B , The lysates ( T ) were sonicated and centrifuged at 100 , 000 g for one hour to separate pellet ( P ) and supernatant ( S ) . The distribution of CPY and Apg8p was examined by immunoblotting with each specific antibody , respectively .

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B, The lysates (T) were sonicated and centrifuged at 100,000 g for one hour to separate pellet (P) and supernatant (S). The distribution of CPY and Apg8p was examined by immunoblotting with each specific antibody, respectively.


C , The sonicated lysates were centrifuged at 100 , 000 g for one hour to generate pellet fraction . The pellet fraction was treated with 2 % Triton X - 100 ( TX - 100 ) for 30 min on ice . The sample was centrifuged at 100 , 000 g for one hour and separated to supernatant ( S ) and pellet ( P ) fractions . The distribution of Apg8p was examined by immunoblotting with the anti - Apg8p antibody .

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C, The sonicated lysates were centrifuged at 100,000 g for one hour to generate pellet fraction. The pellet fraction was treated with 2% Triton X-100 (TX-100) for 30 min on ice. The sample was centrifuged at 100,000 g for one hour and separated to supernatant (S) and pellet (P) fractions. The distribution of Apg8p was examined by immunoblotting with the anti-Apg8p antibody.


Immunofluorescent staining of Δapg8 cells expressing 3 × HA - Apg8p . A , The Δapg8 cells harboring 3 × HA - tagged APG8 plasmid , TK114 cells , were grown until logarithmic phase . The cells were fixed by formaldehyde and were treated with Zymolyase 100T to generate spheroplasts . The spheroplasts were permeabilized by 0 . 5 % Triton X - 100 , and incubated with the anti - HA antibody , 16B12 , followed with the FITC - conjugated anti - mouse IgG . Left , Fluorescence image of 3 × HA - Apg8p ; right , Nomarski image of the cells . Arrows show punctate signals that are a little larger than tiny dot signals . Bars , 10 μm . B , Immunofluorescent staining of YW5 - 1B cells before ( 0 h ) and after ( 3 h ) shift to starvation ( negative control ) . Left , Fluorescence image ; right , Nomarski image . Bars , 10 μm . C , The cells were shifted to starvation for 0 , 0 . 5 , and 3 h . Upper panels , Fluorescence images of 3 × HA - Apg8p ; lower panels , Nomarski images of the cell . Bars , 5 μm .

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Immunofluorescent staining of Δapg8 cells expressing 3 × HA-Apg8p. A, The Δapg8 cells harboring 3 × HA-tagged APG8 plasmid, TK114 cells, were grown until logarithmic phase. The cells were fixed by formaldehyde and were treated with Zymolyase 100T to generate spheroplasts. The spheroplasts were permeabilized by 0.5% Triton X-100, and incubated with the anti-HA antibody, 16B12, followed with the FITC-conjugated anti-mouse IgG. Left, Fluorescence image of 3 × HA-Apg8p; right, Nomarski image of the cells. Arrows show punctate signals that are a little larger than tiny dot signals. Bars, 10 μm. B, Immunofluorescent staining of YW5-1B cells before (0 h) and after (3 h) shift to starvation (negative control). Left, Fluorescence image; right, Nomarski image. Bars, 10 μm. C, The cells were shifted to starvation for 0, 0.5, and 3 h. Upper panels, Fluorescence images of 3 × HA-Apg8p; lower panels, Nomarski images of the cell. Bars, 5 μm.


Change of Apg8p localization in Δpep4 cells after shift to starvation . The Δapg8Δpep4 cells harboring 3 × HA - tagged APG8 plasmid , TK116 cells , were shifted to starvation for 0 , 0 . 5 , 1 , 3 , and 6 h . Immunofluorescence microscopy was performed as described in Fig . 5 . A - E , FITC staining of 3 × HA - Apg8p . F - J , Nomarski images of the cells . Arrows show an autophagic body . Bars , 5 μm .

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Change of Apg8p localization in Δpep4 cells after shift to starvation. The Δapg8Δpep4 cells harboring 3 × HA-tagged APG8 plasmid, TK116 cells, were shifted to starvation for 0, 0.5, 1, 3, and 6 h. Immunofluorescence microscopy was performed as described in Fig. 5. A-E, FITC staining of 3 × HA-Apg8p. F-J, Nomarski images of the cells. Arrows show an autophagic body. Bars, 5 μm.


Immunostaining image of a cell containing autophagic bodies in the vacuole . The Δapg8Δpep4 cells expressing 3 × HA - Apg8p , TK116 cells , were shifted to SD ( − N ) medium for two hours . Cells were immunolabeled with anti - N mAb , 16B12 , followed by 10 - nm gold - conjugated goat anti - mouse IgG . AB , vacuole ; N , nucleus ; V , vacuole .

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Immunostaining image of a cell containing autophagic bodies in the vacuole. The Δapg8Δpep4 cells expressing 3 × HA-Apg8p, TK116 cells, were shifted to SD(−N) medium for two hours. Cells were immunolabeled with anti-N mAb, 16B12, followed by 10-nm gold-conjugated goat anti-mouse IgG. AB, vacuole; N, nucleus; V, vacuole.


Immuno - EM of autophagosomes . A and B , Mature autophagosome . C and D , Premature autophagosome . Arrows show expanded regions of the intramembrane space of the premature autophagosomes . TK116 cells were incubated in SD ( − N ) medium for one hour ( D ) , two hours ( B and C ) , or three hours ( A ) . The localization of 3 × HA - Apg8p was detected with anti - HA antibody , followed by the incubation with 5 - nm gold - ( A and D ) or 10 - nm gold - ( B and C ) conjugated goat anti - mouse IgG . AP , autophagosome ; N , nucleus ; V , vacuole .

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Immuno-EM of autophagosomes. A and B, Mature autophagosome. C and D, Premature autophagosome. Arrows show expanded regions of the intramembrane space of the premature autophagosomes. TK116 cells were incubated in SD(−N) medium for one hour (D), two hours (B and C), or three hours (A). The localization of 3 × HA-Apg8p was detected with anti-HA antibody, followed by the incubation with 5-nm gold- (A and D) or 10-nm gold- (B and C) conjugated goat anti-mouse IgG. AP, autophagosome; N, nucleus; V, vacuole.


Possible intermediate structures of autophagosome . A , A membrane sac under construction . Arrows show gold particles . B and C , Isolation membranes . B , Isolation membrane is detected along the arrows . C , The small arrow shows a semicircular isolation membrane and a large arrow marks its open area . D , Apg8p - residing structures gathered in the area close to the vacuole . TK116 cells were incubated in SD ( − N ) medium for one hour ( A and C ) , or two hours ( B and D ) . The localization of 3 × HA - Apg8p was detected with anti - HA antibody , followed by the incubation with 5 - nm gold - ( A ) or 10 - nm gold - ( B - D ) conjugated goat anti - mouse IgG . V , vacuole .

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

Possible intermediate structures of autophagosome. A, A membrane sac under construction. Arrows show gold particles. B and C, Isolation membranes. B, Isolation membrane is detected along the arrows. C, The small arrow shows a semicircular isolation membrane and a large arrow marks its open area. D, Apg8p-residing structures gathered in the area close to the vacuole. TK116 cells were incubated in SD(−N) medium for one hour (A and C), or two hours (B and D). The localization of 3 × HA-Apg8p was detected with anti-HA antibody, followed by the incubation with 5-nm gold- (A) or 10-nm gold- (B-D) conjugated goat anti-mouse IgG. V, vacuole.


Fine morphology of Δypt7 and Δapg8 cells and proteinase - K accession to proAPI in these mutants . A , EM image of Δypt7 cell starved in SD ( − N ) medium for four hours . Autophagosomes ( AP , arrows ) were accumulated . B , EM image of the starved Δapg8 cell . C - E , The representatives of the membrane structures detected in the starved Δapg8 cells . C , Autophagosome - like structure indistinguishable from autophagosome . D and E , Aberrant multivesicular structures . N , nucleus ; V , vacuole . F , Proteinase K - sensitivity of proAPI . Cell lysates were prepared from Δypt7 and Δapg8 cells starved in SD ( − N ) medium for 4 . 5 h . The lysates were treated with 100 μg / ml proteinase K in the presence or absence of Triton X - 100 . Asterisk shows mature API and digestion product of proAPI by proteinase K .

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Fine morphology of Δypt7 and Δapg8 cells and proteinase-K accession to proAPI in these mutants. A, EM image of Δypt7 cell starved in SD(−N) medium for four hours. Autophagosomes (AP, arrows) were accumulated. B, EM image of the starved Δapg8 cell. C-E, The representatives of the membrane structures detected in the starved Δapg8 cells. C, Autophagosome-like structure indistinguishable from autophagosome. D and E, Aberrant multivesicular structures. N, nucleus; V, vacuole. F, Proteinase K-sensitivity of proAPI. Cell lysates were prepared from Δypt7 and Δapg8 cells starved in SD(−N) medium for 4.5 h. The lysates were treated with 100 μg/ml proteinase K in the presence or absence of Triton X-100. Asterisk shows mature API and digestion product of proAPI by proteinase K.


B The Na + and K + contents and Na + / K + ratio in roots and shoots of three groups of 369 accessions after treatment with 150 mM NaCl for 1 d .

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B The Na+ and K+ contents and Na+/K+ ratio in roots and shoots of three groups of 369 accessions after treatment with 150 mM NaCl for 1 d.


C Linear regression of root Na + / K + ratio under salt stress ( 150 mM NaCl for 1 d ) and fruit weight in 195 accessions from three groups . R2 , coefficient of determination . n is the accession number of the three groups .

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C Linear regression of root Na+/K+ ratio under salt stress (150 mM NaCl for 1 d) and fruit weight in 195 accessions from three groups. R2, coefficient of determination. n is the accession number of the three groups.


D The Na + / K + ratios in roots of TS - 21 , TS - 422 , TS - 577 , and TS - 670 after treatment with 150 mM NaCl for the indicated time ( n = 3 biological repeats ) .

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D The Na+/K+ ratios in roots of TS-21, TS-422, TS-577, and TS-670 after treatment with 150 mM NaCl for the indicated time (n = 3 biological repeats).


E , F Salt - resistance assay of two PIM accessions ( TS - 21 and TS - 422 ) and two BIG ( TS - 577 and TS - 670 ) . 20 - day - old seedlings grown in soil were treated with 200 mM NaCl for 3 weeks ( E ) . The survival rates were obtained from at least 12 plants in three repeated experiments ( F ) .

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E, F Salt-resistance assay of two PIM accessions (TS-21 and TS-422) and two BIG (TS-577 and TS-670). 20-day-old seedlings grown in soil were treated with 200 mM NaCl for 3 weeks (E). The survival rates were obtained from at least 12 plants in three repeated experiments (F).


A Manhattan plot displaying the GWAS results of Na + / K + ratio in root . The red - dashed line indicates the Bonferroni - adjusted significance threshold ( P = 1 . 0 × 10 - 7 ) . Red arrow indicates the significant SNP signal of Na + / K + ratio associated with SlHAK20 .

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A Manhattan plot displaying the GWAS results of Na+/K+ ratio in root. The red-dashed line indicates the Bonferroni-adjusted significance threshold (P = 1.0 × 10-7). Red arrow indicates the significant SNP signal of Na+/K+ ratio associated with SlHAK20.


B The nucleotide diversity ratios between PIM and CER , and between CER and BIG on chromosome 4 . The black - dashed horizontal lines indicate top 10 % threshold for entire chromosome 4 ( 1 . 82 πPIM / πCER for domestication and 4 . 27 πCER / πBIG for improvement ) . The red arrows indicate the position of SlHAK20 in the sweeps . π , nucleotide diversity .

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B The nucleotide diversity ratios between PIM and CER, and between CER and BIG on chromosome 4. The black-dashed horizontal lines indicate top 10% threshold for entire chromosome 4 (1.82 πPIM/πCER for domestication and 4.27 πCER/πBIG for improvement). The red arrows indicate the position of SlHAK20 in the sweeps. π, nucleotide diversity.


C SlHAK20 - based association mapping and pairwise LD analysis . Dots represent SNPs . Indel48 is highlighted in red . The indel - 2381 , indel - 1818 in the promoter region and five nonsynonymous variants are marked in blue . These eight variants are related to the pairwise LD diagram with dashed lines .

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C SlHAK20-based association mapping and pairwise LD analysis. Dots represent SNPs. Indel48 is highlighted in red. The indel-2381, indel-1818 in the promoter region and five nonsynonymous variants are marked in blue. These eight variants are related to the pairwise LD diagram with dashed lines.


D Haplotypes of SlHAK20 among tomato natural variations . The distribution of Na + / K + ratio in each haplotype group is exhibited by a box plot . The n indicates the number of accessions belonging to each haplotype . In the box plots , the middle line indicates the median , the box indicates the range of the percentiles of the total data using Turkey method , the whiskers indicate the interquartile range and the outer dots are outliers . Significant difference was determined by Student ' s t - test .

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D Haplotypes of SlHAK20 among tomato natural variations. The distribution of Na+/K+ ratio in each haplotype group is exhibited by a box plot. The n indicates the number of accessions belonging to each haplotype. In the box plots, the middle line indicates the median, the box indicates the range of the percentiles of the total data using Turkey method, the whiskers indicate the interquartile range and the outer dots are outliers. Significant difference was determined by Student's t-test.


E Allele distribution of the SlHAK20 locus at position in PIM , CER , and BIG groups . The n indicates the accession number .

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E Allele distribution of the SlHAK20 locus at position in PIM, CER, and BIG groups. The n indicates the accession number.


The expression pattern of SlHAK20 in tomato . The GUS activity was examined in seedling ( left ) and root ( right ) of 21 - days - old SlHAK20TS - 21pro : GUS transgenic tomato ( B ) . Bars , 2 cm .

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The expression pattern of SlHAK20 in tomato. The GUS activity was examined in seedling (left) and root (right) of 21-days-old SlHAK20TS-21pro:GUS transgenic tomato (B). Bars, 2 cm.


The expression pattern of SlHAK20 in tomato . Cross - sections of hypocotyl ( C ) of SlHAK20TS - 21pro : GUS transgenic plants . The scale bar is 400 μm in ( C )

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The expression pattern of SlHAK20 in tomato. Cross-sections of hypocotyl (C) of SlHAK20TS-21pro:GUS transgenic plants. The scale bar is 400 μm in (C)


The expression pattern of SlHAK20 in tomato . Cross - sections of root he scale bar is 200 μm in ( D ) .

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The expression pattern of SlHAK20 in tomato. Cross-sections of root he scale bar is 200 μm in (D).


E Subcellular localization of SlHAK20 - YFP in tomato . The SlHAK20Hap1 - YFP ( left ) and SlHAK20Hap2 - YFP ( right ) were localized in the plasma membrane . YFP , yellow florescence protein . FM4 - 64 , a lipophilic styryl compound as a red fluorescent marker of plasma membrane . Bar , 20 μm .

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E Subcellular localization of SlHAK20-YFP in tomato. The SlHAK20Hap1-YFP (left) and SlHAK20Hap2-YFP (right) were localized in the plasma membrane. YFP, yellow florescence protein. FM4-64, a lipophilic styryl compound as a red fluorescent marker of plasma membrane. Bar, 20 μm.


F SlHAK20 complements the K + uptake - defective yeast mutant assayed using a dose - response method . The K + uptake - defective yeast mutant R5421 transformed with SlHAK20Hap1 , SlHAK20Hap2 , and vector ( p416 - GPD ) were cultured in AP medium containing indicated concentrations of K + for 24 h . The data points represent three replicates .

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F SlHAK20 complements the K+ uptake-defective yeast mutant assayed using a dose-response method. The K+ uptake-defective yeast mutant R5421 transformed with SlHAK20Hap1, SlHAK20Hap2, and vector (p416-GPD) were cultured in AP medium containing indicated concentrations of K+ for 24 h. The data points represent three replicates.


G Na + uptake analysis of yeast cells expressing SlHAK20 . Shown are the Na + concentrations in the medium over the time of culture of the ANT3 yeast cells transformed with SlHAK20Hap1 , SlHAK20Hap2 , or empty vector . The initial NaCl concentration in the medium was 60 μM . The data are shown as the means ± SD ( n = 4 ) .

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G Na+ uptake analysis of yeast cells expressing SlHAK20. Shown are the Na+ concentrations in the medium over the time of culture of the ANT3 yeast cells transformed with SlHAK20Hap1, SlHAK20Hap2, or empty vector. The initial NaCl concentration in the medium was 60 μM. The data are shown as the means ± SD (n = 4).


H Na + uptake kinetic analysis of SlHAK20 in yeast cells . The curves show concentration - dependent Na + inﬂux in yeast expressing SlHAK20Hap1 or SlHAK20Hap2 in the presence of 0 , 5 , 10 , 20 , 30 , 40 , 50 , 60 , 100 , 150 or 200 μM NaCl in the medium . The data are expressed as mean ± SD ( n = 3 ) .

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H Na+ uptake kinetic analysis of SlHAK20 in yeast cells. The curves show concentration- dependent Na+ inﬂux in yeast expressing SlHAK20Hap1 or SlHAK20Hap2 in the presence of 0, 5, 10, 20, 30, 40, 50, 60, 100, 150 or 200 μM NaCl in the medium. The data are expressed as mean ± SD (n = 3).


Na + / K + ratio contents in roots of TS - 21 wild type and slhak20 mutants . Data are shown as means ± SD ( n = 4 ) .

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Na+/K+ ratio contents in roots of TS-21 wild type and slhak20 mutants. Data are shown as means ± SD (n = 4).


Na + contents in roots of TS - 21 wild type and slhak20 mutants . Data are shown as means ± SD ( n = 4 ) .

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Na+ contents in roots of TS-21 wild type and slhak20 mutants. Data are shown as means ± SD (n = 4).


K + contents in roots of TS - 21 wild type and slhak20 mutants . Data are shown as means ± SD ( n = 4 ) .

K+ contents in roots of TS-21 wild type and slhak20 mutants. Data are shown as means ± SD (n = 4).


Na + contents in the xylem sap of TS - 21 and slhak20 mutants after 50 mM NaCl treatment for the indicated days . Data represent means ± SD ( n = 4 ) .

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Na+ contents in the xylem sap of TS-21 and slhak20 mutants after 50 mM NaCl treatment for the indicated days. Data represent means ± SD (n = 4).


K + contents in the xylem sap of TS - 21 and slhak20 mutants after 50 mM NaCl treatment for the indicated days . Data represent means ± SD ( n = 4 ) .

K+ contents in the xylem sap of TS-21 and slhak20 mutants after 50 mM NaCl treatment for the indicated days. Data represent means ± SD (n = 4).


G Shoot height of TS - 21 , slhak20 - 1 , and slhak20 - 2 . Plant height of 24 - day - old TS - 21 , slhak20 - 1 , and slhak20 - 2 were measured after growing under normal conditions for 14 days , followed by salt treatment for 14 days , and recovery for 10 days . Values are means ± SD ( n = 10 plants of each genotype ) .

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G Shoot height of TS-21, slhak20-1, and slhak20-2. Plant height of 24-day-old TS-21, slhak20-1, and slhak20-2 were measured after growing under normal conditions for 14 days, followed by salt treatment for 14 days, and recovery for 10 days. Values are means ± SD (n = 10 plants of each genotype).


H Salt resistance of TS - 21 wild type and slhak20 mutants . TS - 21 , slhak20 - 1 , and slhak20 - 2 grown under normal growth conditions for 24 days , followed by a treatment with 200 mM NaCl for 14 days , and then recovered for 10 days . Control plants grown under normal growth conditions for 38 days . Scale bars , 5 cm .

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H Salt resistance of TS-21 wild type and slhak20 mutants. TS-21, slhak20-1, and slhak20-2 grown under normal growth conditions for 24 days, followed by a treatment with 200 mM NaCl for 14 days, and then recovered for 10 days. Control plants grown under normal growth conditions for 38 days. Scale bars, 5 cm.


Salt - tolerance assay of TS - 670 and slhak20 mutant plants . TS - 670 , slhak20 - 3 , and slhak20 - 4 grown under normal growth conditions for 23 days , and then treated with 150 mM NaCl for 5 days . Control plants grown under normal growth conditions for 28 days .

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Salt-tolerance assay of TS-670 and slhak20 mutant plants. TS-670, slhak20-3, and slhak20-4 grown under normal growth conditions for 23 days, and then treated with 150 mM NaCl for 5 days. Control plants grown under normal growth conditions for 28 days.


Plant height and fresh weight of TS - 670 , slhak20 - 3 , and slhak20 - 4 under normal and salt stress conditions . Shoot height f 23 - day - old TS - 670 , slhak20 - 3 , and slhak20 - 4 were quantified after growing under normal conditions for 5 days , or salt treatment with 150 mM NaCl for 5 days . Values are means ± SD ( n = 10 plants of each genotype ) .

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Plant height and fresh weight of TS-670, slhak20-3, and slhak20-4 under normal and salt stress conditions. Shoot height f 23-day-old TS-670, slhak20-3, and slhak20-4 were quantified after growing under normal conditions for 5 days, or salt treatment with 150 mM NaCl for 5 days. Values are means ± SD (n = 10 plants of each genotype).


Plant height and fresh weight of TS - 670 , slhak20 - 3 , and slhak20 - 4 under normal and salt

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Plant height and fresh weight of TS-670, slhak20-3, and slhak20-4 under normal and salt stress


Plant height and fresh weight of TS - 670 , slhak20 - 3 , and slhak20 - 4 under normal and salt stress conditions . resh weight f 23 - day - old TS - 670 , slhak20 - 3 , and slhak20 - 4 were quantified after growing under normal conditions for 5 days , or salt treatment with 150 mM NaCl for 5 days . Values are means ± SD ( n = 10 plants of each genotype ) .

Plant height and fresh weight of TS-670, slhak20-3, and slhak20-4 under normal and salt stress conditions. resh weight f 23-day-old TS-670, slhak20-3, and slhak20-4 were quantified after growing under normal conditions for 5 days, or salt treatment with 150 mM NaCl for 5 days. Values are means ± SD (n = 10 plants of each genotype).


Phenotype of NIP and oshak4 / 17 double mutant plants under normal and salt stress conditions . 20 - day - old seedlings of NIP and oshak4 / 17 mutants were treated with 100 mM NaCl for 8 days . The pictures show representative plants ere analyzed among these genotypes . ontrol plants were grown under normal growth conditions for 28 days

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Phenotype of NIP and oshak4/17 double mutant plants under normal and salt stress conditions. 20-day-old seedlings of NIP and oshak4/17 mutants were treated with 100 mM NaCl for 8 days. The pictures show representative plants ere analyzed among these genotypes. ontrol plants were grown under normal growth conditions for 28 days


Phenotype of NIP and oshak4 / 17 double mutant plants under normal and salt stress conditions . 20 - day - old seedlings of NIP and oshak4 / 17 mutants were treated with 100 mM NaCl for 8 days . The pictures show representative plants ere analyzed among these genotypes . ontrol plants were grown under normal growth conditions for 28

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Phenotype of NIP and oshak4 / 17 double mutant plants under normal and salt stress conditions . 20 - day - old seedlings of NIP and oshak4 / 17 mutants were treated with 100 mM NaCl for 8 days . The pictures show representative fresh weight of shoots were analyzed among these genotypes . Control plants were grown under normal growth conditions for 28 days and the fresh weights of shoots were measured . Values are means ± SD ( n = 12 plants of each genotype for fresh weight

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Phenotype of NIP and oshak4/17 double mutant plants under normal and salt stress conditions. 20-day-old seedlings of NIP and oshak4/17 mutants were treated with 100 mM NaCl for 8 days. The pictures show representative fresh weight of shoots were analyzed among these genotypes. Control plants were grown under normal growth conditions for 28 days and the fresh weights of shoots were measured. Values are means ± SD (n = 12 plants of each genotype for fresh weight


Phenotype of NIP and oshak4 / 17 double mutant plants under normal and salt stress conditions . 20 - day - old seedlings of NIP and oshak4 / 17 mutants were treated with 100 mM NaCl for 8 days . The pictures show representative fresh weight of shoots were analyzed among these genotypes . Control plants were grown under normal growth conditions for 28 days and the fresh weights of shoots were measured . Values are means ± SD ( n = 12 plants of each genotype for fresh

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


Phenotype of NIP and oshak4 / 17 double mutant plants under normal and salt stress conditions . 20 - day - old seedlings of NIP and oshak4 / 17 mutants were treated with 100 mM NaCl for 8 days . The pictures show representative survival rate were analyzed among these genotypes .

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Phenotype of NIP and oshak4/17 double mutant plants under normal and salt stress conditions. 20-day-old seedlings of NIP and oshak4/17 mutants were treated with 100 mM NaCl for 8 days. The pictures show representative survival rate were analyzed among these genotypes.


A The transcript levels of SlHAK20 in TS - 670 and three independent SlHAK20Hap1 - YFP transgenic lines grown under normal growth conditions .

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A The transcript levels of SlHAK20 in TS-670 and three independent SlHAK20Hap1-YFP transgenic lines grown under normal growth conditions.


B The protein levels of SlHAK20Hap1 - YFP in TS - 670 and transgenic plants . ACTIN was used as loading control .

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B The protein levels of SlHAK20Hap1-YFP in TS-670 and transgenic plants. ACTIN was used as loading control.


Phenotypes of transgenic lines in soil under salt stress . The survival rates were obtained from at least 8 plants in four repeated

Phenotypes of transgenic lines in soil under salt stress. The survival rates were obtained from at least 8 plants in four repeated experiments


38 - day - old plants grown in soil as control . 20 - day - old TS - 670 and Hap1OE - 1 to - 3 were treated with 175 mM NaCl for 3 weeks and recovered for 2

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38-day-old plants grown in soil as control. 20-day-old TS-670 and Hap1OE-1 to -3 were treated with 175 mM NaCl for 3 weeks and recovered for 2 weeks


The Na + in shoots and roots of TS - 670 and three independent SlHAK20Hap1 - YFP transgenic lines during salt stress . The 21 - day - old TS - 670 , Hap1OE - 1 , Hap1OE - 2 and Hap1OE - 3 plants grown in 0 . 25 × Hoagland which were then treated with 50 mM NaCl for additional 0 and 7 days .

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The Na+ in shoots and roots of TS-670 and three independent SlHAK20Hap1-YFP transgenic lines during salt stress. The 21-day-old TS-670, Hap1OE-1, Hap1OE-2 and Hap1OE-3 plants grown in 0.25× Hoagland which were then treated with 50 mM NaCl for additional 0 and 7 days.


The K + in shoots and roots of TS - 670 and three independent SlHAK20Hap1 - YFP transgenic lines during salt stress . The 21 - day - old TS - 670 , Hap1OE - 1 , Hap1OE - 2 and Hap1OE - 3 plants grown in 0 . 25 × Hoagland which were then treated with 50 mM NaCl for additional 0 and 7 days .

The K+ in shoots and roots of TS-670 and three independent SlHAK20Hap1-YFP transgenic lines during salt stress. The 21-day-old TS-670, Hap1OE-1, Hap1OE-2 and Hap1OE-3 plants grown in 0.25× Hoagland which were then treated with 50 mM NaCl for additional 0 and 7 days.


The Na + / K + ratio in shoots and roots of TS - 670 and three independent SlHAK20Hap1 - YFP transgenic lines during salt stress . The 21 - day - old TS - 670 , Hap1OE - 1 , Hap1OE - 2 and Hap1OE - 3 plants grown in 0 . 25 × Hoagland which were then treated with 50 mM NaCl for additional 0 and 7 days .

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

The Na+/K+ ratio in shoots and roots of TS-670 and three independent SlHAK20Hap1-YFP transgenic lines during salt stress. The 21-day-old TS-670, Hap1OE-1, Hap1OE-2 and Hap1OE-3 plants grown in 0.25× Hoagland which were then treated with 50 mM NaCl for additional 0 and 7 days.


A . Quantitative analysis of developmental expression of Lrig1 mRNA in rat hippocampus by real - time RT - PCR . The results are shown as mean SEM of n = 3 independent assays . The levels of Lrig1 mRNA were normalized using the expression of the housekeeping gene Tbp ( TATA binding protein ) . The insert shows the expression of Lrig1 in embryonic E17 . 5 rat hippocampus examined by RT - PCR . Control sample without reverse transcriptase ( - RT ) is also shown .

A. Quantitative analysis of developmental expression of Lrig1 mRNA in rat hippocampus by real-time RT-PCR. The results are shown as mean SEM of n=3 independent assays. The levels of Lrig1 mRNA were normalized using the expression of the housekeeping gene Tbp (TATA binding protein). The insert shows the expression of Lrig1 in embryonic E17.5 rat hippocampus examined by RT-PCR. Control sample without reverse transcriptase (-RT) is also shown.


B . Localization of Lrig1 ( red ) in coronal sections from P15 ratbrain by immunofluorescence . Anti - Lrig1ECD antibodies label dentate gyrus cells , CA1 - CA3 hippocampalneurons and pyramidal corticalneurons ( * ) . Scale bar , 400 m .

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B. Localization of Lrig1 (red) in coronal sections from P15 ratbrain by immunofluorescence. Anti-Lrig1ECD antibodies label dentate gyrus cells, CA1-CA3 hippocampalneurons and pyramidal corticalneurons (*). Scale bar, 400 m.


C - E . Confocal image of cortical ( C ) and hippocampal ( D - E ) pyramidal neurons stained with rabbit anti - Lrig1ECD antibody . Arrows indicate Lrig1 staining in proximal segments of apical dendrites of CA1 - CA3hippocampal and pyramidal cortical neurons ( layer V ) . Scale bars , 20 m .

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C-E. Confocal image of cortical (C) and hippocampal (D-E) pyramidal neurons stained with rabbit anti-Lrig1ECD antibody. Arrows indicate Lrig1 staining in proximal segments of apical dendrites of CA1-CA3hippocampal and pyramidal cortical neurons (layer V). Scale bars, 20 m.


F . Immunofluorescence staining of Lrig1 ( red ) with anti - Lrig1ECD antibody and the neuronal marker ßIII tubulin ( green ) in dissociated hippocampal cells cultured for 7 DIV . Yellow indicates neuronal expression of Lrig1 . Scale bar , 15 m .

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F. Immunofluorescence staining of Lrig1 (red) with anti-Lrig1ECD antibody and the neuronal marker ßIII tubulin (green) in dissociated hippocampal cells cultured for 7 DIV. Yellow indicates neuronal expression of Lrig1. Scale bar, 15 m.


G . Immunofluorescence staining of Lrig1 ( red ) with anti - Lrig1ECD antibody and the astrocytic marker S100 ( green ) in dissociated hippocampal cells cultured for 7 DIV . Scale bar , 20 m .

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G. Immunofluorescence staining of Lrig1 (red) with anti-Lrig1ECD antibody and the astrocytic marker S100 (green) in dissociated hippocampal cells cultured for 7 DIV. Scale bar, 20 m.


H . Localization of Lrig1 ( red ) with anti - Lrig1ECD antibody and the somatodendritic marker MAP - 2 ( blue ) by immunocytochemistry in dissociated rat hippocampalneurons after 12 DIV . Scale bar , 20 m .

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H. Localization of Lrig1 (red) with anti-Lrig1ECD antibody and the somatodendritic marker MAP-2 (blue) by immunocytochemistry in dissociated rat hippocampalneurons after 12 DIV. Scale bar, 20 m.


A . Representative images of mouse hippocampal neurons transfected with either GFP - expressing control or Lrig1 shRNA vector at 9 DIV and maintained for 3 additional days in vitro ( 9 + 3 DIV ) . Scale bar , 15 m . Boxed area represents a higher - magnification image showing the profuse proximal dendritic arborization of Lrig1 - shRNA transfected neurons . B . Sholl analysis of the dendritic arbor from hippocampal neurons transfected with either control or Lrig1 shRNA - GFP vector at 9 DIV and maintained for 3 additional days in vitro ( 9 + 3 DIV ) . Data are shown as mean SEM of n = 3 independent experiments . * p < 0 . 05 and * * p < 0 . 01 by two - way ANOVA followed by Bonferroni multiple comparisons test . C - G . Quantification of primary ( C ) , and secondary ( D ) dendrites as well as total dendritic branching ( E ) , terminal dendritic points ( F ) , and total dendritic length ( G ) of hippocampal neurons transfected with either control or Lrig1 shRNA - GFP vector . The results are shown as mean SEM of n = 3 independent experiments . * p < 0 . 05 by Students t test .

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A. Representative images of mouse hippocampal neurons transfected with either GFP-expressing control or Lrig1 shRNA vector at 9 DIV and maintained for 3 additional days in vitro (9+3 DIV). Scale bar, 15 m. Boxed area represents a higher-magnification image showing the profuse proximal dendritic arborization of Lrig1-shRNA transfected neurons.B. Sholl analysis of the dendritic arbor from hippocampal neurons transfected with either control or Lrig1 shRNA-GFP vector at 9 DIV and maintained for 3 additional days in vitro (9+3 DIV). Data are shown as mean SEM of n=3 independent experiments. *p<0.05 and **p<0.01 by two-way ANOVA followed by Bonferroni multiple comparisons test.C-G. Quantification of primary (C), and secondary (D) dendrites as well as total dendritic branching (E), terminal dendritic points (F), and total dendritic length (G) of hippocampal neurons transfected with either control or Lrig1 shRNA-GFP vector. The results are shown as mean SEM of n=3 independent experiments. *p<0.05 by Students t test.


H . Knockdown efficiency was analyzed by real - time RT - PCR in MN1 cells transfected with control or Lrig1 shRNA vectors . Transfected cells were enriched by puromycin treatment in order to increase the population of cells expressing control or Lrig1 shRNA constructs . Data are shown as individual values of a representative assay measured in triplicates . n = 2 independent experiments were performed .

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H. Knockdown efficiency was analyzed by real-time RT-PCR in MN1 cells transfected with control or Lrig1 shRNA vectors. Transfected cells were enriched by puromycin treatment in order to increase the population of cells expressing control or Lrig1 shRNA constructs. Data are shown as individual values of a representative assay measured in triplicates. n=2 independent experiments were performed.


I . Representative images of MAP - 2 immunostained hippocampalneurons obtained from wild - type and Lrig1 - deficient mice cultured for 7 days in vitro ( 7 DIV ) . Scale bar , 15 m . J . Sholl analysis of the dendritic arbor from MAP - 2 stained hippocampalneurons ( 7 DIV ) isolated from wild type and Lrig1 - deficient mice . Data are shown as mean SEM of n = 3 independent experiments . * p < 0 . 05 by two - way ANOVA followed by Bonferroni multiple comparisons test . K - M . Quantification of the number of primary dendrites ( K ) , secondary dendrites ( L ) and total dendritic branching ( M ) from MAP - 2 stained hippocampalneurons ( 7 DIV ) isolated from wild - type and Lrig1 - deficient mice . The results are shown as mean SEM of n = 3 independent experiments . * p < 0 . 05 by Students t test .

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I. Representative images of MAP-2 immunostained hippocampalneurons obtained from wild-type and Lrig1-deficient mice cultured for 7 days in vitro (7 DIV). Scale bar, 15 m.J. Sholl analysis of the dendritic arbor from MAP-2 stained hippocampalneurons (7 DIV) isolated from wild type and Lrig1-deficient mice. Data are shown as mean SEM of n=3 independent experiments. *p<0.05 by two-way ANOVA followed by Bonferroni multiple comparisons test.K-M. Quantification of the number of primary dendrites (K), secondary dendrites (L) and total dendritic branching (M) from MAP-2 stained hippocampalneurons (7 DIV) isolated from wild-type and Lrig1-deficient mice. The results are shown as mean SEM of n=3 independent experiments. *p<0.05 by Students t test.


A . Representative images and drawings of Golgi - stainedhippocampalCA1pyramidal neurons from 4 weeks - old wild type and Lrig1 - null littermate mice . Scale bar , 15 m . B . Quantification of the number of primary dendrites and branching of apical and basal dendritic arbors of hippocampalCA1pyramidal neurons from 4 - 5 weeks - old control ( wild type / heterozygous ) and Lrig1 - null littermate mice . The results are shown as mean SEM of independent determinations performed in n = 4 mice of each genotype . * p < 0 . 05 , * * p < 0 . 001 by Students t test . NS , not significant . C . Cummulative dendrite crossings of concentric circles of increasing radius ( 10 μm ring interval ) centering the reference point at the cell body . These values were obtained by Sholl analysis and represent the summatory of the dendritic crossings registered within the first 60 m closest to the soma . The results are shown as mean SEM of independent determinations performed in n = 4 mice of each genotype . * p < 0 . 05 by Students t test . D . Sholl analysis of apical and basal dendritic arbors of hippocampalCA1pyramidal neurons from 4 - 5 weeks - old control ( wild type / heterozygous ) and Lrig1 - null littermate mice . The results are shown as mean SEM . * p < 0 . 05 , * * p < 0 . 001 by two - way ANOVA followed by Bonferroni multiple comparisons test . Quantifications showed in B - D were performed in n = 60 neurons from 4 wild type / heterozygous mice and 4 Lrig1 - null littermate mice ( n = 4 ) .

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A. Representative images and drawings of Golgi-stainedhippocampalCA1pyramidal neurons from 4 weeks-old wild type and Lrig1-null littermate mice. Scale bar, 15 m.B. Quantification of the number of primary dendrites and branching of apical and basal dendritic arbors of hippocampalCA1pyramidal neurons from 4-5 weeks-old control (wild type/heterozygous) and Lrig1-null littermate mice. The results are shown as mean SEM of independent determinations performed in n=4 mice of each genotype. *p<0.05, **p<0.001 by Students t test. NS, not significant.C. Cummulative dendrite crossings of concentric circles of increasing radius (10 μm ring interval) centering the reference point at the cell body. These values were obtained by Sholl analysis and represent the summatory of the dendritic crossings registered within the first 60 m closest to the soma. The results are shown as mean SEM of independent determinations performed in n=4 mice of each genotype. *p<0.05 by Students t test.D. Sholl analysis of apical and basal dendritic arbors of hippocampalCA1pyramidal neurons from 4-5 weeks-old control (wild type/heterozygous) and Lrig1-null littermate mice. The results are shown as mean SEM. *p<0.05, **p<0.001 by two-way ANOVA followed by Bonferroni multiple comparisons test.Quantifications showed in B-D were performed in n=60 neurons from 4 wild type/heterozygous mice and 4 Lrig1-null littermate mice (n=4).


A . Schematic diagram of the social interaction device indicating the social and the empty chambers . B , C . Mice were simultaneously exposed to an empty container and a caged unfamiliar juvenile mouse ( social enclosure , stranger 1 ) . Lrig1 - mutant mice exhibit social interaction defects as determined by the time spent interacting ( sniffing ) with the stranger enclosure ( B ) and the percentage of total interaction time with stranger in the three - chamber social interaction test ( C ) . Dashed line in ( C ) represents chance - level performance ( i . e . 50 % ) when mice equally explore the social enclosure and the empty container . Data represent means ± SEM of independent determinations performed in n = 8 - 9 mice of each genotype , and the statistical significance between wt and knockout mice are * p < 0 . 05 by Students t test .

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A. Schematic diagram of the social interaction device indicating the social and the empty chambers.B, C. Mice were simultaneously exposed to an empty container and a caged unfamiliar juvenile mouse (social enclosure, stranger 1). Lrig1-mutant mice exhibit social interaction defects as determined by the time spent interacting (sniffing) with the stranger enclosure (B) and the percentage of total interaction time with stranger in the three-chamber social interaction test (C). Dashed line in (C) represents chance-level performance (i.e. 50%) when mice equally explore the social enclosure and the empty container. Data represent means ± SEM of independent determinations performed in n=8-9 mice of each genotype, and the statistical significance between wt and knockout mice are *p<0.05 by Students t test.


D . Schematic diagram of the social novelty test . In the test for social novelty , a second stranger ( stranger 2 ) mouse was introduced in the empty container . E . In the preference for social novelty task , wt mice showed a preference for social novelty , while mutant mice showed no significant preference for novel target ( stranger 2 ) . Mutants also spent significantly less time interacting with the novel mice compared to controls . Data represent means ± SEM of independent determinations performed in n = 8 - 9 mice of each genotype , and the statistical significance are as follow : * p < 0 . 05 and * * p < 0 . 005 by ANOVA followed by Student - Neuman Keuls multiple comparison test .

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D. Schematic diagram of the social novelty test. In the test for social novelty, a second stranger (stranger 2) mouse was introduced in the empty container.E. In the preference for social novelty task, wt mice showed a preference for social novelty, while mutant mice showed no significant preference for novel target (stranger 2). Mutants also spent significantly less time interacting with the novel mice compared to controls. Data represent means ± SEM of independent determinations performed in n=8-9 mice of each genotype, and the statistical significance are as follow: *p<0.05 and **p<0.005 by ANOVA followed by Student-Neuman Keuls multiple comparison test.


A . Schematic representation of Lrig1 mutants are shown on the left . Expression levels of these mutants were analysed in transfected cell extracts by immunoblotting with anti - Flag antibodies .

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A. Schematic representation of Lrig1 mutants are shown on the left. Expression levels of these mutants were analysed in transfected cell extracts by immunoblotting with anti-Flag antibodies.


B . Representative images of rathippocampal neurons transfected at DIV8 with empty vector , full length ( FL ) Flag - tagged Lrig1 , or Lrig1 mutant lacking the LRR domain ( LRR ) in combination with an enhanced green fluorescent protein ( GFP ) expression vector . After transfection at 9 DIV , neurons were cultured in the absence or in the presence of BDNF ( 30 ng / ml ) for 48 h . Then , hippocampal cultures at 11 DIV were fixed and stained with anti - Flag antibodies to control Lrig1 expression . Scale bar , 15 m .

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B. Representative images of rathippocampal neurons transfected at DIV8 with empty vector, full length (FL) Flag-tagged Lrig1, or Lrig1 mutant lacking the LRR domain (LRR) in combination with an enhanced green fluorescent protein (GFP) expression vector. After transfection at 9 DIV, neurons were cultured in the absence or in the presence of BDNF (30 ng/ml) for 48 h. Then, hippocampal cultures at 11 DIV were fixed and stained with anti-Flag antibodies to control Lrig1 expression. Scale bar, 15 m.


C , D . Quantification of the effects of Flag - Lrig1 constructs on BDNF - induced primary ( C ) and secondary ( D ) dendrite formation of hippocampal neurons treated as indicated in A . The results are shown as mean SEM of 3 independent experiments . * p < 0 . 05 , * * p < 0 . 01 by one - way ANOVA followed by Tukey ´ s multiple comparison test . NS , not significant .

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C, D. Quantification of the effects of Flag-Lrig1 constructs on BDNF-induced primary (C) and secondary (D) dendrite formation of hippocampal neurons treated as indicated in A. The results are shown as mean SEM of 3 independent experiments. *p<0.05, **p<0.01 by one-way ANOVA followed by Tukey´s multiple comparison test. NS, not significant.


E . Representative confocal images of dendritic shafts containing spines from hippocampal neurons transfected at 15 DIV with either control vector or Flag - Lrig1 construct together with GFP . After transfection , neurons were cultured in the absence or in the presence of BDNF ( 30 ng / ml ) for 48 h ( 15 + 3 DIV ) . Scale bar , 5 m . F . Quantification of the effect of Lrig1 overexpression on neurotrophin - induced spine density . The results are shown as mean SEM of n = 3 independent experiments . * p < 0 . 05 by one - way ANOVA followed by Student - Neuman Keuls multiple comparison test .

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E. Representative confocal images of dendritic shafts containing spines from hippocampal neurons transfected at 15 DIV with either control vector or Flag-Lrig1 construct together with GFP. After transfection, neurons were cultured in the absence or in the presence of BDNF (30 ng/ml) for 48 h (15+3 DIV). Scale bar, 5 m.F. Quantification of the effect of Lrig1 overexpression on neurotrophin-induced spine density. The results are shown as mean SEM of n=3 independent experiments. *p<0.05 by one-way ANOVA followed by Student-Neuman Keuls multiple comparison test.


A . Coexpression of Lrig1 ( red ) and TrkB ( green ) in primary rat hippocampal neurons . Boxed area represents a higher - magnification image showing a high colocalization between TrkB and Lrig1 in a pyramidal neuron . Yellow indicates regions of colocalization . Scale bar , 30 m . Data represent n = 3 independent experiments .

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

A. Coexpression of Lrig1 (red) and TrkB (green) in primary rat hippocampal neurons. Boxed area represents a higher-magnification image showing a high colocalization between TrkB and Lrig1 in a pyramidal neuron. Yellow indicates regions of colocalization. Scale bar, 30 m. Data represent n=3 independent experiments.


B . Quantitative analysis of Lrig1 mRNA expression by real - time RT - PCR . Rat hippocampal cultures ( 10 DIV ) were treated with BDNF ( 50 ng / ml ) during the indicated times . The levels were normalized using the expression of the housekeeping gene Tbp . The results are shown as mean SD of n = 3 independent experiments . * p < 0 . 05 vs . control group by one - way ANOVA followed by Dunnetts test .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Quantitative analysis of Lrig1 mRNA expression by real-time RT-PCR. Rat hippocampal cultures (10 DIV) were treated with BDNF (50 ng/ml) during the indicated times. The levels were normalized using the expression of the housekeeping gene Tbp. The results are shown as mean SD of n=3 independent experiments. *p<0.05 vs. control group by one-way ANOVA followed by Dunnetts test.


C , D . Coimmunoprecipitation between Flag - Lrig1 and HA - TrkB ( C ) or between Flag - Lrig3 and HA - TrkB ( D ) overexpressed in HEK293 cells . Cell extracts were analysed by immunoprecipitation with anti - Flag antibodies followed by immunoblot ( IB ) with antibodies against HA . Reprobing of the same blots with anti - Flag antibodies is shown below . The bottom panel shows HA expression in total lysates . Data represent n = 3 independent experiments .

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

C, D. Coimmunoprecipitation between Flag-Lrig1 and HA-TrkB (C) or between Flag-Lrig3 and HA-TrkB (D) overexpressed in HEK293 cells. Cell extracts were analysed by immunoprecipitation with anti-Flag antibodies followed by immunoblot (IB) with antibodies against HA. Reprobing of the same blots with anti-Flag antibodies is shown below. The bottom panel shows HA expression in total lysates. Data represent n=3 independent experiments.


E . Coimmunoprecipitation between HA - TrkB and Flag - Lrig1 or between HA - TrkB and HA - Lrig2 exogenously expressed in HEK293 cells . Cell extracts were analysed by IP with anti - pan - Trk antibodies followed by IB with antibodies against Flag or Lrig2 . Reprobing of the same blots with anti - TrkB antibodies is shown below . The bottom panels show Flag and Lrig2 expression in total lysates . Data represent n = 2 independent assays .

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

E. Coimmunoprecipitation between HA-TrkB and Flag-Lrig1 or between HA-TrkB and HA-Lrig2 exogenously expressed in HEK293 cells. Cell extracts were analysed by IP with anti-pan-Trk antibodies followed by IB with antibodies against Flag or Lrig2. Reprobing of the same blots with anti-TrkB antibodies is shown below. The bottom panels show Flag and Lrig2 expression in total lysates. Data represent n=2 independent assays.


F . In vivo interaction between Lrig1 and TrkB . Coimmunoprecipitation between Lrig1 and TrkB receptor endogenously expressed from P15 rat hippocampal tissue extracts . Samples were equally divided in two parts and then the analysis was done by immunoprecipitation with control ( anti - HA epitope tag antibody ) or anti - Lrig1 antibodies , followed by immunoblotting with anti - TrkB antibody . Reprobing of the same blot with anti - Lrig1 antibody is also shown . Expression of TrkB in one aliquot of the starting material ( indicated as lysate ) is included . Arrow indicates the band of TrkB coimmunoprecipitated with anti - Lrig1 antibody . Similar results were obtained in n = 3 independent assays .

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

F. In vivo interaction between Lrig1 and TrkB. Coimmunoprecipitation between Lrig1 and TrkB receptor endogenously expressed from P15 rat hippocampal tissue extracts. Samples were equally divided in two parts and then the analysis was done by immunoprecipitation with control (anti-HA epitope tag antibody) or anti-Lrig1 antibodies, followed by immunoblotting with anti-TrkB antibody. Reprobing of the same blot with anti-Lrig1 antibody is also shown. Expression of TrkB in one aliquot of the starting material (indicated as lysate) is included. Arrow indicates the band of TrkB coimmunoprecipitated with anti-Lrig1 antibody. Similar results were obtained in n=3 independent assays.


G . Ligand - dependent activation of TrkB ( p - TrkB ) was evaluated by transient transfection of HA - TrkB plasmid with either a control or a Flag - Lrig1 vector into HEK cells . After 36 h cells were serum - starved and stimulated with or without BDNF ( 30 ng / ml ) for 15 min . The level of TrkB activation ( p - TrkB ) was evaluated in total cell lysates by immunoblotting ( IB ) with a specific antibody that recognizes TrkB phosphorylated in tyrosine 705 ( pY705 ) . Reprobing of the same blot with anti - HA and anti - Flag antibodies is shown . Fold of p - TrkB change relative to total TrkB is indicated . Similar results were obtained in n = 3 independent assays .

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

G. Ligand-dependent activation of TrkB (p-TrkB) was evaluated by transient transfection of HA-TrkB plasmid with either a control or a Flag- Lrig1 vector into HEK cells. After 36 h cells were serum-starved and stimulated with or without BDNF (30 ng/ml) for 15 min. The level of TrkB activation (p-TrkB) was evaluated in total cell lysates by immunoblotting (IB) with a specific antibody that recognizes TrkB phosphorylated in tyrosine 705 (pY705). Reprobing of the same blot with anti-HA and anti-Flag antibodies is shown. Fold of p-TrkB change relative to total TrkB is indicated. Similar results were obtained in n=3 independent assays.


H . TrkB ubiquitination was evaluated by transient transfection of HA - TrkB plasmid with either a control or a Flag - Lrig1 vector into MN1 cells . After 36 h , cells were serum - starved , pre - treated with the cell - permeable proteasome inhibitor MG - 132 ( 20 M ) and stimulated with BDNF for 15 min . Total lysates were immunoprecipitated with anti - HA antibodies followed by immunoblot ( IB ) with antibodies against ubiquitin . Reprobing of the same blot with anti - HA antibodies is also shown . TrkB activation ( p - TrkB ) was evaluated in cell lysates . Reprobing of the same blot with anti - TrkB and anti - Flag antibodies is also shown . Fold of p - TrkB ( p - Y705 ) change relative to total TrkB is indicated . Data represent n = 3 independent assays .

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

H. TrkB ubiquitination was evaluated by transient transfection of HA-TrkB plasmid with either a control or a Flag-Lrig1 vector into MN1 cells. After 36 h, cells were serum-starved, pre-treated with the cell-permeable proteasome inhibitor MG-132 (20 M) and stimulated with BDNF for 15 min. Total lysates were immunoprecipitated with anti-HA antibodies followed by immunoblot (IB) with antibodies against ubiquitin. Reprobing of the same blot with anti-HA antibodies is also shown. TrkB activation (p-TrkB) was evaluated in cell lysates. Reprobing of the same blot with anti-TrkB and anti-Flag antibodies is also shown. Fold of p-TrkB (p-Y705) change relative to total TrkB is indicated. Data represent n=3 independent assays.


A . Immunoblot showing TrkB activation in hippocampal neurons cultured from Lrig1 heterozygous ( + / - ) and Lrig1 knockout ( - / - ) mice littermates treated in the absence or in the presence of BDNF ( 30 ng / ml ) for 30 minutes . Reprobing of the same blot with anti - ßIII - Tubulin is shown as loading control . B . Fold of TrkB activation relative to unstimulated control group ( phospho - TrkB at Tyr705 ) in hippocampal neurons cultured from Lrig1 ( + / + ; + / - ) and Lrig1 ( - / - ) mice treated in the absence or in the presence of BDNF ( 30 ng / ml ) for 30 minutes . Results are presented as mean SEM of n = 4 independent experiments . ( * p < 0 . 05 by Students t test ) .

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

A. Immunoblot showing TrkB activation in hippocampal neurons cultured from Lrig1 heterozygous (+/-) and Lrig1 knockout (-/-) mice littermates treated in the absence or in the presence of BDNF (30 ng/ml) for 30 minutes. Reprobing of the same blot with anti- ßIII-Tubulin is shown as loading control.B. Fold of TrkB activation relative to unstimulated control group (phospho-TrkB at Tyr705) in hippocampal neurons cultured from Lrig1 (+/+; +/-) and Lrig1 (-/-) mice treated in the absence or in the presence of BDNF (30 ng/ml) for 30 minutes. Results are presented as mean SEM of n=4 independent experiments. (*p<0.05 by Students t test).


C . Immunoblot showing MAPK activation in hippocampal neurons cultured from Lrig1 wild - type ( + / + ) and Lrig1 - deficient ( - / - ) mice littermates treated in the absence or in the presence of BDNF ( 30 ng / ml ) for the indicated times . Reprobing of the same blot with anti - ßIII - Tubulin is shown as loading control . Fold of MAPK activation relative to tubulin is indicated . D . Fold of MAPK activation ( P - MAPK ) relative to untreated control group in hippocampal neurons cultured from Lrig1 ( + / + ; + / - ) and Lrig1 ( - / - ) mice treated in the absence or in the presence of BDNF ( 30 ng / ml ) for 30 minutes . Results are presented as mean SEM of n = 4 independent experiments . ( * p < 0 . 05 by Students t test ) .

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

C. Immunoblot showing MAPK activation in hippocampal neurons cultured from Lrig1 wild-type (+/+) and Lrig1-deficient (-/-) mice littermates treated in the absence or in the presence of BDNF (30 ng/ml) for the indicated times. Reprobing of the same blot with anti- ßIII-Tubulin is shown as loading control. Fold of MAPK activation relative to tubulin is indicated.D. Fold of MAPK activation (P-MAPK) relative to untreated control group in hippocampal neurons cultured from Lrig1 (+/+; +/-) and Lrig1 (-/-) mice treated in the absence or in the presence of BDNF (30 ng/ml) for 30 minutes. Results are presented as mean SEM of n=4 independent experiments. (*p<0.05 by Students t test).


E . Representative images of mousehippocampal neurons transfected at 4 DIV with either GFP - expressing control vector or Lrig1shRNA - GFP plasmid . After transfection , neurons were cultured in the absence or in the presence of BDNF ( 25 ng / ml ) for 48 h ( 7 DIV ) . Arrows indicate branching points along the principal dendrite . Scale bar , 10 m .

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

E. Representative images of mousehippocampal neurons transfected at 4 DIV with either GFP-expressing control vector or Lrig1shRNA-GFP plasmid. After transfection, neurons were cultured in the absence or in the presence of BDNF (25 ng/ml) for 48 h (7 DIV). Arrows indicate branching points along the principal dendrite. Scale bar, 10 m.


F - I . Quantification of primary dendrites ( F ) , secondary dendrites ( G ) , total branching ( H ) and branchig of the principal dendrite ( I ) of hippocampal neurons treated as indicated in E . The results are shown as individual values and the means of a representative assay . A total of 30 neurons were analyzed per experimental condition . Similar results were obtained in n = 3 independent experiments .

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

F-I. Quantification of primary dendrites (F), secondary dendrites (G), total branching (H) and branchig of the principal dendrite (I) of hippocampal neurons treated as indicated in E. The results are shown as individual values and the means of a representative assay. A total of 30 neurons were analyzed per experimental condition. Similar results were obtained in n=3 independent experiments.


J . Lrig1 + / + and Lrig1 - / - hippocampal lysates from postnatal day 11 ( P11 ) mice were immunoblotted against phospho - TrkB ( p - TrkB ) at Tyr705 and total TrkB . Quantification ( mean SD ) of the p - TrkB / TrkB ratio between Lrig1 + / + ( n = 4 ) and Lrig1 - / - ( n = 3 ) hippocampi is also shown .

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

J. Lrig1+/+ and Lrig1-/- hippocampal lysates from postnatal day 11 (P11) mice were immunoblotted against phospho-TrkB (p-TrkB) at Tyr705 and total TrkB. Quantification (mean SD) of the p-TrkB/TrkB ratio between Lrig1+/+ (n=4) and Lrig1-/- (n=3) hippocampi is also shown.


( A ) Heteroplasmy levels by PCR - RFLP at position 3243 in 82 hiPSCs clones at passage 2 , derived from three MELAS patients ( MitoA , MitoB , MitoC ) . The black dots represent the mutation load for a specific hiPSC clone ; n is the total number of individual hiPSC clones analyzed per patient . The red stars represent the heteroplasmy levels for the parental fibroblasts .

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

(A) Heteroplasmy levels by PCR-RFLP at position 3243 in 82 hiPSCs clones at passage 2, derived from three MELAS patients (MitoA, MitoB, MitoC). The black dots represent the mutation load for a specific hiPSC clone; n is the total number of individual hiPSC clones analyzed per patient. The red stars represent the heteroplasmy levels for the parental fibroblasts.


( A ) mtDNA NGS of MitoA fibroblasts ( MitoA FB at passage 5 ) and MitoA - derived hiPSCs ( passages 7 - 9 ) ; n = 13 . ( B ) mtDNA NGS of MitoB fibroblasts ( MitoB FB at passage 3 ) and MitoB - derived hiPSCs ( passages 5 - 11 ) ; n = 12 . ( C ) mtDNA NGS of MitoC fibroblasts ( MitoC FB at passage 7 ) and MitoC - derived hiPSCs ( passages 5 - 11 ) ; n = 11 . GPMs are represented by black diamonds at the specific mtDNA positions ; small black diamonds denote mutation load between 1 and 10 % , medium black diamonds 10 to 40 % , and large black diamonds > 40 % . Red diamonds : point mutation A3243G ( MELAS causing disease mutation ) . Numbers beside diamonds indicate the specific mutation load at that position .

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

(A) mtDNA NGS of MitoA fibroblasts (MitoA FB at passage 5) and MitoA-derived hiPSCs (passages 7-9); n=13.(B) mtDNA NGS of MitoB fibroblasts (MitoB FB at passage 3) and MitoB-derived hiPSCs (passages 5-11); n=12.(C) mtDNA NGS of MitoC fibroblasts (MitoC FB at passage 7) and MitoC-derived hiPSCs (passages 5-11); n=11.GPMs are represented by black diamonds at the specific mtDNA positions; small black diamonds denote mutation load between 1 and 10%, medium black diamonds 10 to 40%, and large black diamonds > 40%. Red diamonds: point mutation A3243G (MELAS causing disease mutation). Numbers beside diamonds indicate the specific mutation load at that position.


( B ) Distribution of GPMs with mutation load lower or greater than 10 % , in control and MELAS groups . The majority of GPMs are present at levels lower than 10 % in either group .

O O O O O O O O O O O O O O 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) Distribution of GPMs with mutation load lower or greater than 10%, in control and MELAS groups. The majority of GPMs are present at levels lower than 10% in either group.


( C ) Location of GPMs greater than 10 % , in mtDNA .

(C) Location of GPMs greater than 10%, in mtDNA.


( B ) mtDNA NGS showing the position and mutation load of GPMs in seven isogenic MitoA - derived hiPSCs clones and their differentiated counterparts . The mtDNA genotype is stable during in vitro cardiac differentiation and lactate enrichment . MitoA hiPSCs 57 and 65 ( wild - type ) are labelled in black , MitoA hiPSCs 61 and 69 ( m . 3243A > G mutant ) are labelled in red and MitoA hiPSCs 58 , 59 and 217 ( with amplified GPMs in nuclear reprogramming ) are labelled in blue .

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(B) mtDNA NGS showing the position and mutation load of GPMs in seven isogenic MitoA-derived hiPSCs clones and their differentiated counterparts. The mtDNA genotype is stable during in vitro cardiac differentiation and lactate enrichment. MitoA hiPSCs 57 and 65 (wild-type) are labelled in black, MitoA hiPSCs 61 and 69 (m.3243A>G mutant) are labelled in red and MitoA hiPSCs 58, 59 and 217 (with amplified GPMs in nuclear reprogramming) are labelled in blue.


( C ) Quantification of the percentage of mature hiPSCs - derived cardiomyocytes in direct differentiation of hiPSC clones . Representative histograms showing the percentage of the cells expressing mature cardiac markers : myosin heavy chain ( MF20 ) , troponin T ( cTnT ) and troponin I ( cTnI ) by flow cytometry , in MitoA - 57 - derived cardiomyocytes ( wild - type ) and MitoA - 69 - derived cardiomyocytes ( 83 % m . 3243A > G ) . For complete quantification of cardiac markers in all hiPSCs - derived cardiomyocytes generated .

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

(C) Quantification of the percentage of mature hiPSCs-derived cardiomyocytes in direct differentiation of hiPSC clones. Representative histograms showing the percentage of the cells expressing mature cardiac markers: myosin heavy chain (MF20), troponin T (cTnT) and troponin I (cTnI) by flow cytometry, in MitoA-57-derived cardiomyocytes (wild-type) and MitoA-69-derived cardiomyocytes (83% m.3243A>G). For complete quantification of cardiac markers in all hiPSCs-derived cardiomyocytes generated.


( D ) Immunofluorescence images of cardiac markers ( cTnT and MF20 ) showing similar sarcomere structures in hiPSC - derived cardiomyocytes wild - type ( MitoA - 57 ) and mutant ( MitoA - 69 ) .

(D) Immunofluorescence images of cardiac markers (cTnT and MF20) showing similar sarcomere structures in hiPSC-derived cardiomyocytes wild-type (MitoA-57) and mutant (MitoA-69).


( E ) Oxygen consumption rate ( OCR ) in hiPSCs . OCR reported as a % of that in MitoA - 57 hiPSC , the control line . MitoA - 57 and MitoA - 65 hiPSCs wild - type ( in black ) , MitoA - 61 and MitoA - 69 m . 3243A > G mutant ( in red ) , MitoA - 58 , MitoA - 59 and MitoA - 217 hiPSCs amplified GPMs ( in blue ) . Error bars are mean ± SD and OCR data are representative 3 biological replicates in all samples . ( F ) Oxygen consumption rate in hiPSCs - derived cardiomyocytes at day 19 of cardiac differentiation . OCR reported as % of that in MitoA - 57 - derived cardiomyocytes , the control line . MitoA - 57 and MitoA - 65 derived cardiomyocytes wild - type ( in black ) , MitoA - 61 and MitoA - 69 derived cardiomyocytes mutants m . 3243A > G ( in red ) , MitoA - 58 , MitoA - 59 and MitoA - 217 derived cardiomyocytes with amplified GPMs ( in blue ) . Error bars are mean ± SD and OCR data are representative of two independent in vitro cardiac differentiation experiments , with at least 3 biological replicates in each sample , and significance established using Student ' s t - test .

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

(E) Oxygen consumption rate (OCR) in hiPSCs. OCR reported as a % of that in MitoA-57 hiPSC, the control line. MitoA-57 and MitoA-65 hiPSCs wild-type (in black), MitoA-61 and MitoA-69 m.3243A>G mutant (in red), MitoA-58, MitoA-59 and MitoA-217 hiPSCs amplified GPMs (in blue). Error bars are mean ± SD and OCR data are representative 3 biological replicates in all samples.(F) Oxygen consumption rate in hiPSCs-derived cardiomyocytes at day 19 of cardiac differentiation. OCR reported as % of that in MitoA-57-derived cardiomyocytes, the control line. MitoA-57 and MitoA-65 derived cardiomyocytes wild-type (in black), MitoA-61 and MitoA-69 derived cardiomyocytes mutants m.3243A>G (in red), MitoA-58, MitoA-59 and MitoA-217 derived cardiomyocytes with amplified GPMs (in blue). Error bars are mean ± SD and OCR data are representative of two independent in vitro cardiac differentiation experiments, with at least 3 biological replicates in each sample, and significance established using Student's t-test.


a - b , Representative images of cilia ( a ) and dot plots ( b ) showing primary cilia length variation at different time points after serum starvation ( SS ) . Markers in ( a ) show ARL13B ( cilia ) and PCNT ( Basal body ) . The graph shows individual length of cilia from 3 biological replicates ( n > 150 cilia per sample and experiment ) . Scale bar : 1µm . Data shown in include mean ± s . d . are calculated by two - tailed unpaired student t - test .

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a-b, Representative images of cilia (a) and dot plots (b) showing primary cilia length variation at different time points after serum starvation (SS). Markers in (a) show ARL13B (cilia) and PCNT (Basal body). The graph shows individual length of cilia from 3 biological replicates (n>150 cilia per sample and experiment). Scale bar: 1µm. Data shown in include mean ±s.d. are calculated by two-tailed unpaired student t-test.


d , Representative images show cilia segmentation based on acetylated tubulin ( Ac tub ) , polyglutamylated tubulin ( Glu tub ) , ARL13B and PCNT . The cartoon on the left indicates proximal segments ( PS , Glu tub rich - region ) and distal segments ( DS ) . DS were determined by calculating the difference between PS and total cilia length based on ARL13B staining as depicted by the dashed lines on the images . Scale bar : 1 . 5µm . e , Quantification of d . Comparative analysis of average cilia length based on Ac tubulin and ARL13B ( cilia ) staining shown for specified cilia length groups ( 2 < , 2 - 3 , 3 - 4 and > 4µm ) . Three biological replicates , n = 400 cilia per sample and repetition . The numbers above the cartoons show the ratio of the calculated Ac tub / whole cilia length . Data shown include mean ± s . d . are calculated by two - tailed unpaired student t - test .

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

d, Representative images show cilia segmentation based on acetylated tubulin (Ac tub), polyglutamylated tubulin (Glu tub), ARL13B and PCNT. The cartoon on the left indicates proximal segments (PS, Glu tub rich-region) and distal segments (DS). DS were determined by calculating the difference between PS and total cilia length based on ARL13B staining as depicted by the dashed lines on the images. Scale bar: 1.5µm. e, Quantification of d. Comparative analysis of average cilia length based on Ac tubulin and ARL13B (cilia) staining shown for specified cilia length groups (2<, 2-3, 3-4 and >4µm). Three biological replicates, n=400 cilia per sample and repetition. The numbers above the cartoons show the ratio of the calculated Ac tub/whole cilia length. Data shown include mean ±s.d. are calculated by two-tailed unpaired student t-test.


a , Representative images of 48h serum starved RPE1 cells treated with solvent control ( DMSO ) , cytochalasin D ( CytoD ) and / or nocodazole ( Noco ) for 3h before immunostaining with the indicated antibodies . Scale bar : 3µm . b , Quantification of ( a ) showing average length of proximal segment ( PS , Glu tub ) and whole cilia ( ARL13B ) . The numbers above the cartoons show the ratio of the calculated distal segment ( DS ) / whole cilia length . Three biological replicates , n = 100 cilia per sample and repetition . Data shown include mean ± s . d . ; P values are calculated by two - tailed unpaired student t - test .

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

a, Representative images of 48h serum starved RPE1 cells treated with solvent control (DMSO), cytochalasin D (CytoD) and/or nocodazole (Noco) for 3h before immunostaining with the indicated antibodies. Scale bar: 3µm. b, Quantification of (a) showing average length of proximal segment (PS, Glu tub) and whole cilia (ARL13B). The numbers above the cartoons show the ratio of the calculated distal segment (DS)/whole cilia length. Three biological replicates, n=100 cilia per sample and repetition. Data shown include mean ±s.d.; P values are calculated by two-tailed unpaired student t-test.


RPE1 cells were treated with the indicated siRNAs before serum starvation for 48h . Representative immunostained images with indicated antibodies Scale bar : 2µm .

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RPE1 cells were treated with the indicated siRNAs before serum starvation for 48h. Representative immunostained images with indicated antibodies Scale bar: 2µm.


e - f , Cells were treated with the indicated siRNAs before serum starvation for 48h and treatment with DMSO or Noco for 3h before immunostaining with the indicated antibodies . Representative images ( e ) and quantification of cilia length based on ARL13B ( f ) are shown . Scale bar : 2µm . Three biological replicates , n = 100 cilia per sample and repetition . Data shown include mean ± s . d . ; P values are calculated by two - tailed unpaired student t - test .

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e-f, Cells were treated with the indicated siRNAs before serum starvation for 48h and treatment with DMSO or Noco for 3h before immunostaining with the indicated antibodies. Representative images (e) and quantification of cilia length based on ARL13B (f) are shown. Scale bar: 2µm. Three biological replicates, n=100 cilia per sample and repetition. Data shown include mean ±s.d.; P values are calculated by two-tailed unpaired student t-test.


a , Images show the localisation of endogeneous SEPT2 and SEPT7 in RPE1 ARLB13B - mRuby2 cells with basal body staining . The insets to the right show enlargements of the cilium . Scale bar : 4µm ( large ) and 2µm ( small ) . b , STED microscopy images showing ciliated RPE1 cells expressing EGFP - SEPT2 or SEPT7 - EGFP and stained for ARL13B . Scale bar : 1µm .

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a, Images show the localisation of endogeneous SEPT2 and SEPT7 in RPE1 ARLB13B-mRuby2 cells with basal body staining. The insets to the right show enlargements of the cilium. Scale bar: 4µm (large) and 2µm (small). b, STED microscopy images showing ciliated RPE1 cells expressing EGFP-SEPT2 or SEPT7-EGFP and stained for ARL13B. Scale bar: 1µm.


d , Representative images showing co - localisation of MKS3 or CEP290 in ciliated RPE1 ARLB13B - mRuby2 and GFP - SEPT2 cells . The white arrows point to the specific MKS3 or CEP290 signals . Scale bar : 1µm . e , Line scan measurements show the normalised signal intensities along the cilium for the indicated proteins as specified in ( d ) ( dashed lines ) .

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d, Representative images showing co-localisation of MKS3 or CEP290 in ciliated RPE1 ARLB13B-mRuby2 and GFP-SEPT2 cells. The white arrows point to the specific MKS3 or CEP290 signals. Scale bar: 1µm. e, Line scan measurements show the normalised signal intensities along the cilium for the indicated proteins as specified in (d) (dashed lines).


f , Effect of nocodazole ( Noco ) treatment ( 3h ) on the localisation of endogenous SEPT2 and SEPT7 at cilia in RPE1 cells . The insets show magnifications of the cilium . Scale bar : 5µm ( large ) and 2 . 5µm ( small ) . g , Relative signal intensity of endogenous SEPT2 or SEPT7 along the cilium from ( f ) . SEPT2 or SEPT7 signals at cilia were normalized to the total cilia length . Three biological replicates , n > 90 cilia per sample and repetition . Data show in ( g ) includes mean ± s . d . ; P values are calculated by two - tailed unpaired student t - test . See source data .

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

f, Effect of nocodazole (Noco) treatment (3h) on the localisation of endogenous SEPT2 and SEPT7 at cilia in RPE1 cells. The insets show magnifications of the cilium. Scale bar: 5µm (large) and 2.5µm (small). g, Relative signal intensity of endogenous SEPT2 or SEPT7 along the cilium from (f). SEPT2 or SEPT7 signals at cilia were normalized to the total cilia length. Three biological replicates, n>90 cilia per sample and repetition. Data show in (g) includes mean ±s.d.; P values are calculated by two-tailed unpaired student t-test. See source data.


g , Electron microscopy of RPE1 WT and SEPT2 - KO cilia . Black arrows point to the axoneme and basal body ( BB ) . Scale bar : 400nm . h , Quantification of ( g ) . Only cilia detected from bottom ( BB ) to tip in serial sections were quantified . N > 15 cilia from two independent experiments . Data include mean ± s . d . ; P values are calculated by unpaired Wilcoxon - Mann - Whitney Rank Sum

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g, Electron microscopy of RPE1 WT and SEPT2-KO cilia. Black arrows point to the axoneme and basal body (BB). Scale bar: 400nm. h, Quantification of (g). Only cilia detected from bottom (BB) to tip in serial sections were quantified. N>15 cilia from two independent experiments. Data include mean ±s.d.; P values are calculated by unpaired Wilcoxon-Mann-Whitney Rank Sum Test


i - j , RPE1 WT and SEPT2 - KO cells after 48h SS were treated with solvent control ( DMSO ) , cytochalasin D ( CytoD ) and / or nocodazole ( Noco ) for 3h before immunostaining with the indicated antibodies . Representative images ( i ) and quantifications of PS ( Glu tub ) and whole cilia ( ARL13B ) length ( j ) are shown . The numbers above the cartoons in ( j ) show the ratio of the calculated DS / whole cilia length . Scale bar : 3µm . Three biological replicates , n = 100 cilia per sample and repetition . Data show include mean ± s . d . ; P values are calculated by two - tailed unpaired student t -

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

i-j, RPE1 WT and SEPT2-KO cells after 48h SS were treated with solvent control (DMSO), cytochalasin D (CytoD) and/or nocodazole (Noco) for 3h before immunostaining with the indicated antibodies. Representative images (i) and quantifications of PS (Glu tub) and whole cilia (ARL13B) length (j) are shown. The numbers above the cartoons in (j) show the ratio of the calculated DS/whole cilia length. Scale bar: 3µm. Three biological replicates, n=100 cilia per sample and repetition. Data show include mean ±s.d.; P values are calculated by two-tailed unpaired student t-test


quantification of the relative signal intensities for the indicated proteins ( b ) . IFT88 and KIF17 signal intensities were normalized to the whole cilia length . KIF7 signal intensity was quantified at ciliary tip or base Three biological replicates , n > 80 cilia per sample and repetition . Data include mean ± s . d . and P values are calculated by two - tailed unpaired student t - test .

quantification of the relative signal intensities for the indicated proteins (b). IFT88 and KIF17 signal intensities were normalized to the whole cilia length. KIF7 signal intensity was quantified at ciliary tip or base Three biological replicates, n>80 cilia per sample and repetition. Data include mean ±s.d. and P values are calculated by two-tailed unpaired student t-test.


c - d , KIF7 localisation at the tip and base of cilia ( indicated by arrows ) in RPE1 cells after 48h SS treated for 3h with solvent control ( DMSO ) or nocodazole ( Noco ) and quantification of the relative signal intensities for the ciliary base and tip of KIF7 ( d ) . Scale bar : 2µm . KIF7 signal intensity was quantified at the ciliary tip or base as indicated in ( c ) ( white arrows ) . Three biological replicates , at least 80 cilia per sample and repetition . Data include mean ± s . d . and P values are calculated by two - tailed unpaired student t - test .

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

c-d, KIF7 localisation at the tip and base of cilia (indicated by arrows) in RPE1 cells after 48h SS treated for 3h with solvent control (DMSO) or nocodazole (Noco) and quantification of the relative signal intensities for the ciliary base and tip of KIF7 (d). Scale bar: 2µm. KIF7 signal intensity was quantified at the ciliary tip or base as indicated in (c) (white arrows). Three biological replicates, at least 80 cilia per sample and repetition. Data include mean ±s.d. and P values are calculated by two-tailed unpaired student t-test.


e - f , RPE1 WT and SEPT2 - KO cells were treated with control or KIF7 siRNA after 48h SS and immunostained with the indicated antibodies . Representative images ( e ) and quantifications of proximal segment ( Glu tub ) and whole cilia ( ARL13B ) length ( f ) are shown . The numbers above the cartoons in ( f ) show the ratio of the calculated distal segments / whole cilia length . Three biological replicates , n = 100 cilia per sample and repetition . Scale bar : 2µm . Data include mean ± s . d . and P values are calculated by two - tailed unpaired student t - test .

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e-f, RPE1 WT and SEPT2-KO cells were treated with control or KIF7 siRNA after 48h SS and immunostained with the indicated antibodies. Representative images (e) and quantifications of proximal segment (Glu tub) and whole cilia (ARL13B) length (f) are shown. The numbers above the cartoons in (f) show the ratio of the calculated distal segments /whole cilia length. Three biological replicates, n=100 cilia per sample and repetition. Scale bar: 2µm. Data include mean ±s.d. and P values are calculated by two-tailed unpaired student t-test.


d , FRAP experiment of SSTR3 - GFP in RPE1 WT and SEPT2 KO cells . Cells were starved for 48h before the beginning of inspection and imaged every 30sec for 10mins after bleaching . Scale bar : 5µm . e , Percentage of normalised SSTR3 - GFP signal recovery at whole cilia after photobleching from ( d ) . n = 20 cilia are quantified from three biological replicates Data include mean ± s . d . and P values are calculated by two - tailed unpaired student t - test .

d, FRAP experiment of SSTR3-GFP in RPE1 WT and SEPT2 KO cells. Cells were starved for 48h before the beginning of inspection and imaged every 30sec for 10mins after bleaching. Scale bar: 5µm. e, Percentage of normalised SSTR3-GFP signal recovery at whole cilia after photobleching from (d). n=20 cilia are quantified from three biological replicates Data include mean ±s.d. and P values are calculated by two-tailed unpaired student t-test.


f - g , Influence of MKS3 depletion on cilia length of RPE WT and SEPT2 - KO cells . Cells are treated with the indicated siRNA and serum starved ( SS ) for 48h before immunostaining with the indicated antibodies . Representative images ( f ) and quantifications of proximal segment ( Glu tub ) and whole cilia ( ARL13B ) length ( g ) are shown . The numbers above the cartoons in ( g ) show the ratio of the calculated distal segments / whole cilia length . Scale bar : 2µm . Three 3 biological replicates , n = 100 cilia per sample and repetition . Data include mean ± s . d . and P values are calculated by two - tailed unpaired student t - test .

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f-g, Influence of MKS3 depletion on cilia length of RPE WT and SEPT2-KO cells. Cells are treated with the indicated siRNA and serum starved (SS) for 48h before immunostaining with the indicated antibodies. Representative images (f) and quantifications of proximal segment (Glu tub) and whole cilia (ARL13B) length (g) are shown. The numbers above the cartoons in (g) show the ratio of the calculated distal segments/whole cilia length. Scale bar: 2µm. Three 3 biological replicates, n=100 cilia per sample and repetition. Data include mean ±s.d. and P values are calculated by two-tailed unpaired student t-test.


h - i , A functional TZ is required for KIF7 cilia tip localisation in RPE1 cells . KIF7 signal intensities are measured in ciliated RPE1 cells treated with control or MKS3 siRNA and 48h SS . Representative images ( h ) and quantification of KIF7 signal intensity at the base and tip of cilia ( h ) are shown . Scale bar : 2µm . Three biological replicates , at least 60 cilia per sample and repetition . Data include mean ± s . d . and P values are calculated by two - tailed unpaired student t - test .

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h-i, A functional TZ is required for KIF7 cilia tip localisation in RPE1 cells. KIF7 signal intensities are measured in ciliated RPE1 cells treated with control or MKS3 siRNA and 48h SS. Representative images (h) and quantification of KIF7 signal intensity at the base and tip of cilia (h) are shown. Scale bar: 2µm. Three biological replicates, at least 60 cilia per sample and repetition. Data include mean ±s.d. and P values are calculated by two-tailed unpaired student t-test.


a , Time - lapse images of RPE1 WT and SEPT2 - KO cells stably expressing ARL13B - GFP and γ - tubulin - mRuby2 . Cells are starved for 32h before the beginning of inspection and imaged every 15min for 9 . 5h . Scale bar : 5µm . b , Quantification of ( a ) showing changes in cilia length during time - lapse imaging . Three biological replicates , 10 cilia per sample and repetition . c , Time - lapse images show ectocytosis events observed in RPE1 SEPT2 - KO cells . Experiments are done as in ( a ) with exception that cells are imaged every 15min . Scale bar : 2 . 5µm .

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a, Time-lapse images of RPE1 WT and SEPT2-KO cells stably expressing ARL13B-GFP and γ-tubulin-mRuby2. Cells are starved for 32h before the beginning of inspection and imaged every 15min for 9.5h. Scale bar: 5µm. b, Quantification of (a) showing changes in cilia length during time-lapse imaging. Three biological replicates, 10 cilia per sample and repetition. c, Time-lapse images show ectocytosis events observed in RPE1 SEPT2-KO cells. Experiments are done as in (a) with exception that cells are imaged every 15min. Scale bar: 2.5µm.


g - h , RPE1 SEPT2 - KO cells expressing ARL13B - mRuby2 and Neongreen - EFHC1 after 48h SS and immunostained with Glu - tub antibody . Representative images ( g ) and quantifications of proximal segment ( Glu tub ) , EFHC1 and whole cilia ( ARL13B ) length ( h ) are shown . Three biological replicates , n = 100 cilia per sample and repetition . Scale bar : 2µm .

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g-h, RPE1 SEPT2-KO cells expressing ARL13B-mRuby2 and Neongreen-EFHC1 after 48h SS and immunostained with Glu-tub antibody. Representative images (g) and quantifications of proximal segment (Glu tub), EFHC1 and whole cilia (ARL13B) length (h) are shown. Three biological replicates, n=100 cilia per sample and repetition. Scale bar: 2µm.


B HeLa cells were infected with x - light Shigella mCherry for 4 h 40 min for quantitative confocal microscopy . IPTG was added 30 min prior to fixation , and then samples were labelled with antibody for SEPT7 . The scale bar represents 5 μm . Graph represents mean % ± SEM of Shigella responding to IPTG outside ( - ) or inside ( + ) SEPT7 cages from 4 independent experiments . Student ' s t - test , * * * = p < 0 . 001 .

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B HeLa cells were infected with x-light Shigella mCherry for 4 h 40 min for quantitative confocal microscopy. IPTG was added 30 min prior to fixation, and then samples were labelled with antibody for SEPT7. The scale bar represents 5 μm. Graph represents mean % ± SEM of Shigella responding to IPTG outside (-) or inside (+) SEPT7 cages from 4 independent experiments. Student's t-test, *** = p<0.001.