figf3 ( b ) Flow cytometry of splenocytes from recipient mice ( n = 6 ) given adoptive transfer of P14 cells transduced with retrovirus as in a , assessed on days 5 and 8 after infection of recipients with LCMV ( left ) , and quantification of P14 cells per spleen ( right ) . Numbers adjacent to outlined areas ( left ) indicate percent P14 ( CD45 . 1 + ) cells . * P ≤ 0 . 0005 ( unpaired t - test ) . ( c ) Flow cytometry of the size of P14 cells transduced with wild - type mCherry - GFP - LC3b retrovirus ( assessed as in Fig . 1b ) in mice treated as in b , assessed at days 5 and 8 after infection . ( d ) Efficiency of retroviral transduction ( left ) and expression of GFP and mCherry ( right ) in retrovirus - transduced P14 cells , assessed by flow cytometry gated on CD45 . 1 + P14 cells ( left ) or CD45 . 1 + Thy - 1 . 1 + P14 cells ( right ) . Numbers in quadrants indicate percent CD45 . 1 + Thy - 1 . 1 − ( untransduced ) P14 cells ( top left ) or CD45 . 1 + Thy - 1 . 1 + ( transduced ) P14 cells ( top right ; magenta ) ( left ) or percent cells in each ( right ) . ( e ) Ratio of the geometric mean fluorescence intensity ( gMFI ) of GFP to that of mCherry ( GFP / mCherry ) for the cells in d ; each symbol represents an individual mouse ; numbers above lines connecting groups indicate difference in ratio . Data are representative of two independent experiments with three or more mice per group ( error bars ( b ) , s . e . m . ) .

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 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

figf3(b) Flow cytometry of splenocytes from recipient mice (n = 6) given adoptive transfer of P14 cells transduced with retrovirus as in a, assessed on days 5 and 8 after infection of recipients with LCMV (left), and quantification of P14 cells per spleen (right). Numbers adjacent to outlined areas (left) indicate percent P14 (CD45.1+) cells. *P ≤ 0.0005 (unpaired t-test).(c) Flow cytometry of the size of P14 cells transduced with wild-type mCherry-GFP-LC3b retrovirus (assessed as in Fig. 1b) in mice treated as in b, assessed at days 5 and 8 after infection.(d) Efficiency of retroviral transduction (left) and expression of GFP and mCherry (right) in retrovirus-transduced P14 cells, assessed by flow cytometry gated on CD45.1+ P14 cells (left) or CD45.1+Thy-1.1+ P14 cells (right). Numbers in quadrants indicate percent CD45.1+Thy-1.1− (untransduced) P14 cells (top left) or CD45.1+Thy-1.1+ (transduced) P14 cells (top right; magenta) (left) or percent cells in each (right). (e) Ratio of the geometric mean fluorescence intensity (gMFI) of GFP to that of mCherry (GFP/mCherry) for the cells in d; each symbol represents an individual mouse; numbers above lines connecting groups indicate difference in ratio. Data are representative of two independent experiments with three or more mice per group (error bars (b), s.e.m.).


figf4 ( a ) Genomic deletion of Atg7 in H - 2Db - gp33 + T cells and in total activated ( CD44hiCD62Llo ) CD8 + T cells from Atg7fl / fl mice ( Cre − ) and Atg7fl / flGzmb - Cremice ( Cre + ) at day 8 after infection with LCMV Armstrong strain ; results are presented relative to those of Atg7fl / fl mice , set as 100 % . Each symbol represents an individual mouse ; small horizontal lines indicate the mean ( ± s . e . m . ) . ( b ) Immunoblot analysis of Atg7 and the products of its enzymatic activity ( left margin ) in CD44hiCD62Llo CD8 + T cells isolated at day 8 after infection as in a ; β - actin serves as a loading control . ( c ) Flow cytometry of T cells in the peripheral blood at day 8 after infection as in a , gated on CD8 + T cells ( left ) , and of the H - 2Db - gp33 + T cells identified at left , identifying terminal effector ( KLRG1hiCD127lo ) and memory precursor ( KLRG1loCD127hi ) populations ( right ) . Numbers adjacent to outlined areas ( left ) indicate percent H - 2Db - gp33 + T cells ; numbers in quadrants ( right ) indicate percent cells in each . ( d ) Total antigen - specific CD8 + T cells in the spleen , liver and lungs at day 8 after infection as in a . ( e ) Production of IFN - γ and TNF in cells obtained 8 d after infection and stimulated ex vivo with various peptides ( above plots ) , gated on total CD8 + T cells . Numbers in quadrants indicate percent cells in each . ( f ) Flow cytometry of CD8 + T cells in the peripheral blood of mice at day 15 after infection as in a ( numbers in plots as in c , left ) . ( g ) Quantification of tetramer - positive cells in spleens at day 15 after infection as in a . ( h ) Kinetics of the appearance H - 2Db - gp33 + CD8 + T cells among peripheral blood mononuclear cells ( PBMCs ) after infection as in a ( left ) ; each line represents one mouse . Right , frequency of H - 2Db - gp33 + CD8 + T cells remaining on day 15 relative to that on day 8 , set as 100 % . ( i ) Quantification of tetramer - positive cells at day 30 after infection as in a . Horizontal dashed lines ( g , h ) indicate the limit of detection . * P ≤ 0 . 05 , * * P ≤ 0 . 005 and * * * P ≤ 0 . 0005 ( unpaired t - test ) . Data were pooled from two independent experiments ( a ) or at least two independent experiments ( c - i ) with three to seven mice per group ( error bars , s . e . m . ) or are from one experiment with samples obtained from two independent experiments ( b ) .

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 O O 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 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 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 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 B-PANEL_START 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 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

figf4(a) Genomic deletion of Atg7 in H-2Db-gp33+ T cells and in total activated (CD44hiCD62Llo) CD8+ T cells from Atg7fl/fl mice (Cre −) and Atg7fl/flGzmb-Cremice (Cre +) at day 8 after infection with LCMV Armstrong strain; results are presented relative to those of Atg7fl/fl mice, set as 100%. Each symbol represents an individual mouse; small horizontal lines indicate the mean (±s.e.m.).(b) Immunoblot analysis of Atg7 and the products of its enzymatic activity (left margin) in CD44hiCD62Llo CD8+ T cells isolated at day 8 after infection as in a; β-actin serves as a loading control.(c) Flow cytometry of T cells in the peripheral blood at day 8 after infection as in a, gated on CD8+ T cells (left), and of the H-2Db-gp33+ T cells identified at left, identifying terminal effector (KLRG1hiCD127lo) and memory precursor (KLRG1loCD127hi) populations (right). Numbers adjacent to outlined areas (left) indicate percent H-2Db-gp33+ T cells; numbers in quadrants (right) indicate percent cells in each.(d) Total antigen-specific CD8+ T cells in the spleen, liver and lungs at day 8 after infection as in a.(e) Production of IFN-γ and TNF in cells obtained 8 d after infection and stimulated ex vivo with various peptides (above plots), gated on total CD8+ T cells. Numbers in quadrants indicate percent cells in each.(f) Flow cytometry of CD8+ T cells in the peripheral blood of mice at day 15 after infection as in a (numbers in plots as in c, left).(g) Quantification of tetramer-positive cells in spleens at day 15 after infection as in a.(h) Kinetics of the appearance H-2Db-gp33+ CD8+ T cells among peripheral blood mononuclear cells (PBMCs) after infection as in a (left); each line represents one mouse. Right, frequency of H-2Db-gp33+ CD8+ T cells remaining on day 15 relative to that on day 8, set as 100%.(i) Quantification of tetramer-positive cells at day 30 after infection as in a. Horizontal dashed lines (g,h) indicate the limit of detection. *P ≤ 0.05, **P ≤ 0.005 and ***P ≤ 0.0005 (unpaired t-test). Data were pooled from two independent experiments (a) or at least two independent experiments (c-i) with three to seven mice per group (error bars, s.e.m.) or are from one experiment with samples obtained from two independent experiments (b).


figf5 ( a ) Flow cytometry of circulating LCMV - specific CD8 + T cells among peripheral blood mononuclear cells from Atg5fl / fl and Atg5fl / flGzmb - Cremice on days 8 , 15 and 50 after infection with LCMV Armstrong strain . Numbers adjacent to outlined areas indicate percent H - 2Db - gp33 + T cells , gated on CD8 + T cells . ( b ) Longitudinal kinetics of the appearance of H - 2Db - gp33 + T cells in the peripheral blood of mice as in a ( left ) , and percent H - 2Db - gp33 + CD8 + T cells remaining on day 15 ( right ) ( all presented as in Fig . 4h ) . ( c ) Flow cytometry of T cells in tissues on day 279 after infection as in a ( numbers in plots as in a , among total gated CD8 + T cells ) . ( d ) Total T cells specific for H - 2Db - gp33 ( left ) or H - 2Db - NP396 ( right ) in the spleen , liver and lungs of mice as in a . * P ≤ 0 . 05 , * * P ≤ 0 . 005 and * * * P ≤ 0 . 0005 ( unpaired t - test ) . Data are representative of two independent experiments with three to seven mice per group ( error bars ( d ) , s . e . m . ) .

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 O O O O O O O O

figf5(a) Flow cytometry of circulating LCMV-specific CD8+ T cells among peripheral blood mononuclear cells from Atg5fl/fl and Atg5fl/flGzmb-Cremice on days 8, 15 and 50 after infection with LCMV Armstrong strain. Numbers adjacent to outlined areas indicate percent H-2Db-gp33+ T cells, gated on CD8+ T cells. (b) Longitudinal kinetics of the appearance of H-2Db-gp33+ T cells in the peripheral blood of mice as in a (left), and percent H-2Db-gp33+ CD8+ T cells remaining on day 15 (right) (all presented as in Fig. 4h).(c) Flow cytometry of T cells in tissues on day 279 after infection as in a (numbers in plots as in a, among total gated CD8+ T cells). (d) Total T cells specific for H-2Db-gp33 (left) or H-2Db-NP396 (right) in the spleen, liver and lungs of mice as in a. *P ≤ 0.05, **P ≤ 0.005 and ***P ≤ 0.0005 (unpaired t-test). Data are representative of two independent experiments with three to seven mice per group (error bars (d), s.e.m.).


figf6 ( a , b ) Flow cytometry of CD8 + T cell obtained from chimeras generated ( as in Supplementary Fig . 5a ) by reconstitution of wild - type mice with a mixture of bone marrow cells from Atg7fl / fl C57BL / 6 and wild - type C57BL / 6C57BL / 6 ( Atg7fl / fl vs C57BL / 6 ) or from Atg7fl / flGzmb - Cremice and wild - type C57BL / 6mice and ( Atg7fl / flGzmb - Cre vs C57BL / 6 ) , gated ( far left ) as populations specific for H - 2Db - gp33 ( a ) or H - 2Db - NP396 ( b ) and then assessed , on days 8 , 15 and 30 after infection of recipients with LCMV Armstrong infection , as CD45 . 2 + ( Atg7fl / fl or Atg7fl / flGzmb - Cre ) donor cells or CD45 . 1 + ( C57BL / 6 ) donor cells ( middle ) . Far right , appearance of tetramer - positive T cells ( key , donor source ) in the peripheral blood of chimeras from day 8 to day 30 after infection , presented relative to that at day 8 after infection , set as 100 % . ( c ) Frequency of CD45 . 2 + antigen - specific CD8 + T cells in the spleen , liver and lungs on day 30 after infectionwith LCMV Armstrongas in a , b . Data are representative of two independent experiments with three to four mice per group ( error bars , s . e . m . ) .

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 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

figf6(a,b) Flow cytometry of CD8+ T cell obtained from chimeras generated (as in Supplementary Fig. 5a) by reconstitution of wild-type mice with a mixture of bone marrow cells from Atg7fl/fl C57BL/6 and wild-type C57BL/6C57BL/6 (Atg7fl/fl vs C57BL/6) or from Atg7fl/flGzmb-Cremice and wild-type C57BL/6mice and (Atg7fl/flGzmb-Cre vs C57BL/6), gated (far left) as populations specific for H-2Db-gp33 (a) or H-2Db-NP396 (b) and then assessed, on days 8, 15 and 30 after infection of recipients with LCMV Armstrong infection, as CD45.2+ (Atg7fl/fl or Atg7fl/flGzmb-Cre) donor cells or CD45.1+ (C57BL/6) donor cells (middle). Far right, appearance of tetramer-positive T cells (key, donor source) in the peripheral blood of chimeras from day 8 to day 30 after infection, presented relative to that at day 8 after infection, set as 100%.(c) Frequency of CD45.2+ antigen-specific CD8+ T cells in the spleen, liver and lungs on day 30 after infectionwith LCMV Armstrongas in a,b. Data are representative of two independent experiments with three to four mice per group (error bars, s.e.m.).


figf8 ( a ) Kinetics of the appearance of H - 2Db - gp276 + T cells in the peripheral blood of Atg7fl / fl and Atg7fl / flGzmb - Cre mice at various times ( horizontal axis ) after infection with LCMV clone 13 . ( b ) Total IFN - γ - secreting cells in spleens obtained from mice at day 8 or day 15 after infection as in a , assessed after 5 h of stimulation ex vivo with various peptides ( horizontal axes ) . ( c ) Viral titers in serum of mice at various times ( horizontal axis ) after infection with LCMV clone 13 as in a . Each symbol represents an individual mouse ; small horizontal lines indicate the mean ( ± s . e . m . ) . * P ≤ 0 . 05 , * * P ≤ 0 . 005 and * * * P ≤ 0 . 0005 ( unpaired t - test ) . Data are representative of two independent experiments with three to five mice per group ( error bars , s . e . m . ) .

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 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

figf8(a) Kinetics of the appearance of H-2Db-gp276+ T cells in the peripheral blood of Atg7fl/fl and Atg7fl/flGzmb-Cre mice at various times (horizontal axis) after infection with LCMV clone 13.(b) Total IFN-γ-secreting cells in spleens obtained from mice at day 8 or day 15 after infection as in a, assessed after 5 h of stimulation ex vivo with various peptides (horizontal axes).(c) Viral titers in serum of mice at various times (horizontal axis) after infection with LCMV clone 13 as in a. Each symbol represents an individual mouse; small horizontal lines indicate the mean (±s.e.m.). *P ≤ 0.05, **P ≤ 0.005 and ***P ≤ 0.0005 (unpaired t-test). Data are representative of two independent experiments with three to five mice per group (error bars, s.e.m.).


Figure 6The intestine - specific RNAi against irx - 1 reduces the Smurf phenotype at day 8 of adulthood . Mean ± SD . Scale bar : 100 μm . Unpaired t - test , two - tailed . More than 30 worms analysed in each of the three biological replicates . , C The intestine - specific RNAi against irx - 1 reduces the Smurf phenotype at day 8 of adulthood . Mean ± SD . Scale bar : 100 μm . Unpaired t - test , two - tailed . More than 30 worms analysed in each of the three biological replicates .

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 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

Figure 6The intestine-specific RNAi against irx-1 reduces the Smurf phenotype at day 8 of adulthood. Mean ± SD. Scale bar: 100 μm. Unpaired t-test, two-tailed. More than 30 worms analysed in each of the three biological replicates., C The intestine-specific RNAi against irx-1 reduces the Smurf phenotype at day 8 of adulthood. Mean ± SD. Scale bar: 100 μm. Unpaired t-test, two-tailed. More than 30 worms analysed in each of the three biological replicates.


Figure 7B , C The BWM - specific RNAi against myrf - 2 improves sarcomere in the worms at day 8 of adulthood . Arrowheads and arrows respectively denote the gaps of myofilaments and the disorganised myofilaments with GFP aggregations . Scale Bar : 10 μm . Mean ± SD . Unpaired t - test , two - tailed . n > = 50 . D , E Suppressing myrf - 2 in BWM increases worms ' motility at day 5 of adulthood . The tracks were from worms ' movement in 30 sec ( D ) . The speed of worm movement was measured by the ratio of track length versus body length ( E ) . Mean ± SD . n > = 120 . Unpaired t - test , two - tailed .

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

Figure 7B, C The BWM-specific RNAi against myrf-2 improves sarcomere in the worms at day 8 of adulthood. Arrowheads and arrows respectively denote the gaps of myofilaments and the disorganised myofilaments with GFP aggregations. Scale Bar: 10 μm. Mean ± SD. Unpaired t-test, two-tailed. n>=50.D, E Suppressing myrf-2 in BWM increases worms' motility at day 5 of adulthood. The tracks were from worms' movement in 30 sec (D). The speed of worm movement was measured by the ratio of track length versus body length (E). Mean ± SD. n>=120. Unpaired t-test, two-tailed.


Figure 1 ( A ) survival curves of MyD88 flies injected with different doses of A . fumigatus conidia ( c / f : conidia injected per fly ; error bars represent mean ± SD of the survival of biological triplicates of 20 flies each ) . ( B ) fungal loads of single MyD88 mutant and wild - type flies ( 50 conidia injected per fly ) . ( C - D ) GFP - labeled A . fumigatus ( 50 conidia per fly ) injected in wild - type ( C ) or MyD88 mutant ( D ) flies form hyphae in the thorax of the flies ( arrows ) . Scale bars 50 μm . ( E ) expression level of Drosomycin induced by different doses of injected A . fumigatus conidia measured by RT - qPCR ; M . luteus ( OD = 10 ) represents the positive control ( pooled data of n = 3 experiments , biological replicates ) . Hayan flies are susceptible to A . fumigatus . Survival ( F ) , of Hayan mutant flies ; Hayan 0h vs . 120h , ( error bars represent mean ± SD of the survival of biological triplicates of 20 flies each ) . time course of fungal loads of single Hayan mutant flies ( 500 conidia per fly ) ( G ) , and fungal load upon death ( 500 conidia per fly ) ( H ) of Hayan mutant flies ; Hayan 0h vs . 120h , ( error bars represent mean ± SD of the survival of biological triplicates of 20 flies each ) .

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 B-PANEL_START 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 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 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

Figure 1(A) survival curves of MyD88 flies injected with different doses of A. fumigatus conidia (c/f: conidia injected per fly; error bars represent mean ± SD of the survival of biological triplicates of 20 flies each).(B) fungal loads of single MyD88 mutant and wild-type flies (50 conidia injected per fly).(C-D) GFP-labeled A. fumigatus (50 conidia per fly) injected in wild-type (C) or MyD88 mutant (D) flies form hyphae in the thorax of the flies (arrows). Scale bars 50 μm.(E) expression level of Drosomycin induced by different doses of injected A. fumigatus conidia measured by RT-qPCR; M. luteus (OD=10) represents the positive control (pooled data of n=3 experiments, biological replicates).Hayan flies are susceptible to A. fumigatus. Survival (F), of Hayan mutant flies; Hayan 0h vs. 120h, (error bars represent mean ± SD of the survival of biological triplicates of 20 flies each).time course of fungal loads of single Hayan mutant flies (500 conidia per fly) (G), and fungal load upon death (500 conidia per fly) (H) of Hayan mutant flies; Hayan 0h vs. 120h, (error bars represent mean ± SD of the survival of biological triplicates of 20 flies each).


Figure 2 ( A ) survival of MyD88 flies injected with ∆ pptA and wild - type ( Af ) A . fumigatus conidia ( error bars represent mean ± SD of the survival of biological triplicates of 20 flies each ) . ( B ) fungal loads of single flies after the injection of 500 ∆ pptA conidia . ( E ) dose response of MyD88 flies after ∆ gliP ( gliotoxin ) mutant or wild - type [ ∆ akuB ] A . fumigatus infection ; error bars represent mean ± SD of the survival of biological triplicates of 20 flies each ; wild - type flies are used as a control for the dose of 250 conidia . ( F - H ) dose response of MyD88 and wild - type flies after gliotoxin ( F ) , fumagillin ( G ) , and helvolic acid ( H ) injection at the indicated concentrations ( 20 flies per condition ) .

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 B-PANEL_START 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

Figure 2(A) survival of MyD88 flies injected with ∆pptA and wild-type (Af) A. fumigatus conidia (error bars represent mean ± SD of the survival of biological triplicates of 20 flies each).(B) fungal loads of single flies after the injection of 500 ∆pptA conidia.(E) dose response of MyD88 flies after ∆gliP (gliotoxin) mutant or wild-type [∆akuB] A. fumigatus infection; error bars represent mean ± SD of the survival of biological triplicates of 20 flies each; wild-type flies are used as a control for the dose of 250 conidia. (F-H) dose response of MyD88 and wild-type flies after gliotoxin (F), fumagillin (G), and helvolic acid (H) injection at the indicated concentrations (20 flies per condition).


Figure 4 ( A ) survival of MyD88 or wild - type flies to 250 injected ∆ aspf1 ( restrictocin mutant ) or wild - type [ ∆ akuB ] A . fumigatus conidia ( 20 flies per condition ) ; MyD88 : ∆ aspf1 vs . ∆ akuB ( * * * p = 0 . 0007 ) . ( B ) survival of MyD88 flies after the injection of different concentrations of restrictocin ( R ) ( 20 flies per condition ) . ( C - D ) survival of antibiotics - treated ( C ) and axenic ( D ) MyD88 mutant flies after restrictocin injection ( 20 flies per condition ) . ( E - F ) ribosomal RNA cleavage measurement after restrictocin or PBS injection in wild - type ( E ) and MyD88 ( F ) flies ; the arrowheads show the position of the 28S RNA - derived α - sarcin fragments whereas arrows on the right show its electrophoretic band position . ( H ) SDS - PAGE analysis of 35S - labelled translated proteins produced in a rabbit reticulocytes lysate from a m7G - capped reporter RNA containing the 5 ' UTR of β - globin followed by the Renilla luciferase coding sequence ( arrow ) , in the presence of increasing concentrations ( 0 . 125 to 25 nM ) of restrictocin .

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 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 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 4(A) survival of MyD88 or wild-type flies to 250 injected ∆aspf1 (restrictocin mutant) or wild-type [∆akuB] A. fumigatus conidia (20 flies per condition); MyD88: ∆aspf1 vs. ∆akuB (***p=0.0007). (B) survival of MyD88 flies after the injection of different concentrations of restrictocin (R) (20 flies per condition ). (C-D) survival of antibiotics-treated (C) and axenic (D) MyD88 mutant flies after restrictocin injection (20 flies per condition).(E-F) ribosomal RNA cleavage measurement after restrictocin or PBS injection in wild-type (E) and MyD88 (F) flies; the arrowheads show the position of the 28S RNA-derived α-sarcin fragments whereas arrows on the right show its electrophoretic band position.(H) SDS-PAGE analysis of 35S-labelled translated proteins produced in a rabbit reticulocytes lysate from a m7G-capped reporter RNA containing the 5'UTR of β-globin followed by the Renilla luciferase coding sequence (arrow), in the presence of increasing concentrations (0.125 to 25 nM) of restrictocin.


Figure 5 ( A - B ) survival ( A ) and fungal load ( B ) of Bom ∆ 55C ( 55C ) deficient flies compared to wild - type and MyD88 after injection of 250 conidia ( error bars represent mean ± SD of the survival of biological triplicates of 20 flies each ) ; * * * * p < 0 . 0001 . ( B ) : the fungal burden does not increase in Bom ∆ 55C - deficient flies ; wt 0h vs . 48h , * * p = 0 . 001 ; 55C 0h vs . 48h , * * p = 0 . 007 ; pooled data of n = 3 experiments , biological replicates . ( C - D ) rescue of the sensitivity of Bom ∆ 55C flies to verruculogen ( C ) or to restrictocin ( D ) by the transgenic expression of individual 55C locus genes ( caption in D also applies to ( C ) ) . 55C flies vs . BomS1 , * p = 0 . 0495 , vs . BomS6 * p = 0 . 011 for verruculogen assay ( C ) ; 55C flies vs . BomBc1 or BomS3 , * * * * p < 0 . 0001 , 55C flies vs . BomS6 , * * p = 0 . 0028 for restrictocin assay ( D ) ( 20 flies per condition ) . ( E - F ) expression levels of BomBc1 , and BomS3 measured by RT - digital PCR 48 hours after challenge ; BomBc1 PBST vs . Af , * p = 0 . 015 , PBST vs . restrictocin ( R ) , * p = 0 . 015 ; BomS3 : PBST vs . Af , * p = 0 . 02 , PBST vs . R , * p = 0 . 03 ( pooled data of n = 3 experiments , biological replicates ) .

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 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 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

Figure 5(A-B) survival (A) and fungal load (B) of Bom∆55C (55C) deficient flies compared to wild-type and MyD88 after injection of 250 conidia (error bars represent mean ± SD of the survival of biological triplicates of 20 flies each); ****p < 0.0001. (B): the fungal burden does not increase in Bom∆55C-deficient flies; wt 0h vs. 48h, **p=0.001; 55C 0h vs. 48h, **p=0.007; pooled data of n=3 experiments, biological replicates.(C-D) rescue of the sensitivity of Bom∆55C flies to verruculogen (C) or to restrictocin (D) by the transgenic expression of individual 55C locus genes (caption in D also applies to (C)). 55C flies vs. BomS1, *p=0.0495, vs. BomS6 *p=0.011 for verruculogen assay (C); 55C flies vs. BomBc1 or BomS3, ****p<0.0001, 55C flies vs. BomS6, **p=0.0028 for restrictocin assay (D) (20 flies per condition).(E-F) expression levels of BomBc1, and BomS3 measured by RT-digital PCR 48 hours after challenge; BomBc1 PBST vs. Af, *p=0.015, PBST vs. restrictocin (R), *p=0.015; BomS3: PBST vs. Af, *p=0.02, PBST vs. R, *p=0.03 (pooled data of n=3 experiments, biological replicates).


Figure 6 ( A - B ) tremor rate ( A ) and survival ( B ) of flies ( 20 flies per condition ) overexpressing Tl [ 10B ] in neurons compared to wild - type after injection of verruculogen . ( A ) Each dot corresponds to the tremor rate measured in a batch of 20 flies ; tremor rate wt vs . elav > UAS - Toll10B , * * p = 0 . 002 . ( C - D ) tremor rate ( C ) and survival ( D ) of flies ( 20 flies per condition ) overexpressing Tl [ 10B ] in glia compared to wild - type after injection of verruculogen . ( C ) Each dot corresponds to the tremor rate measured in a batch of 20 flies ; tremor rate wt vs . repo > UAS - Toll10B , * * p = 0 . 002 . ( D ) Survival wt V vs . repo > UAS - Toll10B V , * * p = 0 . 005 . ( E - G ) recovery time from tremor ( E , F ) and survival ( G ) of single flies overexpressing BomS6 ubiquitously ( E , G ) or in neurons ( F - G ) ( biological replicates ) compared to wild - type after injection of verruculogen ; in ( G ) the inset represents the survival of vehicle control groups . ( H - I ) recovery time from tremor ( H ) and survival ( I ) of single flies overexpressing BomS6 in glia ( pooled data from n = 3 experiments , biological replicates ) compared to wild - type after injection of verruculogen ; in ( I ) the inset represents the survival of vehicle control groups . Recovery time ( H ) repo > mCherry vs . repo > BomS6 , p = 0 . 058 , survival ( I ) repo > mCherry V vs . repo > BomS6 V , * p = 0 . 016 . ( J - M ) expression of BomS4 ( J ) and BomS6 ( K ) in head after verruculogen powder challenge , and BomS4 ( L ) and BomS6 ( M ) in head after A . fumigatus ( Af ) , restrictocin or M . luteus injection ( pooled data from n = 3 experiments , biological replicates ) .

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 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 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 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

Figure 6(A-B) tremor rate (A) and survival (B) of flies (20 flies per condition) overexpressing Tl[10B] in neurons compared to wild-type after injection of verruculogen. (A) Each dot corresponds to the tremor rate measured in a batch of 20 flies; tremor rate wt vs. elav> UAS-Toll10B, **p=0.002. (C-D) tremor rate (C) and survival (D) of flies (20 flies per condition) overexpressing Tl[10B] in glia compared to wild-type after injection of verruculogen. (C) Each dot corresponds to the tremor rate measured in a batch of 20 flies; tremor rate wt vs. repo> UAS-Toll10B, **p=0.002. (D) Survival wt V vs. repo> UAS-Toll10B V, **p=0.005.(E-G) recovery time from tremor (E,F) and survival (G) of single flies overexpressing BomS6 ubiquitously (E,G) or in neurons (F-G) (biological replicates) compared to wild-type after injection of verruculogen; in (G) the inset represents the survival of vehicle control groups.(H-I) recovery time from tremor (H) and survival (I) of single flies overexpressing BomS6 in glia (pooled data from n=3 experiments, biological replicates) compared to wild-type after injection of verruculogen; in (I) the inset represents the survival of vehicle control groups. Recovery time (H) repo>mCherry vs. repo>BomS6, p=0.058, survival (I) repo>mCherry V vs. repo>BomS6 V, *p=0.016.(J-M) expression of BomS4 (J) and BomS6 (K) in head after verruculogen powder challenge, and BomS4 (L) and BomS6 (M) in head after A. fumigatus (Af), restrictocin or M. luteus injection (pooled data from n=3 experiments, biological replicates).


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 ≥ 25 cilia per experimental condition ) . Statistical significance according to Kruskal - Wallis followed by Dunn ' s post - hoc test ( * * 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 O O 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 ≥ 25 cilia per experimental condition). Statistical significance according to Kruskal-Wallis followed by Dunn's post-hoc test (** P = 0.0046).


Figure 1Clustering of metabolome data in patients and controls ; PLS - DA plots ; VIP score plots of top 15 metabolites ; volcano plots of all metabolites in blood of IOSCA ( A ) Data information Colours in VIP score and volcano plots indicate the same most relevant and / or significantly changed metabolites among all patients groups . C3 , component 3 CDCA , chenodeoxycholic acidClustering of metabolome data in patients and controls ; PLS - DA plots ; VIP score plots of top 15 metabolites ; volcano plots of all metabolites in blood of MIRAS ( B ) Data information : a Significantly changed metabolites outside the FDR cut - off . b Metabolites not significantly changed between patients and controls . Colours in VIP score and volcano plots indicate the same most relevant and / or significantly changed metabolites among all patients groups . C3 , component 3 CDCA , chenodeoxycholic acidClustering of metabolome data in patients and controls ; PLS - DA plots ; VIP score plots of top 15 metabolites ; volcano plots of all metabolites in blood of PEO ( C ) Data information : Colours in VIP score and volcano plots indicate the same most relevant and / or significantly changed metabolites among all patients groups . C3 , component 3 ; CDCA , chenodeoxycholic acid ; GABA , γ - aminobutyric acid ; OH - Kyn , 3 - hydroxy - DL - kynurenineA - D Clustering of metabolome data in patients and controls ; PLS - DA plots ; VIP score plots of top 15 metabolites ; volcano plots of all metabolites in blood of MELAS / MIDD ( D ) . Data information : Colours in VIP score and volcano plots indicate the same most relevant and / or significantly changed metabolites among all patients groups . C3 , component 3 HIAA , 5 - Hydroxyindole - 3 - acetic acid ; SDMA , symmetric dimethylarginine ; TCA , taurocholic acid .

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 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 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

Figure 1Clustering of metabolome data in patients and controls; PLS-DA plots; VIP score plots of top 15 metabolites; volcano plots of all metabolites in blood of IOSCA (A) Data information Colours in VIP score and volcano plots indicate the same most relevant and/or significantly changed metabolites among all patients groups. C3, component 3 CDCA, chenodeoxycholic acidClustering of metabolome data in patients and controls; PLS-DA plots; VIP score plots of top 15 metabolites; volcano plots of all metabolites in blood of MIRAS (B) Data information: a Significantly changed metabolites outside the FDR cut-off. b Metabolites not significantly changed between patients and controls. Colours in VIP score and volcano plots indicate the same most relevant and/or significantly changed metabolites among all patients groups. C3, component 3 CDCA, chenodeoxycholic acidClustering of metabolome data in patients and controls; PLS-DA plots; VIP score plots of top 15 metabolites; volcano plots of all metabolites in blood of PEO (C) Data information: Colours in VIP score and volcano plots indicate the same most relevant and/or significantly changed metabolites among all patients groups. C3, component 3; CDCA, chenodeoxycholic acid; GABA, γ-aminobutyric acid; OH-Kyn, 3-hydroxy-DL-kynurenineA - D Clustering of metabolome data in patients and controls; PLS-DA plots; VIP score plots of top 15 metabolites; volcano plots of all metabolites in blood of MELAS/MIDD (D). Data information: Colours in VIP score and volcano plots indicate the same most relevant and/or significantly changed metabolites among all patients groups. C3, component 3 HIAA, 5-Hydroxyindole-3-acetic acid; SDMA, symmetric dimethylarginine; TCA, taurocholic acid.