resin ( ' empty ' ) , but all proteins are pulled down by IFT54 / 20 - GST complex . Note that the IFT20 - GST and IFT38 proteins co - migrate on the gel and cannot be properly resolved . However , the presence of the IFT57 protein in the pull - down sample implies that its binding partner IFT38 is also there .

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(B)Pull-down analysis for the hexameric IFT-B2 complex. A mixture of IFT172ΔC, IFT80, and the IFT57/38 complex ('input') shows only weak non-specific binding to GSH-resin ('empty'), but all proteins are pulled down by IFT54/20-GST complex. Note that the IFT20-GST and IFT38 proteins co-migrate on the gel and cannot be properly resolved. However, the presence of the IFT57 protein in the pull-down sample implies that its binding partner IFT38 is also there.


( C ) ( left ) SEC elution profiles of individual IFT - B2 components and of the assembled hexameric IFT - B2 complex . The elution peak of IFT - B2 is shifted compared to all of the individual components . Note that the peak heights were adjusted for better visualization of the result . ( right ) Coomassie stained SDS PAGE gel of the six main fractions of the IFT - B2 SEC elution peak demonstrating the presence of all six IFT - B2 subunits . Weak contaminating bands are marked with asterisks .

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(C) (left) SEC elution profiles of individual IFT-B2 components and of the assembled hexameric IFT-B2 complex. The elution peak of IFT-B2 is shifted compared to all of the individual components. Note that the peak heights were adjusted for better visualization of the result. (right) Coomassie stained SDS PAGE gel of the six main fractions of the IFT-B2 SEC elution peak demonstrating the presence of all six IFT-B2 subunits. Weak contaminating bands are marked with asterisks.


( A - C ) SEC elution profiles and corresponding SDS PAGE gels of IFT172ΔC , IFT57 / 38 , IFT80 , IFT54 / 20 as well as mixtures of IFT172ΔC / 57 / 38 , IFT80 / 57 / 38 and IFT172ΔC / 80 / 57 / 38 demonstrating that IFT57 / 38 associates with both IFT80 and IFT172ΔC . Only the central peak fraction from each SEC experiment is shown on the SDS PAGE gel . Note that the presence of both IFT172ΔC and IFT80 in a single peak in ( c ) ( peak 3 ) is due to the fact that these two proteins elute in the same position , and not because they physically interact ( see also Appendix Fig . S2B ) .

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(A-C) SEC elution profiles and corresponding SDS PAGE gels of IFT172ΔC, IFT57/38, IFT80, IFT54/20 as well as mixtures of IFT172ΔC/57/38, IFT80/57/38 and IFT172ΔC/80/57/38 demonstrating that IFT57/38 associates with both IFT80 and IFT172ΔC. Only the central peak fraction from each SEC experiment is shown on the SDS PAGE gel. Note that the presence of both IFT172ΔC and IFT80 in a single peak in (c) (peak 3) is due to the fact that these two proteins elute in the same position, and not because they physically interact (see also Appendix Fig. S2B).


( D ) GSH resin pull - down of a mixture of IFT80 and IFT172ΔC ( input , lane 1 ) with GST - tagged CH - domains of IFT57 or IFT38 ( IFT57CH or IFT38CH ) or with the GST - tagged coiled - coil ( CC ) region of IFT57 mixed with His - tagged coiled - coil region of IFT38 . The SDS - PAGE gel shows interaction between IFT57CH - IFT172ΔC , IFT38CH - IFT80 and IFT57CC - IFT38CC .

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(D) GSH resin pull-down of a mixture of IFT80 and IFT172ΔC (input, lane 1) with GST-tagged CH-domains of IFT57 or IFT38 (IFT57CH or IFT38CH) or with the GST-tagged coiled-coil (CC) region of IFT57 mixed with His-tagged coiled-coil region of IFT38. The SDS-PAGE gel shows interaction between IFT57CH-IFT172ΔC, IFT38CH-IFT80 and IFT57CC-IFT38CC.


( E ) Ni - NTA resin pull - down of untagged IFT54 / 20 with His - tagged IFT38 / 38 ( both subunits His - tagged ) . The two bands for IFT38 on the SDS PAGE gel correspond to His - tagged ( upper band ) and proteolytically cleaved untagged IFT38 ( lower band ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Ni-NTA resin pull-down of untagged IFT54/20 with His-tagged IFT38/38 (both subunits His-tagged). The two bands for IFT38 on the SDS PAGE gel correspond to His-tagged (upper band) and proteolytically cleaved untagged IFT38 (lower band).


( F ) Ni - NTA resin pull - down of untagged IFT54 / 20 with His - tagged IFT80 .

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

(F) Ni-NTA resin pull-down of untagged IFT54/20 with His-tagged IFT80.


( A ) Ni2 + - NTA affinity pull - down of unpolymerised bovine αβ - tubulin with His - tagged IFT - B2 ( sub ) - complexes . A Coomassie stained SDS PAGE gel shows the IFT - B2 complexes used for pull - downs ( top ) and a western blot for anti - α - tubulin shows co - precipitated αβ - tubulin ( bottom ) .

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(A) Ni2+-NTA affinity pull-down of unpolymerised bovine αβ-tubulin with His-tagged IFT-B2 (sub)-complexes. A Coomassie stained SDS PAGE gel shows the IFT-B2 complexes used for pull-downs (top) and a western blot for anti-α-tubulin shows co-precipitated αβ-tubulin (bottom).


( F ) Ni2 + - NTA affinity pull - down of unpolymerised bovine αβ - tubulin with WT and mutant MmIFT54CH . ( top ) Coomassie stained SDS PAGE gel of input ( I ) and pulled down ( PD ) proteins and the corresponding western blot to visualize αβ - tubulin .

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(F) Ni2+-NTA affinity pull-down of unpolymerised bovine αβ-tubulin with WT and mutant MmIFT54CH. (top) Coomassie stained SDS PAGE gel of input (I) and pulled down (PD) proteins and the corresponding western blot to visualize αβ-tubulin.


( A ) Coomassie - stained SDS - PAGE gel showing GST - pulldown experiments between the IFT - B2 complex and an IFT88 / 52N subcomplex . The pre - assembled IFT - B2 complex ( lane 1 ) shows no non - specific interaction with empty GSH - beads ( lane 2 ) , but is efficiently pulled down by IFT88 / 52N - GST ( with an IFT52 ( 1 - 335 ) construct comprising the N - terminal domain as well as the IFT88 - binding region ) ( lane 3 ) . Both IFT88 and IFT52N are required for the interaction with IFT - B2 because neither the N - terminal IFT52 domain ( residues 1 - 275 ; lane 4 ) alone , nor an IFT88 / 52 complex lacking the N - terminal IFT52 domain ( with an IFT52 ( 281 - 335 ) construct containing only the IFT88 - binding region ) ( lane 5 ) show efficient pull - down of IFT - B2 . Contaminants from the purification of the complex shown in lane 5 are marked with asterisks .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Coomassie-stained SDS-PAGE gel showing GST-pulldown experiments between the IFT-B2 complex and an IFT88/52N subcomplex. The pre-assembled IFT-B2 complex (lane 1) shows no non-specific interaction with empty GSH-beads (lane 2), but is efficiently pulled down by IFT88/52N-GST (with an IFT52(1-335) construct comprising the N-terminal domain as well as the IFT88-binding region) (lane 3). Both IFT88 and IFT52N are required for the interaction with IFT-B2 because neither the N-terminal IFT52 domain (residues 1-275; lane 4) alone, nor an IFT88/52 complex lacking the N-terminal IFT52 domain (with an IFT52(281-335) construct containing only the IFT88-binding region) (lane 5) show efficient pull-down of IFT-B2. Contaminants from the purification of the complex shown in lane 5 are marked with asterisks.


( B ) Coomassie - stained SDS - PAGE gel showing a salt - stability test of the ( IFT88 / 52N ) - ( IFT - B2 ) interaction . Pre - assembled IFT - B2 complex was bound to IFT88 / 52N - GST immobilized on GSH - beads . The beads were then divided into five aliquots which were washed with buffers containing increasing concentrations of NaCl . After these washes the bound material was eluted and analyzed . IFT172ΔC is the only salt - labile component in this complex and is already washed off at a NaCl - concentration of 150 - 200 mM , whereas all the other IFT - B2 proteins remain associated even after washing with 1 M NaCl .

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 B-CONTROLLED_VAR 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 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 B-CONTROLLED_VAR O

(B) Coomassie-stained SDS-PAGE gel showing a salt-stability test of the (IFT88/52N)-(IFT-B2) interaction. Pre-assembled IFT-B2 complex was bound to IFT88/52N-GST immobilized on GSH-beads. The beads were then divided into five aliquots which were washed with buffers containing increasing concentrations of NaCl. After these washes the bound material was eluted and analyzed. IFT172ΔC is the only salt-labile component in this complex and is already washed off at a NaCl-concentration of 150-200 mM, whereas all the other IFT-B2 proteins remain associated even after washing with 1 M NaCl.


( C ) Coomassie - stained SDS - PAGE gels showing GST - pulldown experiments between IFT88 / 52N - GST complex immobilized on GSH beads and various combinations of IFT - B2 proteins . The left gel shows the IFT - B2 input mixtures , the right gel shows the bound material after washing and elution from the beads . A pentameric IFT - B2 complex ( IFT80 / 57 / 54 / 38 / 20 ) is efficiently pulled down by IFT88 / 52N - GST , but not by empty beads ( compare lanes 1 and 2 ) . Omission of IFT54 / 20 ( lane 4 ) , or IFT80 ( lane 5 ) does not influence the binding of any of the other components , but a lack of IFT57 / 38 in the mixture abolishes detectable pull - downs of IFT54 / 20 and IFT80 ( lane 4 ) , indicating that IFT57 / 38 is the direct interaction partner of IFT88 / 52N . Indeed , IFT57 / 38 alone ( lane 7 ) , but not IFT54 / 20 ( lane 6 ) or IFT80 ( lane 8 ) , is sufficient to be pulled down in this assay . Contaminating bands from the IFT88 / 52N sample are marked with asterisks .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Coomassie-stained SDS-PAGE gels showing GST-pulldown experiments between IFT88/52N-GST complex immobilized on GSH beads and various combinations of IFT-B2 proteins. The left gel shows the IFT-B2 input mixtures, the right gel shows the bound material after washing and elution from the beads. A pentameric IFT-B2 complex (IFT80/57/54/38/20) is efficiently pulled down by IFT88/52N-GST, but not by empty beads (compare lanes 1 and 2). Omission of IFT54/20 (lane 4), or IFT80 (lane 5) does not influence the binding of any of the other components, but a lack of IFT57/38 in the mixture abolishes detectable pull-downs of IFT54/20 and IFT80 (lane 4), indicating that IFT57/38 is the direct interaction partner of IFT88/52N. Indeed, IFT57/38 alone (lane 7), but not IFT54/20 (lane 6) or IFT80 (lane 8), is sufficient to be pulled down in this assay. Contaminating bands from the IFT88/52N sample are marked with asterisks.


( B ) Coomassie stained SDS PAGE gel of a pull - down of IFT - B2 using WT or mutant IFT88 / 521 - 335 - GST . Whereas single point mutations slightly reduced the amounts of pulled down IFT - B2 , the K130E / R204E double mutation reduced it to background levels ( compare lanes 2 and 6 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Coomassie stained SDS PAGE gel of a pull-down of IFT-B2 using WT or mutant IFT88/521-335-GST. Whereas single point mutations slightly reduced the amounts of pulled down IFT-B2, the K130E/R204E double mutation reduced it to background levels (compare lanes 2 and 6).


( A ) SEC - profiles of the IFT - B1 and IFT - B2 complexes as well as the 15 - subunit reconstituted IFT - B complex . IFT - B1 and IFT - B2 elute at similar volumes , but after mixing the resulting high - molecular weight complex is clearly shifted towards a smaller elution volume . The Coomassie - stained SDS - PAGE gel on the right shows the peak fractions of the individual runs .

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(A) SEC-profiles of the IFT-B1 and IFT-B2 complexes as well as the 15-subunit reconstituted IFT-B complex. IFT-B1 and IFT-B2 elute at similar volumes, but after mixing the resulting high-molecular weight complex is clearly shifted towards a smaller elution volume. The Coomassie-stained SDS-PAGE gel on the right shows the peak fractions of the individual runs.


A . Ig sequence diversification in wild type and h3 . 3 . Each line represents an IGVLR gene from a population cells that have expanded for 36 divisions after cloning . Blue bars = gene conversions ; green lollipops = point mutations ; green bars = ambiguous mutations ( Sale et al , 2001 ) . B . Quantification of sequence diversification in wild type and two h3 . 3 clones expressed as unique events ( gene conversions or point mutations ) per kb per generation . ( Sequences analysed : WT n = 142 , for h3 . 3 c20 n = 140 , for h3 . 3 c32 n = 190 ) .

A. Ig sequence diversification in wild type and h3.3. Each line represents an IGVLR gene from a population cells that have expanded for 36 divisions after cloning. Blue bars = gene conversions; green lollipops = point mutations; green bars = ambiguous mutations (Sale et al, 2001).B. Quantification of sequence diversification in wild type and two h3.3 clones expressed as unique events (gene conversions or point mutations) per kb per generation. (Sequences analysed: WT n = 142, for h3.3 c20 n = 140, for h3.3 c32 n = 190).


C . Quantification of endogenous AID mRNA in wild type and h3 . 3 cells . The graph represents the mean and SD of four independent RNA extractions of wild type and h3 . 3c20 . p value calculated with the unpaired T test .

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C. Quantification of endogenous AID mRNA in wild type and h3.3 cells. The graph represents the mean and SD of four independent RNA extractions of wild type and h3.3c20. p value calculated with the unpaired T test.


D . Fluctuation analysis for the formation of sIg - loss variants in wild type ( black circles ) and h3 . 3 clones ( black diamonds ) and in two wild type clones ( blue circles ) , two h3 . 3 clones ( blue diamonds ) expressing hAIDup ( Wang et al , 2009 ) at 15 - 20 times the level of wild type AID . The final column shows h3 . 3 cells with hAIDup complemented with H3 . 3 ( blue triangles ) . Clones were expanded for 36 cell divisions at which point the percentage of sIgM negative cells in each population was determined by flow cytometry . Error bars represent mean and 1 S . D . P values calculated with the Mann - Whitney test .

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D. Fluctuation analysis for the formation of sIg-loss variants in wild type (black circles) and h3.3 clones (black diamonds) and in two wild type clones (blue circles), two h3.3 clones (blue diamonds) expressing hAIDup (Wang et al, 2009) at 15 - 20 times the level of wild type AID. The final column shows h3.3 cells with hAIDup complemented with H3.3 (blue triangles). Clones were expanded for 36 cell divisions at which point the percentage of sIgM negative cells in each population was determined by flow cytometry. Error bars represent mean and 1 S.D. P values calculated with the Mann-Whitney test.


E . Ig sequence diversification in wild type , h3 . 3 and complemented h3 . 3 cells overexpressing hAIDup . Key as in ( A ) plus * = deletion or duplication .

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E. Ig sequence diversification in wild type, h3.3 and complemented h3.3 cells overexpressing hAIDup. Key as in (A) plus * = deletion or duplication.


F . Mutation rate as a function of hAIDup expression in wild type and h3 . 3 . Filled symbols represent clones overexpressing hAIDup , open symbols clones with natural AID expression . Linear regression lines are shown .

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F. Mutation rate as a function of hAIDup expression in wild type and h3.3. Filled symbols represent clones overexpressing hAIDup, open symbols clones with natural AID expression. Linear regression lines are shown.


B . Diversification in wild type and h3 . 3 cells expressing similar levels of hAIDup and UGI . Red lollipops = transitions at C / T ; Black lollipops = other point mutations ; blue bars = gene conversions .

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B. Diversification in wild type and h3.3 cells expressing similar levels of hAIDup and UGI. Red lollipops = transitions at C/T; Black lollipops = other point mutations; blue bars = gene conversions.


C . Mutation rate as a function of hAIDup expression in UGI - expressing wild type and h3 . 3 cells . Each clone was expanded for 36 cell divisions and hAIDup expression determined by qPCR during the last week of culture .

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C. Mutation rate as a function of hAIDup expression in UGI-expressing wild type and h3.3 cells. Each clone was expanded for 36 cell divisions and hAIDup expression determined by qPCR during the last week of culture.


A . Enrichment of H3 . 3 in the rearranged allele of the Ig light chain ( IGVLR ) compared with the unrearranged allele ( IGVLUR ) . Each bar represents the mean of 3 independent ChIPs each assayed with 3 repeat qPCRs ( * * * * = p < 0 . 0001 , unpaired T - test ) .

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A. Enrichment of H3.3 in the rearranged allele of the Ig light chain (IGVLR) compared with the unrearranged allele (IGVLUR). Each bar represents the mean of 3 independent ChIPs each assayed with 3 repeat qPCRs (**** = p<0.0001, unpaired T-test).


B . Igλ mRNA levels in wild type and h3 . 3 cells determined by qPCR , normalised to EF - 1α expression and to WT . n = 3 ( ns = not significant , unpaired T - test ) .

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B. Igλ mRNA levels in wild type and h3.3 cells determined by qPCR, normalised to EF-1α expression and to WT. n = 3 (ns = not significant, unpaired T-test).


C . Surface Ig ( sIg ) in multiple clones of wild type ( blue ) and h3 . 3 ( red ) cells . Green = unstained control .

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C. Surface Ig (sIg) in multiple clones of wild type (blue) and h3.3 (red) cells. Green = unstained control.


D . ChIP for H3K4me3 and H3K36me3 in IGVLR and IGVLUR . Each bar represents the mean of 3 ChIPs for H3K4me3 and IgG and 2 for H3K36me3 , each assayed with 3 qPCR repeats ( statistical analysis as in Panel A ) .

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D. ChIP for H3K4me3 and H3K36me3 in IGVLR and IGVLUR. Each bar represents the mean of 3 ChIPs for H3K4me3 and IgG and 2 for H3K36me3, each assayed with 3 qPCR repeats (statistical analysis as in Panel A).


E . 4sUDRB mapping of transcription kinetics through IGVL . Representative locations of qPCR amplicons ( blue ) are shown relative to the TSS of IGVL ( distance is shown in parenthesis ) . qPCR signals were normalised to the enrichment at time zero ( before release ) . Error bars represent 1SD of qPCR triplicates .

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E. 4sUDRB mapping of transcription kinetics through IGVL. Representative locations of qPCR amplicons (blue) are shown relative to the TSS of IGVL (distance is shown in parenthesis). qPCR signals were normalised to the enrichment at time zero (before release). Error bars represent 1SD of qPCR triplicates.


A . DNA - RNA hybrids in IGVL and IGCL in wild type and h3 . 3 . The DRIP signal is normalised to total input and to wild type IgVλ ( * * * = p < 0 . 001 , ns = not significant , unpaired T - test ) .

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A. DNA-RNA hybrids in IGVL and IGCL in wild type and h3.3. The DRIP signal is normalised to total input and to wild type IgVλ (*** = p<0.001, ns = not significant, unpaired T-test).


B . MNase mapping reveals that loss of H3 . 3 does not affect average Igλ nucleosome positioning . The red bars indicate the qPCR amplicons for the MNase mapping . The qPCR signals are normalised to the first amplicon at the TSS . Error bars represent 1SD of 2 experimental repeats .

B. MNase mapping reveals that loss of H3.3 does not affect average Igλ nucleosome positioning. The red bars indicate the qPCR amplicons for the MNase mapping. The qPCR signals are normalised to the first amplicon at the TSS. Error bars represent 1SD of 2 experimental repeats.


C . DNA - RNA hybrids in IGVL of wild type clones either without overexpression of RNAseHI ( red , clones 1 and 2 , each assayed with three qPCR repeats ) or with overexpression of RNAseHI ( blue , clones 3 and 4 , each assayed with three qPCR repeats ) ( * * * = p < 0 . 001 , * * = p < 0 . 01 ; unpaired T - test ) . Expression of hAIDup - FLAG and chickenRNAseHI - YFP in these clones is shown in Figure EV6 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. DNA-RNA hybrids in IGVL of wild type clones either without overexpression of RNAseHI (red, clones 1 and 2, each assayed with three qPCR repeats) or with overexpression of RNAseHI (blue, clones 3 and 4, each assayed with three qPCR repeats) (*** = p<0.001, ** = p < 0.01; unpaired T-test). Expression of hAIDup-FLAG and chickenRNAseHI-YFP in these clones is shown in Figure EV6.


D . Rate of Ig diversification in wild type cells with and without overexpression of RNAseHI .

D. Rate of Ig diversification in wild type cells with and without overexpression of RNAseHI.


A . Location of ssDNA tracts ≥ 8nt in IGVL in wild type ( WT ) and h3 . 3 cells detected by bisulphite sequencing . Data is from three independent experiments , the results from each being shown as black , red and green bars respectively . ssDNA on the coding strand is shown above the marker line ( numbered relative to the TSS at 0 ) and on the template strand below . In the centre , the percentage of G and C bases on the coding strand in a 30 bp rolling average window is shown . B . Quantitation of the bisulphite detection of single stranded DNA in wild type , h3 . 3 and h3 . 3 complemented cells , normalised to wild type . p - values calculated with a  test . Sequences analysed : Wild type n = 322 , h3 . 3n = 320 and h3 . 3 + H3 . 3 n = 95 .

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 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 I-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 O O O O O O

A. Location of ssDNA tracts ≥ 8nt in IGVL in wild type (WT) and h3.3 cells detected by bisulphite sequencing. Data is from three independent experiments, the results from each being shown as black, red and green bars respectively. ssDNA on the coding strand is shown above the marker line (numbered relative to the TSS at 0) and on the template strand below. In the centre, the percentage of G and C bases on the coding strand in a 30 bp rolling average window is shown.B. Quantitation of the bisulphite detection of single stranded DNA in wild type, h3.3 and h3.3 complemented cells, normalised to wild type. p-values calculated with a  test. Sequences analysed: Wild type n = 322, h3.3n = 320 and h3.3 + H3.3 n = 95.


C . Bisulphite detection of single stranded DNA in wild type cells with and without RNAseHI overexpression . p - value calculated with a chi ‐ square test . Sequences analysed : RNAseH1 + ve n = 94 , RNAseH1 - ve n = 90 .

O O O O O B-MEASURED_VAR I-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

C. Bisulphite detection of single stranded DNA in wild type cells with and without RNAseHI overexpression. p-value calculated with a chi‐square test. Sequences analysed: RNAseH1+ve n = 94, RNAseH1-ve n = 90.


Bacterial growth assessed by color change due to medium acidification for both the undefined ATCC 1161 and semi - defined CSY media with or without sucrose , 4 % and 1 % respectively . ( + ) , inoculated wells ; ( - ) , non - inoculated control . The picture was taken after a 24 - hour incubation period .

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Bacterial growth assessed by color change due to medium acidification for both the undefined ATCC 1161 and semi-defined CSY media with or without sucrose, 4% and 1% respectively. (+), inoculated wells; (-), non-inoculated control. The picture was taken after a 24-hour incubation period.


Impact of horse serum ( HS ) and yeast extract ( YE ) dilution on M . florum doubling time . The green and red dotted lines indicate HS and YE concentrations found in ATCC 1161 medium ( 20 % HS , 1 . 35 % YE ) and CSY medium ( 0 . 313 % HS , 0 . 02 % YE ) , respectively . Doubling times were measured in a CMRL - 1066 base medium using colorimetric assays . Dots and error bars indicate the mean and standard deviation calculated from three biological replicates .

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

Impact of horse serum (HS) and yeast extract (YE) dilution on M. florum doubling time. The green and red dotted lines indicate HS and YE concentrations found in ATCC 1161 medium (20% HS, 1.35% YE) and CSY medium (0.313% HS, 0.02% YE), respectively. Doubling times were measured in a CMRL-1066 base medium using colorimetric assays. Dots and error bars indicate the mean and standard deviation calculated from three biological replicates.


The maximal biomass concentration observed for M . florum cultures growing in CSY medium with varying initial concentrations of sucrose . Biomass was measured using colony forming units ( CFU / ml ; left axis ) and converted to grams of dry weight ( gDW / L ; right axis ) . A rectangular hyperbola fit is shown ( yellow ) , and the dotted line represents the maximal biomass value predicted by the fit ( 1e8 x 5 . 95 CFU / ml ; 0 . 013 gDW / L ) .

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

The maximal biomass concentration observed for M. florum cultures growing in CSY medium with varying initial concentrations of sucrose. Biomass was measured using colony forming units (CFU/ml; left axis) and converted to grams of dry weight (gDW/L; right axis). A rectangular hyperbola fit is shown (yellow), and the dotted line represents the maximal biomass value predicted by the fit (1e8 x 5.95 CFU/ml; 0.013 gDW/L).


Sucrose specific uptake rate ( e ) and combined lactate / acetate specific secretion rates ( f ) at varying initial sucrose concentrations in CSY medium . Boxplots represent the median and interquartile range of uptake or secretion rate values calculated at different time intervals during the exponential growth phase of M . florum cultures . Whiskers indicate minimal and maximal values . Dotted lines indicate the selected uptake ( - 5 . 26 mmol ∙ gDW - 1 ∙ hr - 1 ) and secretion rate ( 8 . 69 mmol ∙ gDW - 1 ∙ hr - 1 ) values used for modeling . Sucrose quantifications were performed in technical duplicate , whereas lactate and acetate quantifications were performed in single replicates . See Appendix Figure S5 for further details .

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

Sucrose specific uptake rate (e) and combined lactate/acetate specific secretion rates (f) at varying initial sucrose concentrations in CSY medium. Boxplots represent the median and interquartile range of uptake or secretion rate values calculated at different time intervals during the exponential growth phase of M. florum cultures. Whiskers indicate minimal and maximal values. Dotted lines indicate the selected uptake (-5.26 mmol∙gDW-1∙hr-1) and secretion rate (8.69 mmol∙gDW-1∙hr-1) values used for modeling. Sucrose quantifications were performed in technical duplicate, whereas lactate and acetate quantifications were performed in single replicates. See Appendix Figure S5 for further details.


( A - B ) Representative MA currents recorded in whole cell patch clamp experiments at - 60 mV in large DRG neurons in response to repetitive mechanical indentations with a blunt glass probe ; experiments were performed as described in the methods section . The applications of 25 μM baclofen ( B ) or vehicle ( A ) are indicated by the red horizontal lines . The downwards spikes represent individual MA currents induced by repetitive mechanical stimuli every 30 s for 200 ms stimulus duration .

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

(A-B) Representative MA currents recorded in whole cell patch clamp experiments at -60 mV in large DRG neurons in response to repetitive mechanical indentations with a blunt glass probe; experiments were performed as described in the methods section. The applications of 25 μM baclofen (B) or vehicle (A) are indicated by the red horizontal lines. The downwards spikes represent individual MA currents induced by repetitive mechanical stimuli every 30 s for 200 ms stimulus duration.


( C ) Quantification of normalized MA current amplitudes from DRG neurons , vehicle ( white , n = 7 ) , neurons responding to baclofen ( R , red ) ( n = 6 , * * P < 0 . 01 , Paired t - test and non - responding DRG neurons ( NR , blue , n = 6 ) . Striped column shows the average normalized current amplitudes 3 - 10 minutes after removal of baclofen in responding neurons ( Washout ) ( n = 6 ) . Data are shown as mean ± SEM and scatter plots .

O 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 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

(C) Quantification of normalized MA current amplitudes from DRG neurons, vehicle (white, n=7), neurons responding to baclofen (R, red) (n=6, **P<0.01, Paired t-test and non-responding DRG neurons (NR, blue, n=6). Striped column shows the average normalized current amplitudes 3-10 minutes after removal of baclofen in responding neurons (Washout) (n=6). Data are shown as mean ± SEM and scatter plots.


( D - E ) Representative MA currents recorded at - 60 mV in large DRG neurons in response to repetitive mechanical stimulation with a blunt glass probe displaced 2 - 6 µm in 0 . 4 µm increments every 15 s ( step protocol ) before ( black ) and after ( red ) exposure to 25 µM Baclofen . Not every trace is shown for clarity . Inset above the current traces show the mechanical step protocol indicating the displacement of the mechanical probe .

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

(D-E) Representative MA currents recorded at -60 mV in large DRG neurons in response to repetitive mechanical stimulation with a blunt glass probe displaced 2-6 µm in 0.4 µm increments every 15 s (step protocol) before (black) and after (red) exposure to 25 µM Baclofen. Not every trace is shown for clarity. Inset above the current traces show the mechanical step protocol indicating the displacement of the mechanical probe.


( F ) Quantification of normalized MA currents of responding large DRG neurons [ n = 13 , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , before ( black ) and after ( red ) baclofen treatment . Dashed grey line shows average MA currents 3 - 15 minutes after washout of baclofen . Repeated measures ANOVA with student ' s t - test no corrections was used to compare baseline and baclofen treatment . Data are shown as mean ± SEM .

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

(F) Quantification of normalized MA currents of responding large DRG neurons [n=13, *P<0.05, **P<0.01, ***P<0.001, before (black) and after (red) baclofen treatment. Dashed grey line shows average MA currents 3-15 minutes after washout of baclofen. Repeated measures ANOVA with student's t-test no corrections was used to compare baseline and baclofen treatment. Data are shown as mean ± SEM.


( G ) Quantification of normalized MA currents of non - responding DRG neurons ( n = 9 ) . Data are shown as mean ± SEM .

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(G) Quantification of normalized MA currents of non-responding DRG neurons (n=9). Data are shown as mean ± SEM.


( H - I ) Quantification of MA current inactivation rate ( Tau ) measured for responding ( R ) large DRG neurons ( n = 13 , * * P < 0 . 01 , Paired t - test ) and non - responding large ( NR ) DRG neurons ( n = 9 , NS , not significant ) before ( gray ) and after ( red ) baclofen application . Data are shown as mean ± SEM and scatter plots .

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

(H-I) Quantification of MA current inactivation rate (Tau) measured for responding (R) large DRG neurons (n=13, **P<0.01, Paired t-test) and non-responding large (NR) DRG neurons (n=9, NS, not significant) before (gray) and after (red) baclofen application. Data are shown as mean ± SEM and scatter plots.


( A - B ) Representative MA currents recorded in whole cell patch clamp experiments at - 60 mV in HEK293 cells transiently transfected with Piezo2 + GABAB1 + GABAB2 + GFP ( for visualization ) in response to repetitive mechanical stimulation with a blunt glass probe displaced 2 - 5 . 6 µm in 0 . 4 μm increments every 15 s before ( black ) and after ( red ) exposure to 25 µM baclofen . Not every trace is shown for clarity .

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

(A-B) Representative MA currents recorded in whole cell patch clamp experiments at -60 mV in HEK293 cells transiently transfected with Piezo2 +GABAB1+ GABAB2 + GFP (for visualization) in response to repetitive mechanical stimulation with a blunt glass probe displaced 2-5.6 µm in 0.4 μm increments every 15 s before (black) and after (red) exposure to 25 µM baclofen. Not every trace is shown for clarity.


( C ) Quantification of normalized MA currents of HEK293 cells transfected with Piezo2 + GABAB1 + GABAB2 + GFP [ C , n = 13 , * P < 0 . 05 , * * P < 0 . 01 , before ( black ) and after ( red ) baclofen treatment , Repeated measures ANOVA with student ' s t - test no corrections ] . Data are shown as mean ± SEM .

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(C) Quantification of normalized MA currents of HEK293 cells transfected with Piezo2+GABAB1+ GABAB2 + GFP [C, n=13, *P<0.05, **P<0.01, before (black) and after (red) baclofen treatment, Repeated measures ANOVA with student's t-test no corrections]. Data are shown as mean ± SEM.


( D ) Quantification of MA current inactivation rate ( Tau ) in HEK293 cells transfected with Piezo2 + GABAB1 + GABAB2 + GFP before ( gray ) and after ( red ) baclofen application ( n = 13 , NS , not significant ; Paired t - test ) . Data are shown as mean ± SEM and scatter plots .

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(D) Quantification of MA current inactivation rate (Tau) in HEK293 cells transfected with Piezo2+GABAB1+ GABAB2 + GFP before (gray) and after (red) baclofen application (n=13, NS, not significant; Paired t-test). Data are shown as mean ± SEM and scatter plots.


Quantification of normalized MA currents of HEK293 cells transfected Piezo2 + GFP ( E , n = 7 ) before ( black ) and after ( red ) application of 25 µM baclofen . Data are shown as mean ± SEM .

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Quantification of normalized MA currents of HEK293 cells transfected Piezo2 + GFP (E, n=7) before (black) and after (red) application of 25 µM baclofen. Data are shown as mean ± SEM.


Quantification of normalized MA currents of HEK293 cells transfected Piezo2 + GABAB1 + GABAB2 + βARKct - mCherry ( F , n = 8 ) before ( black ) and after ( red ) application of 25 µM baclofen . Data are shown as mean ± SEM .

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Quantification of normalized MA currents of HEK293 cells transfected Piezo2+GABAB1+ GABAB2 + βARKct-mCherry (F, n=8) before (black) and after (red) application of 25 µM baclofen. Data are shown as mean ± SEM.


Quantification of normalized MA Piezo2 currents in HEK293 cells transiently transfected with Piezo2 + D2RS - YFP ( A , n = 13 ) before ( black ) and after ( red ) application of quinpirole 200 nM . * P < 0 . 05 , Repeated measures ANOVA with student ' s t - test no corrections . Data are shown as mean ± SEM .

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Quantification of normalized MA Piezo2 currents in HEK293 cells transiently transfected with Piezo2+D2RS-YFP (A, n=13) before (black) and after (red) application of quinpirole 200 nM. *P<0.05, Repeated measures ANOVA with student's t-test no corrections. Data are shown as mean ± SEM.


Quantification of normalized MA Piezo2 currents in HEK293 cells transfected with Piezo2 and GFP ( B , n = 5 ) before ( black ) and after ( red ) application of quinpirole 200 nM . * P < 0 . 05 , Repeated measures ANOVA with student ' s t - test no corrections . Data are shown as mean ± SEM .

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Quantification of normalized MA Piezo2 currents in HEK293 cells transfected with Piezo2 and GFP (B, n=5) before (black) and after (red) application of quinpirole 200 nM. *P<0.05, Repeated measures ANOVA with student's t-test no corrections. Data are shown as mean ± SEM.


( C ) Quantification of Piezo2 current inactivation rate ( Tau ) in HEK cells transfected with Piezo2 + D2RS - YFP . Data are shown as mean ± SEM and scatter plots ( n = 13 )

O O O O O O O O O O O O O O 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) Quantification of Piezo2 current inactivation rate (Tau) in HEK cells transfected with Piezo2+D2RS-YFP. Data are shown as mean ± SEM and scatter plots (n=13)


( D , E ) Quantification of normalized MA currents in responding large DRG neurons ( D , n = 5 ) versus non - responding DRG neurons ( E , n = 3 ) before ( black ) and after ( red ) application of sumatriptan 10 µM to activate the Gi - coupled Htr1d receptors . * P < 0 . 05 , Repeated measures ANOVA with student ' s t - test no corrections . Data are shown as mean ± SEM .

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(D,E) Quantification of normalized MA currents in responding large DRG neurons (D, n=5) versus non-responding DRG neurons (E, n=3) before (black) and after (red) application of sumatriptan 10 µM to activate the Gi-coupled Htr1d receptors. *P<0.05, Repeated measures ANOVA with student's t-test no corrections. Data are shown as mean ± SEM.


( F ) Quantification of MA current inactivation rate ( Tau ) measured for responding ( R ) large DRG neurons ( n = 5 , Paired t - test ) . Data are shown as mean ± SEM and scatter plots .

O O O O O O O O O O O O O O O O O O O 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) Quantification of MA current inactivation rate (Tau) measured for responding (R) large DRG neurons (n=5, Paired t-test). Data are shown as mean ± SEM and scatter plots.


( G - H ) Quantification of normalized MA currents recorded from large DRG neurons dialyzed with 50 ng / ml Gβγ ( G , n = 7 ) or with 0 . 0001 % Lubrol ( vehicle for Gβγ ) ( H , n = 6 ) . * P < 0 . 05 , Repeated measures ANOVA with student ' s t - test no corrections . Data are shown as mean ± SEM .

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

(G-H) Quantification of normalized MA currents recorded from large DRG neurons dialyzed with 50 ng/ml Gβγ (G, n=7) or with 0.0001% Lubrol (vehicle for Gβγ) (H, n=6). *P<0.05, Repeated measures ANOVA with student's t-test no corrections. Data are shown as mean ± SEM.


( I , J ) Quantification of MA current inactivation rate ( Tau ) measured for large DRG neurons dialyzed with Gβγ ( n = 7 , * * P < 0 . 01 , Paired t - test ) and vehicle ( Paired t - test ) . Data are shown as mean ± SEM and scatter plots .

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

(I,J) Quantification of MA current inactivation rate (Tau) measured for large DRG neurons dialyzed with Gβγ (n=7, **P<0.01, Paired t-test) and vehicle (Paired t-test). Data are shown as mean ± SEM and scatter plots.


Quantification of normalized MA Piezo2 currents in HEK293 cells transiently transfected with Piezo2 + GABAB1 + GABAB2 + GFP ( A , n = 12 )

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

Quantification of normalized MA Piezo2 currents in HEK293 cells transiently transfected with Piezo2+ GABAB1+ GABAB2 + GFP (A, n=12)


Quantification of normalized MA Piezo2 currents in HEK293 cells transiently transfected with Piezo2 + GABAB1 + GABAB2 + GFP ( A , n = 12 ) , treated with the PI3K inhibitor Wortmannin 50 nM ( B , n = 10 ) Cells were treated with wortmannin 30 min before the experiments , and were present throughout the measurements .

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

Quantification of normalized MA Piezo2 currents in HEK293 cells transiently transfected with Piezo2+ GABAB1+ GABAB2 + GFP (A, n=12), treated with the PI3K inhibitor Wortmannin 50 nM (B, n=10) Cells were treated with wortmannin 30 min before the experiments, and were present throughout the measurements.


Quantification of normalized MA Piezo2 currents in HEK293 cells transiently transfected with Piezo2 + GABAB1 + GABAB2 + GFP ( A , n = 12 ) , treated with the MAPK inhibitor U0126 10 µM ( C , n = 7 ) . Cells were treated with U0126 for 30 min before the experiments , and were present throughout the measurements .

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Quantification of normalized MA Piezo2 currents in HEK293 cells transiently transfected with Piezo2+ GABAB1+ GABAB2 + GFP (A, n=12), treated with the MAPK inhibitor U0126 10 µM (C, n=7). Cells were treated with U0126 for 30 min before the experiments, and were present throughout the measurements.


Mice were injected in the plantar surface of the hind paw with sumatriptan , or vehicle ± the combination of wortmannin and U0126 , as described in the methods section . Mechanical sensitivity was assessed 30 min later using von Frey filaments in an ascending order . ( A - C ) Percentage of withdrawal response for female mice using von Frey filaments at : 0 . 07 g ( A ) , 0 . 16 g ( B ) and 0 . 4 g ( C ) . ( n = 11 - 16 per group , * * * P < 0 . 001 , * * * * P < 0 . 0001 , One way - ANOVA with Tukey ' s multiple comparison test ) . Data are shown as mean ± SEM and scatter plots .

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

Mice were injected in the plantar surface of the hind paw with sumatriptan, or vehicle ± the combination of wortmannin and U0126, as described in the methods section. Mechanical sensitivity was assessed 30 min later using von Frey filaments in an ascending order. (A-C) Percentage of withdrawal response for female mice using von Frey filaments at: 0.07 g (A), 0.16 g (B) and 0.4 g (C). (n= 11-16 per group, ***P<0.001, ****P<0.0001, One way- ANOVA with Tukey's multiple comparison test). Data are shown as mean ± SEM and scatter plots.


Mice were injected in the plantar surface of the hind paw with sumatriptan , or vehicle ± the combination of wortmannin and U0126 , as described in the methods section . Mechanical sensitivity was assessed 30 min later using von Frey filaments in an ascending order . ( D - F ) Percentage of withdrawal response for male mice using von Frey filaments at : 0 . 07 g ( D ) , 0 . 16 g ( E ) and 0 . 4 g ( F ) . ( n = 12 - 16 per group , * * * P < 0 . 001 , * * * * P < 0 . 0001 , One way - ANOVA with Tukey ' s multiple comparison test ) . Data are shown as mean ± SEM and scatter plots .

Mice were injected in the plantar surface of the hind paw with sumatriptan, or vehicle ± the combination of wortmannin and U0126, as described in the methods section. Mechanical sensitivity was assessed 30 min later using von Frey filaments in an ascending order. (D-F) Percentage of withdrawal response for male mice using von Frey filaments at: 0.07 g (D), 0.16 g (E) and 0.4 g (F). (n= 12-16 per group, ***P<0.001, ****P<0.0001, One way- ANOVA with Tukey's multiple comparison test). Data are shown as mean ± SEM and scatter plots.


( A ) Purification of the Halo - tagged cohesin tetramer . Human cohesin tetramer tagged by Halo was purified from insect cells and Alexa488 dye was conjugated to Halo - tag ( cohesinHalo488 ) .

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(A) Purification of the Halo-tagged cohesin tetramer. Human cohesin tetramer tagged by Halo was purified from insect cells and Alexa488 dye was conjugated to Halo-tag (cohesinHalo488).


( B ) CohesinHalo488 loaded onto DNA in the presence of Scc2 - Scc4 was incubated in the buffer with or without TEV protease . After high - salt washing , CohesinHalo488 intensities on DNA were measured . Red bars denote the median , lower , and upper quartile values ( n ≥ 98 ; * p < 0 . 0001 , two - tailed Mann - Whitney test ) .

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(B) CohesinHalo488 loaded onto DNA in the presence of Scc2-Scc4 was incubated in the buffer with or without TEV protease. After high-salt washing, CohesinHalo488 intensities on DNA were measured. Red bars denote the median, lower, and upper quartile values (n ≥ 98; *p < 0.0001, two-tailed Mann-Whitney test).


( C ) Kymograph of a single DNA - interacting cohesinHalo488 particle . CohesinHalo488 is loaded onto DNA in the presence of Scc2 - Scc4 and , after high - salt washing , single cohesin particles were observed in the presence of ATP and 100 mM KCl . Scale bar , 2 μm . ( D ) Histogram of the net displacement ( xfinal - xinitial ) of cohesin particles treated as in ( C ) . The magenta curve represents the Gaussian fit .

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(C) Kymograph of a single DNA-interacting cohesinHalo488 particle. CohesinHalo488 is loaded onto DNA in the presence of Scc2-Scc4 and, after high-salt washing, single cohesin particles were observed in the presence of ATP and 100 mM KCl. Scale bar, 2 μm.(D) Histogram of the net displacement (xfinal - xinitial) of cohesin particles treated as in (C). The magenta curve represents the Gaussian fit.


( E ) CohesinHalo488 was loaded and washed in high salt as in ( C ) , and the cohesin particles were observed in the presence of ATP and 100 mM or 750 mM KCl . Kymograph of DNA - associated cohesin particles under high - salt conditions ( 750 mM KCl , right ) and the mean square displacement ( MSD ) versus time ( left ) are shown . Dotted lines are regression lines used to estimate the diffusion coefficient ( D ) . Scale bar , 2 μm .

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(E) CohesinHalo488 was loaded and washed in high salt as in (C), and the cohesin particles were observed in the presence of ATP and 100 mM or 750 mM KCl. Kymograph of DNA-associated cohesin particles under high-salt conditions (750 mM KCl, right) and the mean square displacement (MSD) versus time (left) are shown. Dotted lines are regression lines used to estimate the diffusion coefficient (D). Scale bar, 2 μm.


( F ) MSD versus time for DNA - bound cohesinWT - Halo488 or cohesinKA - Halo488 particles . CohesinWT - Halo488 or cohesinKA - Halo488 was loaded onto DNA in the presence of Scc2 - 4 and ATP . After high - salt washing , the cohesin particles on DNA were observed in the presence of 1 mM AMP - PCP or ATP and 100 mM KCl . D indicates the diffusion coefficient ( n = 45 , mean ± s . e . m . ) .

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(F) MSD versus time for DNA-bound cohesinWT-Halo488 or cohesinKA-Halo488 particles. CohesinWT-Halo488 or cohesinKA-Halo488 was loaded onto DNA in the presence of Scc2-4 and ATP. After high-salt washing, the cohesin particles on DNA were observed in the presence of 1 mM AMP-PCP or ATP and 100 mM KCl. D indicates the diffusion coefficient (n = 45, mean ± s.e.m.).


( A ) Kymograph of CohesinHalo488 in the presence or absence of Wapl - Pds5 . CohesinHalo488 was loaded onto DNA in the presence of Scc2 - Scc4 and washed in high - salt buffer . DNA - bound cohesin was further incubated with or without Wapl - Pds5 , and then cohesinHalo488 particles were observed in the presence of ATP and 100 mM KCl . Scale bar , 1 μm . ( B ) MSD versus time of cohesin particles in ( A ) are shown . D indicates the diffusion coefficient ( n = 45 , mean ± s . e . m . ) .

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(A) Kymograph of CohesinHalo488 in the presence or absence of Wapl-Pds5. CohesinHalo488 was loaded onto DNA in the presence of Scc2-Scc4 and washed in high-salt buffer. DNA-bound cohesin was further incubated with or without Wapl-Pds5, and then cohesinHalo488 particles were observed in the presence of ATP and 100 mM KCl. Scale bar, 1 μm.(B) MSD versus time of cohesin particles in (A) are shown. D indicates the diffusion coefficient (n = 45, mean ± s.e.m.).


( C ) Acetylated - or unacetylated - cohesinHalo488 was loaded onto DNA in the presence of Scc2 - Scc4 and washed in high - salt buffer . DNA - bound cohesin was further incubated with Wapl - Pds5 , and then cohesinHalo488 particles were observed in the presence of ATP and 100 mM KCl . MSD versus time is shown . D indicates the diffusion coefficient ( n = 45 , mean ± s . e . m . ) .

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(C) Acetylated- or unacetylated-cohesinHalo488 was loaded onto DNA in the presence of Scc2-Scc4 and washed in high-salt buffer. DNA-bound cohesin was further incubated with Wapl-Pds5, and then cohesinHalo488 particles were observed in the presence of ATP and 100 mM KCl. MSD versus time is shown. D indicates the diffusion coefficient (n = 45, mean ± s.e.m.).


( D ) Acetylated cohesinHalo488 was treated as in ( C ) , the DNA - bound cohesin was further incubated with buffer , Wapl - Pds5 , or Wapl - Pds5 plus Sororin , and then cohesinHalo488 particles were observed in the presence of ATP and 100 mM KCl . MSD versus time is shown . D indicates the diffusion coefficient ( n = 45 , mean ± s . e . m . ) .

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(D) Acetylated cohesinHalo488 was treated as in (C), the DNA-bound cohesin was further incubated with buffer, Wapl-Pds5, or Wapl-Pds5 plus Sororin, and then cohesinHalo488 particles were observed in the presence of ATP and 100 mM KCl. MSD versus time is shown. D indicates the diffusion coefficient (n = 45, mean ± s.e.m.).


( A ) Acetylated cohesinHalo488 was loaded onto DNA in the presence of Scc2 - Scc4 and washed in high - salt buffer . DNA - bound cohesin was further incubated with Wapl - Pds5 plus Sororin , and then treated with buffer , Plk1 , or Plk1 plus BI4834 . CohesinHalo488 particles were observed in the presence of ATP and 100 mM KCl . MSD versus time is shown . D indicates the diffusion coefficient ( n = 45 , mean ± s . e . m . ) .

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(A) Acetylated cohesinHalo488 was loaded onto DNA in the presence of Scc2-Scc4 and washed in high-salt buffer. DNA-bound cohesin was further incubated with Wapl-Pds5 plus Sororin, and then treated with buffer, Plk1, or Plk1 plus BI4834. CohesinHalo488 particles were observed in the presence of ATP and 100 mM KCl. MSD versus time is shown. D indicates the diffusion coefficient (n = 45, mean ± s.e.m.).


( B ) Acetylated cohesinHalo488 was treated as in ( A ) , or treated with CDK1 or Aurora B rather than Plk1 . Sororin intensity on DNA was measured after immunofluorescence microscopy with anti - Sororin antibody ( n > 100 , * p < 0 . 0001 , two - tailed Mann - Whitney test ) .

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(B) Acetylated cohesinHalo488 was treated as in (A), or treated with CDK1 or Aurora B rather than Plk1. Sororin intensity on DNA was measured after immunofluorescence microscopy with anti-Sororin antibody (n > 100, *p < 0.0001, two-tailed Mann-Whitney test).


( C ) Acetylated cohesinHalo488 was treated as in ( A ) but treated with Aurora B rather than Plk1 . CohesinHalo488 particles were observed in the presence of ATP and 100 mM KCl . MSD versus time is shown . D indicates the diffusion coefficient ( n = 45 , mean ± s . e . m . ) .

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(C) Acetylated cohesinHalo488 was treated as in (A) but treated with Aurora B rather than Plk1. CohesinHalo488 particles were observed in the presence of ATP and 100 mM KCl. MSD versus time is shown. D indicates the diffusion coefficient (n = 45, mean ± s.e.m.).


( A ) Stretched DNA was incubated in the HSS in the presence or absence of geminin . After washing the HSS , endogenous Smc3 ( green ) and Mcm2 ( magenta ) were detected by immunofluorescence staining . DNA was counterstained with SYTOX ( gray ) . Scale bar , 2 µm . ( B ) Numbers of Smc3 and Mcm2 signals were counted on 10 μm DNA shown in ( A ) . ( 22 DNAs per condition , n = 3 , mean ± s . e . m . * p < 0 . 0001 , two - tailed Mann - Whitney test ) .

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(A) Stretched DNA was incubated in the HSS in the presence or absence of geminin. After washing the HSS, endogenous Smc3 (green) and Mcm2 (magenta) were detected by immunofluorescence staining. DNA was counterstained with SYTOX (gray). Scale bar, 2 µm.(B) Numbers of Smc3 and Mcm2 signals were counted on 10 μm DNA shown in (A). (22 DNAs per condition, n = 3, mean ± s.e.m. *p < 0.0001, two-tailed Mann-Whitney test).


( C ) Chromatin formed in the HSS in the presence of geminin , and cohesinHalo488 ( green ) was loaded onto the chromatin with Scc2 - Scc4 . Chromatin was detected by immunostaining of histone H3 ( magenta ) . DNA was counterstained with SYTOX ( gray ) . The distributions of cohesin particles and histone H3 are shown as line - scan plots ( bottom ) . Scale bar , 5 μm . ( D ) Cohesin particles observed in ( C ) were categorized as indicated ( > 37 particles per condition . n = 6 , mean ± s . e . m . ) . ( E ) Cohesin particle numbers for each condition . Red bars denote the median , lower , and upper quartile values ( n ≥ 12 , * p < 0 . 0004 , unpaired t - test ) . ( F ) Colocalization of Mcm2 with Smc3 was quantified ( 50 Mcm2 particles per condition , n = 3 , mean ± s . e . m . )

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(C) Chromatin formed in the HSS in the presence of geminin, and cohesinHalo488 (green) was loaded onto the chromatin with Scc2-Scc4. Chromatin was detected by immunostaining of histone H3 (magenta). DNA was counterstained with SYTOX (gray). The distributions of cohesin particles and histone H3 are shown as line-scan plots (bottom). Scale bar, 5 μm.(D) Cohesin particles observed in (C) were categorized as indicated (> 37 particles per condition. n = 6, mean ± s.e.m.).(E) Cohesin particle numbers for each condition. Red bars denote the median, lower, and upper quartile values (n ≥ 12, *p < 0.0004, unpaired t-test).(F) Colocalization of Mcm2 with Smc3 was quantified (50 Mcm2 particles per condition, n = 3, mean ± s.e.m.)


( A ) Stretched DNA was incubated in the HSS . Endogenous Smc3 ( green ) and Mcm2 ( magenta ) were detected by immunofluorescence staining . DNA was counterstained with SYTOX ( gray ) . Colocalization of Smc3 and Mcm2 was quantified ( bottom , n = 3 , mean ± s . e . m . ) . Scale bar , 2 μm .

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(A) Stretched DNA was incubated in the HSS. Endogenous Smc3 (green) and Mcm2 (magenta) were detected by immunofluorescence staining. DNA was counterstained with SYTOX (gray). Colocalization of Smc3 and Mcm2 was quantified (bottom, n = 3, mean ± s.e.m.). Scale bar, 2 μm.


( B ) Fluorescently labeled Xenopus cohesins ( xCohesinHaloTMR ) were loaded onto DNA in the HSS , and DNA - bound cohesin particles were observed in the HSS with or without 5 mM AMP - PNP supplementation . On the kymograph , the magenta lines indicate the duration of DNA binding of each cohesin particle . DNA was counterstained with SYTOX . Scale bar , 5 μm .

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(B) Fluorescently labeled Xenopus cohesins (xCohesinHaloTMR) were loaded onto DNA in the HSS, and DNA-bound cohesin particles were observed in the HSS with or without 5 mM AMP-PNP supplementation. On the kymograph, the magenta lines indicate the duration of DNA binding of each cohesin particle. DNA was counterstained with SYTOX. Scale bar, 5 μm.


( C ) Kymographs of DNA - bound xCohesinHaloTMR ( green ) in HSS . After time - lapse imaging of xCohesinHaloTMR , nucleosomes were immunostained by anti - H3 antibody ( magenta ) . Result of H3 immunostaining was merged with the kymograph . DNA was counterstained with SYTOX . Scale bar , 5 μm .

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(C) Kymographs of DNA-bound xCohesinHaloTMR (green) in HSS. After time-lapse imaging of xCohesinHaloTMR, nucleosomes were immunostained by anti-H3 antibody (magenta). Result of H3 immunostaining was merged with the kymograph. DNA was counterstained with SYTOX. Scale bar, 5 μm.


( D ) HSS were subjected to mock or XEco2 immunodepletion and human Esco1 - His ( hEsco1 ) was added back to XEco2 - depleted HSS . Sperm chromatin was incubated in these extracts and chromatin - bound proteins were analyzed by immunoblotting .

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(D) HSS were subjected to mock or XEco2 immunodepletion and human Esco1-His (hEsco1) was added back to XEco2-depleted HSS. Sperm chromatin was incubated in these extracts and chromatin-bound proteins were analyzed by immunoblotting.


( E ) Kymographs of DNA - bound xCohesinHaloTMR in mock - , XEco2 - depleted , or hEsco1 - added XEco2 - depleted HSS . Scale bar , 2 μm . ( F ) MSD versus time of cohesin particle in the experiment in ( E ) . D indicates the diffusion coefficient ( n = 15 , mean ± s . e . m . ) .

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(E) Kymographs of DNA-bound xCohesinHaloTMR in mock-, XEco2-depleted, or hEsco1-added XEco2-depleted HSS. Scale bar, 2 μm.(F) MSD versus time of cohesin particle in the experiment in (E). D indicates the diffusion coefficient (n = 15, mean ± s.e.m.).


( B ) Kymographs of examples for cohesin motion during DNA replication . xCohesinHaloTMR ( magenta ) was loaded onto DNA in the HSS , and NPE was introduced to allow DNA replication . DNA replication was monitored by xFen1D179A - PAGFP ( green ) . Thirthy - four particles were categorized into five groups . Arrowheads indicate the time points when cohesin was dissociated from chromatin . Scale bar , 2μm .

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(B) Kymographs of examples for cohesin motion during DNA replication. xCohesinHaloTMR (magenta) was loaded onto DNA in the HSS, and NPE was introduced to allow DNA replication. DNA replication was monitored by xFen1D179A-PAGFP (green). Thirthy-four particles were categorized into five groups. Arrowheads indicate the time points when cohesin was dissociated from chromatin. Scale bar, 2μm.


B Analysis of cell differentiation ( left ) and proliferation ( right ) in the intestine after CDK8 deletion as in ( A ) . Olfm4 , Lysozyme , PAS and Dclk1 staining was used to reveal , respectively , stem , Paneth , goblet and tuft cells . β - Catenin staining allows detection of cancer cells ( cytoplasmic vs nuclear localisation ) . Cell proliferation was assessed by PCNA , Ki - 67 and BrdU ( 2h pulse ) staining . Scatter plots represent the percentage of the area stained by each antibody ( relative to the area occupied by hematoxylin ) . For Paneth cells , BrdU , PCNA and Ki67 , only crypts were analysed . For goblet cells , crypts and villi were analysed . For Tuft cell quantification , Dclk1 - positive cells were counted in 50 villi . Colour code depicts small intestine ( green ) , proximal colon ( blue ) , and distant colon ( red ) . Mean ± SEM is shown . P - value of unpaired two - tailed t - test is indicated ( ns , not significant ; p > 0 . 05 ) . Scale bars , 25μm ( Olfm4 , Lysozyme , Dclk1 and β - Catenin ) and 50μm ( PAS , BrdU , Ki - 67 and PCNA ) . ( n = 9 biological replicates )

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B Analysis of cell differentiation (left) and proliferation (right) in the intestine after CDK8 deletion as in (A). Olfm4, Lysozyme, PAS and Dclk1 staining was used to reveal, respectively, stem, Paneth, goblet and tuft cells. β-Catenin staining allows detection of cancer cells (cytoplasmic vs nuclear localisation). Cell proliferation was assessed by PCNA, Ki-67 and BrdU (2h pulse) staining. Scatter plots represent the percentage of the area stained by each antibody (relative to the area occupied by hematoxylin). For Paneth cells, BrdU, PCNA and Ki67, only crypts were analysed. For goblet cells, crypts and villi were analysed. For Tuft cell quantification, Dclk1-positive cells were counted in 50 villi. Colour code depicts small intestine (green), proximal colon (blue), and distant colon (red). Mean ± SEM is shown. P-value of unpaired two-tailed t-test is indicated (ns, not significant; p > 0.05). Scale bars, 25μm (Olfm4, Lysozyme, Dclk1 and β-Catenin) and 50μm (PAS, BrdU, Ki-67 and PCNA). (n=9 biological replicates)


C Analysis of mouse colon after AOM / DSS treatment . Quantification of the number of neoplastic lesions ( n = 10 for Cdk8 + / + , and n = 7 for Cdk8 - / - mice ) . P - value of unpaired t - test is indicated : ns , not significant ( p > 0 . 05 ) . Mean ± SD is shown .

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C Analysis of mouse colon after AOM/DSS treatment. Quantification of the number of neoplastic lesions (n = 10 for Cdk8+/+, and n = 7 for Cdk8 -/- mice). P-value of unpaired t-test is indicated: ns, not significant (p > 0.05). Mean ± SD is shown.