Activation of pS6 ( Ser235 / 236 ) and ( Ser240 / 244 ) ( M ) . Data shown in bar graphs are mean fluorescence intensity of indicated protein expression in PKCδ cKO HSPCs subsets relative to WT controls ( n = 4 - 6 mice per genotype per target ) . Data information : The statistical significance of difference was assessed using Two - tailed Student ' s unpaired t - test analysis for comparison of control and PKCδ cKO HSPCs . All data are presented as mean ± SEM . , * p < 0 . 05 , * * p < 0 . 001 .

Activation of pS6 (Ser235/236) and (Ser240/244) (M). Data shown in bar graphs are mean fluorescence intensity of indicated protein expression in PKCδ cKO HSPCs subsets relative to WT controls (n=4-6 mice per genotype per target). Data information: The statistical significance of difference was assessed using Two-tailed Student's unpaired t-test analysis for comparison of control and PKCδ cKO HSPCs. All data are presented as mean± SEM., *p<0.05, **p<0.001.


A Germination of dog1 - 3 ( knock - out ) , WT , and dog1 - 5 ( DOG1 upregulation ) seeds in media supplemented with NaCl for the indicated number of days after stratification ( DAS ) . Scale bar represents 5 mm . B Germination rate in 100 mM NaCl 7 days after stratification for seeds of different genotypes . Lines represent the fitted curves with a 95 % confidence interval ( grey area ) . ∗ p - value < 0 . 05 from two - tailed Student ' s t - test . Data for WT is the same as plotted in Fig 3C .

O O O 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 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

A Germination of dog1-3 (knock-out), WT, and dog1-5 (DOG1 upregulation) seeds in media supplemented with NaCl for the indicated number of days after stratification (DAS). Scale bar represents 5 mm. B Germination rate in 100 mM NaCl 7 days after stratification for seeds of different genotypes. Lines represent the fitted curves with a 95% confidence interval (grey area). ∗ p-value < 0.05 from two-tailed Student's t-test. Data for WT is the same as plotted in Fig 3C. C


C shDOG1 expression levels normalized to UBC21 ( AT5G25760 ) in dog1 - 3 and dog1 - 5 relative to WT . D shDOG1 and lgDOG1 expression levels normalized to UBC21 in seeds treated with 100 mM NaCl relative to mock .

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

C shDOG1 expression levels normalized to UBC21 (AT5G25760) in dog1-3 and dog1-5 relative to WT. D shDOG1 and lgDOG1 expression levels normalized to UBC21 in seeds treated with 100 mM NaCl relative to mock. E


A 3 ' RNA - seq read coverage on DOG1 locus . Black colour is used for coverage within the scale on the right - hand side ( 0 to 500 ) , and red is used for coverage within the scale on the left - hand side ( 0 to 50 ) . Above is a schematic representation of the annotated transcripts from the DOG1 locus and the chromosome coordinates .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 3'RNA-seq read coverage on DOG1 locus. Black colour is used for coverage within the scale on the right-hand side (0 to 500), and red is used for coverage within the scale on the left-hand side (0 to 50). Above is a schematic representation of the annotated transcripts from the DOG1 locus and the chromosome coordinates.


Germination time - course in 100 mM NaCl after stratification for puppies - 1 ( C ) relative to WT . Lines represent fitted curves with a 95 % confidence interval ( grey area ) , dots represent data points , ∗ p - value < 0 . 05 from the two - tailed Student ' s t - test . ( C ) Data for WT is the same as

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

Germination time-course in 100 mM NaCl after stratification for puppies-1 (C) relative to WT. Lines represent fitted curves with a 95% confidence interval (grey area), dots represent data points, ∗ p-value < 0.05 from the two-tailed Student's t-test. (C) Data for WT is the same as plotted


F RT - qPCR fold - change induction of PUPPIES - uns , PUPPIES - prom , PUPPIES - fusion , shDOG1 , and lgDOG1 in puppies - ox relative to WT ( blue dashed line ) , in dry ( light grey ) and imbibed seeds in the presence of 100 mM NaCl ( red ) . Significance from the two - tailed Student ' s t - test for comparing dry seeds of WT and puppies - ox is represented with light grey asterisks , and salt - imbibed seeds of WT and puppies - ox are represented with red asterisks . Black asterisks represent significance from two - way ANOVA with Tukey ' s multiple comparisons test for dry seeds vs imbibed seeds . ns p - value > 0 . 05 , ∗ p - value < 0 . 05 , ∗ ∗ p - value < 0 . 01 , ∗ ∗ ∗ p - value < 0 . 001 . Error bars represent the mean ± SD . n = 4 biological replicates .

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

F RT-qPCR fold-change induction of PUPPIES-uns, PUPPIES-prom, PUPPIES-fusion, shDOG1, and lgDOG1 in puppies-ox relative to WT (blue dashed line), in dry (light grey) and imbibed seeds in the presence of 100 mM NaCl (red). Significance from the two-tailed Student's t-test for comparing dry seeds of WT and puppies-ox is represented with light grey asterisks, and salt-imbibed seeds of WT and puppies-ox are represented with red asterisks. Black asterisks represent significance from two-way ANOVA with Tukey's multiple comparisons test for dry seeds vs imbibed seeds. ns p-value > 0.05, ∗ p-value < 0.05, ∗∗ p-value < 0.01, ∗∗∗ p-value < 0.001. Error bars represent the mean ± SD. n = 4 biological replicates.


G RT - qPCR with primers for shDOG1 and lgDOG1 on nascent RNA from seeds imbibed in 100 mM NaCl from puppies - ox relative to WT . Nascent RNA levels were normalized to UBC21 . Bars and error bars represent the mean ± SD . Points represent biological replicates . Statistical significance from two - tailed Student ' s t - test . ∗ p - value < 0 . 05 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 RT-qPCR with primers for shDOG1 and lgDOG1 on nascent RNA from seeds imbibed in 100 mM NaCl from puppies-ox relative to WT. Nascent RNA levels were normalized to UBC21. Bars and error bars represent the mean ± SD. Points represent biological replicates. Statistical significance from two-tailed Student's t-test. ∗ p-value < 0.05.


A z - stack max - projection image of smFISH for DOG1 RNA . The " Inferno " colour scale is used for the intensity of fluorescence from Quasar670 fluorophore ( DOG1 ) . The blue colour shows fluorescence from DAPI ( nuclei staining ) . Arrowheads point to foci corresponding to transcription sites ( TS ) . The scale bar is 20 μm . B Distribution of cytoplasmic DOG1 foci per cell in WT . The blue vertical dashed line indicates the average .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 z-stack max-projection image of smFISH for DOG1 RNA. The "Inferno" colour scale is used for the intensity of fluorescence from Quasar670 fluorophore (DOG1). The blue colour shows fluorescence from DAPI (nuclei staining). Arrowheads point to foci corresponding to transcription sites (TS). The scale bar is 20 μm. B Distribution of cytoplasmic DOG1 foci per cell in WT. The blue vertical dashed line indicates the average. C


G z - stack max - projection images from seeds imbibed in 100 mM NaCl of WT ( left ) versus puppies - ox ( right ) . Arrowheads point to foci corresponding to TS .

O O O O O O O O O O O O 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 z-stack max-projection images from seeds imbibed in 100 mM NaCl of WT (left) versus puppies-ox (right). Arrowheads point to foci corresponding to TS.


A - E The profile of Pol II during DOG1 transcription . Plots show the fraction of unique reads from targeted NET - Seq using rolling median ( 11 nt ) . Targeted libraries for sequencing of DOG1 locus were obtained by usage of 5 primers across the gene . Pol II dynamics is analyzed separately for each region sequenced in exon 1 ( A ) , exon 2 ( B ) , exon 3 ( C ) , intron 1 ( D ) , and intron 2 ( E ) . The x - axis shows the distance in bp from DOG1 . A light red colour fill highlights the regions where Pol II density is higher in puppies - ox . A light blue colour fill highlights the regions where Pol II density is higher in WT . For each plot , coloured tiles represent the fold - change ( puppies - ox / WT ) on a logarithmic scale . Greyscale tiles represent the result of a statistical test for the difference between genotypes from the two - tailed Student ' s t - test after Bonferroni correction . Tiles correspond to a 25 bp bin each . Vertical dashed lines connect the exon boundaries on the plots with the schematics of the DOG1 gene with grey boxes representing exons and black lines representing introns .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-E The profile of Pol II during DOG1 transcription. Plots show the fraction of unique reads from targeted NET-Seq using rolling median (11 nt). Targeted libraries for sequencing of DOG1 locus were obtained by usage of 5 primers across the gene. Pol II dynamics is analyzed separately for each region sequenced in exon 1 (A), exon 2 (B), exon 3 (C), intron 1 (D), and intron 2 (E). The x-axis shows the distance in bp from DOG1. A light red colour fill highlights the regions where Pol II density is higher in puppies-ox. A light blue colour fill highlights the regions where Pol II density is higher in WT. For each plot, coloured tiles represent the fold-change (puppies-ox / WT) on a logarithmic scale. Greyscale tiles represent the result of a statistical test for the difference between genotypes from the two-tailed Student's t-test after Bonferroni correction. Tiles correspond to a 25 bp bin each. Vertical dashed lines connect the exon boundaries on the plots with the schematics of the DOG1 gene with grey boxes representing exons and black lines representing introns.


F , G RT - qPCR measurement of alternative splicing of lgDOG1 alpha and beta isoforms and unspliced intron 1 DOG1 isoforms levels in puppies - ox , and puppies - 1 relative to WT , in seeds treated with 100 mM NaCl .

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

F, G RT-qPCR measurement of alternative splicing of lgDOG1 alpha and beta isoforms and unspliced intron 1 DOG1 isoforms levels in puppies-ox, and puppies-1 relative to WT, in seeds treated with 100 mM NaCl.


B . The in vitro synthesized [ 35S ] Met - labeled single - cysteine Bax proteins were activated and targeted to the mitochondria that were pretreated with NEM to block the sulfhydryls of mitochondrial proteins . The resulting mitochondria were isolated and oxidized by CuPhe for 30 min . NEM and EDTA were then added to stop the oxidation . For the " 0 min " controls , NEM and EDTA were added before the addition of CuPhe . The resulting samples were analyzed by phosphor - imaging after non - reducing or reducing SDS - PAGE ( see Appendix Figure S2A ) .

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

B. The in vitro synthesized [35S]Met-labeled single-cysteine Bax proteins were activated and targeted to the mitochondria that were pretreated with NEM to block the sulfhydryls of mitochondrial proteins. The resulting mitochondria were isolated and oxidized by CuPhe for 30 min. NEM and EDTA were then added to stop the oxidation. For the "0 min" controls, NEM and EDTA were added before the addition of CuPhe. The resulting samples were analyzed by phosphor-imaging after non-reducing or reducing SDS-PAGE (see Appendix Figure S2A).


C . Oxidized mitochondria with the radioactive single - cysteine Bax protein pair or double - cysteine Bax protein were prepared and analyzed as in ( B ) .

O O O O O 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. Oxidized mitochondria with the radioactive single-cysteine Bax protein pair or double-cysteine Bax protein were prepared and analyzed as in (B).


D . Oxidized mitochondria with the radioactive single - cysteine Bax protein pair or double - cysteine Bax protein were prepared as in ( B ) . After an aliquot was withdrawn as input , another aliquot was extracted by Na2CO3 ( pH 11 . 5 ) and centrifuged through a sucrose cushion to separate the integral proteins in the membrane pellet from the soluble and peripheral proteins in the supernatant . The input , pellet and supernatant were analyzed by non - reducing SDS - PAGE and phosphor - imaging . In a parallel control experiment , the pellet and supernatant were analyzed by reducing SDS - PAGE and immunoblotting with an antibody specific to PDHE1α , a soluble mitochondrial matrix protein .

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

D. Oxidized mitochondria with the radioactive single-cysteine Bax protein pair or double-cysteine Bax protein were prepared as in (B). After an aliquot was withdrawn as input, another aliquot was extracted by Na2CO3 (pH 11.5) and centrifuged through a sucrose cushion to separate the integral proteins in the membrane pellet from the soluble and peripheral proteins in the supernatant. The input, pellet and supernatant were analyzed by non-reducing SDS-PAGE and phosphor-imaging. In a parallel control experiment, the pellet and supernatant were analyzed by reducing SDS-PAGE and immunoblotting with an antibody specific to PDHE1α, a soluble mitochondrial matrix protein.


B . The in vitro synthesized radioactive Bax proteins , each with a single cysteine positioned in helix α2 , α3 , α4 or α5 , were activated and targeted to the mitochondria . The resulting mitochondria were isolated and treated with IASD in the absence or presence of CHAPS , urea , or both . After 30 min , the labeling reactions were stopped by mercaptoethanol . For the " 0 min " controls , the samples were pretreated with mercaptoethanol before addition of IASD . The IASD - labeled radioactive Bax proteins were resolved from the unlabeled ones using either isoelectric focusing ( IEF ; as indicated ) or gradient SDS - PAGE and detected by phosphor - imaging . Triangles and arrows indicate the unlabeled and IASD - labeled Bax proteins , respectively . n = 3 for V111C , and K119C ; 4 for W107C , and A117C ; 2 for other mutants . C - D . The phosphor - imaging data for IASD labeling of Bax mutants in ( B ) and the similar data from the independent replicates were quantified to derive the membrane or protein burial indices as described and shown in Appendix Figure S3 . The membrane burial indices were normalized by that of G179C in α9 , a reference for the membrane - buried residues . The resulting relative membrane burial indices are shown in ( C ) . The residues with the relative membrane burial index ≥ 40 % of that of Gly179 are considered as buried in the MOM . Similarly , the relative protein burial indices shown in ( D ) were obtained by using Y115C in α5 as a reference for the protein - buried residues . The residues with the relative protein burial index ≥ 40 % of that of Tyr115 are considered as buried in the protein or its complex .

O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. The in vitro synthesized radioactive Bax proteins, each with a single cysteine positioned in helix α2, α3, α4 or α5, were activated and targeted to the mitochondria. The resulting mitochondria were isolated and treated with IASD in the absence or presence of CHAPS, urea, or both. After 30 min, the labeling reactions were stopped by mercaptoethanol. For the "0 min" controls, the samples were pretreated with mercaptoethanol before addition of IASD. The IASD-labeled radioactive Bax proteins were resolved from the unlabeled ones using either isoelectric focusing (IEF; as indicated) or gradient SDS-PAGE and detected by phosphor-imaging. Triangles and arrows indicate the unlabeled and IASD-labeled Bax proteins, respectively. n = 3 for V111C, and K119C; 4 for W107C, and A117C; 2 for other mutants.C-D. The phosphor-imaging data for IASD labeling of Bax mutants in (B) and the similar data from the independent replicates were quantified to derive the membrane or protein burial indices as described and shown in Appendix Figure S3. The membrane burial indices were normalized by that of G179C in α9, a reference for the membrane-buried residues. The resulting relative membrane burial indices are shown in (C). The residues with the relative membrane burial index ≥ 40% of that of Gly179 are considered as buried in the MOM. Similarly, the relative protein burial indices shown in (D) were obtained by using Y115C in α5 as a reference for the protein-buried residues. The residues with the relative protein burial index ≥ 40% of that of Tyr115 are considered as buried in the protein or its complex.


A - B . Oxidized mitochondria with the radioactive single - or double - cysteine Bax proteins , or the single - cysteine Bax protein pairs were prepared and analyzed as in Figure 1B .

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

A-B. Oxidized mitochondria with the radioactive single- or double-cysteine Bax proteins, or the single-cysteine Bax protein pairs were prepared and analyzed as in Figure 1B.


C . Oxidized mitochondria with the radioactive double - cysteine Bax protein were prepared , subjected to Na2CO3 extraction and analyzed as in Figure 1D .

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

C. Oxidized mitochondria with the radioactive double-cysteine Bax protein were prepared, subjected to Na2CO3 extraction and analyzed as in Figure 1D.


B and D . The radioactive single - or double - cysteine Bax proteins with or without the indicated mutations were activated and targeted to the mitochondria that were either untreated ( mito ) or pretreated with NEM ( NEM - mito ) , then oxidized and analyzed as in Figure 1B . Protein standards , and monomers and disulfide - linked dimers of the Bax proteins are indicated as in Figure 1B . n = 2 for all mutants .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 and D. The radioactive single- or double-cysteine Bax proteins with or without the indicated mutations were activated and targeted to the mitochondria that were either untreated (mito) or pretreated with NEM (NEM-mito), then oxidized and analyzed as in Figure 1B. Protein standards, and monomers and disulfide-linked dimers of the Bax proteins are indicated as in Figure 1B. n = 2 for all mutants.


C . The in vitro synthesized [ 35S ] Met - labeled Bax E69C protein with or without the additional mutations ( G108E and / or S184V ) were activated by the in vitro synthesized [ 35S ] Met - labeled tBid L105C protein and targeted to the Bax - / - / Bak - / - mitochondria that were pretreated with NEM . The resulting mitochondria were isolated and oxidized by CuPhe for 0 or 30 min . The resulting " 0 min " samples ( 1 equivalent each ) and " 30 min " samples ( 1 equivalent each ) were analyzed by non - reducing SDS - PAGE and phosphor - imaging . The remaining " 30 min " samples ( 4 equivalent each ) were immunoprecipitated ( IP ) by either Bax - or tBid - specific antibody , and then analyzed by non - reducing SDS - PAGE and phosphor - imaging . The identities of the four major products , indicated on the right side of the image , were based on their Mr and recognition by the respective antibody . n = 2 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O 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. The in vitro synthesized [35S]Met-labeled Bax E69C protein with or without the additional mutations (G108E and/or S184V) were activated by the in vitro synthesized [35S]Met-labeled tBid L105C protein and targeted to the Bax-/-/Bak-/- mitochondria that were pretreated with NEM. The resulting mitochondria were isolated and oxidized by CuPhe for 0 or 30 min. The resulting "0 min" samples (1 equivalent each) and "30 min" samples (1 equivalent each) were analyzed by non-reducing SDS-PAGE and phosphor-imaging. The remaining "30 min" samples (4 equivalent each) were immunoprecipitated (IP) by either Bax- or tBid-specific antibody, and then analyzed by non-reducing SDS-PAGE and phosphor-imaging. The identities of the four major products, indicated on the right side of the image, were based on their Mr and recognition by the respective antibody. n = 2.


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


A . The in vitro synthesized radioactive Bax proteins , each with a single cysteine positioned in α9 , were activated and targeted to the mitochondria , and labeled with IASD as in Figure 2B . The IASD - labeled and unlabeled Bax proteins were resolved using gradient SDS - PAGE , detected by phosphor - imaging and indicated by triangles and arrows , respectively . For all mutants , n = 2 , except for A183C , n = 3 . B - C . The IASD labeling data in ( A ) and the similar data from the independent replicates were quantified to derive the membrane or protein burial indices shown in Appendix Figure S5 , from which the relative membrane ( B ) or protein ( C ) burial indices were obtained as described in Figure 2C - D , and shown . D . The structure of α9 ( extracted from the NMR structure of Bax monomer , PDB entry 1F16 ) is shown with the residues that are buried in the MOM or exposed to the aqueous milieu , as the IASD labeling data in ( A - C ) suggested , presented in yellow or cyan stick form , respectively .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. The in vitro synthesized radioactive Bax proteins, each with a single cysteine positioned in α9, were activated and targeted to the mitochondria, and labeled with IASD as in Figure 2B. The IASD-labeled and unlabeled Bax proteins were resolved using gradient SDS-PAGE, detected by phosphor-imaging and indicated by triangles and arrows, respectively. For all mutants, n = 2, except for A183C, n = 3.B-C. The IASD labeling data in (A) and the similar data from the independent replicates were quantified to derive the membrane or protein burial indices shown in Appendix Figure S5, from which the relative membrane (B) or protein (C) burial indices were obtained as described in Figure 2C-D, and shown.D. The structure of α9 (extracted from the NMR structure of Bax monomer, PDB entry 1F16) is shown with the residues that are buried in the MOM or exposed to the aqueous milieu, as the IASD labeling data in (A-C) suggested, presented in yellow or cyan stick form, respectively.


B and D . The oxidized mitochondria with radioactive single - cysteine Bax proteins were prepared and analyzed as in Figure 1B .

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

B and D. The oxidized mitochondria with radioactive single-cysteine Bax proteins were prepared and analyzed as in Figure 1B.


C and E . The oxidized mitochondria with the radioactive single - cysteine Bax proteins were prepared , subjected to Na2CO3 extraction , and analyzed as in Figure 1D .

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

C and E. The oxidized mitochondria with the radioactive single-cysteine Bax proteins were prepared, subjected to Na2CO3 extraction, and analyzed as in Figure 1D.


B - D . The in vitro synthesized radioactive single or double - cysteine Bax proteins with or without the indicated mutations were activated and targeted to the mitochondria , oxidized , and analyzed as in Figure 1B . Protein standards , and monomers and disulfide - linked dimers of the Bax proteins are indicated as in Figure 1B . n = 2 for all mutants .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-D. The in vitro synthesized radioactive single or double-cysteine Bax proteins with or without the indicated mutations were activated and targeted to the mitochondria, oxidized, and analyzed as in Figure 1B. Protein standards, and monomers and disulfide-linked dimers of the Bax proteins are indicated as in Figure 1B. n = 2 for all mutants.


A . The oxidized mitochondria with radioactive single , double , or triple - cysteine Bax proteins were prepared and analyzed by non - reducing or reducing SDS - PAGE as in Figure 1B . Protein standards , Bax monomers and disulfide - linked Bax homodimers are also indicated as in Figure 1B . Closed triangles indicate disulfide - linked higher order Bax homo - oligomers . Open triangles indicate a background band that could not be reduced , and hence is not a disulfide - linked complex .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. The oxidized mitochondria with radioactive single, double, or triple-cysteine Bax proteins were prepared and analyzed by non-reducing or reducing SDS-PAGE as in Figure 1B. Protein standards, Bax monomers and disulfide-linked Bax homodimers are also indicated as in Figure 1B. Closed triangles indicate disulfide-linked higher order Bax homo-oligomers. Open triangles indicate a background band that could not be reduced, and hence is not a disulfide-linked complex.


B . The BMH - treated mitochondria with the radioactive single - and double - cysteine Bax proteins were prepared and analyzed as in Figure EV3G , except that closed triangles indicate the BMH - linked Bax oligomers .

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 B-CONTROLLED_VAR O O O O O

B. The BMH-treated mitochondria with the radioactive single- and double-cysteine Bax proteins were prepared and analyzed as in Figure EV3G, except that closed triangles indicate the BMH-linked Bax oligomers.


A - C . The single or double - cysteine Bax proteins with or without the indicated mutations were synthesized in vitro , activated by tBid protein and targeted to the Bax - / - / Bak - / - mitochondria . Cytochrome c release from the mitochondria was measured using ELISA . The dots are the fractions of cytochrome c release from two independent replicates after they were corrected as described in Figure EV1B , and the lines are the means . The amount of the Bax mutants bound to the mitochondria in the assay was determined and the results are shown in Appendix Figure S1 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-C. The single or double-cysteine Bax proteins with or without the indicated mutations were synthesized in vitro, activated by tBid protein and targeted to the Bax-/-/Bak-/- mitochondria. Cytochrome c release from the mitochondria was measured using ELISA. The dots are the fractions of cytochrome c release from two independent replicates after they were corrected as described in Figure EV1B, and the lines are the means. The amount of the Bax mutants bound to the mitochondria in the assay was determined and the results are shown in Appendix Figure S1.


A . The cytochrome c release by the indicated Bax mutants was measured in two independent replicates , corrected and shown as in Figure 9 . The amount of the Bax mutants bound to the mitochondria in the assay was determined and the results are shown in Appendix Figure S1 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. The cytochrome c release by the indicated Bax mutants was measured in two independent replicates, corrected and shown as in Figure 9. The amount of the Bax mutants bound to the mitochondria in the assay was determined and the results are shown in Appendix Figure S1.


B . Purified wild - type ( WT ) or mutant ( G179I or T182I ) Bax protein of the indicated concentration was incubated with the Bax - / - / Bak - / - mitochondria in the absence or presence of tBid protein . The proteins released from and those remaining in the mitochondria were separated and analyzed by SDS - PAGE and immunoblotting with antibody specific to either cytochrome c or Smac . n = 2 .

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B. Purified wild-type (WT) or mutant (G179I or T182I) Bax protein of the indicated concentration was incubated with the Bax-/-/Bak-/- mitochondria in the absence or presence of tBid protein. The proteins released from and those remaining in the mitochondria were separated and analyzed by SDS-PAGE and immunoblotting with antibody specific to either cytochrome c or Smac. n = 2.


C . Purified wild - type or mutant Bax protein of the indicated concentration alone or together with purified cBid was incubated with the Bax - / - / Bak - / - mitochondria containing Smac - mCherry protein . Release of Smac - mCherry was detected by measuring the fluorescence in the supernatant after the mitochondria were pelleted by centrifugation . The dots are the fractions of Smac - mCherry release from three independent replicates , and the lines are the means .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Purified wild-type or mutant Bax protein of the indicated concentration alone or together with purified cBid was incubated with the Bax-/-/Bak-/- mitochondria containing Smac-mCherry protein. Release of Smac-mCherry was detected by measuring the fluorescence in the supernatant after the mitochondria were pelleted by centrifugation. The dots are the fractions of Smac-mCherry release from three independent replicates, and the lines are the means.


D . The in vitro synthesized radioactive Bax proteins with indicated mutations were targeted to the mitochondria in the absence or presence of purified tBid protein , and then labeled with IASD , as in Figure 2B . The IASD - labeled and unlabeled Bax proteins were resolved using either gradient SDS - PAGE or IEF , detected by phosphor - imaging , and indicated by triangles and arrows , respectively . n = 3 for A178C , G179I ; 2 for other mutants . E . The IASD labeling data in ( D ) and the similar data from the independent replicates were quantified to derive the membrane burial indices shown in Appendix Figure S7A , from which the relative membrane burial indices were obtained as described in Figure 2C , and 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. The in vitro synthesized radioactive Bax proteins with indicated mutations were targeted to the mitochondria in the absence or presence of purified tBid protein, and then labeled with IASD, as in Figure 2B. The IASD-labeled and unlabeled Bax proteins were resolved using either gradient SDS-PAGE or IEF, detected by phosphor-imaging, and indicated by triangles and arrows, respectively. n = 3 for A178C,G179I; 2 for other mutants.E. The IASD labeling data in (D) and the similar data from the independent replicates were quantified to derive the membrane burial indices shown in Appendix Figure S7A, from which the relative membrane burial indices were obtained as described in Figure 2C, and shown.


Representative IF staining of Sox2 ( green ) and Ki67 ( red ) on the P7 CLE stem cell and transit amplifying cell regions and counterstained with DAPI ( blue ) on a sagittal section . Dotted lines , basement membrane ; yellow arrowheads mark approximate stem cell boundaries . SCs , stem cells ; TACs , transit amplifying cells ; Ant , Anterior ; Post , Posterior .

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

Representative IF staining of Sox2 (green) and Ki67 (red) on the P7 CLE stem cell and transit amplifying cell regions and counterstained with DAPI (blue) on a sagittal section. Dotted lines, basement membrane; yellow arrowheads mark approximate stem cell boundaries. SCs, stem cells; TACs, transit amplifying cells; Ant, Anterior; Post, Posterior.


The mRNA expression profiling on specific markers of stem cells ( B ) and transit amplifying cells captured on P7 incisors CLE followed by analysis using real - time RT - PCR ( C ) . qRT - PCR results are in arbitrary values after normalization for GapDH . Statistics was performed on triplicates using one - way ANOVA followed by Bonferroni ' s test .

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The mRNA expression profiling on specific markers of stem cells (B) and transit amplifying cells captured on P7 incisors CLE followed by analysis using real-time RT-PCR (C). qRT- PCR results are in arbitrary values after normalization for GapDH. Statistics was performed on triplicates using one-way ANOVA followed by Bonferroni's test.


Representative IF double staining of E - Cadherin ( ECad , green , for cell border ) and AcTub ( red , for the cores of primary cilia ) on a sagittal section . Sample is counterstained with DAPI ( blue ) .

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Representative IF double staining of E-Cadherin (ECad, green, for cell border) and AcTub (red, for the cores of primary cilia) on a sagittal section. Sample is counterstained with DAPI (blue).


3D reconstructions of ciliary cores of the stem cells and transit amplifying cells based on AcTub immunostaining .

3D reconstructions of ciliary cores of the stem cells and transit amplifying cells based on AcTub immunostaining.


Quantification of cilia length ( F ) and volume ( G ) based on 3D reconstruction ( E ) and comparison between those in stem cell and transit amplifying cell regions . Numbers of quantified cilia were indicated ( n ) . Statistics was performed using one - way ANOVA : All error bars were defined in Statistical Analysis section : All quantification and real - time RT - PCR results were presented using style of mean and standard deviation .

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

Quantification of cilia length (F) and volume (G) based on 3D reconstruction (E) and comparison between those in stem cell and transit amplifying cell regions. Numbers of quantified cilia were indicated (n). Statistics was performed using one-way ANOVA: All error bars were defined in Statistical Analysis section: All quantification and real-time RT-PCR results were presented using style of mean and standard deviation.


Tamoxifen induced IFT88 deletion strategy ( top panel ) and representative images of Sox2 ( green ) and Ki67 ( red ) immunostaining on CLE regions of the indicated mouse strains ( bottom panels ) . Dotted lines , basement membrane . Note the significant reduction of both Sox2 and Ki67 immunolabeling upon deletion of IFT88 .

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Tamoxifen induced IFT88 deletion strategy (top panel) and representative images of Sox2 (green) and Ki67 (red) immunostaining on CLE regions of the indicated mouse strains (bottom panels). Dotted lines, basement membrane. Note the significant reduction of both Sox2 and Ki67 immunolabeling upon deletion of IFT88.


3D reconstruction of Sox2 - positive regions based on its immunolabeling of the indicated mouse strains ( I ) and their volume comparison between mouse strains as indicated ( J ) . Statistics was performed using Student ' s t - test : * * , p < 0 . 01 . Data are represented as mean and standard deviation .

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3D reconstruction of Sox2-positive regions based on its immunolabeling of the indicated mouse strains (I) and their volume comparison between mouse strains as indicated (J). Statistics was performed using Student's t-test: **, p < 0.01. Data are represented as mean and standard deviation.


Representative IF double staining of ADP - ribosylation factor - like protein 13B ( Arl13b , red , K ) or histone deacetylase 6 ( Hdac6 , red , L ) with AcTub ( green ) as a marker of the primary cilium core following stem cell to transit amplifying cell transition .

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Representative IF double staining of ADP-ribosylation factor-like protein 13B (Arl13b, red, K) or histone deacetylase 6 (Hdac6, red, L) with AcTub (green) as a marker of the primary cilium core following stem cell to transit amplifying cell transition.


Representative IF staining of Prom1 using specific antibody clone 13A4 targeting extracellular loop ( green ) on the stem cell and transit amplifying cell regions of lower incisor CLE at P7 . Sample is counterstained with DAPI ( blue ) . Dotted lines , basement membrane . SCs , stem cells ; TACs , transit amplifying cells ; Ams , ameloblasts .

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Representative IF staining of Prom1 using specific antibody clone 13A4 targeting extracellular loop (green) on the stem cell and transit amplifying cell regions of lower incisor CLE at P7. Sample is counterstained with DAPI (blue). Dotted lines, basement membrane. SCs, stem cells; TACs, transit amplifying cells; Ams, ameloblasts.


3D reconstruction showing the association of Prom1 ( green ) with AcTub - labeled ( red ) primary cilia in stem cell and transit amplifying cell regions . Note that the expression of Prom1 is not limited primary cilium but also to microvilli . A representative example of Prom1 association with one primary cilium at the stem cell to transit amplifying cell transition region . Green channel transparency was set up to 70 % .

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

3D reconstruction showing the association of Prom1 (green) with AcTub-labeled (red) primary cilia in stem cell and transit amplifying cell regions. Note that the expression of Prom1 is not limited primary cilium but also to microvilli. A representative example of Prom1 association with one primary cilium at the stem cell to transit amplifying cell transition region. Green channel transparency was set up to 70%.


Representative IF staining of Prom1 using antibodies directed either its extracellular loop ( clone 13A4 , green ) or cytoplasmic C - terminal end ( Biorbyt , Orb129549 , red ) on transit amplifying cell regions of the WT vs . Prom1 KO mice . Samples are counterstained with DAPI ( blue ) . Note the lack of Prom1 labeling in Prom1 KO mice .

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

Representative IF staining of Prom1 using antibodies directed either its extracellular loop (clone 13A4, green) or cytoplasmic C-terminal end (Biorbyt, Orb129549, red) on transit amplifying cell regions of the WT vs. Prom1 KO mice. Samples are counterstained with DAPI (blue). Note the lack of Prom1 labeling in Prom1 KO mice.


The mRNA profiling on shRNA - meditated Prom1 knockdown ( 3 different shRNAs were used , marked as NO . 1 , 2 and 3 ) in cultured CLESCs . qRT - PCR results are in arbitrary values after normalization for GapDH .

The mRNA profiling on shRNA-meditated Prom1 knockdown (3 different shRNAs were used, marked as NO.1, 2 and 3) in cultured CLESCs. qRT- PCR results are in arbitrary values after normalization for GapDH.


protein profiling on shRNA - meditated Prom1 knockdown ( 3 different shRNAs were used , marked as NO . 1 , 2 and 3 ) in cultured CLESCs . The immunoblotting was performed with anti - Prom1 C - terminal antibody . β - actin was used as loading control ( F ) . Molecular mass markers are indicated .

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protein profiling on shRNA-meditated Prom1 knockdown (3 different shRNAs were used, marked as NO.1, 2 and 3) in cultured CLESCs. The immunoblotting was performed with anti-Prom1 C-terminal antibody. β-actin was used as loading control (F). Molecular mass markers are indicated.


Representative IF staining of Prom1 ( red ) and Sox2 ( green ) in CLESCs in vitro . Sample is counterstained with DAPI ( blue ) . Arrows indicate colocalisation of Prom1 with low - medium level of Sox2 in the cell nuclei .

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

Representative IF staining of Prom1 (red) and Sox2 (green) in CLESCs in vitro. Sample is counterstained with DAPI (blue). Arrows indicate colocalisation of Prom1 with low-medium level of Sox2 in the cell nuclei.


Subcellular fractionation of CLESCs into cytoplasm / membrane ( C + M ) and nuclear ( N ) fractions . Materials were analyzed by immunoblotting for Prom1 ( H ) and Arl13b ( I ) . As controls , membranes were blotted for anti - Lamin A / C ( nuclei markers ) and GapDH ( cytoplasm marker ) . Molecular mass markers are indicated . Note the presence of full - length Prom1 as well as Arl13b in nuclear fraction .

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

Subcellular fractionation of CLESCs into cytoplasm/membrane (C+M) and nuclear (N) fractions. Materials were analyzed by immunoblotting for Prom1 (H) and Arl13b (I). As controls, membranes were blotted for anti-Lamin A/C (nuclei markers) and GapDH (cytoplasm marker). Molecular mass markers are indicated. Note the presence of full-length Prom1 as well as Arl13b in nuclear fraction.


Representative images ( A ) and quantitative analysis ( B ) of IF staining and 3D reconstruction on Sox2 - positive region for WT vs . Prom1 KO mice . Ant : Anterior ; Post : Posterior ( A ) . Statistics was performed using Student ' s t - test : * * , p < 0 . 01 . Data are represented as mean and standard deviation .

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Representative images (A) and quantitative analysis (B) of IF staining and 3D reconstruction on Sox2-positive region for WT vs. Prom1 KO mice. Ant: Anterior; Post: Posterior (A). Statistics was performed using Student's t-test: **, p < 0.01. Data are represented as mean and standard deviation.


Representative IF double staining of Sox 2 ( green ) and Ki67 ( red ) on the and transit amplifying cell regions of WT vs . Prom1 KO mice on frontal sections . Samples are counterstained with DAPI ( blue ) . Quantification of Ki67 + cell number in Sox2 - positive region based on IF data from WT and Prom1 KO mice ( C ) .

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Representative IF double staining of Sox 2 (green) and Ki67 (red) on the and transit amplifying cell regions of WT vs. Prom1 KO mice on frontal sections. Samples are counterstained with DAPI (blue). Quantification of Ki67+ cell number in Sox2-positive region based on IF data from WT and Prom1 KO mice (C).


Stereo images of three explants cultured with indicated recombination conditions before harvesting at day 3 . Dotted line indicates the CLE .

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Stereo images of three explants cultured with indicated recombination conditions before harvesting at day 3. Dotted line indicates the CLE.


Passaging capacity analysis of the CLESCs derived from P7 WT and Prom1 KO mice . Note the passages of cells derived from WT animals are still ongoing while those from Prom1 KO mice were arrested at the first passage . Arrows indicated cells were still under passaging while blunt bars represent cells could not be passaged and stopped at the indicated passage .

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Passaging capacity analysis of the CLESCs derived from P7 WT and Prom1 KO mice. Note the passages of cells derived from WT animals are still ongoing while those from Prom1 KO mice were arrested at the first passage. Arrows indicated cells were still under passaging while blunt bars represent cells could not be passaged and stopped at the indicated passage.


Clonogenicity assay for colony number ( B ) and size ( C ) after the first the passage of CLESCs derived from WT vs . Prom1 KO mice in the absence or presence of SHH ( 100ng / ml ) . Cells were seeded in triplicate on 24 - well plates as 5 , 000 cell / per well .

Clonogenicity assay for colony number (B) and size (C) after the first the passage of CLESCs derived from WT vs. Prom1 KO mice in the absence or presence of SHH (100ng/ml). Cells were seeded in triplicate on 24-well plates as 5,000 cell/per well.


Sphenoid analyses . For these assays , epithelial and mesenchymal cells were mixed at ratio of 5 , 000 : 5 , 000 per well in triplicates on 96 - well plates and grown for 3 days on Matrigel . Representative micrographs of entire well under indicated conditions are shown . Quantification of sphere diameter on samples presented in ( D ) .

Sphenoid analyses. For these assays, epithelial and mesenchymal cells were mixed at ratio of 5,000:5,000 per well in triplicates on 96-well plates and grown for 3 days on Matrigel. Representative micrographs of entire well under indicated conditions are shown. Quantification of sphere diameter on samples presented in (D).


Representative image of CLESCs harbouring the Ki67 - Fucci system showing colored cells at different cell cycle phases .

Representative image of CLESCs harbouring the Ki67-Fucci system showing colored cells at different cell cycle phases.


Flow cytometry analysis of Ki67 - Fucci CLESCs at different cell cycle stages in cells transfected with empty vector or full length Prom1 . As a result , > 95 % of the events have been included in the analysis . The accuracy of the strategy has been validated by two independent researchers for IF quantification using Fiji 1 . 0 software .

Flow cytometry analysis of Ki67-Fucci CLESCs at different cell cycle stages in cells transfected with empty vector or full length Prom1. As a result, >95% of the events have been included in the analysis. The accuracy of the strategy has been validated by two independent researchers for IF quantification using Fiji 1.0 software.


Representative IF staining of AcTub ( blue ) on Ki67 - Fucci CLESCs without and with the presence of SHH . Note that the cilium could be visualised ( arrows ) solely when SHH is present .

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Representative IF staining of AcTub (blue) on Ki67-Fucci CLESCs without and with the presence of SHH. Note that the cilium could be visualised (arrows) solely when SHH is present.


Representative IF double staining of either AcTub ( D , red ) or Arl13b ( E , red ) with Sox2 ( green ) or Ki67 ( green ) in CLESCs in the presence of SHH . Note the expression levels of Sox2 and Ki67 distinguished stem cells and transit amplifying cells , respectively .

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Representative IF double staining of either AcTub (D, red) or Arl13b (E, red) with Sox2 (green) or Ki67 (green) in CLESCs in the presence of SHH. Note the expression levels of Sox2 and Ki67 distinguished stem cells and transit amplifying cells, respectively.


Size of individual cilia in stem cell and transit amplifying cell regions of WT and Prom1 KO mice ( A ) and their density in the respective regions ( B ) . 3 animals were analyzed for each genotype . The number of quantified cilia is indicated ( n ) .

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

Size of individual cilia in stem cell and transit amplifying cell regions of WT and Prom1 KO mice (A) and their density in the respective regions (B). 3 animals were analyzed for each genotype. The number of quantified cilia is indicated (n).


Clonogenicity analysis of CLESCs treated with either Hdac6 inhibitor ( Tubacin ) and appropriate controls ( Nitubacin and DMSO , C ) Colonies were stained with crystal

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

Clonogenicity analysis of CLESCs treated with either Hdac6 inhibitor (Tubacin) and appropriate controls (Nitubacin and DMSO, C) Colonies were stained with crystal violet


Clonogenicity analysis of CLESCs treated with siArl13b and its siControl . Colonies were stained with crystal

Clonogenicity analysis of CLESCs treated with siArl13b and its siControl. Colonies were stained with crystal violet


siRNA knockdown efficiency of Arl13b ( E ) in Ki67 FUCCI - CLESCs was evaluated real - time RT - PCR analysis . The results are in arbitrary values after normalization for

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

siRNA knockdown efficiency of Arl13b (E) in Ki67 FUCCI-CLESCs was evaluated real-time RT-PCR analysis. The results are in arbitrary values after normalization for GapDH


siRNA knockdown efficiency of Hdac6 ( F ) in Ki67 FUCCI - CLESCs was evaluated real - time RT - PCR analysis . The results are in arbitrary values after normalization for

siRNA knockdown efficiency of Hdac6 (F) in Ki67 FUCCI-CLESCs was evaluated real-time RT-PCR analysis. The results are in arbitrary values after normalization for GapDH


Flow cytometry analysis and quantification of samples indicated in ( E and F ) .

Flow cytometry analysis and quantification of samples indicated in (E and F).


Representative IF double staining and 3D reconstruction of the Prom1 ( green ) and AcTub ( red ) at the apical domain of polarized MDCK cells expressing Prom1 and its K138Q mutant ( top panels ) . Black - white inserts show Prom1 signals only . Same cells were analyzed by SEM ( bottom panels ) . Note that Prom1 is asymmetrically distributed along the primary cilium ( white arrows ) as observed in vivo ( see Fig 2C ) and mutation K138Q significantly impacts on the primary cilium length . MV , microvilli .

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Representative IF double staining and 3D reconstruction of the Prom1 (green) and AcTub (red) at the apical domain of polarized MDCK cells expressing Prom1 and its K138Q mutant (top panels). Black-white inserts show Prom1 signals only. Same cells were analyzed by SEM (bottom panels). Note that Prom1 is asymmetrically distributed along the primary cilium (white arrows) as observed in vivo (see Fig 2C) and mutation K138Q significantly impacts on the primary cilium length. MV, microvilli.


IF staining and 3D reconstruction of AcTub - labeled ( red ) primary cilium at the apical membrane of parental MDCK cells ( MDCK ) and those cells transfected with Prom1 or its K138Q mutant . The images were used to quantify the primary cilium length ( Appendix Figs S6B and C ) . Arrows indicate primary cilia with more than 4 μm in length .

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IF staining and 3D reconstruction of AcTub-labeled (red) primary cilium at the apical membrane of parental MDCK cells (MDCK) and those cells transfected with Prom1 or its K138Q mutant. The images were used to quantify the primary cilium length (Appendix Figs S6B and C). Arrows indicate primary cilia with more than 4 μm in length.


Co - immunoisolation ( IS ) analyses were performed with paramagnetic beads using antibodies against either Arl13b ( K ) Recovered materials were analyzed by immunoblotting against the indicated proteins . Brackets and asterisks indicate the plasma membrane and endoplasmic reticulum - associated forms of Prom1 , respectively , while arrows show Arl13b . Arrowhead , immunoglobulin .

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Co-immunoisolation (IS) analyses were performed with paramagnetic beads using antibodies against either Arl13b (K) Recovered materials were analyzed by immunoblotting against the indicated proteins. Brackets and asterisks indicate the plasma membrane and endoplasmic reticulum-associated forms of Prom1, respectively, while arrows show Arl13b. Arrowhead, immunoglobulin.


Co - immunoisolation ( IS ) analyses were performed with paramagnetic beads using antibodies against either Prom1 ( AC133 ) or with anti - FITC antibody as control ( L ) . Recovered materials were analyzed by immunoblotting against the indicated proteins . Brackets and asterisks indicate the plasma membrane and endoplasmic reticulum - associated forms of Prom1 , respectively , while arrows show Arl13b . Arrowhead , immunoglobulin .

Co-immunoisolation (IS) analyses were performed with paramagnetic beads using antibodies against either Prom1 (AC133) or with anti-FITC antibody as control (L). Recovered materials were analyzed by immunoblotting against the indicated proteins. Brackets and asterisks indicate the plasma membrane and endoplasmic reticulum-associated forms of Prom1, respectively, while arrows show Arl13b. Arrowhead, immunoglobulin.


The ratio Arl13b / Prom1 immunoreactivities was quantified ( n = 3 ) ( M ) .

The ratio Arl13b/Prom1 immunoreactivities was quantified (n = 3) (M).


Parental MDCK cells or those transfected with Prom1 or K138Q mutant were grown for 7 dpc Cells were observed by phase - contrast microscopy ( A ) Box - whisker plots show the number of domes per 600 mm2 ( A ) . Three images were analyzed per experiment ( n = 4 ) . 4X magnification ( inset in panel A ) .

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Parental MDCK cells or those transfected with Prom1 or K138Q mutant were grown for 7 dpc Cells were observed by phase-contrast microscopy (A) Box-whisker plots show the number of domes per 600 mm2 (A). Three images were analyzed per experiment (n = 4). 4X magnification (inset in panel A).


Parental MDCK cells or those transfected with Prom1 or K138Q mutant were grown for 7 dpc Cells were observed by SEM ( B , top panels ) or confocal laser scanning microscopy after DAPI staining ( B , bottom panels , Individual cells within a given dome are indicated ( B , top panels ) and were quantified using three different planes ( bottom , centre and top ) of DAPI - labeled nuclei ( B , bottom panels ) .

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Parental MDCK cells or those transfected with Prom1 or K138Q mutant were grown for 7 dpc Cells were observed by SEM (B, top panels) or confocal laser scanning microscopy after DAPI staining (B, bottom panels, Individual cells within a given dome are indicated (B, top panels) and were quantified using three different planes (bottom, centre and top) of DAPI-labeled nuclei (B, bottom panels).


Parental MDCK cells or those transfected with Prom1 or K138Q mutant were grown for 7 dpc ( A - C ) or as indicated ( D ) . confocal laser scanning microscopy after DAPI staining Domes of similar size were evaluated ( C ) . Means and standard deviations are displayed . 15 domes per independent experiment were examined ( n = 3 ) . Cell density at a given time of culture ( 7 , 10 and 14 days ) was determined by counting DAPI - labeled nuclei per 0 . 1 mm2 ( D , n = 4 ) .

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Parental MDCK cells or those transfected with Prom1 or K138Q mutant were grown for 7 dpc (A-C) or as indicated (D). confocal laser scanning microscopy after DAPI staining Domes of similar size were evaluated (C). Means and standard deviations are displayed. 15 domes per independent experiment were examined (n = 3). Cell density at a given time of culture (7, 10 and 14 days) was determined by counting DAPI-labeled nuclei per 0.1 mm2 (D, n = 4).


Cilia with a length of > 4 µm were rarely observed in K138Q mutant and displayed an irregular shape with constrictions along their distal part as observed by SEM ( arrows ) .

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

Cilia with a length of >4 µm were rarely observed in K138Q mutant and displayed an irregular shape with constrictions along their distal part as observed by SEM (arrows).


Mutation in the Hdac6 - binding site of Prom1 increases the release of Prom1 + vesicles . 16 h - conditioned medium was subjected to differential centrifugation for 5 min at 300 x g ( P1 ) ; 20 min at 1 , 200 x g ( P2 ) ; 30 min at 10 , 000 x g ( P3 ) and 1 h at 200 , 000 x g ( P4 ) . In parallel , detergent cell lysates were prepared . The entire pellets and 1 / 40 of cell lysates were analyzed by immunoblotting using antibodies against human Prom1 , AcTub and β - actin ( F ) . Molecular mass markers are indicated . All three proteins were detected in the vesicle fractions ( P3 and P4 ) suggesting Prom1 + vesicles are released from primary cilia and microvilli .

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

Mutation in the Hdac6-binding site of Prom1 increases the release of Prom1+ vesicles. 16 h-conditioned medium was subjected to differential centrifugation for 5 min at 300 x g (P1); 20 min at 1,200 x g (P2); 30 min at 10,000 x g (P3) and 1 h at 200,000 x g (P4). In parallel, detergent cell lysates were prepared. The entire pellets and 1/40 of cell lysates were analyzed by immunoblotting using antibodies against human Prom1, AcTub and β-actin (F). Molecular mass markers are indicated. All three proteins were detected in the vesicle fractions (P3 and P4) suggesting Prom1+ vesicles are released from primary cilia and microvilli.


Mutation in the Hdac6 - binding site of Prom1 increases the release of Prom1 + vesicles . 16 h - conditioned medium was subjected to differential centrifugation for 5 min at 300 x g ( P1 ) ; 20 min at 1 , 200 x g ( P2 ) ; 30 min at 10 , 000 x g ( P3 ) and 1 h at 200 , 000 x g ( P4 ) . In parallel , detergent cell lysates were prepared . The release of Prom1 + vesicles was quantified using its immunoreactivities associated with cell and vesicle fractions ( G )

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

Mutation in the Hdac6-binding site of Prom1 increases the release of Prom1+ vesicles. 16 h-conditioned medium was subjected to differential centrifugation for 5 min at 300 x g (P1); 20 min at 1,200 x g (P2); 30 min at 10,000 x g (P3) and 1 h at 200,000 x g (P4). In parallel, detergent cell lysates were prepared. The release of Prom1+ vesicles was quantified using its immunoreactivities associated with cell and vesicle fractions (G)


The mRNA expression of SHH effector genes ( Gli1 / 2 and Glis1 - 3 ) on laser microdissection captured samples were evaluated by real - time RT - PCR analysis . The results are in arbitrary values after normalization for GapDH . Each sample was isolated from distinct mice ( n = 3 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 mRNA expression of SHH effector genes (Gli1/2 and Glis1-3) on laser microdissection captured samples were evaluated by real-time RT-PCR analysis. The results are in arbitrary values after normalization for GapDH. Each sample was isolated from distinct mice (n = 3).


Representative IF staining of Gli1 ( left panel , red ) and Glis2 ( right panel , red ) on the stem cell and transit amplifying cell regions of lower incisor CLE at P7 . Samples were counterstained with DAPI ( blue ) . Dotted lines , basement membrane .

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

Representative IF staining of Gli1 (left panel, red) and Glis2 (right panel, red) on the stem cell and transit amplifying cell regions of lower incisor CLE at P7. Samples were counterstained with DAPI (blue). Dotted lines, basement membrane.


3D reconstruction of Glis2 staining ( see B , red ) and its association with DAPI - labeled nuclei ( blue ) . Nuclear transparency was set at 0 or 70 % as indicated .

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

3D reconstruction of Glis2 staining (see B, red) and its association with DAPI-labeled nuclei (blue). Nuclear transparency was set at 0 or 70% as indicated.


Representative IF double staining of Glis2 ( red , bottom panel ) and AcTub ( green , top and bottom panels ) along stem cell - transit amplifying cell axis in lower incisor CLE at P7 . Samples were counterstained with DAPI ( blue ) . Note the association of Glis2 with primary cilia particularly in the transition zone between stem cell and transit amplifying cell regions .

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

Representative IF double staining of Glis2 (red, bottom panel) and AcTub (green, top and bottom panels) along stem cell-transit amplifying cell axis in lower incisor CLE at P7. Samples were counterstained with DAPI (blue). Note the association of Glis2 with primary cilia particularly in the transition zone between stem cell and transit amplifying cell regions.


Volume of Sox2 - positive region on the lower incisor CLE at P7 was evaluated in WT vs . Glis2 KO mice upon IF staining . Number of animals analyzed is indicated ( n ) .

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

Volume of Sox2-positive region on the lower incisor CLE at P7 was evaluated in WT vs. Glis2 KO mice upon IF staining. Number of animals analyzed is indicated (n).


Representative IF staining of Prom1 using specific antibodies targeting either its extracellular loop ( top panels , green ) or cytoplasmic C - terminal end ( bottom panels , red ) on the stem cell and transit amplifying cell regions of lower incisor CLE at P7 WT and Glis2 KO mice . Samples are counterstained with DAPI ( blue ) . Dotted lines , basement membrane . Arrowheads marks approximate boundary of stem cell regions .

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

Representative IF staining of Prom1 using specific antibodies targeting either its extracellular loop (top panels, green) or cytoplasmic C-terminal end (bottom panels, red) on the stem cell and transit amplifying cell regions of lower incisor CLE at P7 WT and Glis2 KO mice. Samples are counterstained with DAPI (blue). Dotted lines, basement membrane. Arrowheads marks approximate boundary of stem cell regions.


Representative IF double staining of Glis2 ( red ) and AcTub ( green ) as a marker of primary cilia in stem cell region of WT vs . Prom1 KO mice . Note the reduction of Glis2 expression in Prom1 - deficient animals .

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

Representative IF double staining of Glis2 (red) and AcTub (green) as a marker of primary cilia in stem cell region of WT vs. Prom1 KO mice. Note the reduction of Glis2 expression in Prom1-deficient animals.


Proximity ligation assay analysis of Prom1 and Glis2 association ( red ) in stem cell and transit amplifying cell regions of lower incisor CLE at P7 . As negative control , one conjugated primary antibody ( anti - Glis2 ) was omitted . SCs , stem cells ; TACs , transit amplifying cells .

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

Proximity ligation assay analysis of Prom1 and Glis2 association (red) in stem cell and transit amplifying cell regions of lower incisor CLE at P7. As negative control, one conjugated primary antibody (anti-Glis2) was omitted. SCs, stem cells; TACs, transit amplifying cells.


Time dependent Prom1 and Glis2 co - expression ( proximity ligation assay ) in the cultured CLESCs starved for 4 days , and then treated with SHH ( 100 ng / ml ) and importazole ( 40 µM ) , an inhibitor of importin β1function . DMSO was used as vehicle control . Data are represented triplicated samples as mean and standard deviation .

O O O O O O O O O O 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

Time dependent Prom1 and Glis2 co-expression (proximity ligation assay) in the cultured CLESCs starved for 4 days, and then treated with SHH (100 ng/ml) and importazole (40 µM), an inhibitor of importin β1function. DMSO was used as vehicle control. Data are represented triplicated samples as mean and standard deviation.


PCR array analyses were performed on LCM captured P7 CLE of Prom1 and Glis2 KO vs . WT mice ( n = 3 ) . Arrows indicated genes with expression changed for more than 2 folder increase or decrease .

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

PCR array analyses were performed on LCM captured P7 CLE of Prom1 and Glis2 KO vs. WT mice (n = 3). Arrows indicated genes with expression changed for more than 2 folder increase or decrease.


PCR array analyses were performed on LCM captured P7 CLE of Prom1 and Glis2 KO vs . WT mice ( n = 3 ) . The mRNA expression of gene significantly changed in the PCR array ( in ( C ) ) ( D ) was evaluated in WT , Prom1 and Glis2 KO mice by real - time RT - PCR analysis . Each sample was isolated from distinct mice ( n = 3 ) . The results are in arbitrary values after normalization for GapDH .

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

PCR array analyses were performed on LCM captured P7 CLE of Prom1 and Glis2 KO vs. WT mice (n = 3). The mRNA expression of gene significantly changed in the PCR array (in (C)) (D) was evaluated in WT, Prom1 and Glis2 KO mice by real-time RT-PCR analysis. Each sample was isolated from distinct mice (n = 3). The results are in arbitrary values after normalization for GapDH.


PCR array analyses were performed on LCM captured P7 CLE of Prom1 and Glis2 KO vs . WT mice ( n = 3 ) . The mRNA expression of gene significantly changed in the Stat pathway members ( E ) was evaluated in WT , Prom1 and Glis2 KO mice by real - time RT - PCR analysis . Each sample was isolated from distinct mice ( n = 3 ) . The results are in arbitrary values after normalization for GapDH .

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

PCR array analyses were performed on LCM captured P7 CLE of Prom1 and Glis2 KO vs. WT mice (n = 3). The mRNA expression of gene significantly changed in the Stat pathway members (E) was evaluated in WT, Prom1 and Glis2 KO mice by real-time RT-PCR analysis. Each sample was isolated from distinct mice (n = 3). The results are in arbitrary values after normalization for GapDH.


Chromatin IP analysis of binding of Glis2 to Stat3 promoter ( see Appendix Fig S9D ) in the absence or presence of SHH at different concentration as indicated on triplicated samples , using specific anti - Glis2 antibodies and rabbit IgG as control .

Chromatin IP analysis of binding of Glis2 to Stat3 promoter (see Appendix Fig S9D) in the absence or presence of SHH at different concentration as indicated on triplicated samples, using specific anti-Glis2 antibodies and rabbit IgG as control.


( A ) Three glycosylated forms of endogenous SMO protein are shown . NIH3T3 cells were harvested and treated with indicated glycosidases . Black bracket indicates mature glycosylated form and grey bracket indicates core glycosylated form . The representative results of three independent experiments are 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

(A) Three glycosylated forms of endogenous SMO protein are shown. NIH3T3 cells were harvested and treated with indicated glycosidases. Black bracket indicates mature glycosylated form and grey bracket indicates core glycosylated form. The representative results of three independent experiments are shown.


( C ) PTCH1 inhibited the Endo H - resistant glycosylation of SMO . HEK293T cells were co - transfected with the SMO and PTCH1 expression plasmids . After 48 h , the cells were harvested , lysed , and treated with indicated glycosidases . Results shown are representative of three independent experiments .

(C) PTCH1 inhibited the Endo H-resistant glycosylation of SMO. HEK293T cells were co-transfected with the SMO and PTCH1 expression plasmids. After 48 h, the cells were harvested, lysed, and treated with indicated glycosidases. Results shown are representative of three independent experiments.


( D ) Quantification of Endo H - resistant form of SMO shown in ( C ) . Three independent experiments were quantified by Image J and expressed as a percentage of the total SMO protein .

O O O O O O O O O O O O O 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) Quantification of Endo H-resistant form of SMO shown in (C). Three independent experiments were quantified by Image J and expressed as a percentage of the total SMO protein.


( E ) Shh increased the Endo H - resistant form of SMO . Cells were transfected as indicated for 24 h , and incubated with ShhN conditioned medium for an additional 24 h . Cells were harvested and analyzed as in ( C ) . Results shown are representative of three 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

(E) Shh increased the Endo H-resistant form of SMO. Cells were transfected as indicated for 24 h, and incubated with ShhN conditioned medium for an additional 24 h. Cells were harvested and analyzed as in (C). Results shown are representative of three independent experiments.


( F ) Quantification of Endo H - resistant form of SMO shown in ( E ) . Three independent experiments were quantified by Image J and expressed as a percentage of the total SMO protein .

(F) Quantification of Endo H-resistant form of SMO shown in (E). Three independent experiments were quantified by Image J and expressed as a percentage of the total SMO protein.


( G ) Shh increased the Endo H - resistant form of endogenous SMO in MEF cells . Results shown are representative of three 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

(G) Shh increased the Endo H-resistant form of endogenous SMO in MEF cells. Results shown are representative of three independent experiments.


( H ) Densitometric analysis of Endo H - resistant form of SMO shown in ( G ) . Three independent experiments were quantified by Image J and expressed as a percentage of the total SMO protein .

O O O O O O O O O O O O O O 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) Densitometric analysis of Endo H-resistant form of SMO shown in (G). Three independent experiments were quantified by Image J and expressed as a percentage of the total SMO protein.


( A ) Biotinylation of SMO by SAR1B - BirA * ( asterisk denotes the R118G mutation in BirA ) in a proximity - dependent biotinylation assay . The cells were transfected with plasmids encoding SMO and SAR1B - BirA * - FLAG . After 48 h , cells were treated with different doses of biotin for 4 h . Cells were harvested and lysates were dialyzed to remove free biotin . Biotinylated proteins were pulled down by streptavidin beads and subjected to immunoblotting analysis . ( B ) The cells were treated with different doses of biotin for 24 h . Cells were harvested , lysed , and subjected to procedure as shown in Fig . 2A .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Biotinylation of SMO by SAR1B-BirA* (asterisk denotes the R118G mutation in BirA) in a proximity-dependent biotinylation assay. The cells were transfected with plasmids encoding SMO and SAR1B-BirA*-FLAG. After 48 h, cells were treated with different doses of biotin for 4 h. Cells were harvested and lysates were dialyzed to remove free biotin. Biotinylated proteins were pulled down by streptavidin beads and subjected to immunoblotting analysis. (B) The cells were treated with different doses of biotin for 24 h. Cells were harvested, lysed, and subjected to procedure as shown in Fig. 2A.


( B ) The cells were treated with different doses of biotin for 24 h . Cells were harvested , lysed , and subjected to procedure as shown in Fig . 2A .

O O O O O O O O O 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) The cells were treated with different doses of biotin for 24 h. Cells were harvested, lysed, and subjected to procedure as shown in Fig. 2A.


( C ) The GDP - locked SAR1B ( G37A ) - BirA * failed to label SMO , while the GTP - locked SAR1B ( H79G ) - BirA * could efficiently biotinylate SMO . The cells were transfected with plasmids expressing SMO and indicated SAR1B - BirA * . After 48 h , cells were treated with 15 μM biotin for 4 h . Then experiments were carried out as described in Fig . 2A .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) The GDP-locked SAR1B(G37A)-BirA* failed to label SMO, while the GTP-locked SAR1B(H79G)-BirA* could efficiently biotinylate SMO. The cells were transfected with plasmids expressing SMO and indicated SAR1B-BirA*. After 48 h, cells were treated with 15 μM biotin for 4 h. Then experiments were carried out as described in Fig. 2A.


( D ) PTCH1 decreased SAR1B - BirA * - mediated biotinylation of SMO , but not LMAN1 . The experiments were done similarly in Fig . 2A .

O O O O O 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) PTCH1 decreased SAR1B-BirA*-mediated biotinylation of SMO, but not LMAN1. The experiments were done similarly in Fig. 2A.


( C ) The GDP - locked SAR1B ( G37A ) - BirA * failed to label SMO , while the GTP - locked SAR1B ( H79G ) - BirA * could efficiently biotinylate SMO . The cells were transfected with plasmids expressing SMO and indicated SAR1B - BirA * . After 48 h , cells were treated with 15 μM biotin for 4 h . Then experiments were carried out as described in Fig . 2A . ( D ) PTCH1 decreased SAR1B - BirA * - mediated biotinylation of SMO , but not LMAN1 . The experiments were done similarly in Fig . 2A . ( E ) The Shh increased the binding of SAR1B to SMO . After transfection for 24 h , cells were incubated with the ShhN conditioned medium for an additional 24 h . Then 15 μM biotin was added into the culture medium for 4 h . The cells were harvested and analyzed as described in Fig . 2A .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) The GDP-locked SAR1B(G37A)-BirA* failed to label SMO, while the GTP-locked SAR1B(H79G)-BirA* could efficiently biotinylate SMO. The cells were transfected with plasmids expressing SMO and indicated SAR1B-BirA*. After 48 h, cells were treated with 15 μM biotin for 4 h. Then experiments were carried out as described in Fig. 2A. (D) PTCH1 decreased SAR1B-BirA*-mediated biotinylation of SMO, but not LMAN1. The experiments were done similarly in Fig. 2A. (E) The Shh increased the binding of SAR1B to SMO. After transfection for 24 h, cells were incubated with the ShhN conditioned medium for an additional 24 h. Then 15 μM biotin was added into the culture medium for 4 h. The cells were harvested and analyzed as described in Fig. 2A.


( B ) Treatment with glycosidase demonstrated that SMO ( 4A , in which RRTWCR was mutated to AATACA ) was sensitive to Endo H . The experiments were done similarly in Fig . 2A . Black bracket indicates mature glycosylated form .

O O O O O O O O O O O O O O O O O O O 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) Treatment with glycosidase demonstrated that SMO (4A, in which RRTWCR was mutated to AATACA) was sensitive to Endo H. The experiments were done similarly in Fig. 2A. Black bracket indicates mature glycosylated form.


( C ) SAR1B - BirA * - mediated biotinylation of the SMO variants . The experiments were done similarly in Fig . 2A .

O 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) SAR1B-BirA*-mediated biotinylation of the SMO variants. The experiments were done similarly in Fig. 2A.


( D ) In vitro budding of SMO . Membranes were prepared and incubated in vitro in the presence of cytosol , ATP , GTP , and an ATP - regenerating system as described in experimental procedures . Then vesicle and membrane fractions were separated by centrifugation and subjected to immunoblotting analysis .

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 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

(D) In vitro budding of SMO. Membranes were prepared and incubated in vitro in the presence of cytosol, ATP, GTP, and an ATP-regenerating system as described in experimental procedures. Then vesicle and membrane fractions were separated by centrifugation and subjected to immunoblotting analysis.


( E ) In vitro budding of the SMO variants . The experiments were done as same as in ( D ) .

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

(E) In vitro budding of the SMO variants. The experiments were done as same as in (D).


( F ) The ciliary localization of SMO variants . Smo - KO NIH3T3 cells were transfected with the EGFP fused SMO variants , incubated with serum starvation medium for 24 h to fully induce cilia formation , and treated with or without ShhN conditioned medium for 4 h . Cells were immunostained with the acetylated tubulin ( red ) . Results shown are representative of three independent experiments . Scale bars : 5μm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) The ciliary localization of SMO variants. Smo-KO NIH3T3 cells were transfected with the EGFP fused SMO variants, incubated with serum starvation medium for 24 h to fully induce cilia formation, and treated with or without ShhN conditioned medium for 4 h. Cells were immunostained with the acetylated tubulin (red). Results shown are representative of three independent experiments. Scale bars: 5μm.


( G ) Quantitative assessment of the intensity of ciliary SMO in ( F ) by Image J . More than 45 cells were randomly selected and counted for each 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

(G) Quantitative assessment of the intensity of ciliary SMO in (F) by Image J. More than 45 cells were randomly selected and counted for each group.


( H ) Quantification of the percentage of cells with ciliary SMO . 80 - 90 cilia were analyzed in each group in each experiment .

O O 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) Quantification of the percentage of cells with ciliary SMO. 80-90 cilia were analyzed in each group in each experiment.


( I ) Gli - luciferase reporter assay in Smo - KO NIH3T3 cells . The cells were transfected with plasmids encoding SMO or SMO variants , GLI - promoted firefly luciferase and renilla luciferase . After 24 h , cells were incubated with a medium containing different amounts of ShhN for another 24 h .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Gli-luciferase reporter assay in Smo-KO NIH3T3 cells. The cells were transfected with plasmids encoding SMO or SMO variants, GLI-promoted firefly luciferase and renilla luciferase. After 24 h, cells were incubated with a medium containing different amounts of ShhN for another 24 h.


( A ) Shh increased cholesterylation of SMO . HEK293T cells were transfected , harvested , and

(A) Shh increased cholesterylation of SMO. HEK293T cells were transfected, harvested, and analyzed


( B ) The SMO cholesterylation of Ptch1 - KO NIH3T3 cells were increased comparing with WT cells .

(B) The SMO cholesterylation of Ptch1-KO NIH3T3 cells were increased comparing with WT cells.


( C ) The CP - modified SMO was mainly resistant to Endo H . The CP - modified SMO proteins were pulled down by streptavidin beads and digested with Endo H or PNGase F at 37 ℃ . Then proteins were eluted with SDS - PAGE sample buffer and were subjected to immunoblotting analysis . Results shown are representative of two 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

(C) The CP-modified SMO was mainly resistant to Endo H. The CP-modified SMO proteins were pulled down by streptavidin beads and digested with Endo H or PNGase F at 37 ℃. Then proteins were eluted with SDS-PAGE sample buffer and were subjected to immunoblotting analysis. Results shown are representative of two independent experiments.


( D ) Cholesterylation of SMO ( 4A ) was inhibited by PTCH1 and increased by ShhN .

(D) Cholesterylation of SMO(4A) was inhibited by PTCH1 and increased by ShhN.


( E ) The CP - modified SMO ( 4A ) was sensitive to Endo H treatment .

(E) The CP-modified SMO(4A) was sensitive to Endo H treatment.


( F ) The levels of CP - modified WT SMO and SMO ( 4A ) were compared . 1 . 39 % of SMO ( WT ) pellet samples and 11 . 1 % of SMO ( 4A ) pellet samples were loaded . Results shown are representative of three 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

(F) The levels of CP-modified WT SMO and SMO(4A) were compared. 1.39% of SMO(WT) pellet samples and 11.1% of SMO(4A) pellet samples were loaded. Results shown are representative of three independent experiments.


( G ) Quantification of CP - modified SMO shown in ( F ) . Three independent experiments were quantified by Image J and expressed as a percentage of the total SMO .

(G) Quantification of CP-modified SMO shown in (F). Three independent experiments were quantified by Image J and expressed as a percentage of the total SMO.


( H , I ) The stimulative / inhibitory effect of Shh / PTCH1 on SMO and SAR1B interaction was compromised by D95E mutation . The experiments were done the same as Fig . 2E .

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(H, I) The stimulative /inhibitory effect of Shh/PTCH1 on SMO and SAR1B interaction was compromised by D95E mutation. The experiments were done the same as Fig. 2E.


ALOD4 inhibited the cholesterol modification of SMO Cells were treated with ALOD4 for 1 h , and then incubated with a medium containing ShhN and CP for 2 and 3 h . Cells were harvested and

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ALOD4 inhibited the cholesterol modification of SMO Cells were treated with ALOD4 for 1 h, and then incubated with a medium containing ShhN and CP for 2 and 3 h. Cells were harvested and analyzed


ALOD4 inhibited the cholesterol modification of SMO ( 4A ) . Cells were treated with ALOD4 for 1 h In C , ShhN and CP added for 2 h . Cells were harvested and

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ALOD4 inhibited the cholesterol modification of SMO(4A). Cells were treated with ALOD4 for 1 h In C, ShhN and CP added for 2 h. Cells were harvested and analyzed


( D ) Ciliary localization of endogenous SMO treated with or without ALOD4 . Cells were treated with ALOD4 for 1 h , and then incubated with ShhN conditioned medium for 2 h . Results shown are representative of three independent experiments .

(D) Ciliary localization of endogenous SMO treated with or without ALOD4. Cells were treated with ALOD4 for 1 h, and then incubated with ShhN conditioned medium for 2 h. Results shown are representative of three independent experiments.


( E ) Quantitative assessment of the intensity of ciliary SMO in ( D ) . More than 80 cells were randomly selected and counted for each group . Data are presented as median with interquartile .

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(E) Quantitative assessment of the intensity of ciliary SMO in (D). More than 80 cells were randomly selected and counted for each group. Data are presented as median with interquartile.


( F ) Quantification of the percentage of cells with ciliary SMO in ( D ) . Data are presented as mean ± SEM from three biological replicates ( * * * * p < 0 . 0001 , one - way ANOVA with Dunnett ' s multiple comparison test ) . 90 - 100 cilia were analyzed in each group in each experiment .

(F) Quantification of the percentage of cells with ciliary SMO in (D). Data are presented as mean ± SEM from three biological replicates (****p < 0.0001, one-way ANOVA with Dunnett's multiple comparison test). 90-100 cilia were analyzed in each group in each experiment.


( G ) Effect of ALOD4 on the expression of Gli1 was measured by qRT - PCR . The cells were treated with ALOD4 for 1 h , and then incubated with ShhN conditioned medium for 5 h .

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(G) Effect of ALOD4 on the expression of Gli1 was measured by qRT-PCR. The cells were treated with ALOD4 for 1 h, and then incubated with ShhN conditioned medium for 5 h.


( A , B ) GRAMD1B increased cholesterylation of SMO . HEK293T cells were co - transfected with the SMO or SMO ( 4A ) and GRAMD1B protein expression plasmids for 24 h , and then incubated with a cholesterol depletion medium ( DMEM supplemented with 5 % lipoprotein - deficient serum , 1 μM lovastatin and 10 μM mevalonate ) for 12 h , followed by 2 μg / mL of CP treatment . The cells were harvested and analyzed as Results shown are representative of two independent experiments .

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(A, B) GRAMD1B increased cholesterylation of SMO. HEK293T cells were co-transfected with the SMO or SMO (4A) and GRAMD1B protein expression plasmids for 24 h, and then incubated with a cholesterol depletion medium (DMEM supplemented with 5% lipoprotein-deficient serum, 1 μM lovastatin and 10 μM mevalonate) for 12 h, followed by 2 μg/mL of CP treatment. The cells were harvested and analyzed as Results shown are representative of two independent experiments.


( C , D ) GRAMD1B promoted SMO maturation . The expression plasmids of SMO or SMO ( D95E ) and GRAMD1B were transfected into SMO - KO HEK293T cells . After 12 h , cells were switched to a cholesterol depletion medium for 16 h and then added 50 μM MβCD - cholesterol for 8 h . Black bracket indicates mature glycosylated form .

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(C, D) GRAMD1B promoted SMO maturation. The expression plasmids of SMO or SMO(D95E) and GRAMD1B were transfected into SMO-KO HEK293T cells. After 12 h, cells were switched to a cholesterol depletion medium for 16 h and then added 50 μM MβCD-cholesterol for 8 h. Black bracket indicates mature glycosylated form.


( E ) Expression of Gli1 mRNAs measured by qRT - PCR in NIH3T3 cells stably expressing GRAMD1B . After incubating in serum starvation medium for 16 h , cells were depleted of cholesterol by treatment with 1 . 5 % HPCD for 0 . 5 h and then incubated in serum starvation medium containing 1 μM lovastatin , 10 μM mevalonate and 10 μM MβCD - cholesterol , ShhN as indicated for 24 h .

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(E) Expression of Gli1 mRNAs measured by qRT-PCR in NIH3T3 cells stably expressing GRAMD1B. After incubating in serum starvation medium for 16 h, cells were depleted of cholesterol by treatment with 1.5% HPCD for 0.5 h and then incubated in serum starvation medium containing 1 μM lovastatin, 10 μM mevalonate and 10 μM MβCD-cholesterol, ShhN as indicated for 24 h.


( F ) Confocal images of NIH3T3 cells stably expressing mCherry - GRAMD1B . PM was labeled with wheat germ agglutinin ( WGA ) - CF ® 488A conjugate . Cells were incubated in a cholesterol depletion medium for 16 h and switched to the same medium containing 50 μM MβCD - cholesterol for 1 h . Cells were then treated with or without ShhN conditioned medium for another 2 h . Results shown are representative of three independent experiments .

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(F) Confocal images of NIH3T3 cells stably expressing mCherry-GRAMD1B. PM was labeled with wheat germ agglutinin (WGA)-CF®488A conjugate. Cells were incubated in a cholesterol depletion medium for 16 h and switched to the same medium containing 50 μM MβCD-cholesterol for 1 h. Cells were then treated with or without ShhN conditioned medium for another 2 h. Results shown are representative of three independent experiments.


( G ) The percentage of cells with GRAMD1B localization on PM . 60 - 70 cells were analyzed in each group in each experiment .

(G) The percentage of cells with GRAMD1B localization on PM. 60-70 cells were analyzed in each group in each experiment.


( A ) The cholesterol modification of endogenous SMO was decreased in NIH3T3 cells lacking Gramd1a , 1b , 1c , 2 , and 3 ( Gramd - 5KO ) . The cells were incubated in a cholesterol depletion medium for 12 h , followed by 2 μg / mL of CP and ShhN for an additional 12h .

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(A) The cholesterol modification of endogenous SMO was decreased in NIH3T3 cells lacking Gramd1a, 1b, 1c, 2, and 3 (Gramd-5KO). The cells were incubated in a cholesterol depletion medium for 12 h, followed by 2 μg/mL of CP and ShhN for an additional 12h.


( B ) The glycosylation of endogenous SMO was decreased in Gramd - 5KO NIH3T3 cells . The ShhN conditioned medium was applied for 24 h . Black bracket indicates mature glycosylated form .

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(B) The glycosylation of endogenous SMO was decreased in Gramd-5KO NIH3T3 cells. The ShhN conditioned medium was applied for 24 h. Black bracket indicates mature glycosylated form.


( C ) The ciliary localization of endogenous SMO in WT or Gramd - 5KO NIH3T3 cells . Cells were incubated in serum starvation medium for 24 h to fully induce cilia formation and then incubated with or without ShhN conditioned medium for 2 h . Results shown are representative of three independent experiments .

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(C) The ciliary localization of endogenous SMO in WT or Gramd-5KO NIH3T3 cells. Cells were incubated in serum starvation medium for 24 h to fully induce cilia formation and then incubated with or without ShhN conditioned medium for 2 h. Results shown are representative of three independent experiments.


( D ) Quantitative assessment of the intensity of ciliary SMO in ( C ) . More than 100 cells were randomly selected and counted for each group . Data are presented as median with interquartile .

(D) Quantitative assessment of the intensity of ciliary SMO in (C). More than 100 cells were randomly selected and counted for each group. Data are presented as median with interquartile.


( E ) Quantification of the percentage of cells with ciliary SMO . 86 - 100 cilia were analyzed in each group in each experiment .

(E) Quantification of the percentage of cells with ciliary SMO. 86-100 cilia were analyzed in each group in each experiment.


( F ) The protein levels of Gli1 was significantly reduced in Gramd - 5KO cells . The cells were incubated in a serum starvation medium with or without ShhN for 24 h . Results shown are representative of two independent experiments .

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(F) The protein levels of Gli1 was significantly reduced in Gramd-5KO cells. The cells were incubated in a serum starvation medium with or without ShhN for 24 h. Results shown are representative of two independent experiments.


( G ) GLI - luciferase reporter assay in WT or Gramd - 5KO NIH3T3 cells . Data are presented as mean ± SEM from three technical replicates ( * * * * p < 0 . 0001 ; * * * p < 0 . 001 ; * p < 0 . 05 , two - way ANOVA with Sidak ' s multiple comparison test ) .

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(G) GLI-luciferase reporter assay in WT or Gramd-5KO NIH3T3 cells. Data are presented as mean ± SEM from three technical replicates (****p < 0.0001; ***p < 0.001; *p < 0.05, two-way ANOVA with Sidak's multiple comparison test).


( H ) The Gli1 mRNA levels were measured in WT , Gramd - 5KO , Gramd - 5KO overexpression GRAMD1A , 1B , 1C NIH3T3 cells by qRT - PCR after treatment with or without ShhN conditioned medium for 8 h . Data are expressed as mean ± SEM from three technical replicates ( * * * * p < 0 . 0001 , two - way ANOVA with Tukey ' s multiple comparisons test ) .

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(H) The Gli1 mRNA levels were measured in WT, Gramd-5KO, Gramd-5KO overexpression GRAMD1A, 1B, 1C NIH3T3 cells by qRT-PCR after treatment with or without ShhN conditioned medium for 8 h. Data are expressed as mean ± SEM from three technical replicates (****p < 0.0001, two-way ANOVA with Tukey's multiple comparisons test).


( I ) The GRAMD inhibitor AI3d inhibited cholesterol modification of SMO in NIH3T3 cells . The cells were incubated in cholesterol depletion medium for 16 h and then treated with 3 μM AI3d for 6 h . Next , cells were incubated in the same medium containing ShhN and 2 μg / mL of CP for an additional 2 h .

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(I) The GRAMD inhibitor AI3d inhibited cholesterol modification of SMO in NIH3T3 cells. The cells were incubated in cholesterol depletion medium for 16 h and then treated with 3 μM AI3d for 6 h. Next, cells were incubated in the same medium containing ShhN and 2 μg/mL of CP for an additional 2 h.