Figure 4A , C , E , G , I Fat bodies dissected from 4 hour starved animals expressing mCherry - Atg8a in all cells , and ( A ) sec5IR , ( C ) sec15IR , ( E ) sec3IR , ( G ) sec8IR , ( I ) exo84IR specifically in GFP - marked clone cells ( yw , hsflp / w ; pmCherry - Atg8a / + ; act < FRT , cd2 , FRT > GAL4 , UAS - GFP / + ) analyzed for mCherry - Atg8a puncta . Scale bars represent 20µm . B , D , F , H , J Quantification of data from ( A , C , E , G , I ) . Data are represented as means ± SEM ; n ≥ 10 . Statistical significance was determined using a Student ' s t test .

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

Figure 4A, C, E, G, I Fat bodies dissected from 4 hour starved animals expressing mCherry-Atg8a in all cells, and (A) sec5IR, (C) sec15IR, (E) sec3IR, (G) sec8IR, (I) exo84IR specifically in GFP-marked clone cells (yw, hsflp/w; pmCherry-Atg8a/+; act< FRT, cd2, FRT> GAL4, UAS-GFP/+) analyzed for mCherry-Atg8a puncta. Scale bars represent 20µm.B, D, F, H, J Quantification of data from (A, C, E, G, I). Data are represented as means ± SEM; n≥ 10. Statistical significance was determined using a Student's t test.


Figure 5A Salivary glands dissected from 14 hours after puparium formation ( top left ) control ( fkh - GAL4 / + ; pmCherry - Atg8a / + ) , and those with salivary gland - specific knockdown of ( middle left ) sec5 ( fkh - GAL4 / w ; pmCherry - Atg8a / + ; UAS - sec5IR / + ) , ( bottom left ) sec15 ( fkh - GAL4 / w ; pmCherry - Atg8a / + ; UAS - sec15IR / + ) , ( top right ) sec3 ( fkh - GAL4 / w ; pmCherry - Atg8a / UAS - sec3IR ) , ( middle right ) sec8 ( fkh - GAL4 / w ; pmCherry - Atg8a / UAS - sec8IR ) , ( bottom right ) exo84 ( fkh - GAL4 / w ; pmCherry - Atg8a / UAS - exo84IR ) analyzed for mCherry - Atg8a puncta . Scale bars represent 20µm . B Quantification of data from ( A ) . Data are represented as means ± SEM ; n ≥ 10 . Statistical significance was determined using a Student ' s t test ( * * p < 0 . 001 , * * * p < 0 . 0001 ) .

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

Figure 5A Salivary glands dissected from 14 hours after puparium formation (top left) control (fkh-GAL4/+; pmCherry-Atg8a/+), and those with salivary gland-specific knockdown of (middle left) sec5 (fkh-GAL4/w; pmCherry-Atg8a/+; UAS-sec5IR/+), (bottom left) sec15 (fkh-GAL4/w; pmCherry-Atg8a/+; UAS-sec15IR/+), (top right) sec3 (fkh-GAL4/w; pmCherry-Atg8a/UAS-sec3IR), (middle right) sec8 (fkh-GAL4/w; pmCherry-Atg8a/UAS-sec8IR), (bottom right) exo84 (fkh-GAL4/w; pmCherry-Atg8a/UAS-exo84IR) analyzed for mCherry-Atg8a puncta. Scale bars represent 20µm.B Quantification of data from (A). Data are represented as means ± SEM; n≥ 10. Statistical significance was determined using a Student's t test (** p< 0.001, *** p< 0.0001).


Figure 6A Control animals ( + / w ; UAS - sec5IR / + ) , n = 19 , and those with salivary gland - specific knockdown of sec5 ( fkh - GAL4 / w ; UAS - sec5IR / + ) , n = 20 , were analyzed by histology for the presence of salivary gland material ( red dotted circle ) 24 hours after puparium formation . B Quantification of data from ( A ) . Data are represented as means . Statistical significance was determined using a Chi - square test . C Control animals ( + / w ; UAS - sec15IR / + ) , n = 20 , and those with salivary gland - specific expression sec15 ( fkh - GAL4 / w ; UAS - sec15IR / + ) , n = 20 , were analyzed by histology for the presence of salivary gland material ( red dotted circle ) 24 hours after puparium formation . D Quantification of data from ( C ) . Data are represented as means . Statistical significance was determined using a Chi - square test . E Control animals ( + / w ; UAS - sec3IR / + ) , n = 20 , and those with salivary gland - specific expression sec3 ( fkh - GAL4 / w ; UAS - sec3IR / + ) , n = 20 , were analyzed by histology for the presence of salivary gland material ( red dotted circle ) 24 hours after puparium formation . F Quantification of data from ( E ) . Data are represented as means . Statistical significance was determined using a Chi - square test . G Control animals ( + / w ; UAS - sec8IR / + ) , n = 20 , and those with salivary gland - specific knockdown of sec8 ( fkh - GAL4 / w ; UAS - sec8IR / + ) , n = 19 , were analyzed by histology for the presence of salivary gland material ( red dotted circle ) 24 hours after puparium formation . H Quantification of data from ( G ) . Data are represented as means . Statistical significance was determined using a Chi - square test . I Control animals ( + / w ; UAS - exo84IR / + ) , n = 19 , and those with salivary gland - specific knockdown of exo84 ( fkh - GAL4 / w ; UAS - exo84IR / + ) , n = 20 , were analyzed by histology for the presence of salivary gland material ( red dotted circle ) 24 hours after puparium formation . J Quantification of data from ( I ) . Data are represented as means . Statistical significance was determined using a Chi - square test .

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

Figure 6A Control animals (+/w; UAS-sec5IR/+), n= 19, and those with salivary gland-specific knockdown of sec5 (fkh-GAL4/w; UAS-sec5IR/+), n=20, were analyzed by histology for the presence of salivary gland material (red dotted circle) 24 hours after puparium formation.B Quantification of data from (A). Data are represented as means. Statistical significance was determined using a Chi-square test.C Control animals (+/w; UAS-sec15IR/+), n= 20, and those with salivary gland-specific expression sec15 (fkh-GAL4/w; UAS-sec15IR/+), n= 20, were analyzed by histology for the presence of salivary gland material (red dotted circle) 24 hours after puparium formation.D Quantification of data from (C). Data are represented as means. Statistical significance was determined using a Chi-square test.E Control animals (+/w; UAS-sec3IR/+), n= 20, and those with salivary gland-specific expression sec3 (fkh-GAL4/w; UAS-sec3IR/+), n= 20, were analyzed by histology for the presence of salivary gland material (red dotted circle) 24 hours after puparium formation.F Quantification of data from (E). Data are represented as means. Statistical significance was determined using a Chi-square test.G Control animals (+/w; UAS-sec8IR/+), n= 20, and those with salivary gland-specific knockdown of sec8 (fkh-GAL4/w; UAS-sec8IR/+), n=19, were analyzed by histology for the presence of salivary gland material (red dotted circle) 24 hours after puparium formation.H Quantification of data from (G). Data are represented as means. Statistical significance was determined using a Chi-square test.I Control animals (+/w; UAS-exo84IR/+), n= 19, and those with salivary gland-specific knockdown of exo84 (fkh-GAL4/w; UAS-exo84IR/+), n=20, were analyzed by histology for the presence of salivary gland material (red dotted circle) 24 hours after puparium formation.J Quantification of data from (I). Data are represented as means. Statistical significance was determined using a Chi-square test.


Figure 7A Salivary glands dissected from late 3rd instar larvae ( Gbe Su ( H ) - lac - Z / w - ; + ; Fkh - GAL4 / + ) show less notch activity ( left ) compared to glands from 14 hours after puparium formation ( APF ) animals ( middle ) . Down - regulation of ral in salivary glands from 14 hours APF animals ( Gbe Su ( H ) - lac - Z / w - ; UAS - ralIR / + ; Fkh - GAL4 / + ) reduces the levels of notch activity ( right ) . Scale bar represents 20µm . B Quantification of data from ( A ) . Data are represented as means ± SEM ; n ≥ 4 . Statistical significance was determined using a Student ' s t test . C Salivary glands dissected from 14 hours after puparium formation animals expressing mCherry - Atg8a in all cells , and notchIR specifically in GFP - marked clone cells ( yw , hsflp / w , UAS - notchIR ; pmCherry - Atg8a / + ; act < FRT , cd2 , FRT > GAL4 , UAS - GFP / + ) analyzed for mCherry - Atg8a puncta . Scale bar represents 20µm . D Quantification of data from ( C ) . Data are represented as means ± SEM ; n > 10 . Statistical significance was determined using a Student ' s t test .

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

Figure 7A Salivary glands dissected from late 3rd instar larvae (Gbe Su(H)-lac-Z/w-;+;Fkh-GAL4/+) show less notch activity (left) compared to glands from 14 hours after puparium formation (APF) animals (middle). Down-regulation of ral in salivary glands from 14 hours APF animals (Gbe Su(H)-lac-Z/w-;UAS-ralIR/+;Fkh-GAL4/+) reduces the levels of notch activity (right). Scale bar represents 20µm.B Quantification of data from (A). Data are represented as means ± SEM; n≥ 4. Statistical significance was determined using a Student's t test.C Salivary glands dissected from 14 hours after puparium formation animals expressing mCherry-Atg8a in all cells, and notchIR specifically in GFP-marked clone cells (yw, hsflp/w,UAS-notchIR; pmCherry-Atg8a/+; act< FRT, cd2, FRT> GAL4, UAS-GFP/+) analyzed for mCherry-Atg8a puncta. Scale bar represents 20µm.D Quantification of data from (C). Data are represented as means ± SEM; n> 10. Statistical significance was determined using a Student's t test.


Figure 1D Zygotic grk2i283 mutant at 4dpf compared to wild type ( N = 80 ) . Arrows indicate the position of swim bladder . Scale bar ,

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Figure 1D Zygotic grk2i283 mutant at 4dpf compared to wild type (N=80). Arrows indicate the position of swim bladder. Scale bar, 1mm


Figure 2A Western blot analysis of the different forms of Gli2a in MZgrk2 embryos compared to wild type , shh mRNA and dnPKA mRNA injected wild type and cyclopamine ( Cyc ( A ) ) treated wild type 20hpf embryos . Gli2a - FL levels are low relative to Gli2aR levels in wild type embryos , but are elevated in response to pathway activation ( shh and dnPKA mRNA injected ) . Gli2aR levels are increased while Gli2a - FL is undetectable in CycA treated and MZgrk2 mutant embryos . Probing the same blot with rabbit anti - GRK3 ( which recognizes the zebrafish Grk2 protein ) reveals a complete loss of full length Grk2 in MZgrk2 embryos . Probing with anti - γ - tubulin was performed as a loading control . Three biological replicates of this analysis were performed . B Phenotype of wild type , MZgrk2 , and grk2 - GFP mRNA injected MZgrk2 embryos at 24hpf ( n = 20 for each sample ) . The white lines indicate the shape of the somites ( middle panels ) and the separation of the eyes ( right hand panels ) . Scale bar , 200μmC In situ hybridisation of ptch2 , nkx2 . 2a and olig2 transcripts in wild type , MZgrk2 and grk2 - GFP injected MZgrk2 24hpf embryos . Each panel shows a full view of the embryo on the left and a cross sectional view of a somite on the right . ( n = 30 for each sample ) . Scale bars , 200μm ( whole mounts ) , 50μm ( sections ) . D Expression of Prox1a and Engrailed proteins ( Eng ) in somites of wild - type , MZgrk2 and grk2 - GFP mRNA injected MZgrk2 embryos at 30hpf . Each panel shows Prox1a in green , Eng in red and the merge images with DAPI staining in blue . ( n = 10 for each sample ) . Scale bar , 50μm .

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

Figure 2A Western blot analysis of the different forms of Gli2a in MZgrk2 embryos compared to wild type, shh mRNA and dnPKA mRNA injected wild type and cyclopamine (Cyc(A)) treated wild type 20hpf embryos. Gli2a-FL levels are low relative to Gli2aR levels in wild type embryos, but are elevated in response to pathway activation (shh and dnPKA mRNA injected). Gli2aR levels are increased while Gli2a-FL is undetectable in CycA treated and MZgrk2 mutant embryos. Probing the same blot with rabbit anti-GRK3 (which recognizes the zebrafish Grk2 protein) reveals a complete loss of full length Grk2 in MZgrk2 embryos. Probing with anti-γ-tubulin was performed as a loading control. Three biological replicates of this analysis were performed.B Phenotype of wild type, MZgrk2, and grk2-GFP mRNA injected MZgrk2 embryos at 24hpf (n=20 for each sample). The white lines indicate the shape of the somites (middle panels) and the separation of the eyes (right hand panels). Scale bar, 200μmC In situ hybridisation of ptch2, nkx2.2a and olig2 transcripts in wild type, MZgrk2 and grk2-GFP injected MZgrk2 24hpf embryos. Each panel shows a full view of the embryo on the left and a cross sectional view of a somite on the right. (n=30 for each sample). Scale bars, 200μm (whole mounts), 50μm (sections).D Expression of Prox1a and Engrailed proteins (Eng) in somites of wild-type, MZgrk2 and grk2-GFP mRNA injected MZgrk2 embryos at 30hpf. Each panel shows Prox1a in green, Eng in red and the merge images with DAPI staining in blue. (n=10 for each sample). Scale bar, 50μm.


Figure 3A Prox1a and Eng expression in the myotome of 30hpf wild type and MZgrk2 embryos injected with mRNAs encoding Shh , dnPKA or mSmoA1 - GFP . Each panel shows Prox1a in green , Eng in red , and the merged images with DAPI staining in blue . The co - labelling for Prox1a and Eng ( orange ) is indicative of MP differentiation ( n = 30 for each sample ) Scale bar , 50μm . B Western blot showing levels of Gli2a - FL and Gli2a - R forms in 20hpf wild type and MZgrk2 embryos injected with mRNA encoding Shh , dnPKA or mSmoA1 - GFP , respectively . γ - tubulin was used as loading control . Three biological replicates of this analysis were performed .

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

Figure 3A Prox1a and Eng expression in the myotome of 30hpf wild type and MZgrk2 embryos injected with mRNAs encoding Shh, dnPKA or mSmoA1-GFP. Each panel shows Prox1a in green, Eng in red, and the merged images with DAPI staining in blue. The co-labelling for Prox1a and Eng (orange) is indicative of MP differentiation (n=30 for each sample) Scale bar, 50μm.B Western blot showing levels of Gli2a-FL and Gli2a-R forms in 20hpf wild type and MZgrk2 embryos injected with mRNA encoding Shh, dnPKA or mSmoA1-GFP, respectively. γ-tubulin was used as loading control. Three biological replicates of this analysis were performed.


Figure 4A Western blot analysis of GRK2 , GLI1 , PTCH1 , and GLI3 protein levels in Flp - In - 3T3 cells with lentiCRISPR of 4 different guide RNAs in presence or absence of SAG . α - tubulin was used as loading control . Guide RNA 2 was found to remove Grk2 efficiently and used for subsequent experiments . GLI3 panel shows both full - length ( GLI3FL ) and repressor form of GLI3 ( GLI3R ) . ( n = 3 ) B , C Hh reporter activity assay for wild type and Grk2 - / - cells . Cells were treated with PKA peptide inhibitor ( PKI ) and mutant form of PKA peptide inhibitor ( PKI - M ) respectively , in presence or absence of SHH ( B ) Cells were transfected with mSmoA1 - GFP expressing constructs in the presence of SHH or SAG ( C ) . Data represent the mean and ± SD ( n = 3 ) . Unpaired Students t - test was used for analysis . * * * P < 0 . 001 ; * * P < 0 . 01 ; * P < 0 . 05 and n . s . ( not significant ) .

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

Figure 4A Western blot analysis of GRK2, GLI1, PTCH1, and GLI3 protein levels in Flp-In-3T3 cells with lentiCRISPR of 4 different guide RNAs in presence or absence of SAG. α-tubulin was used as loading control. Guide RNA 2 was found to remove Grk2 efficiently and used for subsequent experiments. GLI3 panel shows both full-length (GLI3FL) and repressor form of GLI3 (GLI3R). (n=3)B, C Hh reporter activity assay for wild type and Grk2-/- cells. Cells were treated with PKA peptide inhibitor (PKI) and mutant form of PKA peptide inhibitor (PKI-M) respectively, in presence or absence of SHH (B) Cells were transfected with mSmoA1-GFP expressing constructs in the presence of SHH or SAG (C). Data represent the mean and ± SD (n=3). Unpaired Students t-test was used for analysis. ***P<0.001; **P<0.01; *P<0.05 and n.s. (not significant).


Figure 5A Expression of Prox1a ( green ) and the eng2a : GFP reporter ( red ) in otherwise wild - type 30hpf embryos injected with mRNA encoding wild type and mutant forms of mouse Smo . Ectopic MPs are indicated by fibres co - labelled with Prox1a and GFP ; ectopic MFFs are labeled only with GFP . Images are representative of embryos in the following proportions of each sample : 28 / 28 ( mSmo ; mSmoA1 ; mSmoSD ; mSmoKRA ) ; 9 / 28 ( mSmoA1SA ) ; 12 / 28 ( mSmoA1SD ) . Scale bar , 50μm . B Average number of Prox1a + ve slow fibres in wild type embryos injected with mRNA encoding different forms of mSmo . Prox1 positive cells were quantified in 4 somites in each of 4 embryos for each sample . The error bars indicate SD . Unpaired Student ' s t - test was used to determine the statistical significance between uninjected embryos and the various Smo mutants ( black asterisk ) ; and between mSmoA1 and mSmoA1SA or mSmoA1SD ( red asterisk ) . * * * P < 0 . 001 ; * * P < 0 . 01 ; * P < 0 . 05 and n . s . ( not significant ) . C Prox1a and Eng expression in smohi1640 mutant embryos injected with mRNA encoding mSmo ( n = 10 ) , mSmoSA ( n = 9 ) and mSmo14SA ( n = 6 ) . Note the full recovery of SSFs and MPs compared to the un - injected controls . Scale bar , 50μm . D In situ hybridisation for transcripts of ptch2 , olig2 and nkx2 . 2 in 24hpf smohi1640 mutant embryos injected with mRNA encoding mSmo and mSmoSA ( n = 6 for each sample ) . Scale bars , are 100μm ( lateral view ) , 50μm ( sections ) . E Hh reporter assay of the activity of wild type and mutant forms of mSmo in Smo - / - MEFs in response to Shh or SAG stimulation . Note that mSmoA1 shows constitutive activity in the absence of either Shh or SAG whereas mSmoKRA dose not ; all mutants affecting phosphorylation failed to restore the response to Shh or SAG . Data represent the mean and ± SD ( n = 3 ) . Unpaired Students t - test was used for analysis . * * * * P < 0 . 0001 ; * * P < 0 . 01 ; and n . s . ( not significant ) .

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

Figure 5A Expression of Prox1a (green) and the eng2a:GFP reporter (red) in otherwise wild-type 30hpf embryos injected with mRNA encoding wild type and mutant forms of mouse Smo. Ectopic MPs are indicated by fibres co-labelled with Prox1a and GFP; ectopic MFFs are labeled only with GFP. Images are representative of embryos in the following proportions of each sample: 28/28 (mSmo; mSmoA1; mSmoSD; mSmoKRA); 9/28 (mSmoA1SA); 12/28 (mSmoA1SD). Scale bar, 50μm.B Average number of Prox1a+ve slow fibres in wild type embryos injected with mRNA encoding different forms of mSmo. Prox1 positive cells were quantified in 4 somites in each of 4 embryos for each sample. The error bars indicate SD. Unpaired Student's t-test was used to determine the statistical significance between uninjected embryos and the various Smo mutants (black asterisk); and between mSmoA1 and mSmoA1SA or mSmoA1SD (red asterisk). ***P<0.001; **P<0.01; *P<0.05 and n.s. (not significant).C Prox1a and Eng expression in smohi1640 mutant embryos injected with mRNA encoding mSmo (n=10), mSmoSA (n=9) and mSmo14SA (n=6). Note the full recovery of SSFs and MPs compared to the un-injected controls. Scale bar, 50μm.D In situ hybridisation for transcripts of ptch2, olig2 and nkx2.2 in 24hpf smohi1640 mutant embryos injected with mRNA encoding mSmo and mSmoSA (n=6 for each sample). Scale bars, are 100μm (lateral view), 50μm (sections).E Hh reporter assay of the activity of wild type and mutant forms of mSmo in Smo-/- MEFs in response to Shh or SAG stimulation. Note that mSmoA1 shows constitutive activity in the absence of either Shh or SAG whereas mSmoKRA dose not; all mutants affecting phosphorylation failed to restore the response to Shh or SAG. Data represent the mean and ± SD (n=3). Unpaired Students t-test was used for analysis. ****P<0.0001; **P<0.01; and n.s. (not significant).


Figure 6A Wild type 18hpf embryos injected with mRNA encoding GFP - tagged mSmo ( green ) showing localisation to the PC of myotomal cells labeled with anti - Acetylated tubulin ( AcTub ; red ) , stimulated in response to Shh injection ( n = 4 ) . Differences in PC distribution are due to morphological changes in the myotome induced by ectopic expression of Shh as revealed by the distribution of nuclei ( DAPI stained ; blue ) . Scale bar , 10μm . B Notochord cells of wild type embryos expressing GFP tagged wild - type and mutant forms of mSmo . Note the localisation to the PC ( labeled with anti - AcTub ; red ) in each case ( n = 4 for each sample ) . Scale bar , 10μm . C MZgrk2 18hpf embryos injected with mRNA encoding GFP - tagged wild type mSmo or mSmoSA ( green ) showing localisation to the PC ( labeled with anti - AcTub ; red ) in myotomal cells . More Smo is localised to the PC in MZgrk2 mutants compared to wild type ( panel A ) . Scale bar , 10μm .

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Figure 6A Wild type 18hpf embryos injected with mRNA encoding GFP-tagged mSmo (green) showing localisation to the PC of myotomal cells labeled with anti-Acetylated tubulin (AcTub; red), stimulated in response to Shh injection (n = 4). Differences in PC distribution are due to morphological changes in the myotome induced by ectopic expression of Shh as revealed by the distribution of nuclei (DAPI stained; blue). Scale bar, 10μm.B Notochord cells of wild type embryos expressing GFP tagged wild-type and mutant forms of mSmo. Note the localisation to the PC (labeled with anti-AcTub; red) in each case (n=4 for each sample). Scale bar, 10μm.C MZgrk2 18hpf embryos injected with mRNA encoding GFP-tagged wild type mSmo or mSmoSA (green) showing localisation to the PC (labeled with anti- AcTub; red) in myotomal cells. More Smo is localised to the PC in MZgrk2 mutants compared to wild type (panel A). Scale bar, 10μm.


Figure 7B Prox1a ( green ) and Eng ( red ) expression in the myotome of 30hpf MZgrk2 embryos that were injected with mRNA encoding : Grk2K220R - GFP ; Grk2K220M - GFP ; Grk2D335N - GFP ; Grk2ΔN7aa - GFP and mGrk2L4AV7AL8A - GFP . Merged panels show nuclear staining with DAPI in blue . ( n = 20 for each sample ) . Scale bar , 50μm . C Hh reporter activity assay in wild type and Grk2 - / - 3T3 cells transfected with the equivalent wild type and mutant kinase domain forms of mouse Grk2 , Grk2K220R , Grk2K220M and Grk2D335N after 24h Shh - N and SAG treatment . Data represent the mean and ± SD ( n = 3 ) . Unpaired Students t - test was used for analysis . * * * * P < 0 . 0001 ; * * P < 0 . 01 ; * P < 0 . 05 ; and n . s . ( not significant ) .

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Figure 7B Prox1a (green) and Eng (red) expression in the myotome of 30hpf MZgrk2 embryos that were injected with mRNA encoding: Grk2K220R-GFP; Grk2K220M-GFP; Grk2D335N-GFP; Grk2ΔN7aa-GFP and mGrk2L4AV7AL8A-GFP. Merged panels show nuclear staining with DAPI in blue. (n=20 for each sample). Scale bar, 50μm.C Hh reporter activity assay in wild type and Grk2-/- 3T3 cells transfected with the equivalent wild type and mutant kinase domain forms of mouse Grk2, Grk2K220R, Grk2K220M and Grk2D335N after 24h Shh-N and SAG treatment. Data represent the mean and ± SD (n=3). Unpaired Students t-test was used for analysis. ****P<0.0001; **P<0.01; *P<0.05; and n.s. (not significant).


Figure 1Immunoblot analysis of protein lysates from ARPE - 19 cells expressing TFEB - WT - FLAG or TFEB - S401A - FLAG treated with 200 μM NaAsO2 or 250 nM Torin - 1 for 1 h . Two different rabbit polyclonal antibodies raised against a TFEB - S401 phospho - specific peptide were tested . Immunoblot analysis of protein lysates from ARPE - 19 cells expressing TFEB - WT - FLAG , TFEB - S3A / R4A - FLAG or TFEB - R245 - 247A - FLAG treated with 200 μM NaAsO2 for 1 h . Immunoblots are representative of at least three independent experiments .

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Figure 1Immunoblot analysis of protein lysates from ARPE-19 cells expressing TFEB-WT-FLAG or TFEB-S401A-FLAG treated with 200 μM NaAsO2 or 250 nM Torin-1 for 1 h. Two different rabbit polyclonal antibodies raised against a TFEB-S401 phospho-specific peptide were tested.Immunoblot analysis of protein lysates from ARPE-19 cells expressing TFEB-WT-FLAG, TFEB-S3A/R4A-FLAG or TFEB-R245-247A-FLAG treated with 200 μM NaAsO2 for 1 h. Immunoblots are representative of at least three independent experiments.


Figure 2Immunoblot analysis of protein lysates from HeLa cells stably expressing TFEB - WT - FLAG incubated with the indicated kinase inhibitors for 1 h prior to the addition of 200 μM NaAsO2 for 1 h . Quantification of immunoblot data shown in ( A ) . Data are presented as mean ± SD using one - way ANOVA ( unpaired ) followed by Tukey ' s multiple comparisons test , ( ns ) not significant , and ( * * * * ) p < 0 . 0001 from three independent experiments . Data information : n = 3 biological replicates ( each dot represents a biological replicate ) . Immunoblot analysis of protein lysates from HeLa cells stably expressing TFEB - WT - FLAG incubated with either 200 μM NaAsO2 for 1 h , EBSS for 4 h , 100 ng / ml EGF for the indicated times or 37 μM Anisomycin for 1 h . Before the addition of EGF or Anisomycin , cells were serum starved for 8 h . Immunoblot analysis of protein lysates from HeLa cells stably expressing TFEB - WT - FLAG exposed to 30J / m2 of UV - C and allowed to recover in complete medium for the indicated times . Cells were incubated with p38 MAPK inhibitor ( 20 μM , SB203580 ) for 1 h before UV - C irradiation and allowed to recover for 30 min in the presence of the inhibitor . Immunoblot analysis of protein lysates from HeLa cells stably expressing TFEB - WT - FLAG depleted of either p38 MAPK ( α + β ) or JNK1 or JNK2 or JNK ( 1 + 2 ) and incubated with 200 μM NaAsO2 for 1 h . Immunoblots are representative of at least three independent experiments . Immunoblot analysis of in vitro p38 MAPK kinase assay using GST - TFEB - PRD as substrate in the presence or absence of either recombinant human active p38α MAPK or ATP . Quantification of immunoblot data shown in ( F ) . Data are presented as mean ± SD using one - way ANOVA ( unpaired ) followed by Tukey ' s multiple comparisons test , ( * ) p < 0 . 05 from three independent experiments . Data information : n = 3 biological replicates ( each dot represents a biological replicate ) .

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Figure 2Immunoblot analysis of protein lysates from HeLa cells stably expressing TFEB-WT-FLAG incubated with the indicated kinase inhibitors for 1 h prior to the addition of 200 μM NaAsO2 for 1 h. Quantification of immunoblot data shown in (A). Data are presented as mean ± SD using one-way ANOVA (unpaired) followed by Tukey's multiple comparisons test, (ns) not significant, and (****)p<0.0001 from three independent experiments. Data information: n = 3 biological replicates (each dot represents a biological replicate).Immunoblot analysis of protein lysates from HeLa cells stably expressing TFEB-WT-FLAG incubated with either 200 μM NaAsO2 for 1 h, EBSS for 4 h, 100 ng/ml EGF for the indicated times or 37 μM Anisomycin for 1 h. Before the addition of EGF or Anisomycin, cells were serum starved for 8 h.Immunoblot analysis of protein lysates from HeLa cells stably expressing TFEB-WT-FLAG exposed to 30J/m2 of UV-C and allowed to recover in complete medium for the indicated times. Cells were incubated with p38 MAPK inhibitor (20 μM, SB203580) for 1 h before UV-C irradiation and allowed to recover for 30 min in the presence of the inhibitor.Immunoblot analysis of protein lysates from HeLa cells stably expressing TFEB-WT-FLAG depleted of either p38 MAPK (α+β) or JNK1 or JNK2 or JNK(1+2) and incubated with 200 μM NaAsO2 for 1 h. Immunoblots are representative of at least three independent experiments.Immunoblot analysis of in vitro p38 MAPK kinase assay using GST-TFEB-PRD as substrate in the presence or absence of either recombinant human active p38α MAPK or ATP. Quantification of immunoblot data shown in (F). Data are presented as mean ± SD using one-way ANOVA (unpaired) followed by Tukey's multiple comparisons test, (*)p<0.05 from three independent experiments. Data information: n = 3 biological replicates (each dot represents a biological replicate).


Figure 3Immunoblot analysis of protein lysates from Raw 264 . 7 cells stably expressing TFEB - WT - FLAG incubated with 1μg / ml LPS for the indicated times . Immunoblot analysis of protein lysates from Raw 264 . 7 cells stably expressing TFEB - WT - FLAG incubated with either 20 μM SB203580 or 250 nM Torin - 1 for 1 h prior to the addition of 1 μg / ml LPS for 30 min . Quantification of immunoblot data shown in ( B ) . Data are presented as mean ± SD using one - way ANOVA ( unpaired ) followed by Tukey ' s multiple comparisons test , ( ns ) not significant , ( * ) p < 0 . 05 and ( * * ) p < 0 . 01 from three independent experiments . Data information : n = 3 biological replicates ( each dot represents a biological replicate ) . Immunoblot analysis of protein lysates from THP1 cells incubated with 1 μg / ml LPS for the indicated times . Immunoblot analysis of protein lysates from THP1 cells incubated with either vehicle ( DMSO ) or 20 μM SB203580 for 1 h prior to the addition of 1μg / ml LPS for the indicated times . Immunoblot analysis of protein lysates from THP1 cells depleted of p38 MAPK ( α ) , p38 MAPK ( β ) or p38 MAPK ( α + β ) and incubated with 1 μg / ml LPS for 1 h . Immunoblots are representative of at least three independent experiments . Quantification of immunoblot data shown in ( F ) . Data are presented as mean ± SD using one - way ANOVA ( unpaired ) followed by Tukey ' s multiple comparisons test , ( * ) p < 0 . 05 from three independent experiments . Data information : n = 3 biological replicates ( each dot represents a biological replicate ) .

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Figure 3Immunoblot analysis of protein lysates from Raw 264.7 cells stably expressing TFEB-WT-FLAG incubated with 1μg/ml LPS for the indicated times.Immunoblot analysis of protein lysates from Raw 264.7 cells stably expressing TFEB-WT-FLAG incubated with either 20 μM SB203580 or 250 nM Torin-1 for 1 h prior to the addition of 1 μg/ml LPS for 30 min.Quantification of immunoblot data shown in (B). Data are presented as mean ± SD using one-way ANOVA (unpaired) followed by Tukey's multiple comparisons test, (ns) not significant, (*)p<0.05 and (**)p<0.01 from three independent experiments. Data information: n = 3 biological replicates (each dot represents a biological replicate).Immunoblot analysis of protein lysates from THP1 cells incubated with 1 μg/ml LPS for the indicated times.Immunoblot analysis of protein lysates from THP1 cells incubated with either vehicle (DMSO) or 20 μM SB203580 for 1 h prior to the addition of 1μg/ml LPS for the indicated times.Immunoblot analysis of protein lysates from THP1 cells depleted of p38 MAPK (α), p38 MAPK (β) or p38 MAPK (α+β) and incubated with 1 μg/ml LPS for 1 h. Immunoblots are representative of at least three independent experiments. Quantification of immunoblot data shown in (F). Data are presented as mean ± SD using one-way ANOVA (unpaired) followed by Tukey's multiple comparisons test, (*)p<0.05 from three independent experiments. Data information: n = 3 biological replicates (each dot represents a biological replicate).


Figure 4Immunoblot analysis of protein lysates from naïve THP1 - WT or TFEB - S401A knock - in ( clone M17 ) cells treated with 50 ng / ml PMA for the indicated times , PMA - differentiated THP1 ( Rested ) cells treated without or with 250 nM Torin - 1 for 1 h . Immunoblot analysis of proteins from nuclear and cytosolic fractions from naïve THP1 - WT or TFEB - S401A knock - ins ( clones I11 and M17 ) cells treated with 50 ng / ml PMA for the indicated times and PMA - differentiated THP1 ( Rested ) cells . Immunoblot analysis of proteins from nuclear and cytosolic fractions from naïve THP1 - WT or TFEB - S401A knock - in ( clone I11 ) cells treated with 50 ng / ml PMA for the indicated times . Immunoblot analysis of proteins from nuclear fractions from naïve THP1 - WT or TFEB - S401A knock - in ( clone I11 ) cells treated with 50 ng / ml PMA or 250 nM Torin - 1 for 1 h . Immunoblots are representative of at least three independent experiments .

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Figure 4Immunoblot analysis of protein lysates from naïve THP1-WT or TFEB-S401A knock-in (clone M17) cells treated with 50 ng/ml PMA for the indicated times, PMA-differentiated THP1 (Rested) cells treated without or with 250 nM Torin-1 for 1 h.Immunoblot analysis of proteins from nuclear and cytosolic fractions from naïve THP1-WT or TFEB-S401A knock-ins (clones I11 and M17) cells treated with 50 ng/ml PMA for the indicated times and PMA-differentiated THP1 (Rested) cells.Immunoblot analysis of proteins from nuclear and cytosolic fractions from naïve THP1-WT or TFEB-S401A knock-in (clone I11) cells treated with 50 ng/ml PMA for the indicated times.Immunoblot analysis of proteins from nuclear fractions from naïve THP1-WT or TFEB-S401A knock-in (clone I11) cells treated with 50 ng/ml PMA or 250 nM Torin-1 for 1 h. Immunoblots are representative of at least three independent experiments.


Figure 5Immunoblot analysis of proteins from nuclear and cytosolic fractions from naïve THP1 - WT or TFEB - S401A knock - in ( clone I11 ) cells treated with 50 ng / ml PMA for 6 h .

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Figure 5Immunoblot analysis of proteins from nuclear and cytosolic fractions from naïve THP1-WT or TFEB-S401A knock-in (clone I11) cells treated with 50 ng/ml PMA for 6 h.


Figure 6Immunoblot analysis of protein lysates and cell culture medium from PMA - differentiated ( Rested ) THP1 - WT or TFEB - S401A knock - ins ( clones I11 and M17 ) cells incubated with 0 . 1 μg / ml LPS for 4 h prior to the addition of 15 μM Nigericin for 45 min . C - F . Quantification of immunoblot data shown in ( B ) . Data are presented as mean ± SD using one - way ANOVA ( unpaired ) followed by Tukey ' s multiple comparisons test , ( ns ) not significant , ( * ) p < 0 . 05 , ( * * ) p < 0 . 01 , ( * * * ) p < 0 . 001 and ( * * * * ) p < 0 . 0001 from three independent experiments . Data information : n ≥ 3 biological replicates ( each dot represents a biological replicate ) . Fluorescence confocal microscopy of PMA - differentiated ( Rested ) THP1 - WT or TFEB - S401A knock - ins ( clones I11 and M17 ) cells incubated with 1 μg / ml LPS for 6 h prior to the detection of dead cells with LIVE / DEAD fixable blue dead cell stain . Scale bars , 10 μm . Quantification of the percentage of dead cells shown in ( G ) . Data are presented as mean ± SD using one - way ANOVA ( unpaired ) followed by Tukey ' s multiple comparisons test , ( ns ) not significant and ( * * * * ) p < 0 . 0001 as compared to their corresponding untreated ( Control ) cells , with > 200 cells counted per trial from three independent experiments . Data information : n ≥ 3 biological replicates ( each dot represents a biological replicate ) .

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Figure 6Immunoblot analysis of protein lysates and cell culture medium from PMA-differentiated (Rested) THP1-WT or TFEB-S401A knock-ins (clones I11 and M17) cells incubated with 0.1 μg/ml LPS for 4 h prior to the addition of 15 μM Nigericin for 45 min. C-F. Quantification of immunoblot data shown in (B). Data are presented as mean ± SD using one-way ANOVA (unpaired) followed by Tukey's multiple comparisons test, (ns) not significant, (*)p<0.05, (**)p<0.01, (***)p< 0.001 and (****)p< 0.0001 from three independent experiments. Data information: n ≥ 3 biological replicates (each dot represents a biological replicate).Fluorescence confocal microscopy of PMA-differentiated (Rested) THP1-WT or TFEB-S401A knock-ins (clones I11 and M17) cells incubated with 1 μg/ml LPS for 6 h prior to the detection of dead cells with LIVE/DEAD fixable blue dead cell stain. Scale bars, 10 μm. Quantification of the percentage of dead cells shown in (G). Data are presented as mean ± SD using one-way ANOVA (unpaired) followed by Tukey's multiple comparisons test, (ns) not significant and (****)p< 0.0001 as compared to their corresponding untreated (Control) cells, with >200 cells counted per trial from three independent experiments. Data information: n ≥ 3 biological replicates (each dot represents a biological replicate).


Figure 1A . From left to right , Surface representation of the three - dimensional structures of C3b - MCP ( CCP1 - 4 ) ( blue ) , C3b - SPICE ( CCP1 - 4 ) ( green ) , C3b - FH ( CCP1 - 4 ) ( orange ) , C3b - CR1 ( CCP15 - 17 ) ( dark red ) , C3b - DAF ( CCP2 - 4 ) ( purple ) . B . Cartoon representation of the five regulators . The width of the cartoon is proportional to the B - factors observed in the structural data . The B - factor coloring ( from blue to red ) is in relative scale according to the resolution of the crystal structures . C . Cartoon representation of the five C3b molecules in the C3b - regulator complexes . Coloring and style as in B . D . Highlight of electron density maps ( 2Fo - Fc , blue , contour level 1 . 2σ ; Fo - Fc , green and red , contour level 3σ and - 3σ , resp . ) for the regions of the C3b - regulator complexes showing flexibility . From left to right : the putative site for MCP CCP2 , SPICE CCP4 , CR1 CCP15 , and DAF CCP2 .

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Figure 1A. From left to right, Surface representation of the three-dimensional structures of C3b-MCP (CCP1-4) (blue), C3b-SPICE (CCP1-4) (green), C3b-FH (CCP1-4) (orange), C3b-CR1 (CCP15-17) (dark red), C3b-DAF (CCP2-4) (purple).B. Cartoon representation of the five regulators. The width of the cartoon is proportional to the B-factors observed in the structural data. The B-factor coloring (from blue to red) is in relative scale according to the resolution of the crystal structures.C. Cartoon representation of the five C3b molecules in the C3b-regulator complexes. Coloring and style as in B.D. Highlight of electron density maps (2Fo-Fc, blue, contour level 1.2σ; Fo-Fc, green and red, contour level 3σ and -3σ, resp.) for the regions of the C3b-regulator complexes showing flexibility. From left to right: the putative site for MCP CCP2, SPICE CCP4, CR1 CCP15, and DAF CCP2.


Figure 2A . " Open - book " surface representation of C3b and regulator molecules , highlighting their domain organizations using different colors as shown in the neighboring schemes . Domains in complement regulators are labeled using domain numbering from CCPi to CCPiv based on their contact sites on C3b . The corresponding CCP domains in each regulator described in this work are shown in the bottom - left scheme . Domains CCP1 - 2 of MCP are shown in shaded grey because they are not involved in C3b binding . The table reports the extent of the regulator contact surface on C3b and the associated buried surface area . B . Opened view of the molecular footprint of the regulator - C3b interaction ( as shown in Fig . 2A ) , highlighting the C3b domains on the regulator surface ( top ) and the regulator domains on C3b ( bottom ) .

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Figure 2A. "Open-book" surface representation of C3b and regulator molecules, highlighting their domain organizations using different colors as shown in the neighboring schemes. Domains in complement regulators are labeled using domain numbering from CCPi to CCPiv based on their contact sites on C3b. The corresponding CCP domains in each regulator described in this work are shown in the bottom-left scheme. Domains CCP1-2 of MCP are shown in shaded grey because they are not involved in C3b binding. The table reports the extent of the regulator contact surface on C3b and the associated buried surface area.B. Opened view of the molecular footprint of the regulator-C3b interaction (as shown in Fig. 2A), highlighting the C3b domains on the regulator surface (top) and the regulator domains on C3b (bottom).


Figure 3A . Superposition of MCP and FH domains CCP3 - 4 ( shown as blue and orange cartoons , resp . ) on the surface of C3b . B . Cartoon representation of the superposition of domains CCP3 - 4 of MCP ( blue ) , FH ( orange ) and SPICE ( green ) . Despite the strong structural similarity and the conserved angle between domains CCP3 and CCP4 , MCP and SPICE show a rotation of approximately 100 ° in the position of CCP4 compared to FH . C . The reported mutagenesis data on MCP CCP3 and CCP4 ( data from Liszewki et al . ( Liszewski et al , 2000 ) ) are consistent with the crystal structure . Shown are two orientations of the surface of MCP CCP3 - 4 as in the crystal structure of the C3b - MCP complex , highlighting the contact sites with the C3b platform ( purple ) , the MCP mutations identified as critical for C3b binding ( red ) and those not affecting the interaction ( blue ) . The GB24 antibody epitope is shown as yellow contour on the MCP surface . D . Model of the putative positioning of the flexible CCP1 - 2 domains of MCP . The model has been generated by superposing the structure of CCP1 - 4 of MCP from PDB 3O8E ( Persson et al , 2010 ) onto the structure of the C3b - MCP ( CCP1 - 4 ) complex . CCP1 - 2 domains of MCP , not visible in the C3b - MCP ( CCP1 - 4 ) structure , are shown in grey . E . Highlight of the primary sequence differences between SPICE and VCP . The 11 mutations distinguishing the two viral regulators are shown on the surface of SPICE in purple . The contact surface of C3b on the regulator in the C3b - SPICE ( CCP1 - 4 ) complex is shown in green on the right panel .

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Figure 3A. Superposition of MCP and FH domains CCP3-4 (shown as blue and orange cartoons, resp.) on the surface of C3b.B. Cartoon representation of the superposition of domains CCP3-4 of MCP (blue), FH (orange) and SPICE (green). Despite the strong structural similarity and the conserved angle between domains CCP3 and CCP4, MCP and SPICE show a rotation of approximately 100° in the position of CCP4 compared to FH.C. The reported mutagenesis data on MCP CCP3 and CCP4 (data from Liszewki et al. (Liszewski et al, 2000)) are consistent with the crystal structure. Shown are two orientations of the surface of MCP CCP3-4 as in the crystal structure of the C3b-MCP complex, highlighting the contact sites with the C3b platform (purple), the MCP mutations identified as critical for C3b binding (red) and those not affecting the interaction (blue). The GB24 antibody epitope is shown as yellow contour on the MCP surface.D. Model of the putative positioning of the flexible CCP1-2 domains of MCP. The model has been generated by superposing the structure of CCP1-4 of MCP from PDB 3O8E (Persson et al, 2010) onto the structure of the C3b-MCP (CCP1-4) complex. CCP1-2 domains of MCP, not visible in the C3b-MCP (CCP1-4) structure, are shown in grey.E. Highlight of the primary sequence differences between SPICE and VCP. The 11 mutations distinguishing the two viral regulators are shown on the surface of SPICE in purple. The contact surface of C3b on the regulator in the C3b-SPICE (CCP1-4) complex is shown in green on the right panel.


Figure 4 ( A ) Mapping of known disease - related mutations on C3b ( Table 3 ) . The high - resolution C3b three - dimensional structure is used as structural template , shown as white surface . The regulator contact surfaces ( based on C3b - FH ( CCP1 - 4 ) ( Wu et al , 2009 ) and C3d - FH ( CCP19 - 20 ) ( Kajander et al , 2011 ; Morgan et al , 2011 ) ) are shown as black contour . All mutations reported in Table 3 are shown in blue on the C3b surface . Disease - related mutants reported to affect regulator binding are shown in red and labeled in the right magnified panel . ( B ) Mapping of disease - related mutations on the FH ( CCP1 - 4 ) surface using data from Table 4 . Colors as in Fig . 4A . ( C ) Mapping of disease - related mutations on the MCP ( CCP3 - 4 ) surface using data from Table 4 . Colors as in Fig . 4A .

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Figure 4(A) Mapping of known disease-related mutations on C3b (Table 3). The high-resolution C3b three-dimensional structure is used as structural template, shown as white surface. The regulator contact surfaces (based on C3b-FH (CCP1-4) (Wu et al, 2009) and C3d-FH (CCP19-20) (Kajander et al, 2011; Morgan et al, 2011)) are shown as black contour. All mutations reported in Table 3 are shown in blue on the C3b surface. Disease-related mutants reported to affect regulator binding are shown in red and labeled in the right magnified panel.(B) Mapping of disease-related mutations on the FH (CCP1-4) surface using data from Table 4. Colors as in Fig. 4A.(C) Mapping of disease-related mutations on the MCP (CCP3-4) surface using data from Table 4. Colors as in Fig. 4A.


Figure 1 ( A ) N - glycan composition of PD - 1 protein expressed from 293T cells by ESI - MS analysis . N - glycans of representative peaks from ESI - MS were identified by MS / MS analysis . H , mannose ; N , N - acetylglucosamine ; F , fucose . Structural formulas : blue square , N - acetylglucosamine ; red triangle , fucose ; green circle , mannose ; yellow circle , galactose . ( B , C ) Accelerated thermal stability of PD - 1 proteins obtained from 293T cells ( green line ) , insect cells ( pink line ) or E . coli ( blue line ) is characterized in all - in - one UNcle platform . Protein unfolding is observed as an increase in barycentric mean ( B ) while small particle formation of the samples is observed as an increase in SLS intensity at 266 nm ( C ) . ( D ) SPR analysis of the binding characteristics of camrelizumab with PD - 1 expressed in 293T cells , insect cells or E . coli cell , which enabled different levels of glycosylations . The binding affinity is presented as KD . The data presented here is a representative of two independent experiments with similar results .

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Figure 1 (A) N-glycan composition of PD-1 protein expressed from 293T cells by ESI-MS analysis. N-glycans of representative peaks from ESI-MS were identified by MS/MS analysis. H, mannose; N, N-acetylglucosamine; F, fucose. Structural formulas: blue square, N-acetylglucosamine; red triangle, fucose; green circle, mannose; yellow circle, galactose.  (B,C) Accelerated thermal stability of PD-1 proteins obtained from 293T cells (green line), insect cells (pink line) or E. coli (blue line) is characterized in all-in-one UNcle platform. Protein unfolding is observed as an increase in barycentric mean (B) while small particle formation of the samples is observed as an increase in SLS intensity at 266 nm (C).  (D) SPR analysis of the binding characteristics of camrelizumab with PD-1 expressed in 293T cells, insect cells or E. coli cell, which enabled different levels of glycosylations. The binding affinity is presented as KD. The data presented here is a representative of two independent experiments with similar results.


Figure 2 ( A ) Gel filtration profiles of PD - 1 ( orange ) , camrelizumab - scFv ( blue ) , and the PD - 1 / camrelizumab - scFv complex ( red ) are analyzed by size - exclusion chromatography as indicated . The SDS - PAGE analyses are shown with peak 1 of camrelizumab - scFv , peak 2 of camrelizumab - scFv / PD - 1 complex , and peak 3 of PD - 1 .

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Figure 2 (A) Gel filtration profiles of PD-1 (orange), camrelizumab-scFv (blue), and the PD-1/camrelizumab-scFv complex (red) are analyzed by size-exclusion chromatography as indicated. The SDS-PAGE analyses are shown with peak 1 of camrelizumab-scFv, peak 2 of camrelizumab-scFv/PD-1 complex, and peak 3 of PD-1.


Figure 4 ( E ) SPR analysis of the binding of camrelizumab with N58A mutated PD - 1 expressed in 293T cells . ( F ) The SPR analysis of the binding of camrelizumab with WT ( upper ) or PNGase F treated PD - 1 proteins ( lower ) expressed in 293T cells . ( G The SPR analysis of the binding of nivolumab with WT ( upper ) or N58A mutated ( lower ) PD - 1 expressed in 293T cells H ) The SPR analysis of the binding of pembrolizumab with WT ( upper ) or N58A mutated ( lower ) PD - 1 proteins .

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Figure 4(E) SPR analysis of the binding of camrelizumab with N58A mutated PD-1 expressed in 293T cells.(F) The SPR analysis of the binding of camrelizumab with WT (upper) or PNGase F treated PD-1 proteins (lower) expressed in 293T cells. (G The SPR analysis of the binding of nivolumab with WT (upper) or N58A mutated (lower) PD-1 expressed in 293T cells H) The SPR analysis of the binding of pembrolizumab with WT (upper) or N58A mutated (lower) PD-1 proteins.


Figure 5 ( A , B ) The blocking of the binding of WT PD - 1 - mFc ( A ) or N58A mutated PD - 1 - mFc ( B ) to PD - L1 transiently expressed on the surface of 293T cells are analyzed with varied concentrations of camrelizumab . The PD - L1 expressing 293T cells stained with PBS alone is used as negative controls , whereas the cells stained with WT or N58A mutated PD - 1 - mFc proteins are used as a positive control . The frequency of PD - 1 - mFc staining positive cells in PD - L1 - GFP positive cells is labeled in the upright corner . The reduced frequency of the PD - L1 - mFc - staining - positive subpopulation compared with positive control indicates the blockage of the PD - 1 / PD - L1 interaction . The density of events at a given position in the plot is color - coded , with red representing the highest number of events at that point in the graph , while yellow , green , and blue represent progressively lower event densities . The results presented here are representative of three independent experiments with similar results .

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

Figure 5 (A,B) The blocking of the binding of WT PD-1-mFc (A) or N58A mutated PD-1-mFc (B) to PD-L1 transiently expressed on the surface of 293T cells are analyzed with varied concentrations of camrelizumab. The PD-L1 expressing 293T cells stained with PBS alone is used as negative controls, whereas the cells stained with WT or N58A mutated PD-1-mFc proteins are used as a positive control. The frequency of PD-1-mFc staining positive cells in PD-L1-GFP positive cells is labeled in the upright corner. The reduced frequency of the PD-L1-mFc-staining-positive subpopulation compared with positive control indicates the blockage of the PD-1/PD-L1 interaction. The density of events at a given position in the plot is color-coded, with red representing the highest number of events at that point in the graph, while yellow, green, and blue represent progressively lower event densities. The results presented here are representative of three independent experiments with similar results.


Figure 1A . Volcano blot representation of the differently expressed genes in FCRLS - and CD11b - positive Grn - / - microglia in comparison to wt microglia isolated from 5 . 5 - month - old mice ( male , n = 5 ) . 116 genes out of 529 genes analyzed , are significantly changed , and from these 58 genes are up - and 58 genes are down regulated . Eight genes with the highest fold change are highlighted . B . Heatmap of significantly affected genes ( p < 0 . 05 ) in FCRLS - and CD11b - positive Grn - / - microglia in comparison to Grn + / + microglia isolated from 5 . 5 - month - old mice ( n = 5 per genotype ) . For the significantly affected genes of the Grn - / - microglia , mRNA expression data of the Trem2 - / - microglia in comparison to the corresponding wt microglia were taken from previously published data ( Mazaheri et al , 2017 ) . The RNA counts for each gene and sample were normalized to the mean value of wt followed by a log2 transformation ( n ≥ 5 per genotype ) . * labelled genes were not analyzed or below detection limit in Trem2 - / - microglia mRNA expression data set ( Mazaheri et al , 2017 ) . D . Gene expression levels of FCRLS - and CD11b - positive Grn - / - microglia normalized to wt in comparison to the published normalized data set of Trem2 - / - microglia ( Mazaheri et al , 2017 ) .

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Figure 1A. Volcano blot representation of the differently expressed genes in FCRLS- and CD11b- positive Grn-/- microglia in comparison to wt microglia isolated from 5.5-month-old mice (male, n = 5). 116 genes out of 529 genes analyzed, are significantly changed, and from these 58 genes are up- and 58 genes are down regulated. Eight genes with the highest fold change are highlighted.B. Heatmap of significantly affected genes (p < 0.05) in FCRLS- and CD11b-positive Grn-/- microglia in comparison to Grn+/+ microglia isolated from 5.5-month-old mice (n = 5 per genotype). For the significantly affected genes of the Grn-/- microglia, mRNA expression data of the Trem2-/- microglia in comparison to the corresponding wt microglia were taken from previously published data (Mazaheri et al, 2017). The RNA counts for each gene and sample were normalized to the mean value of wt followed by a log2 transformation (n ≥ 5 per genotype). * labelled genes were not analyzed or below detection limit in Trem2-/- microglia mRNA expression data set (Mazaheri et al, 2017).D. Gene expression levels of FCRLS- and CD11b-positive Grn-/- microglia normalized to wt in comparison to the published normalized data set of Trem2-/- microglia (Mazaheri et al, 2017).


Figure 2A . Immunoblot analysis of ApoE , CLEC7A , TREM2 and CD68 in lysates of acutely isolated microglia from Grn - / - and Grn + / + mice ( 9 months of age , n = 3 per genotype , female ) . IBA1 was used as loading control . B . Quantification of protein expression normalized to levels of Grn + / + microglia ( n = 3 ) . Data represent the mean ± SD . C . ELISA mediated quantification of sTREM2 in brain homogenates of Grn - / - and Grn + / + mice Data are shown as mean ± SEM ( n = 3 - 6 ) . D . ELISA mediated quantification of sTREM2 in serum of Grn - / - and Grn + / + mice . Data are shown as mean ± SEM ( n = 4 - 13 ) . E . Microglial expression of P2RY12 in cortical sections of wt , Grn - / - and Trem2 - / - mice . ( 9 months of age , female ) F . Quantification of P2RY12 positive microglia . Data are shown as mean ± SD ( n = 3 per genotype , female , except one male for Trem2 - / - ) . G . Immunoblot analysis of secreted PGRN and ApoE ( ApoEmed ) in conditioned media and PGRN , ApoE , CLEC7A , P2RY12 and TREM2 in lysates of BV2 wild type ( Grnwt ) and PGRN deficient ( Grnmut ) cells . Soluble amyloid precursor protein ( APPs ) and calnexin were used as a loading control . H . Quantification of Immunoblots normalized to BV2 Grnwt levels ( n = 3 - 5 ) . Data represent the mean ± SD . I . ELISA mediated quantification of sTREM2 in conditioned media of BV2 Grnwt and Grnmut cells . Data represent the mean ± SDJ . Quantification of relative mRNA levels of Apoe , Clec7a , P2ry12 and Trem2 in BV2 Grnwt and Grnmut cells . Data represent the mean ± SD .

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Figure 2A. Immunoblot analysis of ApoE, CLEC7A, TREM2 and CD68 in lysates of acutely isolated microglia from Grn-/- and Grn+/+ mice (9 months of age, n=3 per genotype, female). IBA1 was used as loading control.B. Quantification of protein expression normalized to levels of Grn+/+ microglia (n=3). Data represent the mean ± SD.C. ELISA mediated quantification of sTREM2 in brain homogenates of Grn-/- and Grn+/+ mice Data are shown as mean ± SEM (n=3-6). D. ELISA mediated quantification of sTREM2 in serum of Grn-/- and Grn+/+ mice. Data are shown as mean ± SEM (n=4-13). E. Microglial expression of P2RY12 in cortical sections of wt, Grn-/-and Trem2-/- mice. (9 months of age, female) F. Quantification of P2RY12 positive microglia. Data are shown as mean ± SD (n=3 per genotype, female, except one male for Trem2-/-). G. Immunoblot analysis of secreted PGRN and ApoE (ApoEmed) in conditioned media and PGRN, ApoE, CLEC7A, P2RY12 and TREM2 in lysates of BV2 wild type (Grnwt) and PGRN deficient (Grnmut) cells. Soluble amyloid precursor protein (APPs) and calnexin were used as a loading control.H. Quantification of Immunoblots normalized to BV2 Grnwt levels (n=3-5). Data represent the mean ± SD.I. ELISA mediated quantification of sTREM2 in conditioned media of BV2 Grnwt and Grnmut cells. Data represent the mean ± SDJ. Quantification of relative mRNA levels of Apoe, Clec7a, P2ry12 and Trem2 in BV2 Grnwt and Grnmut cells. Data represent the mean ± SD.


Figure 3A . Flow cytometric analysis of phagocytic capacity in BV2wt and BV2mut cells using pHrodo green E . coli as target particles . Phagocytosis was terminated after 30 and 60 minutes ( min ) of incubation . Data are presented as mean percentage of cells positive for pHrodo uptake ± S . D ( n = 3 ) . B . Enhanced migration of CD68 positive , PGRN deficient cells in an ex vivo model . Immunofluorescence analysis of cultured Grn + / + and Grn - / - organotypic brain at 7 DIV immunostained using microglial marker CD68 . Nuclei were counterstained using DAPI . Images of boxed regions in left panels are shown at higher magnifications in right panels . Scale bars : 500 µm ( left panels ) , 250 µm ( right panels ) . Quantitative analysis reveals that Grn - / - microglia migrate larger distances than Grn + / + . Data are shown as mean ± SD ( n = 3 independent experiments ) Quantification displays an increased number of migrating Grn - / - cells compared to Grn + / + cells . Data are shown as mean ± SD ( n = 3 independent experiments ) E . Enhanced microglial clustering around amyloid plaques in the absence of PGRN . Left : IBA1 - stained microglia clustering around X - 34 - positive amyloid plaque cores in 4 - month - old APPPS1 / Grn + / + mice . Right : Age - matched APPPS1 / Grn - / - mice display microglial hyper - clustering around amyloid plaques compared to APPPS1 / Grn + / + . Dotted white boxes indicate the area that is shown at higher magnification . Scale bars indicate 10µm and 3µm in inset . Immunohistochemical quantification of IBA1 positive microglial cell number per

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

Figure 3A. Flow cytometric analysis of phagocytic capacity in BV2wt and BV2mut cells using pHrodo green E. coli as target particles. Phagocytosis was terminated after 30 and 60 minutes (min) of incubation. Data are presented as mean percentage of cells positive for pHrodo uptake ± S.D (n=3).B. Enhanced migration of CD68 positive, PGRN deficient cells in an ex vivo model. Immunofluorescence analysis of cultured Grn+/+ and Grn-/- organotypic brain at 7 DIV immunostained using microglial marker CD68. Nuclei were counterstained using DAPI. Images of boxed regions in left panels are shown at higher magnifications in right panels. Scale bars: 500 µm (left panels), 250 µm (right panels). Quantitative analysis reveals that Grn-/- microglia migrate larger distances than Grn+/+. Data are shown as mean ± SD (n=3 independent experiments) Quantification displays an increased number of migrating Grn-/- cells compared to Grn+/+ cells. Data are shown as mean ± SD (n=3 independent experiments)E. Enhanced microglial clustering around amyloid plaques in the absence of PGRN. Left: IBA1-stained microglia clustering around X-34-positive amyloid plaque cores in 4-month-old APPPS1/Grn+/+ mice. Right: Age-matched APPPS1/Grn-/- mice display microglial hyper-clustering around amyloid plaques compared to APPPS1/Grn+/+. Dotted white boxes indicate the area that is shown at higher magnification. Scale bars indicate 10µm and 3µm in inset. Immunohistochemical quantification of IBA1 positive microglial cell number per plaqu


Figure 4A . TSPO - µPET in rodents : Axial slices ( A1 , A2 , A3 ) of averaged % - TSPO - PET differences between Grn - / - or Trem2 - / - mice and wt indicate increased microglial activity in the cerebrum of Grn - / - mice ( hot color scale ) and decreased microglial activity in the cerebrum of Trem2 - / - mice ( cold color scale ) . Scatter plot depicts single TSPO - µPET values deriving from a neocortical volume - of - interest . µPET results are illustrated upon an MRI template ; 6 - 10 female mice per group ( Grn - / - n = 6 at 8 months , Trem2 - / - n = 8 at 11 months ; wt n = 10 at 8 - 11 months ) . Statistics derive from one - way ANOVA with Tukey post - hoc test . B . PGRN serum levels of the patients subjected to TSPO - PET confirmed the GRN mutation and non - GRN mutation carrier . PGRN was measured by ELISA and normalized to serum levels of a healthy control . C . TSPO - PET in human FTLD patients : Visual interpretation of axial TSPO - PET SUVR images in upper cranial layers reveals increased microglial activity in frontal and parietal cortices ( white arrows ) of a FTLD patient with a GRN loss of function mutation in comparison to a FTLD patient without a GRN mutation . PET results are illustrated upon an MRI template ; 60 - 80 min p . i . emission recording ; global mean scaling . Both patients were low - affinity binder .

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

Figure 4A. TSPO-µPET in rodents: Axial slices (A1, A2, A3) of averaged %-TSPO-PET differences between Grn-/-or Trem2-/- mice and wt indicate increased microglial activity in the cerebrum of Grn-/- mice (hot color scale) and decreased microglial activity in the cerebrum of Trem2-/- mice (cold color scale). Scatter plot depicts single TSPO-µPET values deriving from a neocortical volume-of-interest. µPET results are illustrated upon an MRI template; 6-10 female mice per group (Grn-/- n=6 at 8 months, Trem2-/- n=8 at 11 months; wt n=10 at 8-11 months). Statistics derive from one-way ANOVA with Tukey post-hoc test.B. PGRN serum levels of the patients subjected to TSPO-PET confirmed the GRN mutation and non-GRN mutation carrier. PGRN was measured by ELISA and normalized to serum levels of a healthy control.C. TSPO-PET in human FTLD patients: Visual interpretation of axial TSPO-PET SUVR images in upper cranial layers reveals increased microglial activity in frontal and parietal cortices (white arrows) of a FTLD patient with a GRN loss of function mutation in comparison to a FTLD patient without a GRN mutation. PET results are illustrated upon an MRI template; 60-80 min p.i. emission recording; global mean scaling. Both patients were low-affinity binder.


Figure 5FDG - µPET in rodents : Axial slices ( as indicated in Fig . 4A ) of averaged % - FDG - µPET differences between Grn - / - or Trem2 - / - mice and wt indicate decreased glucose metabolism in the cerebrum of Grn - / - and Trem2 - / - ( both cold color scale ) when compared to wt . Scatter plot depicts single FDG - PET values deriving from a neocortical volume - of - interest . µPET results are illustrated upon an MRI template ; 6 - 10 female mice per group ( Grn - / - n = 6 at 8 months , Trem2 - / - n = 6 at 11 months ; wt n = 10 at 8 - 11 months ) .

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

Figure 5FDG-µPET in rodents: Axial slices (as indicated in Fig. 4A) of averaged %-FDG-µPET differences between Grn-/-or Trem2-/- mice and wt indicate decreased glucose metabolism in the cerebrum of Grn-/- and Trem2-/- (both cold color scale) when compared to wt. Scatter plot depicts single FDG-PET values deriving from a neocortical volume-of-interest. µPET results are illustrated upon an MRI template; 6-10 female mice per group (Grn-/- n=6 at 8 months, Trem2-/- n=6 at 11 months; wt n=10 at 8-11 months).


Figure 1 ( A _ B ) In situs on adult brain sections reveal that ( A ) opn4 . 1 and ( B ) opn4xb are coexpressed in many brain domains including the epiphysis cerebri ( indicated with E ) or pineal . A horizontal line through a representation of the brain above the sagittal section indicates the location within the brain . Scale bar : 1 . 0 mm .

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

Figure 1(A_B) In situs on adult brain sections reveal that (A) opn4.1 and (B) opn4xb are coexpressed in many brain domains including the epiphysis cerebri (indicated with E) or pineal. A horizontal line through a representation of the brain above the sagittal section indicates the location within the brain. Scale bar: 1.0 mm.


Figure 4 ( J ) ish with asmt probe on larvae sampled on the 6th dpf at ZT3 shows that asmt mRNA is confined to the pineal and indicates that the asmt gene is not ectopically expressed in opn4 dko larvae . The higher asmt mRNA level measured by qPCR in the opn4 dko therefore points at a defect in the pineal . Note that the eyes were removed after the ish for optimal visibility of expression in the brain . Scale bar : 0 . 5 mm . ( N ) ish with ddc probe on wild - type and opn4 dko larvae sampled on the 6th dpf represents ddc expression in pineals . Biological replicates indicated with BR . Scale bar : 0 . 05 mm ( O ) Quantification of the adjusted intensity in the pineals from larvae presented in N , implies a significant higher ddc transcript level in the opn4 dko pineal . Data information : boxplot divides the data in quartiles : the box indicates the interquartile range , with the horizontal line in the box denoting the median of the data set , the whiskers extend to the minimum and maximum , and meet the box at the median of the lower ( quartile 1 ) and median of the upper ( quartile 3 ) half of the dataset . Black dots indicate biological replicates O : n = 8 ) , the red dot indicates the mean , red error bars indicate the confidence interval ( 95 % ) , asterisks indicate significance ( 0 . 01 < p ( ✱ ) < 0 . 05 , 0 . 001 < p ( ✱ ✱ ) < 0 . 01 , p ( ✱ ✱ ✱ ) < 0 . 001 , ns = not significant ) .

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

Figure 4(J) ish with asmt probe on larvae sampled on the 6th dpf at ZT3 shows that asmt mRNA is confined to the pineal and indicates that the asmt gene is not ectopically expressed in opn4 dko larvae. The higher asmt mRNA level measured by qPCR in the opn4 dko therefore points at a defect in the pineal. Note that the eyes were removed after the ish for optimal visibility of expression in the brain. Scale bar: 0.5 mm.(N) ish with ddc probe on wild-type and opn4 dko larvae sampled on the 6th dpf represents ddc expression in pineals. Biological replicates indicated with BR. Scale bar: 0.05 mm (O) Quantification of the adjusted intensity in the pineals from larvae presented in N, implies a significant higher ddc transcript level in the opn4 dko pineal. Data information: boxplot divides the data in quartiles: the box indicates the interquartile range, with the horizontal line in the box denoting the median of the data set, the whiskers extend to the minimum and maximum, and meet the box at the median of the lower (quartile 1) and median of the upper (quartile 3) half of the dataset. Black dots indicate biological replicates O: n=8), the red dot indicates the mean, red error bars indicate the confidence interval (95%), asterisks indicate significance (0.01<p(✱)<0.05, 0.001<p(✱✱)<0.01, p(✱✱✱)<0.001, ns = not significant).


Figure 3Neuroblastoma PDX and SK - N - BE ( 2 ) c cells treated with IBL inhibitors at indicated concentrations for 48 hours . pAkt ( at Ser473 ( A ) and Thr308 ( B ) sites ) levels in LU - NB - 3 and SK - N - BE ( 2 ) c cells determined by western blotting . Total Akt levels were used as loading control . Neuroblastoma PDX cells treated with IBL inhibitors at indicated concentrations for 48 hours . C . p - p70S6K and p - p85S6K levels in LU - NB - 3 cells determined by western blotting . Actin , p70S6K , and p85S6K levels were used as loading controls . D . Brightfield photomicrographs of LU - NB - 3 and SK - N - BE ( 2 ) c cells treated with 0 . 36μM IBL - 202 or 0 . 05 μM IBL - 301 . Scale bars represent 100 μm ( LU - NB - 3 ) or 200 μm ( SK - N - BE ( 2 ) c ) . Arrows indicate neurite outgrowths and asterisks indicate where inserts are magnified . IBL - 301 treated cells were stained for Tuj1 . DAPI was used to visualize nuclei . E . Quantification of neurite outgrowth presented as number of neurites / cell in LU - NB - 3 PDX and SK - N - BE ( 2 ) cells treated with IBL - 301 . For LU - NB - 3 PDX cells , representative areas ( n = 2 ) were used and n = 344 and n = 240 cells / condition for CTRL and IBL - 301 , respectively , were counted . For SK - N - BE ( 2 ) c cells , representative areas ( n = 2 and n = 3 for CTRL and IBL - 301 respectively ) were used and n = 141 and n = 130 cells / condition for CTRL and IBL - 301 , respectively , were counted . Statistical significance was determined by two - sided student ' s t - tes . P = 0 . 003 for LU - NB - 3 and p = 0 . 08 for SK - N - BE ( 2 ) c . Neuroblastoma PDX and SK - N - BE ( 2 ) c cells treated with IBL inhibitors at indicated concentrations for 48 hours . F . Gap43 protein levels in LU - NB - 3 and SK - N - BE ( 2 ) c cells determined by western blotting . SDHA levels were used as loading control . Neuroblastoma PDX cells treated with IBL inhibitors at indicated concentrations for 48 hours . G . N - Myc levels in LU - NB - 2 and LU - NB - 3 cells determined by western blotting . SDHA levels were used as loading control .

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

Figure 3Neuroblastoma PDX and SK-N-BE(2)c cells treated with IBL inhibitors at indicated concentrations for 48 hours. pAkt (at Ser473 (A) and Thr308 (B) sites) levels in LU-NB-3 and SK-N-BE(2)c cells determined by western blotting. Total Akt levels were used as loading control.Neuroblastoma PDX cells treated with IBL inhibitors at indicated concentrations for 48 hours. C. p-p70S6K and p-p85S6K levels in LU-NB-3 cells determined by western blotting. Actin, p70S6K, and p85S6K levels were used as loading controls.D. Brightfield photomicrographs of LU-NB-3 and SK-N-BE(2)c cells treated with 0.36μM IBL-202 or 0.05 μM IBL-301. Scale bars represent 100 μm (LU-NB-3) or 200 μm (SK-N-BE(2)c). Arrows indicate neurite outgrowths and asterisks indicate where inserts are magnified. IBL-301 treated cells were stained for Tuj1. DAPI was used to visualize nuclei. E. Quantification of neurite outgrowth presented as number of neurites/cell in LU-NB-3 PDX and SK-N-BE(2) cells treated with IBL-301. For LU-NB-3 PDX cells, representative areas (n=2) were used and n=344 and n=240 cells/condition for CTRL and IBL-301, respectively, were counted. For SK-N-BE(2)c cells, representative areas (n=2 and n=3 for CTRL and IBL-301 respectively) were used and n=141 and n=130 cells/condition for CTRL and IBL-301, respectively, were counted. Statistical significance was determined by two-sided student's t-tes. P=0.003 for LU-NB-3 and p=0.08 for SK-N-BE(2)c.Neuroblastoma PDX and SK-N-BE(2)c cells treated with IBL inhibitors at indicated concentrations for 48 hours. F. Gap43 protein levels in LU-NB-3 and SK-N-BE(2)c cells determined by western blotting. SDHA levels were used as loading control.Neuroblastoma PDX cells treated with IBL inhibitors at indicated concentrations for 48 hours. G. N-Myc levels in LU-NB-2 and LU-NB-3 cells determined by western blotting. SDHA levels were used as loading control.


Figure 4A . Viability IBL - 202 or IBL - 301 treated cells determined by CellTiterGlo . B - C . Cell cycle distribution of LU - NB - 3 cells determined by flow cytometry . Bars show mean values from two independent experiments . D . Cleaved caspase 3 ( Cl Casp3 ) levels in LU - NB - 3 cells determined by western blotting . SDHA levels were used as loading control . E . Flow cytometry analyses of Annexin V and PI stainings following treatment with 360 nM IBL - 202 or 50 nM IBL - 301 . F . Quantification of live and dead cells from the AnnexinV / PI stainings . Dead cells = PI positive , Live cells = PI negative . Dot plots from two independent experiments and error bars represent SEM .

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

Figure 4A. Viability IBL-202 or IBL-301 treated cells determined by CellTiterGlo.B-C. Cell cycle distribution of LU-NB-3 cells determined by flow cytometry. Bars show mean values from two independent experiments.D. Cleaved caspase 3 (Cl Casp3) levels in LU-NB-3 cells determined by western blotting. SDHA levels were used as loading control.E. Flow cytometry analyses of Annexin V and PI stainings following treatment with 360 nM IBL-202 or 50 nM IBL-301. F. Quantification of live and dead cells from the AnnexinV/PI stainings. Dead cells=PI positive, Live cells=PI negative. Dot plots from two independent experiments and error bars represent SEM.


Figure 5Neuroblastoma cells were treated with indicated concentrations of IBL - 302 for 48 hours A . Expression of pAkt ( Ser473 ) in LU - NB - 3 and SK - N - BE ( 2 ) c cells . Total Akt and actin were used as loading controls . B . Expression of pAkt ( Thr308 ) in LU - NB - 3 and SK - N - BE ( 2 ) c cells . Total Akt and actin were used as loading controls . Neuroblastoma cells were treated with indicated concentrations of IBL - 302 for 48 hours C . Brightfield photomicrographs of LU - NB - 3 and SK - N - BE ( 2 ) c cells treated with 50 nM IBL - 302 . Scale bars represent 100 μm ( LU - NB - 3 ) or 200 μm ( SK - N - BE ( 2 ) c ) . D . Quantification of neurite outgrowth presented as number of neurites / cell in LU - NB - 3 PDX and SK - N - BE ( 2 ) c cells treated with IBL - 302 . For LU - NB - 3 PDX cells , representative areas ( n = 4 ) were used and n = 460 and n = 216 cells / condition for CTRL and IBL - 302 , respectively , were counted . For SK - N - BE ( 2 ) c cells , representative areas ( n = 5 and n = 7 for CTRL and IBL - 302 respectively ) were used and n = 124 and n = 260 cells / condition for CTRL and IBL - 301 , respectively , were counted . Statistical significance was determined by two - sided student ' s t - test . p = 0 . 009 for LU - NB - 3 and p = 0 . 0003 for SK - N - BE ( 2 ) c . Neuroblastoma cells were treated with indicated concentrations of IBL - 302 for 48 hours E . N - Myc protein expression determined by western blotting . SDHA was used as loading control . Neuroblastoma cells were treated with indicated concentrations of IBL - 302 for 72 hours F . Cell viability determined by CellTiterGlo . Neuroblastoma cells were treated with indicated concentrations of IBL - 302 for 48 hours G . Flow cytometry analyses of Annexin V and PI stainings following treatment with 50nM IBL - 302 H . Quantification of live and dead cells . Dead cells = PI positive , Live cells = PI negative . Dot plots from two independent experiments and error bars represent SEM . I . Neuroblastoma SK - N - BE ( 2 ) c carrying mice ( n = 5 in each group ) were treated with vehicle ( CTRL ) or 40 mg / kg IBL - 302 for up to 35 days and tumor growth was followed over time . Asterisks indicate each occasion a mouse within that particular group was sacrificed . J . Kaplan - Meier survival curves comparing mice treated with vehicle ( CTRL ) or IBL - 302 . Log - rank test was used to determine statistical significance . p = 0 . 0577 .

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

Figure 5Neuroblastoma cells were treated with indicated concentrations of IBL-302 for 48 hours A. Expression of pAkt(Ser473) in LU-NB-3 and SK-N-BE(2)c cells. Total Akt and actin were used as loading controls. B. Expression of pAkt(Thr308) in LU-NB-3 and SK-N-BE(2)c cells. Total Akt and actin were used as loading controls.Neuroblastoma cells were treated with indicated concentrations of IBL-302 for 48 hours C. Brightfield photomicrographs of LU-NB-3 and SK-N-BE(2)c cells treated with 50 nM IBL-302. Scale bars represent 100 μm (LU-NB-3) or 200 μm (SK-N-BE(2)c). D. Quantification of neurite outgrowth presented as number of neurites/cell in LU-NB-3 PDX and SK-N-BE(2)c cells treated with IBL-302. For LU-NB-3 PDX cells, representative areas (n=4) were used and n=460 and n=216 cells/condition for CTRL and IBL-302, respectively, were counted. For SK-N-BE(2)c cells, representative areas (n=5 and n=7 for CTRL and IBL-302 respectively) were used and n=124 and n=260 cells/condition for CTRL and IBL-301, respectively, were counted. Statistical significance was determined by two-sided student's t-test. p=0.009 for LU-NB-3 and p=0.0003 for SK-N-BE(2)c.Neuroblastoma cells were treated with indicated concentrations of IBL-302 for 48 hours E. N-Myc protein expression determined by western blotting. SDHA was used as loading control.Neuroblastoma cells were treated with indicated concentrations of IBL-302 for 72 hours F. Cell viability determined by CellTiterGlo.Neuroblastoma cells were treated with indicated concentrations of IBL-302 for 48 hours G. Flow cytometry analyses of Annexin V and PI stainings following treatment with 50nM IBL-302 H. Quantification of live and dead cells. Dead cells=PI positive, Live cells=PI negative. Dot plots from two independent experiments and error bars represent SEM.I. Neuroblastoma SK-N-BE(2)c carrying mice (n=5 in each group) were treated with vehicle (CTRL) or 40 mg/kg IBL-302 for up to 35 days and tumor growth was followed over time. Asterisks indicate each occasion a mouse within that particular group was sacrificed.J. Kaplan-Meier survival curves comparing mice treated with vehicle (CTRL) or IBL-302. Log-rank test was used to determine statistical significance. p=0.0577.


Figure 6A - B . LU - NB - 3 PDX , SK - N - BE ( 2 ) c and SK - N - SH cells were treated with indicated concentrations of cisplatin and / or 0 . 36 μM IBL - 202 , 0 . 05 μM IBL - 301 , or 0 . 05 μM IBL - 302 for 48 hours . Cell viability determined by CellTiterGlo . Graphs show mean values and SEM from three independent experiments . Theoretical additive curves were evaluated by calculating the combination index ( CI ) based on the Bliss Independence model . C - D . Cell cycle distribution in LU - NB - 3 PDX cells after treatment with 320 nM cisplatin , 50 nM IBL - 302 or the combination of these , determined by flow cytometry . Bars show mean values from two independent experiments . E . Tumor size in individual mice from Neuroblastoma PDX carrying mice ( n = 5 in each group ) were treated with vehicle ( CTRL ) , low - dose ( 20mg / kg ) IBL - 302 , low - dose ( 1mg / kg ) cisplatin , or the combination of low - dose IBL - 302 and low - dose cisplatin for up to 70 days . Tumor size was measured over time . Asterisks indicate each occasion a mouse within that particular group was sacrificed . F . Neuroblastoma PDX carrying mice ( n = 5 in each group ) were treated with vehicle ( CTRL ) , low - dose ( 20mg / kg ) IBL - 302 , low - dose ( 1mg / kg ) cisplatin , or the combination of low - dose IBL - 302 and low - dose cisplatin for up to 70 days . Tumor size was measured over time . Asterisks indicate each occasion a mouse within that particular group was sacrificed . G . Kaplan - Meier survival curves comparing mice treated with vehicle ( CTRL ) , IBL - 302 , cisplatin , or the combination . Log - rank test was used to determine statistical significance . p = 0 . 0014 . H . Immunohistochemical staining of N - Myc expression in tumors Scale bars represent 100 μm .

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

Figure 6A-B. LU-NB-3 PDX, SK-N-BE(2)c and SK-N-SH cells were treated with indicated concentrations of cisplatin and/or 0.36 μM IBL-202, 0.05 μM IBL-301, or 0.05 μM IBL-302 for 48 hours. Cell viability determined by CellTiterGlo. Graphs show mean values and SEM from three independent experiments. Theoretical additive curves were evaluated by calculating the combination index (CI) based on the Bliss Independence model.C-D. Cell cycle distribution in LU-NB-3 PDX cells after treatment with 320 nM cisplatin, 50 nM IBL-302 or the combination of these, determined by flow cytometry. Bars show mean values from two independent experiments.E. Tumor size in individual mice from Neuroblastoma PDX carrying mice (n=5 in each group) were treated with vehicle (CTRL), low-dose (20mg/kg) IBL-302, low-dose (1mg/kg) cisplatin, or the combination of low-dose IBL-302 and low-dose cisplatin for up to 70 days. Tumor size was measured over time. Asterisks indicate each occasion a mouse within that particular group was sacrificed.F. Neuroblastoma PDX carrying mice (n=5 in each group) were treated with vehicle (CTRL), low-dose (20mg/kg) IBL-302, low-dose (1mg/kg) cisplatin, or the combination of low-dose IBL-302 and low-dose cisplatin for up to 70 days. Tumor size was measured over time. Asterisks indicate each occasion a mouse within that particular group was sacrificed.G. Kaplan-Meier survival curves comparing mice treated with vehicle (CTRL), IBL-302, cisplatin, or the combination. Log-rank test was used to determine statistical significance. p=0.0014.H. Immunohistochemical staining of N-Myc expression in tumors Scale bars represent 100 μm.


Figure 7LU - NB - 3 PDX cells were treated with 50 nM of IBL - 302 for 48 hours and subsequently analyzed by RNAseq and mass spectrometry . A - C . Heat map of significantly differentially expressed genes across treatment groups from RNAseq data ( FDR < 0 . 1 ) ( A ) . Genes defined as differentially expressed that were also present in MS and phospho - MS are visualized in ( B ) and ( C ) , respectively .

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Figure 7LU-NB-3 PDX cells were treated with 50 nM of IBL-302 for 48 hours and subsequently analyzed by RNAseq and mass spectrometry. A-C. Heat map of significantly differentially expressed genes across treatment groups from RNAseq data (FDR<0.1) (A). Genes defined as differentially expressed that were also present in MS and phospho-MS are visualized in (B) and (C), respectively.