Figure 1A Localisation of transiently expressed GFP - Rab35 in IMCD3 cells after 48h serum starvation and staining for acetylated tubulin ( acetyl . tub . ) and DNA . Insets show higher magnification images of the cilia region . Scale bars , 10 µm . B Localisation of transiently expressed GFP - RAB35 in hTERT - RPE1 cells after 24 h serum starvation and staining for polyglutamylated tubulin ( polyglu . tub . ) and DNA . Insets show higher magnification images of the cilia region . Scale bars , 10 µm . C Super resolution ( FV - OSR ) imaging of hTERT - RPE1 stably expressing GFP - RAB35 after 24 h serum starvation and staining for GFP , ARL13B ( cilia membrane ) , IFT88 ( axoneme ) , acetylated tubulin ( acetyl . tub . ; axoneme ) , and γ - tubulin ( γ - tub ; centrosome ) . Top panels show representative images and bottom panel shows line profile plots of fluorescence intensity ( arbitrary units ; a . u . ) in the distal ( dotted line ) and proximal ( dashed line ) cilia regions . Data are mean ± S . E . M . ( n = 5 cilia ) , with the solid line in the line profile plots indicating mean and the dotted lines indicating S . E . M . values . Scale bars , 1 µm . D Representative FV - OSR images of 24 h serum - starved hTERT - RPE1 stably expressing GFP - RAB35 and stained for GFP , ARL13B and acetylated tubulin . Right panels show a cilium with proximal enrichment of GFP - RAB35 and left panels a cilium with even GFP - RAB35 localisation along the full length . Scale bars ; 1 µm . E Representative FV - OSR images of 24 h serum - starved hTERT - RPE1 stably expressing GFP - RAB35 and stained for GFP , EHD1 and ARL13B . Line profile plots of fluorescence intensity in the proximal cilia region ( dashed line ) are shown to the right . Data are mean ± S . E . M . ( n = 6 cilia ) , with the solid line in the line profile plots indicating mean and the dotted lines indicating S . E . M . values . Scale bars ; 1 µm .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1A Localisation of transiently expressed GFP-Rab35 in IMCD3 cells after 48h serum starvation and staining for acetylated tubulin (acetyl. tub.) and DNA. Insets show higher magnification images of the cilia region. Scale bars, 10 µm.B Localisation of transiently expressed GFP-RAB35 in hTERT-RPE1 cells after 24 h serum starvation and staining for polyglutamylated tubulin (polyglu. tub.) and DNA. Insets show higher magnification images of the cilia region. Scale bars, 10 µm.C Super resolution (FV-OSR) imaging of hTERT-RPE1 stably expressing GFP-RAB35 after 24 h serum starvation and staining for GFP, ARL13B (cilia membrane), IFT88 (axoneme), acetylated tubulin (acetyl. tub.; axoneme), and γ-tubulin (γ-tub; centrosome). Top panels show representative images and bottom panel shows line profile plots of fluorescence intensity (arbitrary units; a. u.) in the distal (dotted line) and proximal (dashed line) cilia regions. Data are mean ± S.E.M. (n = 5 cilia), with the solid line in the line profile plots indicating mean and the dotted lines indicating S.E.M. values. Scale bars, 1 µm.D Representative FV-OSR images of 24 h serum-starved hTERT-RPE1 stably expressing GFP-RAB35 and stained for GFP, ARL13B and acetylated tubulin. Right panels show a cilium with proximal enrichment of GFP-RAB35 and left panels a cilium with even GFP-RAB35 localisation along the full length. Scale bars; 1 µm.E Representative FV-OSR images of 24 h serum-starved hTERT-RPE1 stably expressing GFP-RAB35 and stained for GFP, EHD1 and ARL13B. Line profile plots of fluorescence intensity in the proximal cilia region (dashed line) are shown to the right. Data are mean ± S.E.M. (n = 6 cilia), with the solid line in the line profile plots indicating mean and the dotted lines indicating S.E.M. values. Scale bars; 1 µm.


Figure 2A Immunoblot analysis of RAB35 depletion in hTERT - RPE1 cells transfected with two independent siRNAs targeting RAB35 ( RAB35 - 1 , RAB35 - 2 ) or a non - targeting siRNA control ( Neg ) . 24 h after transfection , cells were serum - starved for further 48 h . β - tubulin served as a loading control . B Quantification of ciliation of hTERT - RPE1 cells treated and using acetylated tubulin staining as a cilia marker . Data are mean ± S . E . M . of 4 independent experiments ( n ≥ 50 cilia per experimental condition ) . C , D hTERT - RPE1 cells and hTERT - RPE1 cells stably expressing siRNA - resistant GFP - RAB35 were treated and stained for GFP , acetylated tubulin ( acetyl . tub . ) and DNA . Representative images in ( C ) of cells treated with Neg or RAB35 siRNA . Regions within white boxes shown at higher magnifications to the right . Scale bars , 10 µm . Cilia length quantifications in ( D ) are shown as box - and - whisker plots . Horizontal lines show 25 , 50 and 75th percentiles ; whiskers extend to minimum and maximum values . One representative experiment 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 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , n . s . : non - significant ; P - values : hTERT - RPE1 - Neg vs . hTERT - RPE1 - RAB35 - 1 P < 0 . 0001 , hTERT - RPE1 - Neg vs . hTERT - RPE1 - RAB35 - 1 P < 0 . 0001 , hTERT - RPE1 - RAB35 - 1 vs . GFP - RAB35 - RAB35 - 1 P = 0 . 0249 , hTERT - RPE1 - RAB35 - 2 vs . GFP - RAB35 - RAB35 - 2 P = 0 . 0056 ) E Immunoblot analysis of Rab35 depletion in IMCD3 cells transfected with pool of siRNAs targeting mouse Rab35 or a non - targeting siRNA control ( Neg ) . 24 h after transfection , cells were serum - starved for further 48 h . GAPDH served as a loading control . F Quantification of ciliation of IMCD3 cells treated and using acetylated tubulin staining as a cilia marker . Data are mean ± S . E . M . of 3 independent experiments ( n ≥ 50 cilia per experimental condition ) . G - H Immunofluorescence of IMCD3 representative images treated cells are stained for acetylated tubulin ( acetyl . tub . ) and DNA . Cilia length quantification in ( H ) is shown as box - and - whisker plots and is the result of 3 independent experiments ( n ≥ 50 cilia per experimental condition ) . Horizontal lines show 25 , 50 and 75th percentiles ; whiskers extend to minimum and maximum values . Statistical significance according to unpaired t - test with Mann - Whitney test ( * * * P = 0 . 0002 ) I Immunoblot analysis of NIH3T3 wild type ( WT ) and Rab35 knockout ( KO ) cell lines . β - tubulin served as a loading control . NIH3T3 WT and Rab35 KO cell lines were serum - starved for 24h and stained for acetylated tubulin ( acetyl . tub . ) and DNA . Quantification of ciliation in ( J ) . Data are mean ± S . E . M . of 3 independent experiments ( n ≥ 100 cilia per experimental condition ) . NIH3T3 WT and Rab35 KO cell lines were serum - starved for 24h and stained for acetylated tubulin ( acetyl . tub . ) and DNA . Representative images in ( K ) . Regions within white boxes shown at higher magnifications at the bottom . Scale bars , 10 µm . Cilia length quantifications in ( L ) are shown as box - and - whisker plots . Horizontal lines show 25 , 50 and 75th percentiles ; whiskers extend to minimum and maximum values . One representative experiment of three is shown ( n ≥ 100 cilia per experimental condition ) . Statistical significance according to Kruskal - Wallis followed by Dunn ' s post - hoc test ( * * * * P < 0 . 0001 ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2A Immunoblot analysis of RAB35 depletion in hTERT-RPE1 cells transfected with two independent siRNAs targeting RAB35 (RAB35-1, RAB35-2) or a non-targeting siRNA control (Neg). 24 h after transfection, cells were serum-starved for further 48 h. β-tubulin served as a loading control.B Quantification of ciliation of hTERT-RPE1 cells treated and using acetylated tubulin staining as a cilia marker. Data are mean ± S.E.M. of 4 independent experiments (n ≥ 50 cilia per experimental condition).C, D hTERT-RPE1 cells and hTERT-RPE1 cells stably expressing siRNA-resistant GFP-RAB35 were treated and stained for GFP, acetylated tubulin (acetyl. tub.) and DNA. Representative images in (C) of cells treated with Neg or RAB35 siRNA. Regions within white boxes shown at higher magnifications to the right. Scale bars, 10 µm. Cilia length quantifications in (D) are shown as box-and-whisker plots. Horizontal lines show 25, 50 and 75th percentiles; whiskers extend to minimum and maximum values. One representative experiment of three is shown (n ≥ 50 cilia per experimental condition). Statistical significance according to Kruskal-Wallis followed by Dunn's post-hoc test (* P < 0.05, ** P < 0.01, *** P < 0.001, n.s.: non-significant; P-values: hTERT-RPE1-Neg vs. hTERT-RPE1-RAB35-1 P < 0.0001, hTERT-RPE1-Neg vs. hTERT-RPE1-RAB35-1 P < 0.0001, hTERT-RPE1-RAB35-1 vs. GFP-RAB35-RAB35-1 P = 0.0249, hTERT-RPE1-RAB35-2 vs. GFP-RAB35-RAB35-2 P = 0.0056)E Immunoblot analysis of Rab35 depletion in IMCD3 cells transfected with pool of siRNAs targeting mouse Rab35 or a non-targeting siRNA control (Neg). 24 h after transfection, cells were serum-starved for further 48 h. GAPDH served as a loading control.F Quantification of ciliation of IMCD3 cells treated and using acetylated tubulin staining as a cilia marker. Data are mean ± S.E.M. of 3 independent experiments (n ≥ 50 cilia per experimental condition).G-H Immunofluorescence of IMCD3 representative images treated cells are stained for acetylated tubulin (acetyl. tub.) and DNA. Cilia length quantification in (H) is shown as box-and-whisker plots and is the result of 3 independent experiments (n ≥ 50 cilia per experimental condition). Horizontal lines show 25, 50 and 75th percentiles; whiskers extend to minimum and maximum values. Statistical significance according to unpaired t-test with Mann-Whitney test (*** P = 0.0002)I Immunoblot analysis of NIH3T3 wild type (WT) and Rab35 knockout (KO) cell lines. β-tubulin served as a loading control.NIH3T3 WT and Rab35 KO cell lines were serum-starved for 24h and stained for acetylated tubulin (acetyl. tub.) and DNA. Quantification of ciliation in (J). Data are mean ± S.E.M. of 3 independent experiments (n ≥ 100 cilia per experimental condition).NIH3T3 WT and Rab35 KO cell lines were serum-starved for 24h and stained for acetylated tubulin (acetyl. tub.) and DNA. Representative images in (K). Regions within white boxes shown at higher magnifications at the bottom. Scale bars, 10 µm. Cilia length quantifications in (L) are shown as box-and-whisker plots. Horizontal lines show 25, 50 and 75th percentiles; whiskers extend to minimum and maximum values. One representative experiment of three is shown (n ≥ 100 cilia per experimental condition). Statistical significance according to Kruskal-Wallis followed by Dunn's post-hoc test (**** P < 0.0001).


Figure 3A Representative images of hTERT - RPE1 cells transiently expressing wild type ( WT ) , GDP - bound ( S22N ) or GTP - bound ( Q67L ) GFP - tagged RAB35 . Cells were serum - starved for 24 h and stained for polyglutamylated tubulin ( polyglu . tub . ) and DNA . Higher magnification images of the cilia region shown in smaller panels . Scale bars ; 10 µm . Quantification of GFP - RAB35 ciliary localisation ( B ) in hTERT - RPE1 cells transiently expressing indicated GFP - RAB35 constructs or GFP . Data are mean ± S . E . M . of 3 independent experiments . Statistical significance according to ANOVA followed by Bonferroni post - hoc test ( * * P < 0 . 01 ; P - values : ( B ) P = 0 . 0015Quantification of ciliation ( C ) in hTERT - RPE1 cells transiently expressing indicated GFP - RAB35 constructs or GFP . Data are mean ± S . E . M . of 3 independent experiments . Statistical significance according to ANOVA followed by Bonferroni post - hoc test ( * * P < 0 . 01 ; P - values : ( C ) P = 0 . 0062 ) . Quantification of ciliary length in hTERT - RPE1 cells transiently expressing indicated GFP - RAB35 constructs or GFP . Cilia length quantification in ( D ) is shown as box - and - whisker plots . 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 . 05 , * * P < 0 . 01 ; P - values : GFP vs . GFP - RAB35 - S22N P = 0 . 0015 , GFP vs . GFP - RAB35 - Q67L P = 0 . 0158 ) Analysis of cilia length in hTERT - RPE1 cells depleted of GEF DENND1B ) and GAP ( TBC1D10A regulators of RAB35 . Cells transfected with indicated siRNAs were serum - starved for 48 h and stained for acetylated tubulin ( acetyl . tub . ) and DNA . Representative images are shown in ( E ) . Regions within white boxes shown at higher magnifications to the right . Scale bars ; 10 µm . Analysis of cilia length in hTERT - RPE1 cells depleted of GEF ( DENND1A , DENND1B ) and GAP ( TBC1D10A , TBC1D10B ) regulators of RAB35 . Cells transfected with indicated siRNAs were serum - starved for 48 h and stained for acetylated tubulin ( acetyl . tub . ) and DNA . Cilia length quantification in ( F ) is shown as box - and - whisker plots . 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 . 01 , * * * P < 0 . 001 ; P - values : Neg vs . DENND1B P = 0 . 0033 , Neg vs . TBC1D10A P = 0 . 0008 ) G Quantification of ciliary localisation of GFP - RAB35 in hTERT - RPE1 cells stably expressing GFP - RAB35 treated Data are mean ± S . E . M . of 5 independent experiments . Statistical significance according to ANOVA followed by Bonferroni post - hoc test ( * P < 0 . 05 , * * * P < 0 . 001 ; P - values : Neg vs . DENND1B P < 0 . 0001 , Neg vs . TBC1D10A P = 0 . 0322 ) . H - J Localisation of transiently expressed GFP - DENND1B ( H ) or GFP - TBC1D10A ( I ) in hTERT - RPE1 cells after 24 h serum starvation and staining with indicated antibodies . Higher magnification images of the cilia region are shown in smaller panels . Scale bars ; 5 µm . Quantification of localisation to ciliary base or along axoneme in ( J ) . Data are mean ± S . E . M . of 3 independent experiments ( n ≥ 30 cilia per experimental condition ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3A Representative images of hTERT-RPE1 cells transiently expressing wild type (WT), GDP-bound (S22N) or GTP-bound (Q67L) GFP-tagged RAB35. Cells were serum-starved for 24 h and stained for polyglutamylated tubulin (polyglu. tub.) and DNA. Higher magnification images of the cilia region shown in smaller panels. Scale bars; 10 µm.Quantification of GFP-RAB35 ciliary localisation (B) in hTERT-RPE1 cells transiently expressing indicated GFP-RAB35 constructs or GFP. Data are mean ± S.E.M. of 3 independent experiments. Statistical significance according to ANOVA followed by Bonferroni post-hoc test ( ** P < 0.01; P-values: (B) P = 0.0015Quantification of ciliation (C) in hTERT-RPE1 cells transiently expressing indicated GFP-RAB35 constructs or GFP. Data are mean ± S.E.M. of 3 independent experiments. Statistical significance according to ANOVA followed by Bonferroni post-hoc test ( ** P < 0.01; P-values: (C) P = 0.0062).Quantification of ciliary length in hTERT-RPE1 cells transiently expressing indicated GFP-RAB35 constructs or GFP. Cilia length quantification in (D) is shown as box-and-whisker plots. 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 < 0.05, ** P < 0.01; P-values: GFP vs. GFP-RAB35-S22N P = 0.0015, GFP vs. GFP-RAB35-Q67L P = 0.0158)Analysis of cilia length in hTERT-RPE1 cells depleted of GEF DENND1B) and GAP (TBC1D10A regulators of RAB35. Cells transfected with indicated siRNAs were serum-starved for 48 h and stained for acetylated tubulin (acetyl. tub.) and DNA. Representative images are shown in (E). Regions within white boxes shown at higher magnifications to the right. Scale bars; 10 µm.Analysis of cilia length in hTERT-RPE1 cells depleted of GEF (DENND1A, DENND1B) and GAP (TBC1D10A, TBC1D10B) regulators of RAB35. Cells transfected with indicated siRNAs were serum-starved for 48 h and stained for acetylated tubulin (acetyl. tub.) and DNA. Cilia length quantification in (F) is shown as box-and-whisker plots. 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 < 0.01, *** P < 0.001; P-values: Neg vs. DENND1B P = 0.0033, Neg vs. TBC1D10A P = 0.0008)G Quantification of ciliary localisation of GFP-RAB35 in hTERT-RPE1 cells stably expressing GFP-RAB35 treated Data are mean ± S.E.M. of 5 independent experiments. Statistical significance according to ANOVA followed by Bonferroni post-hoc test (* P < 0.05, *** P < 0.001; P-values: Neg vs. DENND1B P < 0.0001, Neg vs. TBC1D10A P = 0.0322).H-J Localisation of transiently expressed GFP-DENND1B (H) or GFP-TBC1D10A (I) in hTERT-RPE1 cells after 24 h serum starvation and staining with indicated antibodies. Higher magnification images of the cilia region are shown in smaller panels. Scale bars; 5 µm. Quantification of localisation to ciliary base or along axoneme in (J). Data are mean ± S.E.M. of 3 independent experiments (n ≥ 30 cilia per experimental condition).


Figure 4A Immunostaining of representative images of KV embryos at 8 somite stage in which mRNA encoding for mCherry or mCherry - Rab35 WT was injected . Staining of acetylated tubulin was used as cilia marker . Inset shows a higher magnification image of the cilia region , displaying colocalization of mCherry - Rab35 and acetylated tubulin . Scale bar , 10 µm . B , C Immunoblot analysis with anti - Rab35 and anti - GAPDH antibodies of 40 µg of total lysates of zebrafish embryos injected with Rab35 morpholino ( MO ) at 8 somite stage , compared with non - injected embryos . The plot in ( C ) shows Rab35 protein level in non - injected and Rab35 morphants , normalized to GAPDH protein level . Data are mean ± S . E . M . of 3 independent experiments ( n > 50 embryos per condition ) . D Organ situs was determined by observing the heart position at 30 hours post - fertilisation ( hpf ) , and the liver position at 53 hpf , by in situ hybridisation with a probe for foxa3 , in controls ( Mismatch MO and non - injected [ WT ] ) and Rab35 MO . Situs solitus means left heart and liver ; situs inversus means right heart and liver ; and heterotaxia means any other possible combination of the heart and liver position . E Whole - mount in situ hybridization with the dand5 probe at 8 somite stage ( 13 hpf ) in wild type non - injected embryos and mismatch morphants , both presenting right - sided distribution of dand5 , and Rab35 morphants presenting bilateral localization of the dand5 probe . Scale bar , 50 µm . Graph shows quantification of embryos ( % ) with bilateral or right - sided dand5 localisation from both conditions . Statistical significance according to Fisher Exact test ( * * P = 0 . 0002 ) F - I Immunostaining of representative images of KV embryos at 8 somite stage in which : ( F ) Mismatch or Rab35 MO was injected , as well as a co - injection of Rab35 MO with mRNA encoding mCherry - Rab35 WT [ Rescue ] ; ( H ) mRNA encoding mCherry or mCherry - tagged Rab35 WT , Rab35 - Q67L or Rab35 - S22N were injected . Acetylated tubulin is shown in green as cilia marker . Quantification of KV cilia length in ( F ) and ( H ) is shown respectively in ( G ) and ( I ) as box - and - whisker plots of 3 independent experiments . N ≥ 8 embryos and n > 300 cilia per experimental condition . Horizontal lines show 25 , 50 and 75th percentiles ; whiskers extend to minimum and maximum values . Statistical significance according to Kruskal - Wallis followed by Dunn ' s post - hoc test ( * * P < 0 . 01 , * * * P < 0 . 001 , ns . not significant ; Mismatch vs Rab35 MO , P < 0 . 001 ; Cherry vs Rab35 - SN , P = 0 . 0063 ; ) . Scale bars , 10 µm .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4A Immunostaining of representative images of KV embryos at 8 somite stage in which mRNA encoding for mCherry or mCherry-Rab35 WT was injected. Staining of acetylated tubulin was used as cilia marker. Inset shows a higher magnification image of the cilia region, displaying colocalization of mCherry-Rab35 and acetylated tubulin. Scale bar, 10 µm.B,C Immunoblot analysis with anti-Rab35 and anti-GAPDH antibodies of 40 µg of total lysates of zebrafish embryos injected with Rab35 morpholino (MO) at 8 somite stage, compared with non-injected embryos. The plot in (C) shows Rab35 protein level in non-injected and Rab35 morphants, normalized to GAPDH protein level. Data are mean ± S.E.M. of 3 independent experiments (n > 50 embryos per condition).D Organ situs was determined by observing the heart position at 30 hours post-fertilisation (hpf), and the liver position at 53 hpf, by in situ hybridisation with a probe for foxa3, in controls (Mismatch MO and non-injected [WT]) and Rab35 MO. Situs solitus means left heart and liver; situs inversus means right heart and liver; and heterotaxia means any other possible combination of the heart and liver position.E Whole-mount in situ hybridization with the dand5 probe at 8 somite stage (13 hpf) in wild type non-injected embryos and mismatch morphants, both presenting right-sided distribution of dand5, and Rab35 morphants presenting bilateral localization of the dand5 probe. Scale bar, 50 µm. Graph shows quantification of embryos (%) with bilateral or right-sided dand5 localisation from both conditions. Statistical significance according to Fisher Exact test (** P = 0.0002)F-I Immunostaining of representative images of KV embryos at 8 somite stage in which: (F) Mismatch or Rab35 MO was injected, as well as a co-injection of Rab35 MO with mRNA encoding mCherry-Rab35 WT [Rescue]; (H) mRNA encoding mCherry or mCherry-tagged Rab35 WT, Rab35-Q67L or Rab35-S22N were injected. Acetylated tubulin is shown in green as cilia marker. Quantification of KV cilia length in (F) and (H) is shown respectively in (G) and (I) as box-and-whisker plots of 3 independent experiments. N ≥ 8 embryos and n > 300 cilia per experimental condition. Horizontal lines show 25, 50 and 75th percentiles; whiskers extend to minimum and maximum values. Statistical significance according to Kruskal-Wallis followed by Dunn's post-hoc test (** P < 0.01, *** P < 0.001, ns. not significant; Mismatch vs Rab35 MO, P < 0.001; Cherry vs Rab35-SN, P = 0.0063; ). Scale bars, 10 µm.


Figure 5A , B hTERT - RPE1 cells and hTERT - RPE1 cells stably expressing siRNA - resistant GFP - RAB35 transfected with non - targeting siRNA control ( Neg ) or Rab35 siRNA , were serum - starved for 48 h and stained for ARL13B , GFP , polyglutamylated tubulin ( polyglu . 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 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 . 01 , * * * P < 0 . 001 , n . s . : non - significant ; P - values : hTERT - RPE1 - Neg vs . hTERT - RPE1 - RAB35 - 1 P < 0 . 0001 , hTERT - RPE1 - Neg vs . hTERT - RPE1 - RAB35 - 1 P = 0 . 0001 , hTERT - RPE1 - RAB35 - 1 vs . GFP - RAB35 - RAB35 - 1 P = 0 . 0014 , hTERT - RPE1 - RAB35 - 2 vs . GFP - RAB35 - RAB35 - 2 P = 0 . 0081 ) . C Immunoblot analysis of total ARL13B protein levels in control and RAB35 - depleted cells . β - tubulin served as a loading control . The graph shows ARL13B protein levels normalized ( norm . ) to β - tubulin . Data are mean ± S . E . M . of 4 independent experiments . 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 . 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 . 0005Quantification 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5A, B hTERT-RPE1 cells and hTERT-RPE1 cells stably expressing siRNA-resistant GFP-RAB35 transfected with non-targeting siRNA control (Neg) or Rab35 siRNA, were serum-starved for 48 h and stained for ARL13B, GFP, polyglutamylated tubulin (polyglu. 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 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 < 0.01, *** P < 0.001, n.s.: non-significant; P-values: hTERT-RPE1-Neg vs. hTERT-RPE1-RAB35-1 P < 0.0001, hTERT-RPE1-Neg vs. hTERT-RPE1-RAB35-1 P = 0.0001, hTERT-RPE1-RAB35-1 vs. GFP-RAB35-RAB35-1 P = 0.0014, hTERT-RPE1-RAB35-2 vs. GFP-RAB35-RAB35-2 P = 0.0081).C Immunoblot analysis of total ARL13B protein levels in control and RAB35-depleted cells. β-tubulin served as a loading control. The graph shows ARL13B protein levels normalized (norm.) to β-tubulin. Data are mean ± S.E.M. of 4 independent experiments.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.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 = 0.0005Quantification 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).


Figure 6A 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 ) . 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6A 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).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)


Figure 7A , 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 ) . 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7A, 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).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).


Figure 8hTERT - 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 ) 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 ) . 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 ≥ P = 0 . 0046 ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 8hTERT-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)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).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 ≥P = 0.0046).


Figure 1 ( 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 . ( 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 .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1(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.(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.


Figure 2 ( 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 . ( 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O

Figure 2(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.(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.


Figure 3 ( 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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Figure 3(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.


Figure 5 ( 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 . ( 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5(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.(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.


Figure 6 ( 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6(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).


Figure 7 ( 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 .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7(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.


Figure 1A . 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 . 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 . 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 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1A. 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.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.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.


Figure 2B . 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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Figure 2B. 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.


Figure 3A . 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 ) . 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 ) . 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 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3A. 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).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).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.


Figure 4A . 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 . 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 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O

Figure 4A. 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.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.


Figure 5A . 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5A. 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.


Figure 4Bacterial 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 . 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 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4Bacterial 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.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.


Figure 1 ( 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 . ( 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 . ( 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1(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.(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.(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.