A - C DAPI - staining visualization of sperm formation in wild - type male ( A ) , Mpfgmyb [ X ] ( B ) , and Mpfgmyb [ Y ] ( C ) plants . Note that background DAPI staining visualizes flagella ( arrows ) in addition to nuclei ( arrowheads ) . ( B ′ ) is an enlarged image of the boxed region in ( B ) , visualizing an incompletely condensed nucleus . Scale bars , 5 µm .

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A-C DAPI-staining visualization of sperm formation in wild-type male (A), Mpfgmyb [X] (B), and Mpfgmyb [Y] (C) plants. Note that background DAPI staining visualizes flagella (arrows) in addition to nuclei (arrowheads). (B′) is an enlarged image of the boxed region in (B), visualizing an incompletely condensed nucleus. Scale bars, 5 µm.


D RT - PCR analysis indicating acquisition of male - like autosomal gene expression patterns in Mpfgmyb [ X ] antheridiophores . Two independent Mpfgmyb [ X ] mutant alleles were analyzed .

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D RT-PCR analysis indicating acquisition of male-like autosomal gene expression patterns in Mpfgmyb [X] antheridiophores. Two independent Mpfgmyb [X] mutant alleles were analyzed.


E , F TEM analyses visualizing the abnormal arrangement of axonemal microtubules in Mpfgmyb [ X ] sperm ( F ) , as compared with those of wild - type males ( E ) . Scale bars , 100 nm .

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E, F TEM analyses visualizing the abnormal arrangement of axonemal microtubules in Mpfgmyb [X] sperm (F), as compared with those of wild-type males (E). Scale bars, 100 nm.


A RNA - seq analysis showing male - specific accumulation of lncRNAs derived from the MpFGMYB 3 ′ region . Diagrams illustrate wild - type and mutant MpFGMYB / SUF loci . Folded lines with a ∆ symbol indicate a deletion .

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A RNA-seq analysis showing male-specific accumulation of lncRNAs derived from the MpFGMYB 3′ region. Diagrams illustrate wild-type and mutant MpFGMYB/SUF loci. Folded lines with a ∆ symbol indicate a deletion.


B RT - PCR analysis of wild - type and genetically male suf mutants revealed loss of SUF expression and gain of MpFGMYB expression after induction of reproductive growth by far - red irradiation . Two independent suf mutant alleles were analyzed . The SUF primer pair used here flanked an intron and the duplicated bands of SUF likely represent spliced and unspliced forms .

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B RT-PCR analysis of wild-type and genetically male suf mutants revealed loss of SUF expression and gain of MpFGMYB expression after induction of reproductive growth by far-red irradiation. Two independent suf mutant alleles were analyzed. The SUF primer pair used here flanked an intron and the duplicated bands of SUF likely represent spliced and unspliced forms.


C - H Gametangiophore morphology ( C , E and G ) and gametangium development ( D , F and H ) of plants with the designated genotypes . Scale bars , 1 mm ( C , E and G ) , 20 µm ( D ) , 50 µm ( F ) , 100 µm ( H ) .

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C-H Gametangiophore morphology (C, E and G) and gametangium development (D, F and H) of plants with the designated genotypes. Scale bars, 1 mm (C, E and G), 20 µm (D), 50 µm (F), 100 µm (H).


A gametangiophore of MpEF1pro : SUF / suf - 30ge [ Y ] , indicating the inability of transgenic SUF overexpression in rescuing the feminization phenotype of sufge [ Y ] . Scale bar , 2mm .

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A gametangiophore of MpEF1pro:SUF/suf-30ge [Y], indicating the inability of transgenic SUF overexpression in rescuing the feminization phenotype of sufge [Y]. Scale bar, 2mm.


B Real - time RT - PCR analyses confirming SUF transcript accumulation in SUF - overexpressing lines . Constitutively expressed MpEF1α was used as control . Measurements of six biological replicates for WT [ Y ] , and three biological replicates for each SUF - overexpressing line are plotted . Data information : bars represent mean ± SD . Asterisks indicate significant differences from WT [ Y ] ( B ) ( p < 0 . 05 , two - tailed Student ' s t - test ) .

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B Real-time RT-PCR analyses confirming SUF transcript accumulation in SUF-overexpressing lines. Constitutively expressed MpEF1α was used as control. Measurements of six biological replicates for WT [Y], and three biological replicates for each SUF-overexpressing line are plotted. Data information: bars represent mean ± SD. Asterisks indicate significant differences from WT [Y] (B) (p <0.05, two-tailed Student's t-test).


D Real - time RT - PCR measurement of MpFGMYB transcript levels . Three biological replicates are analyzed for each line . Data information bars represent mean ± SD . Asterisks indicate significant differences from WT [ X ] ( D ) ( p < 0 . 05 , two - tailed Student ' s t - test ) .

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D Real-time RT-PCR measurement of MpFGMYB transcript levels. Three biological replicates are analyzed for each line. Data information bars represent mean ± SD. Asterisks indicate significant differences from WT [X] (D) (p <0.05, two-tailed Student's t-test).


E - H Gametangiophores ( E , G ) and gametangia ( F , H ) of gMpFGMYB - S [ Y ] ( E , F ) and gMpFGMYB - L [ Y ] ( G , H ) . Scale bars , 1 mm ( E , G ) , 50 μm ( F ) , 100 μm ( H ) .

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E-H Gametangiophores (E, G) and gametangia (F, H) of gMpFGMYB-S [Y] (E, F) and gMpFGMYB-L [Y] (G, H). Scale bars, 1 mm (E, G), 50 μm (F), 100 μm (H).


A ) Transmission electron microscopy ( TEM ) of MoDCs infected with P . gingivalis for 2 , 12 and 24 hours ( left , middle and right panels ) . The sections show the intra - and extra - cellular contents of Cont . ( un - infected ) , Pg381 , Mfa1 + Pg and FimA + Pg infected MoDCs for the different time

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A) Transmission electron microscopy (TEM) of MoDCs infected with P. gingivalis for 2, 12 and 24 hours (left, middle and right panels). The sections show the intra-and extra-cellular contents of Cont. (un-infected), Pg381, Mfa1+Pg and FimA+Pg infected MoDCs for the different time points


B ) The figure shows the intracellular content of P . gingivalis strains after 24 hours of infection . MoDCs infected with Pg381 or isogenic mutants strains were lysed and the survived intracellular bacteria were re - suspended and maintained in anaerobic broth for 5 days . The data represents CFU within MoDCs harvested from three healthy individuals . The means ± standard deviation ( in triplicates ) were analyzed by One - way ANOVA of different groups and Tukey ' s test for multiple group comparisons within 3 different experiments ( * statistical significance at the p < 0 . 001 ) .

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B) The figure shows the intracellular content of P. gingivalis strains after 24 hours of infection. MoDCs infected with Pg381 or isogenic mutants strains were lysed and the survived intracellular bacteria were re-suspended and maintained in anaerobic broth for 5 days. The data represents CFU within MoDCs harvested from three healthy individuals. The means ±standard deviation (in triplicates) were analyzed by One-way ANOVA of different groups and Tukey's test for multiple group comparisons within 3 different experiments (* statistical significance at the p<0.001).


A ) DC - SIGN mRNA expression in P . gingivalis - infected MoDCs at 0 . 1 , 1 and 10 MOIs . The figure shows the gene expression after 12 hours of Pg381 and mutant strains infections . The target gene ( DC - SIGN ) was normalized using the endogenous control GAPDH ( ΔCt ) and fold regulations were calculated using 2 - ( ΔΔCt ) method . The statistical analysis was performed using the t - test , which accounts for the clustering of infected and un - infected controls within 3 different experiments ( * p < 0 . 001 ) .

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A) DC-SIGN mRNA expression in P. gingivalis-infected MoDCs at 0.1, 1 and 10 MOIs. The figure shows the gene expression after 12 hours of Pg381 and mutant strains infections. The target gene (DC-SIGN) was normalized using the endogenous control GAPDH (ΔCt) and fold regulations were calculated using 2-(ΔΔCt) method. The statistical analysis was performed using the t-test, which accounts for the clustering of infected and un-infected controls within 3 different experiments (* p<0.001).


B ) Immuno - electron microscopy of un - infected MoDCs ( Cont . ) ( upper panel ) , MoDCs infected with Pg381 ( middle panel ) and Mfa1 + Pg mutants ( lower panel ) . Gold particles ( marked with red rings ) for positive DC - SIGN were detected in the cell membrane and cytoplasm of cells infected with Mfa1 + Pg strains . Minimal positive staining for DC - SIGN was detected in the membranes of MoDCs infected with Pg381 , while no cytoplasmic gold labeling was detected in these cells .

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B) Immuno-electron microscopy of un-infected MoDCs (Cont.) (upper panel), MoDCs infected with Pg381 (middle panel) and Mfa1+Pg mutants (lower panel). Gold particles (marked with red rings) for positive DC-SIGN were detected in the cell membrane and cytoplasm of cells infected with Mfa1+Pg strains. Minimal positive staining for DC-SIGN was detected in the membranes of MoDCs infected with Pg381, while no cytoplasmic gold labeling was detected in these cells.


C ) Flow cytometry analysis of surface DC - SIGN in human MoDCs after infection with Pg381 , Mfa1 + Pg and FimA + Pg . The analysis of the intensity used Kruskal - Wallis test analysis of different groups and Dunn ' s test for multiple comparisons 3 different experiments ( * p < 0 . 01 ) .

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C) Flow cytometry analysis of surface DC-SIGN in human MoDCs after infection with Pg381, Mfa1+Pg and FimA+Pg. The analysis of the intensity used Kruskal-Wallis test analysis of different groups and Dunn's test for multiple comparisons 3 different experiments (* p<0.01).


A ) Pg381 and mutant strains count after 24 hr incubation with human MoDCs with / without Rapamycin treatment . The survived bacteria were measured after maintaining the lysed MoDCs suspension in anaerobic broth for 5 days . The plot represents the means ± standard deviation of CFU within MoDCs harvested from three healthy individuals ( * P < 0 . 001 ) . The analysis of readings used One - way AVOVA analysis of different groups and Tukey ' s test for multiple comparisons .

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A) Pg381 and mutant strains count after 24 hr incubation with human MoDCs with/without Rapamycin treatment. The survived bacteria were measured after maintaining the lysed MoDCs suspension in anaerobic broth for 5 days. The plot represents the means ±standard deviation of CFU within MoDCs harvested from three healthy individuals (* P<0.001). The analysis of readings used One-way AVOVA analysis of different groups and Tukey's test for multiple comparisons.


B ) Epifluorescence microscopy images of MoDCs treated with Rapamycin 1 hour after P . gingivalis infections . LC3 - II ( red - fluorescent dye ) and the bacterial strains ( green CFSE ) were studied in MoDCs 11 hours after Rapamycin treatment ( 12 hours after infections ) .

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B) Epifluorescence microscopy images of MoDCs treated with Rapamycin 1 hour after P. gingivalis infections. LC3-II (red-fluorescent dye) and the bacterial strains (green CFSE) were studied in MoDCs 11 hours after Rapamycin treatment (12 hours after infections).


C ) Quantifications of the fluorescent intensity of CFSE - labeled P . gingivalis and LC3 - II signals within infected MoDCs using NIS - Elements BR software . One - way ANOVA analysis was used to compare the means of intensity of different groups and Tukey ' s test for multiple comparisons ( * P < 0 . 001 ) .

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C) Quantifications of the fluorescent intensity of CFSE-labeled P. gingivalis and LC3-II signals within infected MoDCs using NIS-Elements BR software. One-way ANOVA analysis was used to compare the means of intensity of different groups and Tukey's test for multiple comparisons (* P<0.001).


A ) Survival of P . gingivalis strains within MoDCs after 6 , 12 , 24 and 48 hours . Blue lines show P . gingivalis strains survival within MoDCs and their survival in anaerobic condition in the absence of DCs are showed in black lines .

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A) Survival of P. gingivalis strains within MoDCs after 6, 12, 24 and 48 hours. Blue lines show P. gingivalis strains survival within MoDCs and their survival in anaerobic condition in the absence of DCs are showed in black lines.


The effect of rapamycin on Mfa1 + Pg , Pg381 and FimA + Pg survival within MoDCs are shown in figures B , C and D respectively . A three - factor repeated measures ANOVA using mixed models was used to test the effect of strain and rapamycin treatment over time on OD reading . The survival curves for the strains are showed in blue , while the effect of rapamycin treatments are in red . Bacterial survivals in the absence of MoDCs with and without rapamycin are plotted in grey and black , respectively . Statistical analysis showed that the strain by rapamycin treatment overtime interaction indicates the pattern of means in each strain ( Mfa1 + Pg , Pg381 and FimA + Pg ) between treated ( rapamycin ) and untreated were significantly different overtime ( p - value < 0 . = 001 ) .

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The effect of rapamycin on Mfa1+Pg, Pg381 and FimA+Pg survival within MoDCs are shown in figures B, C and D respectively. A three-factor repeated measures ANOVA using mixed models was used to test the effect of strain and rapamycin treatment over time on OD reading. The survival curves for the strains are showed in blue, while the effect of rapamycin treatments are in red. Bacterial survivals in the absence of MoDCs with and without rapamycin are plotted in grey and black, respectively. Statistical analysis showed that the strain by rapamycin treatment overtime interaction indicates the pattern of means in each strain (Mfa1+Pg, Pg381 and FimA+Pg) between treated (rapamycin) and untreated were significantly different overtime (p-value <0.=001).


A ) Epifluorescence microscopy images of MoDCs infected with Pg381 , Mfa1 + Pg , FimA + Pg and MFB strains after 12 hours . LC3 - II was detected in red - fluorescent ( Texas red ) dye and the bacterial strains were pre - labeled with CFSE ( green ) . Co - localization of P . gingivalis and LC3 - II showed in the right panels .

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A) Epifluorescence microscopy images of MoDCs infected with Pg381, Mfa1+Pg, FimA+Pg and MFB strains after 12 hours. LC3-II was detected in red-fluorescent (Texas red) dye and the bacterial strains were pre-labeled with CFSE (green). Co-localization of P. gingivalis and LC3-II showed in the right panels.


B ) Quantifications of the fluorescent intensity of LC3 - II within infected MoDCs using NIS - Elements BR software . One - way ANOVA analysis was used to compare the means of intensity of different groups and Tukey ' s test for multiple comparisons of three different experiments ( * p < 0 . 001 , # p < 0 . 01 ) .

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B) Quantifications of the fluorescent intensity of LC3-II within infected MoDCs using NIS-Elements BR software. One-way ANOVA analysis was used to compare the means of intensity of different groups and Tukey's test for multiple comparisons of three different experiments (* p<0.001, # p<0.01).


C ) Bacterial uptake by MoDCs was determined by CFSE fluorescent intensity ( * p < 0 . 001 , # p < 0 . 01 ) .

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C) Bacterial uptake by MoDCs was determined by CFSE fluorescent intensity (* p<0.001, # p<0.01).


A ) Immuno - fluorescence images of LC3 - II and P . gingivalis within MoDCs pre - treated with GP120 ( DC - SIGN blocker ) after 12 hours of infection . LC3 - II was detected in red - fluorescent dye and the bacterial strains were pre - labeled with green CFSE .

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A) Immuno-fluorescence images of LC3-II and P. gingivalis within MoDCs pre-treated with GP120 (DC-SIGN blocker) after 12 hours of infection. LC3-II was detected in red-fluorescent dye and the bacterial strains were pre-labeled with green CFSE.


B ) Fluorescent intensity of LC3 - II and P . gingivalis strains signal ( in triplicates ) were statistically analyzed ( * and # p < 0 . 001 ) .

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B) Fluorescent intensity of LC3-II and P. gingivalis strains signal (in triplicates) were statistically analyzed (* and # p<0.001).


C ) Flow cytometry of MoDCs treated with siRNA for DC - SIGN ( si - DC - SIGN ) after 12 hours . Left panels show the decrease of DC - SIGN in Cont . ( un - infected ) , Pg381 and Mfa1 + Pg - infected MoDCs .

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C) Flow cytometry of MoDCs treated with siRNA for DC-SIGN (si-DC-SIGN) after 12 hours. Left panels show the decrease of DC-SIGN in Cont. (un-infected), Pg381 and Mfa1+Pg-infected MoDCs.


D ) Epifluorescence microscopy images of MoDCs ( si - DC - SIGN ) infected with Pg381 and Mfa1 + Pg . LC3 - II was detected in red - fluorescent dye and the bacterial strains were pre - labeled with green CFSE .

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D) Epifluorescence microscopy images of MoDCs (si-DC-SIGN) infected with Pg381 and Mfa1+Pg. LC3-II was detected in red-fluorescent dye and the bacterial strains were pre-labeled with green CFSE.


E ) The figure shows the CFU counts of the P . gingivalis strains with MoDCs that lack DC - SIGN ( si - DC - SIGN ) . The analysis of readings used One - way ANOVA analysis of different groups and Tukey ' s test for multiple comparisons ( * p < 0 . 001 ) .

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E) The figure shows the CFU counts of the P. gingivalis strains with MoDCs that lack DC-SIGN (si-DC-SIGN). The analysis of readings used One-way ANOVA analysis of different groups and Tukey's test for multiple comparisons (* p<0.001).


A ) Western blot detection of LC3 in MoDCs infected with Pg381 and mutant strains for 12 hours . B ) The mean ± standard deviation of LC3 - II intensity represents three different experiments and the values were analyzed using Kruskal - Wallis test analysis of different groups and Dunn ' s test for multiple comparisons .

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A) Western blot detection of LC3 in MoDCs infected with Pg381 and mutant strains for 12 hours. B) The mean ±standard deviation of LC3-II intensity represents three different experiments and the values were analyzed using Kruskal-Wallis test analysis of different groups and Dunn's test for multiple comparisons.


C ) Blot shows the autophagy flux test after blocking the lysosomal fusion with Bafilomycin in MoDCs . Cells were treated with Bafilomycin for 4 hours at 4nM . D ) LC3 - II intensity in MoDCs infected with Pg381 strains with and without Bafilomycin . The analysis of the intensity of three different experiments used Kruskal - Wallis test analysis of different groups and Dunn ' s test for multiple comparisons .

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C) Blot shows the autophagy flux test after blocking the lysosomal fusion with Bafilomycin in MoDCs. Cells were treated with Bafilomycin for 4 hours at 4nM. D) LC3-II intensity in MoDCs infected with Pg381 strains with and without Bafilomycin. The analysis of the intensity of three different experiments used Kruskal-Wallis test analysis of different groups and Dunn's test for multiple comparisons.


A ) Flow cytometry of CD83 on MoDCs after incubation of TLR4 ligand ( E . coli LPS ) and TLR1 and 2 ligand ( Pam3csk4 ) for 4 hour .

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A) Flow cytometry of CD83 on MoDCs after incubation of TLR4 ligand (E. coli LPS) and TLR1 and 2 ligand (Pam3csk4) for 4 hour.


B ) Immuno - fluorescence images of LC3 - II ( red ) within MoDCs after incubation with TLR4 and TLR1 & amp ; amp ; 2 ligands ( E . coli LPS and Pam3csk4 )

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B) Immuno-fluorescence images of LC3-II (red) within MoDCs after incubation with TLR4 and TLR1&amp;2 ligands (E. coli LPS and Pam3csk4)


C ) The plot represents the means ± standard deviation of CFU within MoDCs harvested from three healthy individuals after 24 hours ( * * p < 0 . 001 ) .

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C) The plot represents the means ±standard deviation of CFU within MoDCs harvested from three healthy individuals after 24 hours (** p<0.001).


A ) Scanning electron microscopy ( SEM ) of MoDCs of the early interaction of MoDCs with Pg381 and Mfa1 + Pg ( upper panel 10000x and lower panel 25000x ) . B ) SEM for MoDCs interacting with Pg381 ( green stains for bacteria are computer generated ) .

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A) Scanning electron microscopy (SEM) of MoDCs of the early interaction of MoDCs with Pg381 and Mfa1+Pg (upper panel 10000x and lower panel 25000x). B) SEM for MoDCs interacting with Pg381 (green stains for bacteria are computer generated).


C ) Transmission electron microscopy ( TEM ) of autophagosome like structures within MoDCs infected with Pg381 , Mfa1 + Pg and FimA + Pg for 12 hours ( upper , middle and lower panels , respectively ) . The right and left sections show the different magnifications of randomly selected sections . Pg381 and FimA + Pg strains are mostly enclosed in the characteristics double - membrane intracellular vesicles ( Orange arrows ) . Contrary , Mfa1 + Pg escaped these autophagic ( double - membrane ) vesicles and enclosed within single membrane structures or freely occupy the cytoplasm ( Green arrows ) .

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C) Transmission electron microscopy (TEM) of autophagosome like structures within MoDCs infected with Pg381, Mfa1+Pg and FimA+Pg for 12 hours (upper, middle and lower panels, respectively). The right and left sections show the different magnifications of randomly selected sections. Pg381 and FimA+Pg strains are mostly enclosed in the characteristics double-membrane intracellular vesicles (Orange arrows). Contrary, Mfa1+Pg escaped these autophagic (double-membrane) vesicles and enclosed within single membrane structures or freely occupy the cytoplasm (Green arrows).


D ) The ratio of bacteria included in the double membrane were compared to total number of bacteria within MoDCs and plotted as percentage . Counting of the bacteria included in single or doubled membrane vesicles after 12 hours of infection . Each strain was counted in three randomly selected grids for each sample . The analysis of the bacterial counts used Kruskal - Wallis test of different groups and Dunn ' s test for multiple comparisons ( * p < 0 . 01 ) .

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D) The ratio of bacteria included in the double membrane were compared to total number of bacteria within MoDCs and plotted as percentage. Counting of the bacteria included in single or doubled membrane vesicles after 12 hours of infection. Each strain was counted in three randomly selected grids for each sample. The analysis of the bacterial counts used Kruskal-Wallis test of different groups and Dunn's test for multiple comparisons (*p<0.01).


A ) Epifluorescence microscopy images of MoDCs infected with pre - labeled bacteria ( green - CFSE ) . LAMP1 detected by transduction of red fluorescent protein ( RFP ) chimera using baculovirus transgenes to MoDCs .

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A) Epifluorescence microscopy images of MoDCs infected with pre-labeled bacteria (green-CFSE). LAMP1 detected by transduction of red fluorescent protein (RFP) chimera using baculovirus transgenes to MoDCs.


B ) The mean of fluorescent intensities ± standard deviation of LAMP1 were quantified in three different experiments ( * p < 0 . 001 ) .

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B) The mean of fluorescent intensities ±standard deviation of LAMP1 were quantified in three different experiments (* p<0.001).


C ) The upper scattered plot shows the co - localization of red and green signals in sections of MoDCs infected with Pg381 and mutants strains . The lower plot shows the average of Pearson ' s correlation measures of three randomly selected region of interest ( ROI ) within each field within 3 different experiments ( * p < 0 . 0001 ) ( Table 4 and 5 ) . All analysis of fluorescence intensity used One - way ANOVA analysis of different groups and Tukey ' s test for multiple comparisons .

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C) The upper scattered plot shows the co-localization of red and green signals in sections of MoDCs infected with Pg381 and mutants strains. The lower plot shows the average of Pearson's correlation measures of three randomly selected region of interest (ROI) within each field within 3 different experiments (* p<0.0001) (Table 4 and 5). All analysis of fluorescence intensity used One-way ANOVA analysis of different groups and Tukey's test for multiple comparisons.


A Representative Gli1ZF / DNA structure extrapolated from MD trajectories . Gli1ZF is shown as blue cartoon , residues involved in binding to DNA ( based on single point mutation study ) are shown as magenta sticks , and Zn ions as gray spheres .

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A Representative Gli1ZF/DNA structure extrapolated from MD trajectories. Gli1ZF is shown as blue cartoon, residues involved in binding to DNA (based on single point mutation study) are shown as magenta sticks, and Zn ions as gray spheres.


B Effect of Gli1ZF mutants on the binding affinity to DNA as predicted by in silico alanine scanning . The ∆ ∆ G was calculated along MD trajectories as the difference between the ∆ G of each Gli1ZF alanine mutant and Gli1ZF ‐ WT . Results are shown as ∆ ∆ G values in kcal / mol calculated by means of the MM ‐ PBSA methods ± SEM .

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B Effect of Gli1ZF mutants on the binding affinity to DNA as predicted by in silico alanine scanning. The ∆∆G was calculated along MD trajectories as the difference between the ∆G of each Gli1ZF alanine mutant and Gli1ZF‐WT. Results are shown as ∆∆G values in kcal/mol calculated by means of the MM‐PBSA methods ± SEM.


C Effect of Gli1ZF mutants on Gli1 ‐ dependent transcriptional activation . Luciferase assay was performed in HEK293T cells transfected with 12XGliBS ‐ Luc ( GliBS , Gli binding site ) , pRL ‐ TK Renilla ( normalization control ) , Flag ‐ Gli1 WT or the indicated Flag ‐ Gli1 mutants . Data show the mean ± SD of three independent experiments . * P 0 . 01 ; * * P 0 . 05 versus Gli1 WT . Western blot analysis of Flag ‐ Gli1 WT or the indicated Flag ‐ Gli1 mutant expression levels ( bottom panel ) .

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C Effect of Gli1ZF mutants on Gli1‐dependent transcriptional activation. Luciferase assay was performed in HEK293T cells transfected with 12XGliBS‐Luc (GliBS, Gli binding site), pRL‐TK Renilla (normalization control), Flag‐Gli1 WT or the indicated Flag‐Gli1 mutants. Data show the mean ± SD of three independent experiments. *P 0.01; **P 0.05 versus Gli1 WT. Western blot analysis of Flag‐Gli1 WT or the indicated Flag‐Gli1 mutant expression levels (bottom panel).


E Gli1 / DNA binding . Double ‐ stranded oligonucleotide containing the canonical GliBS sequence ( 5 ′ - TTGCCTACCTGGGTGGTCTCTCCACTT - 3 ′ ) or mutated GliBS sequence used as control ( 5 ′ - TTGCCTACCTCCCACTTCTCTCCACTT - 3 ′ ) was used as probe ( P ) in EMSA experiments . The assay was performed using recombinant GST ‐ Gli1ZF ‐ WT ( Gli1 zinc finger fragment : aa 242 - 424 ) , GST ‐ Gli1ZF ‐ K350A and GST ‐ Gli1ZF ‐ K340A . The graph on the right indicates ratio ( mean arbitrary units ± SD from three independent experiments ) of GST ‐ Gli1ZF ‐ WT or GST ‐ Gli1ZF mutants bound to the labeled GliBS probe / GliBS ‐ free probe normalized to the amount of GST ‐ Gli1ZF ‐ WT / DNA binding ( as described in ) . * P 0 . 05 versus Gli1 WT .

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E Gli1/DNA binding. Double‐stranded oligonucleotide containing the canonical GliBS sequence (5′-TTGCCTACCTGGGTGGTCTCTCCACTT-3′) or mutated GliBS sequence used as control (5′-TTGCCTACCTCCCACTTCTCTCCACTT-3′) was used as probe (P) in EMSA experiments. The assay was performed using recombinant GST‐Gli1ZF‐WT (Gli1 zinc finger fragment: aa 242-424), GST‐Gli1ZF‐K350A and GST‐Gli1ZF‐K340A. The graph on the right indicates ratio (mean arbitrary units ± SD from three independent experiments) of GST‐Gli1ZF‐WT or GST‐Gli1ZF mutants bound to the labeled GliBS probe/GliBS‐free probe normalized to the amount of GST‐Gli1ZF‐WT/DNA binding (as described in ). *P 0.05 versus Gli1 WT.


A Inhibition of Gli1 ‐ induced transcription in transfected HEK293T cells . HEK293T cells were transfected with 12XGliBS ‐ Luc and pRL ‐ TK Renilla ( normalization control ) plus control ( empty ) or Gli1 vector and treated with increasing concentrations of GlaB or GANT61 . Treatment time was 24 h , and control cells were treated with DMSO only .

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A Inhibition of Gli1‐induced transcription in transfected HEK293T cells. HEK293T cells were transfected with 12XGliBS‐Luc and pRL‐TK Renilla (normalization control) plus control (empty) or Gli1 vector and treated with increasing concentrations of GlaB or GANT61. Treatment time was 24 h, and control cells were treated with DMSO only.


B GlaB chemical structure with numbering scheme for NMR analysis .

B GlaB chemical structure with numbering scheme for NMR analysis.


C Inhibition of Gli1 ‐ induced transcription in transfected Smo − / − MEF cells . Smo − / − MEF cells were transfected with 12XGliBS ‐ Luc and pRL ‐ TK Renilla ( normalization control ) plus control ( empty ) or Gli1 vector and treated for 24 h with increasing concentrations of GlaB or DMSO only as control .

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C Inhibition of Gli1‐induced transcription in transfected Smo−/−MEF cells. Smo−/−MEF cells were transfected with 12XGliBS‐Luc and pRL‐TK Renilla (normalization control) plus control (empty) or Gli1 vector and treated for 24 h with increasing concentrations of GlaB or DMSO only as control.


D The graphs show the Hh target gene expression levels in Ptch1 − / − MEFs treated for 48 h with GlaB and DMSO as a control . mRNA levels were determined by quantitative real ‐ time PCR ( qRT - PCR ) normalized to endogenous control ( β2 ‐ microglobulin and HPRT ) . Pfkfb3 gene was used as a negative control .

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D The graphs show the Hh target gene expression levels in Ptch1−/−MEFs treated for 48 h with GlaB and DMSO as a control. mRNA levels were determined by quantitative real‐time PCR (qRT-PCR) normalized to endogenous control (β2‐microglobulin and HPRT). Pfkfb3 gene was used as a negative control.


F SuFu − / − MEFs were treated for 48 h with GlaB and DMSO as a control . Gli1 and Ptch1mRNA levels were determined by qRT - PCR normalized to β2 ‐ microglobulin and HPRT expression . Pfkfb3 gene was used as a negative control .

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F SuFu−/−MEFs were treated for 48 h with GlaB and DMSO as a control. Gli1 and Ptch1mRNA levels were determined by qRT-PCR normalized to β2‐microglobulin and HPRT expression. Pfkfb3 gene was used as a negative control.


Promoter occupancy of Gli1 is prevented by GlaB treatment . MEF WT cells were transfected with Flag ‐ tagged Gli1 or empty vectors , and chromatin immunoprecipitation ( ChIP ) was carried out . qRT - PCR was performed using primers encompassing the Gli ‐ BS of mouse Ptch1 promoter ( right , schematic representation ) . Results are indicated as fold difference , relative to empty ( pcDNA3 ) control .

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Promoter occupancy of Gli1 is prevented by GlaB treatment. MEF WT cells were transfected with Flag‐tagged Gli1 or empty vectors, and chromatin immunoprecipitation (ChIP) was carried out. qRT-PCR was performed using primers encompassing the Gli‐BS of mouse Ptch1 promoter (right, schematic representation). Results are indicated as fold difference, relative to empty (pcDNA3) control.


H Ptch1 mRNA expression levels ( left panel ) were determined by qRT - PCR in Daoy cells transfected with siRNA specific for Gli1 and Gli2 ( siGli1 / 2 ) or a non ‐ specific control siRNA ( siCtr ) and treated for 24 h with GlaB or DMSO as a control . ( right panel ) The graph shows Gli1 and Gli2mRNA expression levels determined by qRT - PCR in Daoy cells transfected with siGli1 / 2 or siCtr . Results are expressed as fold repression relative to control , and data were normalized to GAPDH and HPRT expression .

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H Ptch1 mRNA expression levels (left panel) were determined by qRT-PCR in Daoy cells transfected with siRNA specific for Gli1 and Gli2 (siGli1/2) or a non‐specific control siRNA (siCtr) and treated for 24 h with GlaB or DMSO as a control. (right panel) The graph shows Gli1 and Gli2mRNA expression levels determined by qRT-PCR in Daoy cells transfected with siGli1/2 or siCtr. Results are expressed as fold repression relative to control, and data were normalized to GAPDH and HPRT expression.


A The predicted binding mode of GlaB ( green sticks ) to Gli1ZF ( blue cartoons ) . Residues highlighted by the single ‐ point mutation study to impact on Gli1 binding to DNA are shown as blue sticks ; K340 and K350 of the GlaB binding site are colored magenta . GlaB protons highlighted by the NMR study are shown as orange spheres .

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A The predicted binding mode of GlaB (green sticks) to Gli1ZF (blue cartoons). Residues highlighted by the single‐point mutation study to impact on Gli1 binding to DNA are shown as blue sticks; K340 and K350 of the GlaB binding site are colored magenta. GlaB protons highlighted by the NMR study are shown as orange spheres.


B Detail of GlaB binding to Gli1ZF ‐ WT , Gli1ZF ‐ K340A , GLI1ZF ‐ K350A , GLI1ZF ‐ K340A / K350A mutants and respective theoretical affinity values . Gli1 is shown as blue transparent surface ; K340 and K350 are colored magenta . GlaB is shown as green sticks .

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B Detail of GlaB binding to Gli1ZF‐WT, Gli1ZF‐K340A, GLI1ZF‐K350A, GLI1ZF‐K340A/K350A mutants and respective theoretical affinity values. Gli1 is shown as blue transparent surface; K340 and K350 are colored magenta. GlaB is shown as green sticks.


C Inhibition of Gli1 / DNA binding by GlaB . EMSA using recombinant GST ‐ Gli1ZF ‐ WT or GST ‐ Gli1ZF ‐ K340A in the presence of different concentrations of GlaB or with DMSO only . The shifted complex is competed with a 50 × excess of cold probe . The graph on the right indicates ratio ( mean arbitrary units ± SD from three independent experiments ) of GST ‐ Gli1ZF ‐ WT or GST ‐ Gli1ZF ‐ K340A bound to the labeled GliBS probe / GliBS ‐ free probe normalized to the amount of GST ‐ Gli1ZF ‐ WT / DNA binding in absence of GlaB . * P 0 . 05 versus DMSO ; * * P 0 . 05 K340A + GlaB versus Gli1 WT + GlaB .

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C Inhibition of Gli1/DNA binding by GlaB. EMSA using recombinant GST‐Gli1ZF‐WT or GST‐Gli1ZF‐K340A in the presence of different concentrations of GlaB or with DMSO only. The shifted complex is competed with a 50× excess of cold probe. The graph on the right indicates ratio (mean arbitrary units ± SD from three independent experiments) of GST‐Gli1ZF‐WT or GST‐Gli1ZF‐K340A bound to the labeled GliBS probe/GliBS‐free probe normalized to the amount of GST‐Gli1ZF‐WT/DNA binding in absence of GlaB. *P 0.05 versus DMSO; **P 0.05 K340A + GlaB versus Gli1 WT + GlaB.


D HEK293T cells were transfected with 12XGliBS ‐ Luc and pRL ‐ TK Renilla ( normalization control ) plus control ( empty vector ) or Gli1 or Gli1K340A mutant and treated with increasing concentrations of GlaB . Treatment time was 24 h , and control cells were treated with DMSO only . Data show the mean ± SD of three independent experiments . * P 0 . 05 versus DMSO ; * * P 0 . 05 K340A versus Gli1 WT .

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D HEK293T cells were transfected with 12XGliBS‐Luc and pRL‐TK Renilla (normalization control) plus control (empty vector) or Gli1 or Gli1K340A mutant and treated with increasing concentrations of GlaB. Treatment time was 24 h, and control cells were treated with DMSO only. Data show the mean ± SD of three independent experiments. *P 0.05 versus DMSO; **P 0.05 K340A versus Gli1 WT.


A qRT - PCR shows Hh target mRNA expression levels determined in 6 ‐ day ‐ old mouse cerebellar progenitors after s . c . injections of GlaB .

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A qRT-PCR shows Hh target mRNA expression levels determined in 6‐day‐old mouse cerebellar progenitors after s.c. injections of GlaB.


B Representative EGLHE staining of 6 ‐ day ‐ old mousecerebellum after s . c . injections of GlaB ( upper panel ) ; immunohistochemistry shows Gli1 ( middle panel ) and Ki67 stainings ( lower panel ) . In all images , the scale bar represents 50 μm .

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B Representative EGLHE staining of 6‐day‐old mousecerebellum after s.c. injections of GlaB (upper panel); immunohistochemistry shows Gli1 (middle panel) and Ki67 stainings (lower panel). In all images, the scale bar represents 50 μm.


C The graph shows the percentage of EGL thickness reduction in GlaB ‐ treated mouse cerebella in comparison with vehicle mouse cerebella . Data show the mean ± SD of cerebella ( n = 10 ) for each treatment . * P 0 . 05 versus Ctr .

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C The graph shows the percentage of EGL thickness reduction in GlaB‐treated mouse cerebella in comparison with vehicle mouse cerebella. Data show the mean ± SD of cerebella (n = 10) for each treatment. *P 0.05 versus Ctr.


D BrdU assay in GCPs . Cerebellargranule cell progenitors ( GCPs ) isolated from 4 ‐ day ‐ old mice were treated with Shh ( recombinant mouseSonic Hedgehog , amino ‐ terminal peptide , ShhN , 3 μg / ml ) and with different concentrations of GlaB , as indicated , for 48 h . Inhibition of cell proliferation was measured as percentage of BrdU incorporation in comparison with DMSO ‐ treated sample . Shown is the mean of three independent experiments . Error bars indicate SD . * P 0 . 05 Shh versus DMSO ; * * P 0 . 05 Shh + GlaB versus Shh .

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D BrdU assay in GCPs. Cerebellargranule cell progenitors (GCPs) isolated from 4‐day‐old mice were treated with Shh (recombinant mouseSonic Hedgehog, amino‐terminal peptide, ShhN, 3 μg/ml) and with different concentrations of GlaB, as indicated, for 48 h. Inhibition of cell proliferation was measured as percentage of BrdU incorporation in comparison with DMSO‐treated sample. Shown is the mean of three independent experiments. Error bars indicate SD. *P 0.05 Shh versus DMSO; **P 0.05 Shh + GlaB versus Shh.


E qRT - PCR and show Hh and proliferation target mRNA and protein expression levels determined in ex vivo GCP culture derived from 6 ‐ day ‐ old mousecerebella after s . c . injections of GlaB . In all qRT - PCR experiments , the results were normalized to endogenous control ( β2 ‐ microglobulin and HPRT ) . * P 0 . 05 versus Ctr .

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E qRT-PCR and show Hh and proliferation target mRNA and protein expression levels determined in ex vivo GCP culture derived from 6‐day‐old mousecerebella after s.c. injections of GlaB. In all qRT-PCR experiments, the results were normalized to endogenous control (β2‐microglobulin and HPRT). *P 0.05 versus Ctr.


F Western blot analysis show Hh and proliferation target mRNA and protein expression levels determined in ex vivo GCP culture derived from 6 ‐ day ‐ old mousecerebella after s . c . injections of GlaB .

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F Western blot analysis show Hh and proliferation target mRNA and protein expression levels determined in ex vivo GCP culture derived from 6‐day‐old mousecerebella after s.c. injections of GlaB.


A , B Ex vivo cell cultures from Ptch1 + / − mice MBs were treated with GlaB ( 5 μM ) , GANT61 ( 10 μM ) or DMSO only . After the indicated times , a trypan blue count was performed ( A ) to determine the growth rate of viable cells .

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A, B Ex vivo cell cultures from Ptch1+/−mice MBs were treated with GlaB (5 μM), GANT61 (10 μM) or DMSO only. After the indicated times, a trypan blue count was performed (A) to determine the growth rate of viable cells.


A , B Ex vivo cell cultures from Ptch1 + / − miceMBs were treated with GlaB ( 5 μM ) , GANT61 ( 10 μM ) or DMSO only . After the indicated times , Gli1 mRNA expression levels were determined by qRT - PCR ( B ) normalized to endogenous control ( β2 ‐ microglobulin and HPRT ) .

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A, B Ex vivo cell cultures from Ptch1+/−miceMBs were treated with GlaB (5 μM), GANT61 (10 μM) or DMSO only. After the indicated times, Gli1 mRNA expression levels were determined by qRT-PCR (B) normalized to endogenous control (β2‐microglobulin and HPRT).


C - F GlaB inhibits MB ‐ SCs ' self ‐ renewal and proliferation . ( C ) Suspension of single MB ‐ SCs isolated from Ptch1 + / − mice . MBs were cultured in stem cell medium to allow the formation of primary neurospheres . Primary neurospheres were dissociated and treated with increasing concentrations of GlaB or DMSO only . After 7 days of treatment , the number of secondary neurospheres derived from a known number of single cells was counted . The self ‐ renewal MB ‐ SCs ' capability is expressed as percentage of neurosphere ‐ forming cells ( left ) . Representative bright field images of tumor neurospheres after GlaB treatment are also shown ( right ) .

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C-F GlaB inhibits MB‐SCs' self‐renewal and proliferation. (C) Suspension of single MB‐SCs isolated from Ptch1+/−mice. MBs were cultured in stem cell medium to allow the formation of primary neurospheres. Primary neurospheres were dissociated and treated with increasing concentrations of GlaB or DMSO only. After 7 days of treatment, the number of secondary neurospheres derived from a known number of single cells was counted. The self‐renewal MB‐SCs' capability is expressed as percentage of neurosphere‐forming cells (left). Representative bright field images of tumor neurospheres after GlaB treatment are also shown (right).


C - F GlaB inhibits MB ‐ SCs ' self ‐ renewal and proliferation . ( D , E ) MB ‐ SCs isolated from Ptch1 + / − miceMBs were treated for 48 h with GlaB ( 5 μM ) or DMSO only . qRT - PCR analysis show Hh , proliferation and stemness target mRNA expression levels . For qRT - PCR , results were normalized to endogenous control ( β2 ‐ microglobulin and HPRT ) .

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C-F GlaB inhibits MB‐SCs' self‐renewal and proliferation. (D, E) MB‐SCs isolated from Ptch1+/−miceMBs were treated for 48 h with GlaB (5 μM) or DMSO only. qRT-PCR analysis show Hh, proliferation and stemness target mRNA expression levels. For qRT-PCR, results were normalized to endogenous control (β2‐microglobulin and HPRT).


C - F GlaB inhibits MB ‐ SCs ' self ‐ renewal and proliferation . ( D , E ) MB ‐ SCs isolated from Ptch1 + / − miceMBs were treated for 48 h with GlaB ( 5 μM ) or DMSO only . Western blot analysis show Hh , proliferation and stemness target protein expression levels .

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C-F GlaB inhibits MB‐SCs' self‐renewal and proliferation. (D, E) MB‐SCs isolated from Ptch1+/−miceMBs were treated for 48 h with GlaB (5 μM) or DMSO only. Western blot analysis show Hh, proliferation and stemness target protein expression levels.


C - F GlaB inhibits MB ‐ SCs ' self ‐ renewal and proliferation . ( F ) BrdU assay in MB ‐ SCs treated with GlaB ( 5 μM ) for 24 or 48 h and plated on polylysinated chamber slides . Inhibition of cell proliferation was measured as percentage of BrdU incorporation in comparison with DMSO ‐ treated sample .

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C-F GlaB inhibits MB‐SCs' self‐renewal and proliferation. (F) BrdU assay in MB‐SCs treated with GlaB (5 μM) for 24 or 48 h and plated on polylysinated chamber slides. Inhibition of cell proliferation was measured as percentage of BrdU incorporation in comparison with DMSO‐treated sample.


G TUNEL assay in MB ‐ SCs . MB ‐ SCs isolated from Ptch1 + / − miceMBs were treated with GlaB ( 5 μM ) and compared to DMSO ‐ treated sample . Bottom panel shows a Western blot of caspase ‐ 3 in GlaB ‐ treated versus control cells .

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G TUNEL assay in MB‐SCs. MB‐SCs isolated from Ptch1+/−miceMBs were treated with GlaB (5 μM) and compared to DMSO‐treated sample. Bottom panel shows a Western blot of caspase‐3 in GlaB‐treated versus control cells.


A Change of tumor volume during GlaB or vehicle treatment period ( 18 days ) .

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A Change of tumor volume during GlaB or vehicle treatment period (18 days).


B Representative flank allografts average volumes . Error bars indicate SD . * P 0 . 05 .

B Representative flank allografts average volumes. Error bars indicate SD. *P 0.05.


C Representative Masson ' s trichromestaining of tumors ; immunohistochemistry of Gli1 , Ki67 and TUNEL stainings . Scale bars represent 35 μm for Masson ' s trichromestaining , 25 μm for Gli1 and TUNEL , and 10 μm for Ki67 stainings .

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C Representative Masson's trichromestaining of tumors; immunohistochemistry of Gli1, Ki67 and TUNEL stainings. Scale bars represent 35 μm for Masson's trichromestaining, 25 μm for Gli1 and TUNEL, and 10 μm for Ki67 stainings.


D Quantification of Ki67 and TUNEL stainings from immunohistochemistry shown in ( C ) . Error bars indicate SD . * P 0 . 05 .

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D Quantification of Ki67 and TUNEL stainings from immunohistochemistry shown in (C). Error bars indicate SD. *P 0.05.


E qRT - PCR analysis show Hh and proliferation target mRNA expression levels . For qRT - PCR , results were normalized to endogenous control ( β2 ‐ microglobulin and HPRT ) . Data show the mean ± SD of tumor ( n = 6 ) for each treatment . * P 0 . 05 versus Ctr .

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E qRT-PCR analysis show Hh and proliferation target mRNA expression levels. For qRT-PCR, results were normalized to endogenous control (β2‐microglobulin and HPRT). Data show the mean ± SD of tumor (n = 6) for each treatment. *P 0.05 versus Ctr.


F Western blot analysis show Hh and proliferation target protein expression levels . Data show the mean ± SD of tumor ( n = 6 ) for each treatment . * P 0 . 05 versus Ctr .

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F Western blot analysis show Hh and proliferation target protein expression levels. Data show the mean ± SD of tumor (n = 6) for each treatment. *P 0.05 versus Ctr.


A Representative images ( low and high magnification ) of HE ( upper panel ) and Ki67immunohistochemistry staining ( lower panel ) of a human Daoy MB cell ‐ derived orthotopic tumor in NOD / SCIDmicecerebella after i . p . injections of GlaB . Scale bars represent 500 or 200 μm for HE staining , and 150 μm or 50 μm for Ki67 staining . B Representative tumor average volumes at the end of treatment . Error bars indicate SD . * P 0 . 05 . C Quantification of Ki67 staining from immunohistochemistry shown in ( A ) . Error bars indicate SD . * P 0 . 05 .

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A Representative images (low and high magnification) of HE (upper panel) and Ki67immunohistochemistry staining (lower panel) of a human Daoy MB cell‐derived orthotopic tumor in NOD/SCIDmicecerebella after i.p. injections of GlaB. Scale bars represent 500 or 200 μm for HE staining, and 150 μm or 50 μm for Ki67 staining.B Representative tumor average volumes at the end of treatment. Error bars indicate SD. *P 0.05.C Quantification of Ki67 staining from immunohistochemistry shown in (A). Error bars indicate SD. *P 0.05.


A , B ASZ001 BCC cells were treated with GlaB ( 5 μM ) or DMSO only . After the indicated times , a trypan blue count was performed to determine ( A ) the growth rate and ( B ) the percentage of cell death . Data show the mean ± SD of three independent experiments . * P 0 . 05 versus DMSO .

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A, B ASZ001 BCC cells were treated with GlaB (5 μM) or DMSO only. After the indicated times, a trypan blue count was performed to determine (A) the growth rate and (B) the percentage of cell death. Data show the mean ± SD of three independent experiments. *P 0.05 versus DMSO.


C Gli1 mRNA expression levels were determined by qRT - PCR after treatment of ASZ001 BCC cells with GlaB or DMSO only for the indicated times . Results were normalized to endogenous control ( β2 ‐ microglobulin and HPRT ) . Data show the mean ± SD of three independent experiments . * P 0 . 05 versus DMSO .

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C Gli1 mRNA expression levels were determined by qRT-PCR after treatment of ASZ001 BCC cells with GlaB or DMSO only for the indicated times. Results were normalized to endogenous control (β2‐microglobulin and HPRT). Data show the mean ± SD of three independent experiments. *P 0.05 versus DMSO.


D - H GlaB inhibits Gli1 ‐ dependent BCC tumor growth in ASZ001 BCC allografts in vivo . ( D ) Change of tumor volume during GlaB or vehicle treatment period ( 18 days ) .

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D-H GlaB inhibits Gli1‐dependent BCC tumor growth in ASZ001 BCC allografts in vivo. (D) Change of tumor volume during GlaB or vehicle treatment period (18 days).


D - H GlaB inhibits Gli1 ‐ dependent BCC tumor growth in ASZ001 BCC allografts in vivo . ( E ) Representative flank allografts average volumes ( upper panel ) .

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D-H GlaB inhibits Gli1‐dependent BCC tumor growth in ASZ001 BCC allografts in vivo. (E) Representative flank allografts average volumes (upper panel).


D - H GlaB inhibits Gli1 ‐ dependent BCC tumor growth in ASZ001 BCC allografts in vivo . ( F ) HE and immunohistochemistry staining of Gli1 , Ki67 and TUNEL of allograft tumor samples . Scale bars represent 50 μm for HE , 25 μm for Gli1 , 25 μm for Ki67 and 30 μm for TUNEL stainings . ( G ) Quantification of Ki67 and TUNEL stainings from immunohistochemistry shown in ( F )

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D-H GlaB inhibits Gli1‐dependent BCC tumor growth in ASZ001 BCC allografts in vivo. (F) HE and immunohistochemistry staining of Gli1, Ki67 and TUNEL of allograft tumor samples. Scale bars represent 50 μm for HE, 25 μm for Gli1, 25 μm for Ki67 and 30 μm for TUNEL stainings. (G) Quantification of Ki67 and TUNEL stainings from immunohistochemistry shown in (F)


D - H GlaB inhibits Gli1 ‐ dependent BCC tumor growth in ASZ001 BCC allografts in vivo . ( H ) qRT - PCR of Hh and proliferation target mRNA expression levels . Results were normalized to endogenous control ( β2 ‐ microglobulin and HPRT ) . Shown is the mean ± SD of tumor ( n = 6 ) for each treatment . * P 0 . 05 versus Ctr .

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D-H GlaB inhibits Gli1‐dependent BCC tumor growth in ASZ001 BCC allografts in vivo. (H) qRT-PCR of Hh and proliferation target mRNA expression levels. Results were normalized to endogenous control (β2‐microglobulin and HPRT). Shown is the mean ± SD of tumor (n = 6) for each treatment. *P 0.05 versus Ctr.


Adult mouse back skin sections immunostained for LYVE1 ( red ) , αSMA ( green ) and counterstained with DAPI ( blue ) . n = 3 - 4 skin samples per mouse , n = 3 - 4 mice . Scale bar , 50 μm . LV , lymphatic vessels ; apm , arrector pili muscle .

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Adult mouse back skin sections immunostained for LYVE1 (red), αSMA (green) and counterstained with DAPI (blue). n= 3 - 4 skin samples per mouse, n= 3 - 4 mice. Scale bar, 50 μm. LV, lymphatic vessels; apm, arrector pili muscle.


Back skin sections of P55 mouse skin immunostained for LYVE1 ( red ) , Tenascin - C ( green ) and counterstained with DAPI ( blue ) . n = 3 - 4 skin samples per mouse , n = 3 - 4 mice . Scale bar , 50 μm . TNC , Tenascin - C .

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Back skin sections of P55 mouse skin immunostained for LYVE1 (red), Tenascin-C (green) and counterstained with DAPI (blue). n= 3 - 4 skin samples per mouse, n= 3 - 4 mice. Scale bar, 50 μm. TNC, Tenascin-C.


Adult mouse back skin sections from different postnatal ( P ) days immunostained for LYVE1 ( red ) , Lhx2 ( green ) and counterstained with DAPI ( blue ) . n = 3 - 4 skin samples per mouse , n = 3 - 4 mice . Scale bar , 50 μm . epi , epidermis ; der , dermis .

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Adult mouse back skin sections from different postnatal (P) days immunostained for LYVE1 (red), Lhx2 (green) and counterstained with DAPI (blue). n= 3 - 4 skin samples per mouse, n= 3 - 4 mice. Scale bar, 50 μm. epi, epidermis; der, dermis.


Histogram of the quantification of the LV distance to HFSC positive for Lhx2 . ) . n = 3 - 4 skin samples per mouse , n = 3 - 4 mice .

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Histogram of the quantification of the LV distance to HFSC positive for Lhx2. ). n= 3 - 4 skin samples per mouse, n= 3 - 4 mice.


Back skin sections of P49 mouse skin immunostained for LYVE1 ( red ) , CD34 ( green ) and counterstained with DAPI ( blue ) . n = 3 - 4 skin samples per mouse , n = 3 - 4 mice . Scale bar , 100 μm . epi , epidermis ; der , dermis . White arrowheads denote the proximity of lymphatic capillaries to HFSC .

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Back skin sections of P49 mouse skin immunostained for LYVE1 (red), CD34 (green) and counterstained with DAPI (blue). n= 3 - 4 skin samples per mouse, n= 3 - 4 mice. Scale bar, 100 μm. epi, epidermis; der, dermis. White arrowheads denote the proximity of lymphatic capillaries to HFSC.


Histogram of the quantification of the LV distance to HFSC positive for CD34 . n = 3 - 4 skin samples per mouse , n = 3 - 4 mice . C , Catagen ; Te , early Telogen ; Tm , mid Telogen ; Tl , late Telogen ; A , Anagen .

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Histogram of the quantification of the LV distance to HFSC positive for CD34. n= 3 - 4 skin samples per mouse, n= 3 - 4 mice. C, Catagen; Te, early Telogen; Tm, mid Telogen; Tl, late Telogen; A, Anagen.


Back skin sections of K15CrePR + / T ; Wls ∆ / ∆ and Control K15CrePR + / + ; Wlsflox / flox mice treated with mifepristone from P7 during 12 weeks ( P90 ) , immunostained for LYVE1 ( red ) , and counterstained with DAPI ( blue ) . n = 3 - 4 skin samples per mouse , n = 3 - 4 mice . Scale bar , 100 μm . epi , epidermis ; der , dermis .

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Back skin sections of K15CrePR+/T; Wls∆/∆ and Control K15CrePR+/+; Wlsflox/flox mice treated with mifepristone from P7 during 12 weeks (P90), immunostained for LYVE1 (red), and counterstained with DAPI (blue). n= 3 - 4 skin samples per mouse, n= 3 - 4 mice. Scale bar, 100 μm. epi, epidermis; der, dermis.


Histogram of the quantification of the LV angle relative to the epidermis . n = 3 - 4 skin samples per mouse , n = 3 - 4 mice .

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Histogram of the quantification of the LV angle relative to the epidermis. n= 3 - 4 skin samples per mouse, n= 3 - 4 mice.


Distribution of LV and HF at the mouse embryo stage E15 . 5 ( A ) , E16 . 5 ( B ) and E17 . 5 ( C ) . Maximum projection images of whole mount immunofluorescence analyses using LYVE1 ( red ) as lymphatic endothelial marker and counterstained with DAPI ( blue ) . Red arrowheads denote HF placodes . n = 3 - 4 embryos . Scale bar , 50 μm .

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Distribution of LV and HF at the mouse embryo stage E15.5 (A), E16.5 (B) and E17.5 (C). Maximum projection images of whole mount immunofluorescence analyses using LYVE1 (red) as lymphatic endothelial marker and counterstained with DAPI (blue). Red arrowheads denote HF placodes. n= 3 - 4 embryos. Scale bar, 50 μm.


LV associated with individual HF further organize associating HF triads , which connect with other HF triads across the skin . P70 , 70 d old mice . n = 3 - 4 mice . White arrowheads denote capillaries stemming from HF triads . Scale bar , 100 μm .

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LV associated with individual HF further organize associating HF triads, which connect with other HF triads across the skin. P70, 70 d old mice. n= 3 - 4 mice. White arrowheads denote capillaries stemming from HF triads. Scale bar, 100 μm.


3D reconstructions of whole skin mounts showing a triad of HF connected by LV . n = 3 - 4 mice . Scale bar , 50 μm . White arrowhead denotes an extending lymphatic capillary from a HF triad .

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3D reconstructions of whole skin mounts showing a triad of HF connected by LV. n= 3 - 4 mice. Scale bar, 50 μm. White arrowhead denotes an extending lymphatic capillary from a HF triad.


Images of intravital microscopy analyses showing aligned HF rows in the back skin interconnected by LV in the Prox1CreERT2 ; Rosa - LSL - eYPF mice . Dotted boxes denote HF triads ; White arrowheads denote capillaries stemming from HF triads interconnecting to other HF triads in adjacent rows . n = 3 - 4 mice . Scale bar , 50 μm .

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Images of intravital microscopy analyses showing aligned HF rows in the back skin interconnected by LV in the Prox1CreERT2; Rosa-LSL-eYPF mice. Dotted boxes denote HF triads; White arrowheads denote capillaries stemming from HF triads interconnecting to other HF triads in adjacent rows. n= 3 - 4 mice. Scale bar, 50 μm.


Adult back skin sections from different postnatal ( P ) days immunostained for LYVE1 ( red ) and counterstained with DAPI ( blue ) . n = 3 - 4 skin samples per mouse , n = 3 - 4 mice . Scale bar , 10 μm . White arrowheads denote LV membrane protrusion and fenestrated areas . LV , lymphatic vessels ; HF , hair follicle .

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Adult back skin sections from different postnatal (P) days immunostained for LYVE1 (red) and counterstained with DAPI (blue). n= 3 - 4 skin samples per mouse, n= 3 - 4 mice. Scale bar, 10 μm. White arrowheads denote LV membrane protrusion and fenestrated areas. LV, lymphatic vessels; HF, hair follicle.


Histogram of the caliber ( μm ) of LV at different postnatal days . n = 3 - 4 skin samples per mouse , n = 3 - 4 mice . A , Anagen ; C , Catagen ; T , Telogen ; Te , early Telogen ; Tm , mid Telogen ; Tl , late Telogen .

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Histogram of the caliber (μm) of LV at different postnatal days. n= 3 - 4 skin samples per mouse, n= 3 - 4 mice. A, Anagen; C, Catagen; T, Telogen; Te, early Telogen; Tm, mid Telogen; Tl, late Telogen.


H & E staining of back skin sections from mice treated intradermally with Control or Clodronate liposomes . n = 3 - 4 skin samples per mouse , n = 3 - 4 mice . Scale bar , 200 μm . epi , epidermis ; der , dermis .

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H&E staining of back skin sections from mice treated intradermally with Control or Clodronate liposomes. n= 3 - 4 skin samples per mouse, n= 3 - 4 mice. Scale bar, 200 μm. epi, epidermis; der, dermis.


LYVE1 immunofluorescence ( red ) counterstained with DAPI ( blue ) ( D ) in back skin sections from mice treated intradermally with Control or Clodronate liposomes . n = 3 - 4 skin samples per mouse , n = 3 - 4 mice . Scale bar , 100 μm . epi , epidermis ; der , dermis .

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LYVE1 immunofluorescence (red) counterstained with DAPI (blue) (D) in back skin sections from mice treated intradermally with Control or Clodronate liposomes. n= 3 - 4 skin samples per mouse, n= 3 - 4 mice. Scale bar, 100 μm. epi, epidermis; der, dermis.


Histogram of the caliber ( μm ) of LV ( E ) in back skin sections from mice treated intradermally with Control or Clodronate liposomes .

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Histogram of the caliber (μm) of LV (E) in back skin sections from mice treated intradermally with Control or Clodronate liposomes.


Back skin sections of Controls and K14ΔNβ - cateninER + / T mice immunostained for LYVE1 ( red ) , and counterstained with DAPI ( blue ) . n = 3 - 4 skin samples per mouse , n = 3 - 4 mice . Scale bar , 50 μm . epi , epidermis ; der , dermis .

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Back skin sections of Controls and K14ΔNβ-cateninER+/T mice immunostained for LYVE1 (red), and counterstained with DAPI (blue). n= 3 - 4 skin samples per mouse, n= 3 - 4 mice. Scale bar, 50 μm. epi, epidermis; der, dermis.


Histogram of the LV caliber in skin sections from Controls and K14ΔNβ - cateninER + / T mice . n = 3 - 4 skin samples per mouse , n = 3 - 4 mice .

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Histogram of the LV caliber in skin sections from Controls and K14ΔNβ-cateninER+/T mice. n= 3 - 4 skin samples per mouse, n= 3 - 4 mice.


Histogram of the percentage of HF in Telogen and Anagen present in the skin of control mice or mice treated with Cilostazol . n = 3 - 4 skin samples per mouse , n = 3 - 4 mice .

Histogram of the percentage of HF in Telogen and Anagen present in the skin of control mice or mice treated with Cilostazol. n= 3 - 4 skin samples per mouse, n= 3 - 4 mice.


Gene set enrichment plots ( GSEA ) of LV using public annotations showing the enrichment of selected gene signatures involved in different pathways in late Telogen ( P69 , Tl ) compared with mid Telogen ( P55 , Tm ) . NES : normalized enrichment score . The false discovery rate ( FDR ; q - value ) is indicated .

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Gene set enrichment plots (GSEA) of LV using public annotations showing the enrichment of selected gene signatures involved in different pathways in late Telogen (P69, Tl) compared with mid Telogen (P55, Tm). NES: normalized enrichment score. The false discovery rate (FDR; q-value) is indicated.


Heat map diagram of DEGs in LV during late Telogen compared to mid Telogen , relative to biological processes , analyzed using the Enrichr platform ( Chen et al . , 2013 ; Kuleshov et al . , 2016 ) . Data shown represent the log2 ( FPKM + 1 ) .

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Heat map diagram of DEGs in LV during late Telogen compared to mid Telogen, relative to biological processes, analyzed using the Enrichr platform (Chen et al., 2013; Kuleshov et al., 2016). Data shown represent the log2 (FPKM+1).


Histograms of RNA ISH analyses by RNAscope to quantify the level of expression of ITGA5 ( D ) , DCN ( E ) , PKD1 ( F ) and PLXND1 ( G ) , assessed by the number of mRNA spots present in LYVE1 + cells in the vicinity of HF in P55 and P70 mouse back skin . n = average of 30 LYVE1 + cells / sample , n = 3 mice .

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Histograms of RNA ISH analyses by RNAscope to quantify the level of expression of ITGA5 (D), DCN (E), PKD1 (F) and PLXND1 (G), assessed by the number of mRNA spots present in LYVE1+ cells in the vicinity of HF in P55 and P70 mouse back skin. n= average of 30 LYVE1+ cells / sample, n= 3 mice.


Histograms represent the mean fluorescence intensity for Jup ( H ) and Emilin1 ( I ) in LYVE1 + cells in the vicinity of HF in P55 and P70 mouse back skin . n = 3 - 4 skin samples per mouse , n = 3 - 4 mice .

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Histograms represent the mean fluorescence intensity for Jup (H) and Emilin1 (I) in LYVE1+ cells in the vicinity of HF in P55 and P70 mouse back skin. n= 3 - 4 skin samples per mouse, n= 3 - 4 mice.


H & E staining ( A ) and histogram of the HF length ( B ) in adult back skin sections from Prox1CreERT2 + / + ; LSL - ROSA26 - iDTRKI / KI mice injected with vehicles ( Control ) , Prox1CreERT2 + / T ; LSL - ROSA26 - iDTRKI / KI mice treated with CSA and vehicle ( CSA ) ; and Prox1CreERT2 + / T ; LSL - ROSA26 - iDTRKI / KI treated with tamoxifen , CSA and intradermal Diphtheria Toxin ( CSA + DT ) starting from early Telogen ( P49 ) and analyzed at the end of the treatments ( P58 ) . n = 3 - 4 skin samples per mouse , n = 3 - 4 mice .

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H&E staining (A) and histogram of the HF length (B) in adult back skin sections from Prox1CreERT2+/+; LSL-ROSA26-iDTRKI/KI mice injected with vehicles (Control), Prox1CreERT2+/T; LSL-ROSA26-iDTRKI/KI mice treated with CSA and vehicle (CSA); and Prox1CreERT2+/T; LSL-ROSA26-iDTRKI/KI treated with tamoxifen, CSA and intradermal Diphtheria Toxin (CSA+DT) starting from early Telogen (P49) and analyzed at the end of the treatments (P58). n= 3 - 4 skin samples per mouse, n= 3 - 4 mice.


Ki67 immunostaining ( C ) and histogram of the number of Ki67 + cells per HF ( D ) in adult back skin sections from Prox1CreERT2 + / + ; LSL - ROSA26 - iDTRKI / KI mice injected with vehicles ( Control ) , Prox1CreERT2 + / T ; LSL - ROSA26 - iDTRKI / KI mice treated with CSA and vehicle ; and Prox1CreERT2 + / T ; LSL - ROSA26 - iDTRKI / KI treated with tamoxifen , CSA and intradermal DT , starting from early Telogen ( P49 ) and analyzed at the end of the treatments ( P58 ) . n = 3 - 4 skin samples per mouse , n = 3 - 4 mice .

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Ki67 immunostaining (C) and histogram of the number of Ki67+ cells per HF (D) in adult back skin sections from Prox1CreERT2+/+; LSL-ROSA26-iDTRKI/KI mice injected with vehicles (Control), Prox1CreERT2+/T; LSL-ROSA26-iDTRKI/KI mice treated with CSA and vehicle; and Prox1CreERT2+/T; LSL-ROSA26-iDTRKI/KI treated with tamoxifen, CSA and intradermal DT, starting from early Telogen (P49) and analyzed at the end of the treatments (P58). n= 3 - 4 skin samples per mouse, n= 3 - 4 mice.


LYVE1 immunostaining ( E ) and histogram of the LV caliber ( F ) analyzed in adult back skin sections from Prox1CreERT2 + / + ; LSL - ROSA26 - iDTRKI / KI mice injected with vehicles ( Control ) , Prox1CreERT2 + / T ; LSL - ROSA26 - iDTRKI / KI mice treated with CSA and vehicle ( CSA ) ; and Prox1CreERT2 + / T ; LSL - ROSA26 - iDTRKI / KI mice treated with tamoxifen , CSA and intradermal DT , starting from early Telogen ( P49 ) and analyzed at the end of the treatments ( P58 ) . Scale bar , 10 μm . n = 3 - 4 skin samples per mouse , n = 3 - 4 mice .

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LYVE1 immunostaining (E) and histogram of the LV caliber (F) analyzed in adult back skin sections from Prox1CreERT2+/+; LSL-ROSA26-iDTRKI/KI mice injected with vehicles (Control), Prox1CreERT2+/T; LSL-ROSA26-iDTRKI/KI mice treated with CSA and vehicle (CSA); and Prox1CreERT2+/T; LSL-ROSA26-iDTRKI/KI mice treated with tamoxifen, CSA and intradermal DT, starting from early Telogen (P49) and analyzed at the end of the treatments (P58). Scale bar, 10 μm. n= 3 - 4 skin samples per mouse, n= 3 - 4 mice.


H & E staining ( G ) and histogram of the HF length ( H ) in adult back skin sections from Prox1CreERT2 + / + ; LSL - ROSA26 - iDTRKI / KI mice ( Control ) , and Prox1CreERT2 + / T ; LSL - ROSA26 - iDTRKI / KI mice treated with tamoxifen and intradermal DT , starting from Anagen ( P30 ) and analyzed at the end of the treatments ( P37 ) . n = 3 - 4 skin samples per mouse , n = 3 - 4 mice . Scale bar , 200 μm . Data represent the mean value ± SEM .

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H&E staining (G) and histogram of the HF length (H) in adult back skin sections from Prox1CreERT2+/+; LSL-ROSA26-iDTRKI/KI mice (Control), and Prox1CreERT2+/T; LSL-ROSA26-iDTRKI/KI mice treated with tamoxifen and intradermal DT, starting from Anagen (P30) and analyzed at the end of the treatments (P37). n= 3 - 4 skin samples per mouse, n= 3 - 4 mice. Scale bar, 200 μm. Data represent the mean value ± SEM.


Cleaved caspase 3 immunostaining ( I ) and histogram of the number of cleaved caspase 3 + cells per HF ( J ) in adult back skin sections from Prox1CreERT2 + / + ; LSL - ROSA26 - iDTRKI / KI mice ( Control ) , and Prox1CreERT2 + / T ; LSL - ROSA26 - iDTRKI / KI mice treated with tamoxifen and intradermal DT , starting from Anagen ( P30 ) and analyzed at the end of the treatments ( P37 ) . Scale bar , 50 μm . n = 3 - 4 skin samples per mouse , n = 3 - 4 mice .

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

Cleaved caspase 3 immunostaining (I) and histogram of the number of cleaved caspase 3+ cells per HF (J) in adult back skin sections from Prox1CreERT2+/+; LSL-ROSA26-iDTRKI/KI mice (Control), and Prox1CreERT2+/T; LSL-ROSA26-iDTRKI/KI mice treated with tamoxifen and intradermal DT, starting from Anagen (P30) and analyzed at the end of the treatments (P37). Scale bar, 50 μm. n= 3 - 4 skin samples per mouse, n= 3 - 4 mice.


A , SW620 and HCT116 cells were treated with the indicated concentrations of SB505124 ( SB ) or LGK974 ( LGK ) for 16 h . Total cell extracts were analyzed by WB . Protein quantification was analyzed by densitometry of three different experiments and represented B , SW620 and HCT116 cells expressing a shCtl or shRor2 vectors were analyzed by WB . Quantification of the WBs from three biological replicates are presented in the right panel . Values correspond to fold variation in shRor2 versus shCtl cells ( mean ± SD ) .

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A, SW620 and HCT116 cells were treated with the indicated concentrations of SB505124 (SB) or LGK974 (LGK) for 16 h. Total cell extracts were analyzed by WB. Protein quantification was analyzed by densitometry of three different experiments and represented B, SW620 and HCT116 cells expressing a shCtl or shRor2 vectors were analyzed by WB. Quantification of the WBs from three biological replicates are presented in the right panel. Values correspond to fold variation in shRor2 versus shCtl cells (mean ± SD).


C , RNA was isolated from shCtl or shRor2 SW620 and HCT116 cells and the expression of the indicated genes was assessed by qRT - PCR . Values correspond to fold variation in shRor2 versus shCtl cells . Results are the mean ± SD of at least three biological replicates performed in triplicate .

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C, RNA was isolated from shCtl or shRor2 SW620 and HCT116 cells and the expression of the indicated genes was assessed by qRT-PCR. Values correspond to fold variation in shRor2 versus shCtl cells. Results are the mean ± SD of at least three biological replicates performed in triplicate.


D , SW620 and HCT116 cells expressing a shCtl or shRor2 vectors were analyzed by WB . Quantification of the WBs are presented in Appendix Fig S3D as in B .

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D, SW620 and HCT116 cells expressing a shCtl or shRor2 vectors were analyzed by WB. Quantification of the WBs are presented in Appendix Fig S3D as in B.


E , HCT116 or SW620 cells were treated with conditioned medium from control or Wnt5a - producing cells for 16 h and expression of the indicated genes assessed by qRT - PCR ( E ) ( mean ± SD of three biological replicates performed in triplicate in E ) .

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E, HCT116 or SW620 cells were treated with conditioned medium from control or Wnt5a-producing cells for 16 h and expression of the indicated genes assessed by qRT-PCR (E) (mean ± SD of three biological replicates performed in triplicate in E).


G , WB analysis of HT29 M6 and SW480 overexpressing Snail1 . Quantification is presented in Appendix Fig S5I .

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G, WB analysis of HT29 M6 and SW480 overexpressing Snail1. Quantification is presented in Appendix Fig S5I.


I , SW620 and HCT116 cells were transfected with control or Snail1 siRNA and analyzed by qRT - PCR ( I , mean ± SD of three biological replicates performed in triplicate ) .

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I, SW620 and HCT116 cells were transfected with control or Snail1 siRNA and analyzed by qRT-PCR (I, mean ± SD of three biological replicates performed in triplicate).


A , Smad2 phosphorylation was determined in extracts from shCtl or shRor2 SW620 and HCT116 cells . The quantification is presented in the right panel ( mean ± SD or three biological replicates ) .

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A, Smad2 phosphorylation was determined in extracts from shCtl or shRor2 SW620 and HCT116 cells. The quantification is presented in the right panel (mean ± SD or three biological replicates).


D , MSC were treated with the conditioned media from shCtl or shRor2 SW620 , D ) for the indicated times . TGFβ receptor inhibitor SB505124 ( SB , 5 μM ) was added when indicated in D . Cells were lysed and proteins analyzed by WB . Quantification of the protein levels from D is represented at the lower panel ( mean ± SD of three biological replicates )

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D, MSC were treated with the conditioned media from shCtl or shRor2 SW620 , D) for the indicated times. TGFβ receptor inhibitor SB505124 (SB, 5 μM) was added when indicated in D. Cells were lysed and proteins analyzed by WB. Quantification of the protein levels from D is represented at the lower panel (mean ± SD of three biological replicates)


E , GFP - labelled MSC were seeded in Matrigel - coated transwells with SW620 and HCT116 cells , and supplemented with SB when indicated . After 48 h non - invading cells were removed from the upper surface of the membrane and cells present at the lower surface were fixed . GFP - labelled cells were counted in five different fields per filter by ImageJ software and represented ( bottom ) . A representative picture of SW620 cells in each condition is shown ( top ) . The bar corresponds to 500 μm . The figure shows the mean ± SD of three experiments .

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E, GFP-labelled MSC were seeded in Matrigel-coated transwells with SW620 and HCT116 cells, and supplemented with SB when indicated. After 48 h non-invading cells were removed from the upper surface of the membrane and cells present at the lower surface were fixed. GFP-labelled cells were counted in five different fields per filter by ImageJ software and represented (bottom). A representative picture of SW620 cells in each condition is shown (top). The bar corresponds to 500 μm. The figure shows the mean ± SD of three experiments.


C , Representative micrographs of the effects of Ror2 depletion on HCT116 cells soft - agar colony formation ( 21 days ) ( left ) . Representative micrographs of SW620 cells are shown in Appendix Fig S7B . Results show the mean ± SD of the number of colonies in triplicate samples of three independent experiments ( right ) .

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C, Representative micrographs of the effects of Ror2 depletion on HCT116 cells soft-agar colony formation (21 days) (left). Representative micrographs of SW620 cells are shown in Appendix Fig S7B. Results show the mean ± SD of the number of colonies in triplicate samples of three independent experiments (right).


Cells were seeded for tumor - sphere formation in tumor organoid complete medium . In E , SW620 cells were supplemented with LGK ( 10 μM ) , starting the day of seeding . After four days , the wells were stained ( top ) and the number of colonies counted and represented ( bottom ) ( mean ± SD of three biological replicates ) . A more detailed micrograph of the colonies is shown in Appendix Fig S7D .

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Cells were seeded for tumor-sphere formation in tumor organoid complete medium. In E, SW620 cells were supplemented with LGK (10 μM), starting the day of seeding. After four days, the wells were stained (top) and the number of colonies counted and represented (bottom) (mean ± SD of three biological replicates). A more detailed micrograph of the colonies is shown in Appendix Fig S7D.


F , RNA was isolated from SW620 control , stably - depleted of Ror2 , treated with LGK974 for 16 h or transfected with siSnail1 and the expression of the stem cell gene markers LGR5 , ASCL2 , SMOC2 and the differentiation marker KRT20 was assessed by qRT - PCR . The figure shows the mean ± SD of at least three biological replicates ( performed in triplicate ) represented with respect to the corresponding control ( dashed line ) .

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F, RNA was isolated from SW620 control, stably-depleted of Ror2, treated with LGK974 for 16 h or transfected with siSnail1 and the expression of the stem cell gene markers LGR5, ASCL2, SMOC2 and the differentiation marker KRT20 was assessed by qRT-PCR. The figure shows the mean ± SD of at least three biological replicates (performed in triplicate) represented with respect to the corresponding control (dashed line).


B , C , Representative images of Fibronectin and Vimentin staining in consecutive sections from paraffin - embedded tumors obtained when tumors were initially detected ( days 18 or 22 ) . Staining with an anti - human antibody is also enclosed . The scale bar corresponds to 10 μm ( B ) . Fibronectin and Vimentin staining were quantified as described in Methods ( six tumors , three areas per tumor ) and represented in C ( mean ± SD ) .

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B, C, Representative images of Fibronectin and Vimentin staining in consecutive sections from paraffin-embedded tumors obtained when tumors were initially detected (days 18 or 22). Staining with an anti-human antibody is also enclosed. The scale bar corresponds to 10 μm (B). Fibronectin and Vimentin staining were quantified as described in Methods (six tumors, three areas per tumor) and represented in C (mean ± SD).


D , Representative images of the invading front of the tumors . The scale bar corresponds to 4 μM . E , Percentage of tumors showing muscle invasion .

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D, Representative images of the invading front of the tumors. The scale bar corresponds to 4 μM. E, Percentage of tumors showing muscle invasion.


F , G , In vivo tumor growth of MTO cells subcutaneously injected in NOD / SCID mice ( F ) ( 5x105 cells / flank ; eight tumors per condition ) and treated with WntC59 ( 10 mg / kg / day in the drinking water ) or vehicle . Tumor weight of NOD / SCID mice at day 26 is presented in G . In F and G , the mean ± SD of the eight tumors is shown .

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F, G, In vivo tumor growth of MTO cells subcutaneously injected in NOD/SCID mice (F) (5x105 cells/flank; eight tumors per condition) and treated with WntC59 (10 mg/kg/day in the drinking water) or vehicle. Tumor weight of NOD/SCID mice at day 26 is presented in G. In F and G, the mean ± SD of the eight tumors is shown.


I , J , WB ( I ) and RNA analysis ( J ) of MTO cells infected with shRor2 or shCtl . Quantification of the WB is also presented ( I , right ) . Results are the mean ± SD of three biological replicates ( performed in triplicated in J ) .

I, J, WB (I) and RNA analysis (J) of MTO cells infected with shRor2 or shCtl. Quantification of the WB is also presented (I, right). Results are the mean ± SD of three biological replicates (performed in triplicated in J).


N , O , C57BL6 mice were injected in the spleen with MTO cells and treated since day 1 with WntC59 or vehicle . In N , kinetics of in vivo luminescence ( mean ± SD of the values in five mice ) ; in O , the percentage of mice showing in vivo luminescence .

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N, O, C57BL6 mice were injected in the spleen with MTO cells and treated since day 1 with WntC59 or vehicle. In N, kinetics of in vivo luminescence (mean ± SD of the values in five mice); in O, the percentage of mice showing in vivo luminescence.


A - C , Viability was assayed by MTT in the indicated cells treated with LGK when specified and supplemented with cisplatin for 24 h . In B , cisplatin resistance was assessed in shRor2 cells transfected with a Snail1 expression plasmid or the corresponding empty plasmid . The figure shows the mean ± SD of the results of three experiments .

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A-C, Viability was assayed by MTT in the indicated cells treated with LGK when specified and supplemented with cisplatin for 24 h. In B, cisplatin resistance was assessed in shRor2 cells transfected with a Snail1 expression plasmid or the corresponding empty plasmid. The figure shows the mean ± SD of the results of three experiments.


D , Cells were treated with cisplatin for the indicated times . Total extracts were lysed and analyzed by WB .

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D, Cells were treated with cisplatin for the indicated times. Total extracts were lysed and analyzed by WB.


I , MTO cell viability was determined as above ( mean ± SD of the results of three - four experiments ) .

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I, MTO cell viability was determined as above (mean ± SD of the results of three-four experiments).


A Quantification and representative immunoblots of lamin B1 in the striatum ( 8w , 12w and 20w ; N = 6 for both genotypes ; 30w ; N = 4 and 5 for WT and R6 / 1 mice , respectively ) , hippocampus ( 8w ; N = 8 and 6 for WT and R6 / 1 mice , respectively ; 12w ; N = 7 and 6 for WT and R6 / 1 mice , respectively ; 20w N = 5 and 6 for WT and R6 / 1 mice , respectively ; 30w ; N = 8 and 5 for WT and R6 / 1 mice , respectively ) and cortex ( 8w ; N = 5 and 4 for WT and R6 / 1 mice , respectively ; 12w and 20w ; N = 6 for both genotypes ; 30w ; N = 6 and 5 for WT and R6 / 1 mice , respectively ) . Data information : Data is expressed as a percentage of controls . Each point corresponds to the value from an individual sample . Bars represent the mean ± S . E . M . * P < 0 . 05 , * * P < 0 . 01 and * * * P < 0 . 001 as compared with WT mice ( two - tailed unpaired Student ' s t test ) . Tubulin was used as loading control . Exact P values are reported in Appendix Table S3 .

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A Quantification and representative immunoblots of lamin B1 in the striatum (8w, 12w and 20w; N = 6 for both genotypes; 30w; N = 4 and 5 for WT and R6/1 mice, respectively), hippocampus (8w; N = 8 and 6 for WT and R6/1 mice, respectively; 12w; N = 7 and 6 for WT and R6/1 mice, respectively; 20w N = 5 and 6 for WT and R6/1 mice, respectively; 30w; N = 8 and 5 for WT and R6/1 mice, respectively) and cortex (8w; N = 5 and 4 for WT and R6/1 mice, respectively; 12w and 20w; N = 6 for both genotypes; 30w; N = 6 and 5 for WT and R6/1 mice, respectively). Data information: Data is expressed as a percentage of controls. Each point corresponds to the value from an individual sample. Bars represent the mean ± S.E.M. *P < 0.05, **P < 0.01 and ***P < 0.001 as compared with WT mice (two-tailed unpaired Student's t test). Tubulin was used as loading control. Exact P values are reported in Appendix Table S3.