( A , A ' ) Anti - Spitz ( red ) immunostaining of w1118 adult head . Spitz can be detected in the retina and lamina . ( B , B ' ) The full - length transmembrane form of Spitz - GFP ( mSpi , green ) expressed in the retina in GMR > mSpiGFP flies was predominantly identified in the photoreceptor soma and terminally localized in the lamina cartridge . mSpitz - GFP requires processing by Rho and Star to become a secreted form . Overexpressed mSpitzGFP has been demonstrated to be retained in the perinuclear ER even in the presence of endogenous Rho / Star [ 56 ] ( C ) Knockdown of both EGFR ligands Spi and Krn in R1 - 6 photoreceptors in Rh1 > Dcr2 + Spi - RNAi + Krn - RNAi and ( D ) blockade of Spi processing in Rh1 > lacZ + iRhom exhibited lamina degeneration after shifting to 29 ° C for 14 days . ( E - E ' ) Lamina degeneration in whole eye rho7M43ru1 double mutant clones at 28 ° C for 12 days . The clone is labeled by DsRed ( red ) . ( F ) Spi secretion is inhibited in Rh1 > lacZ + Rab11S25N . ( G ) The percentages of the vacuole areas of ( C , D , F ) in lamina at 29 ° C for 14 days were examined . All P - values were calculated using one - way ANOVA with Tukey ' s post - test . DAPI : nuclei ( white in A ' , B ' , C - F ) . Scale bar : 20 μm .

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(A, A') Anti-Spitz (red) immunostaining of w1118 adult head. Spitz can be detected in the retina and lamina. (B, B') The full-length transmembrane form of Spitz-GFP (mSpi, green) expressed in the retina in GMR>mSpiGFP flies was predominantly identified in the photoreceptor soma and terminally localized in the lamina cartridge. mSpitz-GFP requires processing by Rho and Star to become a secreted form. Overexpressed mSpitzGFP has been demonstrated to be retained in the perinuclear ER even in the presence of endogenous Rho/Star [56] (C) Knockdown of both EGFR ligands Spi and Krn in R1-6 photoreceptors in Rh1>Dcr2+Spi-RNAi+Krn-RNAi and (D) blockade of Spi processing in Rh1>lacZ+iRhom exhibited lamina degeneration after shifting to 29°C for 14 days. (E-E') Lamina degeneration in whole eye rho7M43ru1 double mutant clones at 28°C for 12 days. The clone is labeled by DsRed (red). (F) Spi secretion is inhibited in Rh1>lacZ+Rab11S25N. (G) The percentages of the vacuole areas of (C, D, F) in lamina at 29°C for 14 days were examined. All P-values were calculated using one-way ANOVA with Tukey's post-test. DAPI: nuclei (white in A', B', C-F). Scale bar: 20 μm.


( A , A ' ) Anti - Spitz detected Spitz ( magenta ) colocalizing ( arrowhead ) with epithelialgliacytoplasm marked by anti - Black ( green ) . Scale bar : 5 μm . ( B - B ' ) EGFR reporter pointed - lacZ ( pnt - lacZ ) exhibited expression in the epithelial ( arrow ) and marginal glia ( arrowhead ) . ( C , C ' ) Dominant - negative form of EGFR ( DERDN ) expressed in glia at 28 ° C for 3 days inhibited pnt - lacZ expression . ( D , D ' ) pnt - laZ expression was lost in Shits - expressing glia at 28 ° C for 3 days . ( E , E ' ) The knockdown of Spi in R1 - 6 photoreceptors at 28 ° C for 12 days also inhibited pnt - lacZ expression in the glia . The penetrances of ( B - E ) are shown in the upper right corner of each panel . DAPI : nuclei ( white in B ' - E ' ) . Scale bar : 20 μm .

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(A, A') Anti-Spitz detected Spitz (magenta) colocalizing (arrowhead) with epithelialgliacytoplasm marked by anti-Black (green). Scale bar: 5 μm. (B-B') EGFR reporter pointed-lacZ (pnt-lacZ) exhibited expression in the epithelial (arrow) and marginal glia (arrowhead). (C, C') Dominant-negative form of EGFR (DERDN) expressed in glia at 28°C for 3 days inhibited pnt-lacZ expression. (D, D') pnt-laZ expression was lost in Shits-expressing glia at 28°C for 3 days. (E, E') The knockdown of Spi in R1-6 photoreceptors at 28°C for 12 days also inhibited pnt-lacZ expression in the glia. The penetrances of (B-E) are shown in the upper right corner of each panel. DAPI: nuclei (white in B'-E'). Scale bar: 20 μm.


( A - C ) Immunostaining of TUNEL assay ( red ) , active Caspase - 3 ( magenta , A ' - C ' ) and Repo ( green , A " - C " ) . ( A ) GMR - wIR ; repots > lacZ . ( B ) GMR - wIR ; repots > Shits1 . ( C ) GMR - wIR ; repots > DERDN . GMR - wIR was used to reduce the autofluorescence of the eye pigments . Scale bar : 20 μm . ( D - F ) The in vivo fluorescent sensor of caspase activity ( Apoliner ) indicated there was no active caspase activity in the glia . ( D ) repots > Apoliner + lacZ . ( E ) repots > Apoliner + Shits1 . ( F ) repots > Apoliner + DERDN . All adults were shifted to 28 ° C for 5 days . nls - GFP ( green , D - F ) ; merge of GFP ( green ) and mRFP ( red ) in ( D ' - F ' ) ; merge of nls - GFP and DAPI ( magenta ) in ( D " - F " ) . Scale bar : 10 μm . ( G ) Percentage of vacuole area in the lamina in repots > Shits1flies that coexpressed the anti - apoptotic factors P35 and Diap1 . The adults were shifted to 28 ° C for 21 days . The P - values were calculated using one - way ANOVA with Tukey ' s post - test . ( H ) The cell number of the epithelialglia was not reduced in the vacuolated lamina . The numbers of epithelial glia in the lamina of repo > H2B - RFP and repo > H2B - RFP + Shits1 female adults incubated at 28 ° C for 14 days were examined by counting the nuclear RFP at the epithelial layer from the entire Z - stacks of confocal images . P - values were calculated via Mann - Whitney tests .

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(A-C) Immunostaining of TUNEL assay (red), active Caspase-3 (magenta, A'-C') and Repo (green, A"-C"). (A) GMR-wIR; repots>lacZ. (B) GMR-wIR; repots>Shits1. (C) GMR-wIR; repots>DERDN. GMR-wIR was used to reduce the autofluorescence of the eye pigments. Scale bar: 20 μm. (D-F) The in vivo fluorescent sensor of caspase activity (Apoliner) indicated there was no active caspase activity in the glia. (D) repots>Apoliner+lacZ. (E) repots>Apoliner+Shits1. (F) repots>Apoliner+DERDN. All adults were shifted to 28°C for 5 days. nls-GFP (green, D-F); merge of GFP (green) and mRFP (red) in (D'-F'); merge of nls-GFP and DAPI (magenta) in (D"-F"). Scale bar: 10 μm. (G) Percentage of vacuole area in the lamina in repots>Shits1flies that coexpressed the anti-apoptotic factors P35 and Diap1. The adults were shifted to 28°C for 21 days. The P-values were calculated using one-way ANOVA with Tukey's post-test. (H) The cell number of the epithelialglia was not reduced in the vacuolated lamina. The numbers of epithelial glia in the lamina of repo>H2B-RFP and repo>H2B-RFP+Shits1 female adults incubated at 28°C for 14 days were examined by counting the nuclear RFP at the epithelial layer from the entire Z-stacks of confocal images. P-values were calculated via Mann-Whitney tests.


In repots > GFP - LC3 + DERDN shifted to 28 ° C , the GFP signal ( green ) was weak on day 1 ( A - A " ) and progressively increased on days 2 ( B - B " ) and 3 ( C - C " ) and colocalized with Ref ( 2 ) P ( magenta ) . ( D ) Ref ( 2 ) P ( magenta ) accumulated within the Egfrco mutant MARCM clone ( green ) . The double - tagged GFP - mCherry - Atg8a is used to distinguish the autophagosomes ( GFP and mCherry , yellow ) and autolysosomes ( mCherry , red ) in autophagic flux . ( E ) In repots > GFP - mCherry - Atg8a + dAtg1 , autophagosomes ( arrowhead ) and autolysosomes ( arrow ) were induced in the glia with normal autophagic flux . ( F ) repots > GFP - mCherry - Atg8a . ( G ) repots > GFP - mCherry - Atg8a + Shits1 . Epithelialglial nuclei are indicated ( arrow ) . ( H ) repots > GFP - mCherry - Atg8a + DERDN . The adults were shifted to 28 ° C for 3 days . GFP : green ( E - H ) ; mCherry : red ( E ' - H ' ) ; merge ( E " - H " ) . Scale bar : 10 μm .

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In repots>GFP-LC3+DERDN shifted to 28°C, the GFP signal (green) was weak on day 1 (A-A") and progressively increased on days 2 (B-B") and 3 (C-C") and colocalized with Ref(2)P (magenta). (D) Ref(2)P (magenta) accumulated within the Egfrco mutant MARCM clone (green). The double-tagged GFP-mCherry-Atg8a is used to distinguish the autophagosomes (GFP and mCherry, yellow) and autolysosomes (mCherry, red) in autophagic flux. (E) In repots>GFP-mCherry-Atg8a+dAtg1, autophagosomes (arrowhead) and autolysosomes (arrow) were induced in the glia with normal autophagic flux. (F) repots>GFP-mCherry-Atg8a. (G) repots>GFP-mCherry-Atg8a+Shits1. Epithelialglial nuclei are indicated (arrow). (H) repots>GFP-mCherry-Atg8a+DERDN. The adults were shifted to 28°C for 3 days. GFP: green (E-H); mCherry: red (E'-H'); merge (E"-H"). Scale bar: 10 μm.


( A - C ) repots > GFP - LAMP1 . ( D - F ) repots > GFP - LAMP1 + Shits1 . ( G - I ) repots > GFP - LAMP1 + DERDN . Adults were incubated at 28 ° C for 12 h ( A , D , G ) , 24 h ( B , E , H ) , and 48 h ( C , F , I ) , respectively . The GFP - LAMP1 signal ( green ) was induced at 12 h and progressively enhanced in ( D - F ) and ( G - I ) . ( J ) repots > Rab7 - mCherry + H2B - RFP . ( K ) repots > Rab7 - mCherry + Shits1 . ( L ) repots > Rab7 - mCherry + DERDN . The adults were shifted to 28 ° C for 2 days . Rab7 - mCherry puncta ( red ) were increased in ( K , L ) . DAPI : nuclei ( white in J ' - L ' ) . Scale bar : 20 μm .

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(A-C) repots>GFP-LAMP1. (D-F) repots>GFP-LAMP1+Shits1. (G-I) repots>GFP-LAMP1+DERDN. Adults were incubated at 28°C for 12 h (A, D, G), 24 h (B, E, H), and 48 h (C, F, I), respectively. The GFP-LAMP1 signal (green) was induced at 12 h and progressively enhanced in (D-F) and (G-I). (J) repots>Rab7-mCherry+H2B-RFP. (K) repots>Rab7-mCherry+Shits1. (L) repots>Rab7-mCherry+DERDN. The adults were shifted to 28°C for 2 days. Rab7-mCherry puncta (red) were increased in (K, L). DAPI: nuclei (white in J'-L'). Scale bar: 20 μm.


( A ) repots > Rab5S43N . ( B ) repots > Rab7T22N . ( C ) repots > Rab11S25N . ( D - H ) MARCM clones labeled by RFP or GFP ( green ) of control ( D ) , Rab52 ( E ) , α - Ada3 ( F ) , HrsD28 ( G ) , Rab7KO ( H ) , dor8 ( I ) and carΔ146 ( J ) . The penetrance ( number of samples with vacuole over the number of samples examined ) is indicated in each panel . Adults of all genotypes were incubated at 28 ° C for 14 days . DAPI : nuclei ( white in A - J ) . Scale bar : 20 μm . ( K ) The percentage of the vacuole area in ( A - C ) was summarized . Adults of these genotypes were incubated at 28 ° C for 12 days . P - values were calculated using Kruskal - Wallis with Dunn ' s post - tests .

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(A) repots>Rab5S43N. (B) repots>Rab7T22N. (C) repots>Rab11S25N. (D-H) MARCM clones labeled by RFP or GFP (green) of control (D), Rab52 (E), α-Ada3 (F), HrsD28 (G), Rab7KO (H), dor8 (I) and carΔ146 (J). The penetrance (number of samples with vacuole over the number of samples examined) is indicated in each panel. Adults of all genotypes were incubated at 28°C for 14 days. DAPI: nuclei (white in A-J). Scale bar: 20 μm. (K) The percentage of the vacuole area in (A-C) was summarized. Adults of these genotypes were incubated at 28°C for 12 days. P-values were calculated using Kruskal-Wallis with Dunn's post-tests.


repots > DERDN was coexpressed with ( A ) H2B - RFP , ( B ) Dorwt , ( C ) Dor - RNAi , ( D ) H2B - RFP , ( E ) Car , ( F ) Car - RNAi , ( G ) lacZ , ( H ) Car - RNAi + Dor - RNAi and ( I ) Car + Dorwt . The autophagy reporter Ref ( 2 ) P ( green ) was stained ( G ' - H ' ) . Scale bar : 20 μm . ( J , K , L ) The percentages of the vacuole areas in the lamina of ( A - C , D - F , G - I ) were summarized , respectively . The adults were shifted to 28 ° C for 7 days . DAPI : nuclei ( white in A - I ) . All P - values were calculated using Kruskal - Wallis tests with Dunn ' s post - tests .

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

repots>DERDN was coexpressed with (A) H2B-RFP, (B) Dorwt, (C) Dor-RNAi, (D) H2B-RFP, (E) Car, (F) Car-RNAi, (G) lacZ, (H) Car-RNAi+Dor-RNAi and (I) Car+Dorwt. The autophagy reporter Ref(2)P (green) was stained (G'-H'). Scale bar: 20 μm. (J, K, L) The percentages of the vacuole areas in the lamina of (A-C, D-F, G-I) were summarized, respectively. The adults were shifted to 28°C for 7 days. DAPI: nuclei (white in A-I). All P-values were calculated using Kruskal-Wallis tests with Dunn's post-tests.


A . Typical fluorescence intensity trace collected over 10 seconds .

A. Typical fluorescence intensity trace collected over 10 seconds.


B . Typical normalized FCS curves of Smc3 - GFP at different cell cycle phases . Dotted lines indicate fitting of the one - component anomalous diffusion model to the respective FCS curve .

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B. Typical normalized FCS curves of Smc3-GFP at different cell cycle phases. Dotted lines indicate fitting of the one-component anomalous diffusion model to the respective FCS curve.


C - F . Diffusion times of Smc3 - GFP , Scc3 - GFP , and Scc1 - GFP at the G1 phase ( C ) , S phase ( D ) , G2 phase ( E ) , and mitosis ( F ) . Data shown as mean ± s . e . m . from at least three independent experiments . * : p < 0 . 01 , # : p < 10 - 13 with the Mann - Whitney test .

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C-F. Diffusion times of Smc3-GFP, Scc3-GFP, and Scc1-GFP at the G1 phase (C), S phase (D), G2 phase (E), and mitosis (F). Data shown as mean ± s.e.m. from at least three independent experiments. *: p<0.01, #: p<10-13 with the Mann-Whitney test.


Representative normalized FCS curve of Scc1 - GFP at different cell cycle phases . Dotted lines indicate fitting of one - component anomalous diffusion models to the respective FCS curve .

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Representative normalized FCS curve of Scc1-GFP at different cell cycle phases. Dotted lines indicate fitting of one-component anomalous diffusion models to the respective FCS curve.


The diffusion times of Scc1 - GFP at S phase ( B ) were measured in strains yIO664 ( wild - type ) and yEB005 ( WPL1 ∆ ) , and in auxin - depleted yEB002 ( PDS5 - AID ) , yAM085 ( SCC3 - AID ) and yAM946 ( ECO1 - AID ) cells . Data are shown as mean ± s . e . m . from at least three independent experiments . * : p < 10 - 5 , * * : p < 10 - 13 .

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The diffusion times of Scc1-GFP at S phase (B) were measured in strains yIO664 (wild-type) and yEB005 (WPL1∆), and in auxin-depleted yEB002 (PDS5-AID), yAM085 (SCC3-AID) and yAM946 (ECO1-AID) cells. Data are shown as mean ± s.e.m. from at least three independent experiments. *: p< 10-5, **: p< 10-13.


The diffusion times of Scc1 - GFP at G2 phase ( C ) and M phase ( D ) were measured in strains yIO664 ( wild - type ) and yEB005 ( WPL1 ∆ ) , and in auxin - depleted yEB002 ( PDS5 - AID ) , yAM085 ( SCC3 - AID ) and yAM946 ( ECO1 - AID ) cells . Data are shown as mean ± s . e . m . from at least three independent experiments . * : p < 10 - 5 , * * : p < 10 - 13 .

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The diffusion times of Scc1-GFP at G2 phase (C) and M phase (D) were measured in strains yIO664 (wild-type) and yEB005 (WPL1∆), and in auxin-depleted yEB002 (PDS5-AID), yAM085 (SCC3-AID) and yAM946 (ECO1-AID) cells. Data are shown as mean ± s.e.m. from at least three independent experiments. *: p< 10-5, **: p< 10-13.


A . Typical fitted photon counting histograms of GFP and GFP - GFP in live yeast cells .

A. Typical fitted photon counting histograms of GFP and GFP-GFP in live yeast cells.


B . The molecular brightness of different GFP tagged constructs was obtained with GFP monomer and GFP - GFP dimer as the brightness standard . Cells of strain yIO664 ( SCC1 - GFP ) were analyzed throughout the cell cycle by PCH . To examine the effects of Wpl1 , Pds5 , and Eco1 on cohesin dimerization , the PCH analysis was repeated in strain yEB005 cells ( SCC1 - GFP WPL1 ∆ ) , strain yEB002 cells ( SCC1 - GFP PDS5 - AID ) , and yAM946 ( SCC1 - GFP ECO1 - AID ) in which Pds5 - AID / Eco1 - AID was depleted with auxin .

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B. The molecular brightness of different GFP tagged constructs was obtained with GFP monomer and GFP-GFP dimer as the brightness standard. Cells of strain yIO664 (SCC1-GFP) were analyzed throughout the cell cycle by PCH. To examine the effects of Wpl1, Pds5, and Eco1 on cohesin dimerization, the PCH analysis was repeated in strain yEB005 cells (SCC1-GFP WPL1∆), strain yEB002 cells (SCC1-GFP PDS5-AID), and yAM946 (SCC1-GFP ECO1-AID) in which Pds5-AID/Eco1-AID was depleted with auxin.


C . PCH analysis of strain yAM085 ( SMC3 - GFP ) throughout the cell cycle .

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C. PCH analysis of strain yAM085 (SMC3-GFP) throughout the cell cycle.


D . PCH analysis of strains yAM953 ( SCC1 - GFP SMC3 - GFP ) and yAP941 ( SCC1 - GFP SCC3 - GFP ) containing two GFP tagged subunits . Data shown as boxplot ( + data overlap ) with values of minimum , 5 % percentile , 25 % percentile , median , 75 % percentile , maximum , and mean ( center square marks ) from at least three independent experiments .

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D. PCH analysis of strains yAM953 (SCC1-GFP SMC3-GFP) and yAP941 (SCC1-GFP SCC3-GFP) containing two GFP tagged subunits. Data shown as boxplot (+data overlap) with values of minimum, 5% percentile, 25% percentile, median, 75% percentile, maximum, and mean (center square marks) from at least three independent experiments.


( A ) AP radiograph demonstrates characteristic findings of ATD in the R05 - 365A proband . Note the shortened humeri ( closed arrowhead ) and elongated clavicles ( arrow ) . ( B ) Radiographs of the Cmh001543 - 01 proband showing similar findings .

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(A) AP radiograph demonstrates characteristic findings of ATD in the R05-365A proband. Note the shortened humeri (closed arrowhead) and elongated clavicles (arrow). (B) Radiographs of the Cmh001543-01 proband showing similar findings.


( A ) Sanger sequence trace showing homozygosity for the c . 469C > T mutation , predicting a stop codon in exon 6 of GRK2 ( arrow ) .

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(A) Sanger sequence trace showing homozygosity for the c.469C>T mutation, predicting a stop codon in exon 6 of GRK2 (arrow).


( B ) Exome analysis showing the biallelic changes c . 1348 _ 1349del and c . 555 + 1G > A . Red arrow points to the place of mutation . Orange and green boxes indicate the reference nucleotide and its substitution , respectively .

(B) Exome analysis showing the biallelic changes c. 1348_1349del and c.555 +1G>A. Red arrow points to the place of mutation. Orange and green boxes indicate the reference nucleotide and its substitution, respectively.


( D ) Expression of GRK2 as measured by RT - PCR in three replicates each of control and R05 - 365A fibroblasts , demonstrating absence of GRK2 transcript in ATD cells . GAPDH served as a positive control .

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

(D) Expression of GRK2 as measured by RT-PCR in three replicates each of control and R05-365A fibroblasts, demonstrating absence of GRK2 transcript in ATD cells. GAPDH served as a positive control.


( E ) Two control and ATD fibroblast samples were immunoblotted for GRK2 . Note the absence of GRK2 protein in the ATD cells . Actin served as a loading control . The data shown are representative of three independent experiments .

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(E) Two control and ATD fibroblast samples were immunoblotted for GRK2. Note the absence of GRK2 protein in the ATD cells. Actin served as a loading control. The data shown are representative of three independent experiments.


( A ) GRK2 immunohistochemistry in a control 17 - week gestational age distal femur growth plate demonstrating widespread expression , particularly in the bone ( B ) , proliferating zone of the growth plate ( P ) , and the perichondrium ( arrowheads ) . Non - reactive antibody was used as a control ( Ab control ) . R , reserve zone . H , hypertrophic zone . Scale bars , 100 μm .

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

(A) GRK2 immunohistochemistry in a control 17-week gestational age distal femur growth plate demonstrating widespread expression, particularly in the bone (B), proliferating zone of the growth plate (P), and the perichondrium (arrowheads). Non-reactive antibody was used as a control (Ab control). R, reserve zone. H, hypertrophic zone. Scale bars, 100 μm.


( B ) Picrosirius red staining of the femoral growth plates of a control and the ATD R05 - 365A fetus . The control growth plate shows normal architecture with clearly distinguishable reserve ( R ) , proliferating ( P ) and hypertrophic ( H ) zones . A profound disruption of growth plate architecture is obvious in the GRK2 - / - growth plate , which shows irregularly - shaped reserve zone chondrocytes , a lack of discernable proliferating chondrocytes and a significantly under - developed hypertrophic zone . Scale bars , 100 μm .

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

(B) Picrosirius red staining of the femoral growth plates of a control and the ATD R05-365A fetus. The control growth plate shows normal architecture with clearly distinguishable reserve (R), proliferating (P) and hypertrophic (H) zones. A profound disruption of growth plate architecture is obvious in the GRK2-/- growth plate, which shows irregularly-shaped reserve zone chondrocytes, a lack of discernable proliferating chondrocytes and a significantly under-developed hypertrophic zone. Scale bars, 100 μm.


( C ) Shortened long bones in the chicken wings injected with CMPD101 . The right limb bud was injected with CMPD101 , the left one was left as a control . Note the shortened humerus ( H ) , ulna ( U ) and radius ( R ) in the CMPD101 - injected wing . Mean ± SEM . Mann - Whitney U test ; number of animals is indicated . Scale bar , 150 μm .

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(C) Shortened long bones in the chicken wings injected with CMPD101. The right limb bud was injected with CMPD101, the left one was left as a control. Note the shortened humerus (H), ulna (U) and radius (R) in the CMPD101-injected wing. Mean ± SEM. Mann-Whitney U test; number of animals is indicated. Scale bar, 150 μm.


( D ) Alcian blue staining of the femoral growth plates of a control and the ATD R05 - 365A fetus . Note the significantly lesser blue staining of the R05 - 365A growth plate stained by alcian blue pH 1 . 0 , suggesting impaired sulfation of the extracellular matrix . Scale bars , 200 μm .

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(D) Alcian blue staining of the femoral growth plates of a control and the ATD R05-365A fetus. Note the significantly lesser blue staining of the R05-365A growth plate stained by alcian blue pH 1.0, suggesting impaired sulfation of the extracellular matrix. Scale bars, 200 μm.


( E ) Alcian blue staining of the chicken limb bud micromasses treated with GRK2 inhibitor CMPD101 . Note the gradual inhibition of the cartilage nodules with increasing concentration of CMPD101 , quantified below . Mean ± SEM . Mann - Whitney U test ; number of micromasses is indicated . A representative experiment of four ( pH 2 . 5 ) and three ( pH 1 . 0 ) is shown . Scale bar , 100 μm . ( F ) Alcian blue staining of the micromasses generated from Grk2 + / + and Grk2 - / - NIH3T3 cells . Note the mildly weaker staining by alcian blue pH 2 . 5 and the nearly absent sulfation stained by alcian blue pH 1 . 0 in the Grk2 - / - micromasses . Central band , median . Box , 1st - 3rd quartile . Whiskers , 10 % - 90 % percentile . Mann - Whitney U test ; number of micromasses is indicated . Scale bar , 1 mm .

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 B-CONTROLLED_VAR 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 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

(E) Alcian blue staining of the chicken limb bud micromasses treated with GRK2 inhibitor CMPD101. Note the gradual inhibition of the cartilage nodules with increasing concentration of CMPD101, quantified below. Mean ± SEM. Mann-Whitney U test; number of micromasses is indicated. A representative experiment of four (pH 2.5) and three (pH 1.0) is shown. Scale bar, 100 μm. (F) Alcian blue staining of the micromasses generated from Grk2+/+ and Grk2-/- NIH3T3 cells. Note the mildly weaker staining by alcian blue pH 2.5 and the nearly absent sulfation stained by alcian blue pH 1.0 in the Grk2-/- micromasses. Central band, median. Box, 1st-3rd quartile. Whiskers, 10%-90% percentile. Mann-Whitney U test; number of micromasses is indicated. Scale bar, 1 mm.


( A , B ) Defective GLI3 processing in GRK2 - / - fibroblasts . Cells were serum - starved to induce cilia before treatment with the Hh pathway activator SAG ( Smoothened agonist ) , and the whole cell lysates were immunoblotted for GLI3 . Note the SAG - mediated downregulation of both full - length ( FL ) and repressor ( R ) GLI3 in control fibroblasts , demonstrating normal processing . There was no effect of SAG on GLI3 processing in GRK2 - / - cells . A representative experiment of four is shown . ( B ) Quantitation of the GLI3FL to GLI3R ratio using densitometry . Mean ± SEM . Mann - Whitney U test ; number of biological replicates is indicated .

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

(A,B) Defective GLI3 processing in GRK2-/- fibroblasts. Cells were serum-starved to induce cilia before treatment with the Hh pathway activator SAG (Smoothened agonist), and the whole cell lysates were immunoblotted for GLI3. Note the SAG-mediated downregulation of both full-length (FL) and repressor (R) GLI3 in control fibroblasts, demonstrating normal processing. There was no effect of SAG on GLI3 processing in GRK2-/- cells. A representative experiment of four is shown. (B) Quantitation of the GLI3FL to GLI3R ratio using densitometry. Mean ± SEM. Mann-Whitney U test; number of biological replicates is indicated.


( C ) Defective SAG response in GRK2 - / - fibroblasts . Cells were treated with SAG for 24 hours . Transcript levels of GLI3 targets GLI1 and PTCH1 were determined by qRT - PCR ; GAPDH was used for normalization . Note the SAG - mediated induction of GLI1 and PTCH1 expression in controls but not in GRK2 - / - cells . Mean ± SEM . Mann - Whitney U test ; number of biological replicates is indicated .

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(C) Defective SAG response in GRK2-/- fibroblasts. Cells were treated with SAG for 24 hours. Transcript levels of GLI3 targets GLI1 and PTCH1 were determined by qRT-PCR; GAPDH was used for normalization. Note the SAG-mediated induction of GLI1 and PTCH1 expression in controls but not in GRK2-/- cells. Mean ± SEM. Mann-Whitney U test; number of biological replicates is indicated.


( D ) Under - phosphorylation of Smoothened ( SMO ) in GRK2 - / - fibroblasts . Lysates of SMO - transfected cells were resolved by the phospho ( p ) - shift PAGE and immunoblotted for SMO . The portion of pSMO analyzed by densitometry is shown . GRK2 add - back demonstrates a rescue of the pSMO in GRK2 - / - fibroblasts . Mean ± SEM . Mann - Whitney U test ; number of biological replicates is indicated .

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(D) Under-phosphorylation of Smoothened (SMO) in GRK2-/- fibroblasts. Lysates of SMO-transfected cells were resolved by the phospho(p)-shift PAGE and immunoblotted for SMO. The portion of pSMO analyzed by densitometry is shown. GRK2 add-back demonstrates a rescue of the pSMO in GRK2-/- fibroblasts. Mean ± SEM. Mann-Whitney U test; number of biological replicates is indicated.


Defective SMO accumulation in cilia of GRK2 - / - fibroblasts . Cells were treated with SAG and immunostained for SMO , ARL13B and pericentrin . Note the failure in SMO accumulation in cilia of GRK2 - / - cells ( arrow ) . Scale bar , 2 µm ( E ) .

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

Defective SMO accumulation in cilia of GRK2-/- fibroblasts. Cells were treated with SAG and immunostained for SMO, ARL13B and pericentrin. Note the failure in SMO accumulation in cilia of GRK2-/- cells (arrow). Scale bar, 2 µm (E).


Defective SMO accumulation in cilia of GRK2 - / - fibroblasts . The percentage of SMO - positive cilia was calculated and plotted . Mean ± SEM . Welch ´ s t - test ; number of biological experiments and the total numbers of analyzed cilia are indicated ( F ) .

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

Defective SMO accumulation in cilia of GRK2-/- fibroblasts. The percentage of SMO-positive cilia was calculated and plotted. Mean ± SEM. Welch´s t-test; number of biological experiments and the total numbers of analyzed cilia are indicated (F).


Defective SMO accumulation in cilia of GRK2 - / - fibroblasts . The intensity of ciliary SMO was analyzed and plotted . Central band , median . Box , 1st - 3rd quartile . Whiskers , 10 % - 90 % percentile . Mann - Whitney U test ; number of biological experiments and the total numbers of analyzed cilia are indicated ( G ) .

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

Defective SMO accumulation in cilia of GRK2-/- fibroblasts. The intensity of ciliary SMO was analyzed and plotted. Central band, median. Box, 1st-3rd quartile. Whiskers, 10%-90% percentile. Mann-Whitney U test; number of biological experiments and the total numbers of analyzed cilia are indicated (G).


Defective SMO cilia accumulation and inhibited Hh signaling in chondrocytes treated with GRK2 inhibitors . Control human R00 - 082 chondrocytes were serum starved in the presence of a GRK2 inhibitor , either CMPD101 ( C ) or paroxetine ( P ) , for 24 hours before they were treated with SAG for additional 12 ( A , B ) ( A , B ) Cilia were stained by ARL13B and SMO antibodies , and the intensity of ciliary SMO was analyzed and plotted . Note the impaired SMO accumulation in cilia caused by GRK2 inhibition ( black arrow ) . Central band , median . Box , 1st - 3rd quartile . Whiskers , 10 % - 90 % percentile . Mann - Whitney U test ; number of biological experiments and the total numbers of analyzed cilia are indicated . Scale bar , 1 µm .

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

Defective SMO cilia accumulation and inhibited Hh signaling in chondrocytes treated with GRK2 inhibitors. Control human R00-082 chondrocytes were serum starved in the presence of a GRK2 inhibitor, either CMPD101 (C) or paroxetine (P), for 24 hours before they were treated with SAG for additional 12 (A, B) (A,B) Cilia were stained by ARL13B and SMO antibodies, and the intensity of ciliary SMO was analyzed and plotted. Note the impaired SMO accumulation in cilia caused by GRK2 inhibition (black arrow). Central band, median. Box, 1st-3rd quartile. Whiskers, 10%-90% percentile. Mann-Whitney U test; number of biological experiments and the total numbers of analyzed cilia are indicated. Scale bar, 1 µm.


Defective SMO cilia accumulation and inhibited Hh signaling in chondrocytes treated with GRK2 inhibitors . Control human R00 - 082 chondrocytes were serum starved in the presence of a GRK2 inhibitor , either CMPD101 ( C ) or paroxetine ( P ) , for 24 hours before they were treated with SAG for additional 24 hours ( C D ) . ( C ) Cell lysates were immunoblotted for GLI3 processing and GLI1 upregulation ; actin levels served as a loading control . GLI3FL and GLI3R , full - length and repressor GLI3 variants , respectively . ( D ) Quantification of GLI3 and GLI1 levels by densitometry . Note the impaired GLI3 processing and no GLI1 upregulation in cells treated with CMPD101 or paroxetine . Mean ± SEM . Mann - Whitney U test ( GLI3 ratio ) and Welch ´ s t - test ( GLI1 levels ) ; number of biological experiments is 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O B-MEASURED_VAR O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O B-MEASURED_VAR O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O B-MEASURED_VAR O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O

Defective SMO cilia accumulation and inhibited Hh signaling in chondrocytes treated with GRK2 inhibitors. Control human R00-082 chondrocytes were serum starved in the presence of a GRK2 inhibitor, either CMPD101 (C) or paroxetine (P), for 24 hours before they were treated with SAG for additional 24 hours (C D). (C) Cell lysates were immunoblotted for GLI3 processing and GLI1 upregulation; actin levels served as a loading control. GLI3FL and GLI3R, full-length and repressor GLI3 variants, respectively. (D) Quantification of GLI3 and GLI1 levels by densitometry. Note the impaired GLI3 processing and no GLI1 upregulation in cells treated with CMPD101 or paroxetine. Mean ± SEM. Mann-Whitney U test (GLI3 ratio) and Welch´s t-test (GLI1 levels); number of biological experiments is indicated.


Defective SMO cilia accumulation in chondrocytes with downregulated GRK2 . ( E ) Doxycycline ( DOX ) - inducible GRK2 downregulation in control human R00 - 082 chondrocytes , tested by western blot , normalized to actin and plotted below . Mean ± SEM . Welch ´ s t - test ; number of biological experiments is indicated . shScr , scramble shRNA .

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Defective SMO cilia accumulation in chondrocytes with downregulated GRK2. (E) Doxycycline (DOX)-inducible GRK2 downregulation in control human R00-082 chondrocytes, tested by western blot, normalized to actin and plotted below. Mean ± SEM. Welch´s t-test; number of biological experiments is indicated. shScr, scramble shRNA.


( F ) Cells pre - treated with DOX for three days were serum starved , SAG treated , stained and analyzed as in ( A , B ) , and the intensity of ciliary SMO was analyzed and plotted . Note the impaired SMO translocation to cilia caused by GRK2 loss . Central band , median . Box , 1st - 3rd quartile . Whiskers , 10 % - 90 % percentile . Mann - Whitney U test ; number of biological experiments and the total numbers of analyzed cilia are indicated . Scale bar , 1 µm .

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(F) Cells pre-treated with DOX for three days were serum starved, SAG treated, stained and analyzed as in (A,B), and the intensity of ciliary SMO was analyzed and plotted. Note the impaired SMO translocation to cilia caused by GRK2 loss. Central band, median. Box, 1st-3rd quartile. Whiskers, 10%-90% percentile. Mann-Whitney U test; number of biological experiments and the total numbers of analyzed cilia are indicated. Scale bar, 1 µm.


( A ) Control and GRK2 - / - fibroblasts were transfected with the TOPflash Firefly ( F ) luciferase vector together with a control Renilla ( R ) luciferase vector , and the effect of Wnt3A on TOPflash transcriptional activation was determined by dual - luciferase assay . Note the impaired response to Wnt3A in GRK2 - / - cells . Mean ± SEM . Welch ´ s t - test ; number of biological experiments is indicated .

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(A) Control and GRK2-/- fibroblasts were transfected with the TOPflash Firefly (F) luciferase vector together with a control Renilla (R) luciferase vector, and the effect of Wnt3A on TOPflash transcriptional activation was determined by dual-luciferase assay. Note the impaired response to Wnt3A in GRK2-/- cells. Mean ± SEM. Welch´s t-test; number of biological experiments is indicated.


( B Low phosphorylation ( p ) of Wnt3A co - receptor LRP6 as determined by western blot with pS1490 - and pT1572 - LRP6 specific antibodies , reduced LRP6 and DVL2 expression , and increased steady state phosphorylation of DVL2 . A representative experiment of four is shown . # , nonspecific band .

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(B Low phosphorylation (p) of Wnt3A co-receptor LRP6 as determined by western blot with pS1490- and pT1572-LRP6 specific antibodies, reduced LRP6 and DVL2 expression, and increased steady state phosphorylation of DVL2. A representative experiment of four is shown. #, nonspecific band.


C ) Low phosphorylation ( p ) of Wnt3A co - receptor LRP6 as determined by western blot with pS1490 - and pT1572 - LRP6 specific antibodies , reduced LRP6 and DVL2 expression , and increased steady state phosphorylation of DVL2 . ( C ) Densitometry of western blot results demonstrating decreased levels of LRP6 in GRK2 - / - cells , lower response of GRK2 - / - cells to Wnt3A by LRP6 phosphorylation at S1490 , and tendency towards the similar defect at T1572 . Densitometry also showed less of total DVL2 levels , and increased steady state pDVL2 in GRK2 - / - cells . Mean ± SEM . Mann - Whitney U test ; number of biological experiments is indicated .

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C) Low phosphorylation (p) of Wnt3A co-receptor LRP6 as determined by western blot with pS1490- and pT1572-LRP6 specific antibodies, reduced LRP6 and DVL2 expression, and increased steady state phosphorylation of DVL2. (C) Densitometry of western blot results demonstrating decreased levels of LRP6 in GRK2-/- cells, lower response of GRK2-/- cells to Wnt3A by LRP6 phosphorylation at S1490, and tendency towards the similar defect at T1572. Densitometry also showed less of total DVL2 levels, and increased steady state pDVL2 in GRK2-/- cells. Mean ± SEM. Mann-Whitney U test; number of biological experiments is indicated.


( D ) Loss of interaction between Frizzled 4 ( FZD4 ) and ß - Arrestin 2 ( ARRB2 ) in GRK2 - / - fibroblasts . Proximity ligation assay ( PLA ) between expressed FZD4 - GFP and ARRB2 - Flag showed significantly reduced proximity events between the two proteins in GRK2 - / - cells . White dashed lines outline the transfected cells . Central band , median . Box , 1st - 3rd quartile . Whiskers , 10 % - 90 % percentile . Mann - Whitney U test ; number of biological experiments and the total numbers of analyzed cells are indicated . Scale bar , 10 µm .

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(D) Loss of interaction between Frizzled 4 (FZD4) and ß-Arrestin 2 (ARRB2) in GRK2-/- fibroblasts. Proximity ligation assay (PLA) between expressed FZD4-GFP and ARRB2-Flag showed significantly reduced proximity events between the two proteins in GRK2-/- cells. White dashed lines outline the transfected cells. Central band, median. Box, 1st-3rd quartile. Whiskers, 10%-90% percentile. Mann-Whitney U test; number of biological experiments and the total numbers of analyzed cells are indicated. Scale bar, 10 µm.


( A ) Control ( + / + ) and Grk2 - / - NIH3T3 cells were transfected with the TOPflash Firefly ( F ) luciferase vector together with a control Renilla ( R ) luciferase vector , treated with Wnt3A ( 100 ng / ml ) for 24 hours and subjected to dual - luciferase assay . Note the impaired response to Wnt3A in Grk2 - / - cells . Mean ± SEM . Mann - Whitney U test ; number of biological experiments is indicated .

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(A) Control (+/+) and Grk2-/- NIH3T3 cells were transfected with the TOPflash Firefly (F) luciferase vector together with a control Renilla (R) luciferase vector, treated with Wnt3A (100 ng/ml) for 24 hours and subjected to dual-luciferase assay. Note the impaired response to Wnt3A in Grk2-/- cells. Mean ± SEM. Mann-Whitney U test; number of biological experiments is indicated.


( B ) Control ( + / + ) and Grk2 - / - NIH3T3 cells were treated with 40 ng / ml Wnt3A for 1 hour . Note the lower LRP6 expression and inhibited Wnt3A - induced LRP6 phosphorylation in the Grk2 - / - NIH3T3 cells . Mean ± SEM . Welch ´ s t - test ; number of biological experiments is indicated .

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(B) Control (+/+) and Grk2-/- NIH3T3 cells were treated with 40 ng/ml Wnt3A for 1 hour. Note the lower LRP6 expression and inhibited Wnt3A-induced LRP6 phosphorylation in the Grk2-/- NIH3T3 cells. Mean ± SEM. Welch´s t-test; number of biological experiments is indicated.


Loss of LRP6 phosphorylation in chondrocytes treated with the GRK2 inhibitor CMPD101 . Control human R00 - 082 chondrocytes ( C ) were treated with 20 µM CMPD101 overnight and then treated with 100 ng / ml Wnt3A for 1 hour . Note the less LRP6 phosphorylation ( pS1490 - and pT1572 - LRP6 ) in cells treated with CMPD101 . Mean ± SEM . Mann - Whitney U test ; number of biological experiments is indicated .

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Loss of LRP6 phosphorylation in chondrocytes treated with the GRK2 inhibitor CMPD101. Control human R00-082 chondrocytes (C) were treated with 20 µM CMPD101 overnight and then treated with 100 ng/ml Wnt3A for 1 hour. Note the less LRP6 phosphorylation (pS1490- and pT1572-LRP6) in cells treated with CMPD101. Mean ± SEM. Mann-Whitney U test; number of biological experiments is indicated.


Loss of LRP6 phosphorylation in chondrocytes treated with the GRK2 inhibitor CMPD101 . rat chondrosarcoma ( RCS ) cells ( D ) were treated with 20 µM CMPD101 overnight and then treated with 100 ng / ml Wnt3A for 1 hour . Note the less LRP6 phosphorylation ( pS1490 - and pT1572 - LRP6 ) in cells treated with CMPD101 . Mean ± SEM . Mann - Whitney U test ; number of biological experiments is indicated .

Loss of LRP6 phosphorylation in chondrocytes treated with the GRK2 inhibitor CMPD101. rat chondrosarcoma (RCS) cells (D) were treated with 20 µM CMPD101 overnight and then treated with 100 ng/ml Wnt3A for 1 hour. Note the less LRP6 phosphorylation (pS1490- and pT1572-LRP6) in cells treated with CMPD101. Mean ± SEM. Mann-Whitney U test; number of biological experiments is indicated.


Loss of interaction between Frizzled 4 ( FZD4 ) and ß - Arrestin 2 ( ARRB2 ) in Grk2 - / - NIH3T3 cells . ( E ) Proximity ligation assay ( PLA ) between expressed FZD4 - GFP and ARRB2 - Flag showed significantly reduced proximity events between the two proteins in Grk2 - / - cells . White dashed lines outline the transfected cells . Central band , median . Box , 1st - 3rd quartile . Whiskers , 10 % - 90 % percentile . Mann - Whitney U test ; number of biological experiments and the total numbers of analyzed cells are indicated . Scale bars , 10 µm .

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Loss of interaction between Frizzled 4 (FZD4) and ß-Arrestin 2 (ARRB2) in Grk2-/- NIH3T3 cells. (E) Proximity ligation assay (PLA) between expressed FZD4-GFP and ARRB2-Flag showed significantly reduced proximity events between the two proteins in Grk2-/- cells. White dashed lines outline the transfected cells. Central band, median. Box, 1st-3rd quartile. Whiskers, 10%-90% percentile. Mann-Whitney U test; number of biological experiments and the total numbers of analyzed cells are indicated. Scale bars, 10 µm.


Loss of interaction between Frizzled 4 ( FZD4 ) and ß - Arrestin 2 ( ARRB2 ) in Grk2 - / - NIH3T3 cells . ( F ) GRK2 addback rescued the interaction . White dashed lines outline the transfected cells . Central band , median . Box , 1st - 3rd quartile . Whiskers , 10 % - 90 % percentile . Mann - Whitney U test ; number of biological experiments and the total numbers of analyzed cells are indicated . Scale bars , 10 µm .

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Loss of interaction between Frizzled 4 (FZD4) and ß-Arrestin 2 (ARRB2) in Grk2-/- NIH3T3 cells. (F) GRK2 addback rescued the interaction. White dashed lines outline the transfected cells. Central band, median. Box, 1st-3rd quartile. Whiskers, 10%-90% percentile. Mann-Whitney U test; number of biological experiments and the total numbers of analyzed cells are indicated. Scale bars, 10 µm.


Box - plot showing the enrichment scores ( E - scores ) of all possible 8 - mers containing the G - box ( CACGTG ) , PBE - box ( CATGTG ) , and BRRE elements ( CGTGC / TG ) . Boxes represent the 25 - 75 % quartiles and the black line the median of distribution . Bars indicate the 1 - 25 % ( above ) and 75 - 100 % ( below ) quartiles . E - scores above 0 . 4 denote that binding of the proteins towards the indicated DNA element is statistically significant . Dashed blue line indicates the 0 . 4

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Box-plot showing the enrichment scores (E-scores) of all possible 8-mers containing the G-box (CACGTG), PBE-box (CATGTG), and BRRE elements (CGTGC/TG). Boxes represent the 25-75% quartiles and the black line the median of distribution. Bars indicate the 1-25% (above) and 75-100% (below) quartiles. E-scores above 0.4 denote that binding of the proteins towards the indicated DNA element is statistically significant. Dashed blue line indicates the 0.4 threshold


Protein - binding microarrays ( PBM ) were probed with PIF4 - His , MBP - BES1 and the PIF4 - His / MBP - BES1 complex . For PIF4 - BES1 complex purification , both proteins were co - expressed in E . coli using the double expression pCOLADuet - 1 vector . An anti - MBP antibody was used for BES1 and BES1 - PIF4 signal detection , while PIF4 was detected with an anti - His antibody Electrophoretic mobility shift assays ( EMSA ) showing interaction of the PIF4 , BES1 , and PIF4 - BES1 proteins with the conserved G - box , PBE - , and BRRE - elements in the PIL1 ( At2g46970 ) , PRE5 ( At3g28857 ) , and DWF4 ( At3g50660 ) promoters . Increasing amounts of protein were used for the

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Protein-binding microarrays (PBM) were probed with PIF4-His, MBP-BES1 and the PIF4-His/MBP-BES1 complex. For PIF4-BES1 complex purification, both proteins were co-expressed in E. coli using the double expression pCOLADuet-1 vector. An anti-MBP antibody was used for BES1 and BES1-PIF4 signal detection, while PIF4 was detected with an anti-His antibody Electrophoretic mobility shift assays (EMSA) showing interaction of the PIF4, BES1, and PIF4-BES1 proteins with the conserved G-box, PBE-, and BRRE-elements in the PIL1 (At2g46970), PRE5 (At3g28857), and DWF4 (At3g50660) promoters. Increasing amounts of protein were used for the assay


Protein - binding microarrays ( PBM ) were probed with PIF4 - His , MBP - BES1 and the PIF4 - His / MBP - BES1 complex . For PIF4 - BES1 complex purification , both proteins were co - expressed in E . coli using the double expression pCOLADuet - 1 vector . An anti - MBP antibody was used for BES1 and BES1 - PIF4 signal detection , while PIF4 was detected with an anti - His antibody BES1 binds both BRRE - and G - box elements as a homodimer . A deletion of BES1 ( delN ) fused to MBP ( MBP - delN ) and the complete protein ( MBP - BES1 ) were co - expressed in E . coli . Formation of intermediate mobility bands , indicative of DNA recognition by a dimeric form of the protein was tested in EMSA assays . A signal corresponding to the dimeric full - length MBP - BES1 and MBP - delN complex was detected with both DNA probes . Increasing amounts of the proteins were used in the

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Protein-binding microarrays (PBM) were probed with PIF4-His, MBP-BES1 and the PIF4-His/MBP-BES1 complex. For PIF4-BES1 complex purification, both proteins were co-expressed in E. coli using the double expression pCOLADuet-1 vector. An anti-MBP antibody was used for BES1 and BES1-PIF4 signal detection, while PIF4 was detected with an anti-His antibody BES1 binds both BRRE- and G-box elements as a homodimer. A deletion of BES1 (delN) fused to MBP (MBP-delN) and the complete protein (MBP-BES1) were co-expressed in E.coli. Formation of intermediate mobility bands, indicative of DNA recognition by a dimeric form of the protein was tested in EMSA assays. A signal corresponding to the dimeric full-length MBP-BES1 and MBP-delN complex was detected with both DNA probes. Increasing amounts of the proteins were used in the assay


Co - expression of the PIF4 and BES1 effector constructs leads to synergistic pPIL1 activation , and reverses BES1 - dependent inhibition of the pDWF4 reporter . The pPIL1 and pDWF4 promoters including three G - boxes ( green boxes ) and two BRRE - elements ( orange boxes ) were fused to the firefly luciferase reporter gene ( LUC ) and co - transfected with 35S : : PIF4 , 35S : : BES1 and 35S : : bes1 - D effector constructs into Nicotiana benthamiana leaves . Leaf discs were collected 48 hours after infiltration and luciferase activity was measured in a microplate luminometer .

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Co-expression of the PIF4 and BES1 effector constructs leads to synergistic pPIL1 activation, and reverses BES1-dependent inhibition of the pDWF4 reporter. The pPIL1 and pDWF4 promoters including three G-boxes (green boxes) and two BRRE- elements (orange boxes) were fused to the firefly luciferase reporter gene (LUC) and co-transfected with 35S::PIF4, 35S::BES1 and 35S::bes1-D effector constructs into Nicotiana benthamiana leaves. Leaf discs were collected 48 hours after infiltration and luciferase activity was measured in a microplate luminometer.


Heat map and fold - change graph representation of the genes identified in A . Expression data were obtained from Genevestigator and heatmap and clustering analyses were performed using the MeV software . PIF + BES - UP indicates genes up - regulated by PIF and BES and identified as targets of both the PIF and BES factors . BES - DOWN are BES repressed targets , while BES - UP correspond to BES induced targets . The PIF - UP cluster includes PIF induced genes directly bound by the PIF factors .

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Heat map and fold-change graph representation of the genes identified in A. Expression data were obtained from Genevestigator and heatmap and clustering analyses were performed using the MeV software. PIF+BES-UP indicates genes up-regulated by PIF and BES and identified as targets of both the PIF and BES factors. BES-DOWN are BES repressed targets, while BES-UP correspond to BES induced targets. The PIF-UP cluster includes PIF induced genes directly bound by the PIF factors.


Time course analysis of BES1 protein accumulation levels . pBES1 : : BES1 - GFP seedlings were grown under short days for 6 days and samples were harvested each 2 hours for a 24 h interval . Total protein extracts were used for western blot analysis with an anti - GFP antibody to detect the BES1 - GFP protein . Hybridization with anti - RPT5 antibody is included as a loading control . Dark and light periods are represented as black and white bars , respectively . The experiment was repeated twice with similar

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Time course analysis of BES1 protein accumulation levels. pBES1::BES1-GFP seedlings were grown under short days for 6 days and samples were harvested each 2 hours for a 24 h interval. Total protein extracts were used for western blot analysis with an anti-GFP antibody to detect the BES1-GFP protein. Hybridization with anti-RPT5 antibody is included as a loading control. Dark and light periods are represented as black and white bars, respectively. The experiment was repeated twice with similar results


Diurnal oscillation of BES1 and BZR1 transcripts . Col - 0 seedlings were grown for 6 days under short days and samples were harvested every 3 hours . BES1 and BZR1 expression levels were determined by qRT - PCR analyses and normalized to PP2A . Relative levels are the mean of three technical replicates and error bars represent standard deviation . The experiment was performed three times with similar results . Dark periods are indicated by

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Diurnal oscillation of BES1 and BZR1 transcripts. Col-0 seedlings were grown for 6 days under short days and samples were harvested every 3 hours. BES1 and BZR1 expression levels were determined by qRT-PCR analyses and normalized to PP2A. Relative levels are the mean of three technical replicates and error bars represent standard deviation. The experiment was performed three times with similar results. Dark periods are indicated by shading


Western blot analyses of 35S : : BES - GFP seedlings grown for six days under short days . Samples were collected every two hours , starting two hours before lights on ( ZT - 2 ) to ZT6 . Western blots were hybridized with an anti - GFP antibody to detect the BES1 - GFP protein , and anti - RPT5 as a loading

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Western blot analyses of 35S::BES-GFP seedlings grown for six days under short days. Samples were collected every two hours, starting two hours before lights on (ZT-2) to ZT6. Western blots were hybridized with an anti-GFP antibody to detect the BES1-GFP protein, and anti-RPT5 as a loading control


BES1 but not BZR1 is stabilized in the light . 35S : : BES1 - GFP and 35S : : BZR1 - GFP seedlings were grown for 6 days in continuous white light ( WL ) , red light ( RL ) , and continuous dark ( DARK ) , and the BES1 and BZR1 proteins analyzed by western blot using an anti - GFP antibody . Hybridization with an anti - RPT5 antibody was used as a loading

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BES1 but not BZR1 is stabilized in the light. 35S::BES1-GFP and 35S::BZR1-GFP seedlings were grown for 6 days in continuous white light (WL), red light (RL), and continuous dark (DARK), and the BES1 and BZR1 proteins analyzed by western blot using an anti-GFP antibody. Hybridization with an anti-RPT5 antibody was used as a loading control


BES1 accumulation is independent of brassinosteroid levels . Six day - old 35S : : BES - GFP seedlings were treated overnight with 100 nM epi - brassinolide ( BL ) or 0 . 5 µM brassinazole ( BRZ ) . Material was collected at the indicated time points and analyzed by western blot hybridization with anti - GFP and - RPT5

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BES1 accumulation is independent of brassinosteroid levels. Six day-old 35S::BES-GFP seedlings were treated overnight with 100 nM epi-brassinolide (BL) or 0.5 µM brassinazole (BRZ). Material was collected at the indicated time points and analyzed by western blot hybridization with anti-GFP and -RPT5 antibodies


In vivo analysis of PIF4 - BES1 complex formation . Six day - old pPIF4 : : PIF4 - HA seedlings grown under short day conditions were collected at ZT0 , ZT1 and ZT8 . PIF4 - HA was immunoprecipitated with an anti - HA antibody and the pulled - down BES1 protein immunodetected with an anti - BES antibody . * Indicates a non - specific band by the primary antibody used to immunoprecipitation .

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In vivo analysis of PIF4-BES1 complex formation. Six day-old pPIF4::PIF4-HA seedlings grown under short day conditions were collected at ZT0, ZT1 and ZT8. PIF4-HA was immunoprecipitated with an anti-HA antibody and the pulled-down BES1 protein immunodetected with an anti-BES antibody. * Indicates a non-specific band by the primary antibody used to immunoprecipitation.


Col - 0 ( black ) , PIF4 - OX ( red ) and pifq ( blue ) seedlings were grown for 6 days under short days and samples harvested at the indicated time points . Total RNA was used for qRT - PCR quantification of PIF + BES - UP and the BR biosynthetic BES - DOWN transcripts , using PP2A as endogenous control . Error bars represent SD of three technical replicates . Dark periods are shown in grey . Relative expression of the XTR7 , PRE5 , IAA19 and BR6ox2 genes in PIF4 - OX lines are referred to the right y axis of the graph . The experiment was repeated three times with similar results .

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Col-0 (black), PIF4-OX (red) and pifq (blue) seedlings were grown for 6 days under short days and samples harvested at the indicated time points. Total RNA was used for qRT-PCR quantification of PIF+BES-UP and the BR biosynthetic BES-DOWN transcripts, using PP2A as endogenous control. Error bars represent SD of three technical replicates. Dark periods are shown in grey. Relative expression of the XTR7, PRE5, IAA19 and BR6ox2 genes in PIF4-OX lines are referred to the right y axis of the graph. The experiment was repeated three times with similar results.


Col - 0 and pifq seedlings were grown under short days at 22ºC and 28ºC , and samples were collected at the indicated time points . Expression of BR biosynthetic genes was analyzed by qRT - PCR , using the constitutive PP2A gene as internal control . Error bars represent SD of three technical replicates . The experiment was repeated twice with similar

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Col-0 and pifq seedlings were grown under short days at 22ºC and 28ºC, and samples were collected at the indicated time points. Expression of BR biosynthetic genes was analyzed by qRT-PCR, using the constitutive PP2A gene as internal control. Error bars represent SD of three technical replicates. The experiment was repeated twice with similar results


Activation of the XTR7 PIF + BES - UP target was studied as a positive control of the experiment . Error bars represent SD of three technical replicates . Dark periods are shown in grey . The experiment was performed three times with similar

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Activation of the XTR7 PIF+BES-UP target was studied as a positive control of the experiment. Error bars represent SD of three technical replicates. Dark periods are shown in grey. The experiment was performed three times with similar results


Increased levels of de - phosphorylated BES1 at elevated temperatures depend on PIFs activation of BR synthesis . Col - 0 and pifq seedlings were grown for 6 days under short days and 22ºC or 28ºC , on half strength MS media ( mock ) or media supplemented with 0 . 5 µM BRZ . BES1 was analyzed by western blot hybridization with an anti - BES1 antibody . RPT5 was used as a loading control .

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Increased levels of de-phosphorylated BES1 at elevated temperatures depend on PIFs activation of BR synthesis. Col-0 and pifq seedlings were grown for 6 days under short days and 22ºC or 28ºC, on half strength MS media (mock) or media supplemented with 0.5 µM BRZ. BES1 was analyzed by western blot hybridization with an anti-BES1 antibody. RPT5 was used as a loading control.


( a - d ) Degradation of long - lived proteins in MEFs from wild - type ( 18Q - htt ) and mutant huntingtin knock - in ( 111Q - htt ) mice . ( a , b ) Rates of protein degradation after serum removal ( a )

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(a-d) Degradation of long-lived proteins in MEFs from wild-type (18Q-htt) and mutant huntingtin knock-in (111Q-htt) mice. (a,b) Rates of protein degradation after serum removal (a)


( a - d ) Degradation of long - lived proteins in MEFs from wild - type ( 18Q - htt ) and mutant huntingtin knock - in ( 111Q - htt ) mice . ( a , b ) Rates of protein degradation after treatment with rapamycin ( rapam ) or thapsigargin ( thapsig ) ( b ) .

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(a-d) Degradation of long-lived proteins in MEFs from wild-type (18Q-htt) and mutant huntingtin knock-in (111Q-htt) mice. (a,b) Rates of protein degradation after treatment with rapamycin (rapam) or thapsigargin (thapsig) (b).


( c ) Lysosomal degradation calculated as percentage of protein degradation sensitive to NH4Cl .

(c) Lysosomal degradation calculated as percentage of protein degradation sensitive to NH4Cl.


( d ) Contribution of macroautophagy calculated as percentage of protein degradation sensitive to 3 - methyladenine ( 3 - MA ) .

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(d) Contribution of macroautophagy calculated as percentage of protein degradation sensitive to 3-methyladenine (3-MA).


( e ) Degradation of long - lived proteins in striatal cells from wild - type ( 7Q - htt ) and mutant huntingtin knock - in mice ( 111Q - htt ) in response to different autophagic stimuli .

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(e) Degradation of long-lived proteins in striatal cells from wild-type (7Q-htt) and mutant huntingtin knock-in mice (111Q-htt) in response to different autophagic stimuli.


( f , g ) Rates of protein degradation in wild - type and HD MEFs ( f ) and striatal cells ( g ) in the absence ( control ) or presence ( Atg7 − ) of Atg7 RNA interference .

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(f,g) Rates of protein degradation in wild-type and HD MEFs (f) and striatal cells (g) in the absence (control) or presence (Atg7−) of Atg7 RNA interference.


( h ) Contribution of macroautophagy ( macroaut ) to the degradation of long - lived proteins in lymphoblasts from unaffected controls ( UC ) and people with HD . Mean + s . d . of values from two to four different individuals and three or four different experiments . * P 0 . 05 versus untreated , # P 0 . 05 versus control .

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(h) Contribution of macroautophagy (macroaut) to the degradation of long-lived proteins in lymphoblasts from unaffected controls (UC) and people with HD. Mean + s.d. of values from two to four different individuals and three or four different experiments. *P 0.05 versus untreated, #P 0.05 versus control.


( a ) Top : LC3 immunostaining of 18Q - htt and 111Q - htt MEFs maintained in the presence ( + ) or absence ( - ) of serum and inhibitors of lysosomal proteolysis ( inhib + ) . Bottom : mean number of LC3 + vesicles per cell in cells maintained in the presence ( left ) or absence ( right ) of serum . n = 4 .

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(a) Top: LC3 immunostaining of 18Q-htt and 111Q-htt MEFs maintained in the presence (+) or absence (-) of serum and inhibitors of lysosomal proteolysis (inhib+). Bottom: mean number of LC3+ vesicles per cell in cells maintained in the presence (left) or absence (right) of serum. n = 4.


( b ) Top : LC3 immunoblot of the same cells after serum ( S ) removal or thapsigargin ( TG ) treatment . PI , protease inhibitors . Bottom : LC3 - II levels and LC3 - II flux ( calculated as a multiple of LC3 - II value in absence of PI ; lateral numbers ) . n = 4 .

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(b) Top: LC3 immunoblot of the same cells after serum (S) removal or thapsigargin (TG) treatment. PI, protease inhibitors. Bottom: LC3-II levels and LC3-II flux (calculated as a multiple of LC3-II value in absence of PI; lateral numbers). n = 4.


( c ) LC3 - II values and LC3 - II flux in neuronal cultures from wild - type ( WT ) or HD94 mice ( HD ) grown on wild - type rat astrocyte monolayers , analyzed as in b . n = 4 .

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(c) LC3-II values and LC3-II flux in neuronal cultures from wild-type (WT) or HD94 mice (HD) grown on wild-type rat astrocyte monolayers, analyzed as in b. n = 4.


( d ) LC3 staining of lymphoblasts , from three unaffected controls ( UC ) and people with HD , maintained in the presence or absence of serum . Accession numbers are indicated above panels . Right : mean number of LC3 + vesicles . Extended study in Supplementary Figure 8b , c .

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(d) LC3 staining of lymphoblasts, from three unaffected controls (UC) and people with HD, maintained in the presence or absence of serum. Accession numbers are indicated above panels. Right: mean number of LC3+ vesicles. Extended study in Supplementary Figure 8b,c.


( e ) Immunoblots for LC3 - II in UC and HD lymphoblasts treated or not treated with protease inhibitors . Right : LC3 - II values and LC3 - II flux . Extended study in Supplementary Figure 8a . Mean + s . d . ; # P 0 . 05 . Full - length blots in Supplementary Figure 20 .

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(e) Immunoblots for LC3-II in UC and HD lymphoblasts treated or not treated with protease inhibitors. Right: LC3-II values and LC3-II flux. Extended study in Supplementary Figure 8a. Mean + s.d.; #P 0.05. Full-length blots in Supplementary Figure 20.


( a ) Electron microscopy of striatal neurons from control and HD94 mice grown over a wild - type ratastrocyte monolayer . Higher magnification fields show the double membrane and clear content of the cytosolic vesicles . Top right : number of vacuoles per cell profile ( 13 - 17 cell profiles in triplicate experiments ; * P 0 . 05 ) . Arrows , enlarged electron - clear vesicles .

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(a) Electron microscopy of striatal neurons from control and HD94 mice grown over a wild-type ratastrocyte monolayer. Higher magnification fields show the double membrane and clear content of the cytosolic vesicles. Top right: number of vacuoles per cell profile (13-17 cell profiles in triplicate experiments; *P 0.05). Arrows, enlarged electron-clear vesicles.


( b ) Electron micrographs of striatal cells from mutant huntingtin knock - in ( 111Q - htt ) and wild - type ( 7Q - htt ) mice .

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(b) Electron micrographs of striatal cells from mutant huntingtin knock-in (111Q-htt) and wild-type (7Q-htt) mice.


( c ) Electron micrographs of lymphoblasts from unaffected controls ( UC ) and people with HD , maintained in the presence or absence of serum . Arrows , enlarged electron - clear vesicles .

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(c) Electron micrographs of lymphoblasts from unaffected controls (UC) and people with HD, maintained in the presence or absence of serum. Arrows, enlarged electron-clear vesicles.


( d ) Immunogold for LC3 in striatal cells from mutant huntingtin knock - in mice ( 111Q - htt ) , striatal neurons from HD94 mice and lymphoblasts ( lymph ) from people with HD . Full fields and more details of autophagic vacuoles in Supplementary Figures 10 and 11 .

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(d) Immunogold for LC3 in striatal cells from mutant huntingtin knock-in mice (111Q-htt), striatal neurons from HD94 mice and lymphoblasts (lymph) from people with HD. Full fields and more details of autophagic vacuoles in Supplementary Figures 10 and 11.


( a ) Electron micrographs of fractions enriched in autophagosomes and autophagolysosomes isolated from liver of 18Q - htt and 111Q - htt mice . Insets : higher magnification of single vesicles . Right : percentage of vesicles with electron - clear ( light ) , vesiculated ( multivesicular ) or electron - dense ( dark ) contents . Mean + s . e . m . of three different isolations ( > ∼ 1 , 000 autophagic vacuoles ) .

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(a) Electron micrographs of fractions enriched in autophagosomes and autophagolysosomes isolated from liver of 18Q-htt and 111Q-htt mice. Insets: higher magnification of single vesicles. Right: percentage of vesicles with electron-clear (light), vesiculated (multivesicular) or electron-dense (dark) contents. Mean + s.e.m. of three different isolations (> ∼1,000 autophagic vacuoles).


( b ) Bidimensional electrophoresis and Sypro Ruby staining of these fractions . Left , intact vacuoles ; right , vacuole contents . Horizontal dimension , isoelectric point ( pH ) ; vertical , molecular mass ( kDa ) . * P 0 . 05 .

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(b) Bidimensional electrophoresis and Sypro Ruby staining of these fractions. Left, intact vacuoles; right, vacuole contents. Horizontal dimension, isoelectric point (pH); vertical, molecular mass (kDa). *P 0.05.


( a ) Immunoblot for the indicated proteins in fractions enriched in APHs and autophagolysosomes ( APHL ) isolated from livers of 18Q - htt ( 18Q ) and 111Q - htt ( 111Q ) mice . Cyt , cytochrome ; Dyn IC , dynein intermediate chain ; ADRP , adipose differentiation - related protein ; Polyubq , polyubiquitin . Bottom : amount of each protein in 111Q - htt sample , as a multiple of its amount in 18Q - htt sample . Mean + s . d . ; n = 4 .

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(a) Immunoblot for the indicated proteins in fractions enriched in APHs and autophagolysosomes (APHL) isolated from livers of 18Q-htt (18Q) and 111Q-htt (111Q) mice. Cyt, cytochrome; Dyn IC, dynein intermediate chain; ADRP, adipose differentiation-related protein; Polyubq, polyubiquitin. Bottom: amount of each protein in 111Q-htt sample, as a multiple of its amount in 18Q-htt sample. Mean + s.d.; n = 4.


( b ) Immunoblots for htt and LC3 in homogenates ( Homog ) , cytosol and fractions enriched in APHs and enriched in autophagolysosomes ( APHL ) isolated from livers of wild - type ( 18Q - htt ) and mutant huntingtin knock - in mice ( 111Q - htt ) . Representative one of four experiments with duplicated samples . * P 0 . 05 . Full - length blots in Supplementary Figure 20 .

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(b) Immunoblots for htt and LC3 in homogenates (Homog), cytosol and fractions enriched in APHs and enriched in autophagolysosomes (APHL) isolated from livers of wild-type (18Q-htt) and mutant huntingtin knock-in mice (111Q-htt). Representative one of four experiments with duplicated samples. *P 0.05. Full-length blots in Supplementary Figure 20.


( a ) Immunoblot for htt or p62 from total APHs ( T ) and their corresponding membranes ( Mbr ) and matrices ( Mtx ) isolated from livers of wild - type htt ( 18Q ) and 111Q - htt mutant huntingtin knock - in mice ( 111Q ) . Left , representative immunoblots . Right , distribution of htt and p62 between Mbr and Mtx calculated by densitometric quantification in six such immunoblots . AV , autophagic vacuoles . Mean + s . d . * P 0 . 05 compared to wild - type values .

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(a) Immunoblot for htt or p62 from total APHs (T) and their corresponding membranes (Mbr) and matrices (Mtx) isolated from livers of wild-type htt (18Q) and 111Q-htt mutant huntingtin knock-in mice (111Q). Left, representative immunoblots. Right, distribution of htt and p62 between Mbr and Mtx calculated by densitometric quantification in six such immunoblots. AV, autophagic vacuoles. Mean + s.d. *P 0.05 compared to wild-type values.


( b ) Filter retardation analysis of the same fractions as in a , blotted for htt . Negative signal indicated absence of aggregates retained in the filter .

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(b) Filter retardation analysis of the same fractions as in a, blotted for htt. Negative signal indicated absence of aggregates retained in the filter.


( c ) Immunoblot for ubiquitin in homogenates ( Homog ) , cytosol and the autophagic fractions described in a , using 6 % ( top ) and 16 % ( bottom ) gels . Dotted line separates stacking and running parts of gel .

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(c) Immunoblot for ubiquitin in homogenates (Homog), cytosol and the autophagic fractions described in a, using 6% (top) and 16% (bottom) gels. Dotted line separates stacking and running parts of gel.


( d ) Immunoblot for htt , LC3 and p62 , of brain homogenates ( Homog ) and fractions enriched in APHs from the same mouse groups as in a .

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(d) Immunoblot for htt, LC3 and p62, of brain homogenates (Homog) and fractions enriched in APHs from the same mouse groups as in a.


( e ) Immunoblot for htt and p62 of membranes from autophagic vacuoles shown in d subjected to co - immunoprecipitation for htt under low stringency . Input for 111Q - htt autophagic vacuoles was one - third of that for 18Q - htt to avoid antibody saturation . Levels of htt ( top ) and p62 ( bottom ) in the input , immunoprecipitate ( IP ) and flow through ( FT ) are shown . Full - length blots in Supplementary Figure 21 .

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(e) Immunoblot for htt and p62 of membranes from autophagic vacuoles shown in d subjected to co-immunoprecipitation for htt under low stringency. Input for 111Q-htt autophagic vacuoles was one-third of that for 18Q-htt to avoid antibody saturation. Levels of htt (top) and p62 (bottom) in the input, immunoprecipitate (IP) and flow through (FT) are shown. Full-length blots in Supplementary Figure 21.


( a ) Neutral lipids in MEFs from 18Q - htt and 111Q - htt mice stained with BODIPY 493 / 503 .

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(a) Neutral lipids in MEFs from 18Q-htt and 111Q-htt mice stained with BODIPY 493/503.


( b ) Electron micrographs of livers from 18Q - htt and 111Q - htt mice . Right : number of lipid droplets ( LD ) per cell profile ( prof . ) , mean area of LDs and percentage of cellular area occupied by LDs . Mean + s . d . ; n = 3 .

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(b) Electron micrographs of livers from 18Q-htt and 111Q-htt mice. Right: number of lipid droplets (LD) per cell profile (prof.), mean area of LDs and percentage of cellular area occupied by LDs. Mean + s.d.; n = 3.


( c - e ) Neutral lipids in striatal cells from 7Q - htt and 111Q - httmice ( c ) , grown on a monolayer of their own

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(c-e) Neutral lipids in striatal cells from 7Q-htt and 111Q-httmice (c), grown on a monolayer of their own astrocytes


( c - e ) Neutral lipids in primary striatal neurons from 18Q - htt and 111Q - httmice grown on a monolayer of their own astrocytes ( d )

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(c-e) Neutral lipids in primary striatal neurons from 18Q-htt and 111Q-httmice grown on a monolayer of their own astrocytes (d)


( c - e ) Neutral lipids in lymphoblasts from a control ( UC ) and HD - affected human ( e ) stained with BODIPY 493 / 503 . MAP2 staining highlights neurons . Extended study in Supplementary Figure 15b .

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(c-e) Neutral lipids in lymphoblasts from a control (UC) and HD-affected human (e) stained with BODIPY 493/503. MAP2 staining highlights neurons. Extended study in Supplementary Figure 15b.


( f ) Electron micrographs of a lymphoblast from an individual with HD ( with 78 and 15 polyglutamine repeats in the two alleles ) . Green arrows , LDs .

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(f) Electron micrographs of a lymphoblast from an individual with HD (with 78 and 15 polyglutamine repeats in the two alleles). Green arrows, LDs.


( g ) Fraction of cellular cytosol occupied by LDs quantified in lymphoblasts from three UC and four HD subjects . Numbers of polyglutamine repeats are shown below . Mean + s . d . of > 100 cell profiles . Dotted red line , mean value of the respective samples . * P 0 . 05 .

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(g) Fraction of cellular cytosol occupied by LDs quantified in lymphoblasts from three UC and four HD subjects. Numbers of polyglutamine repeats are shown below. Mean + s.d. of >100 cell profiles. Dotted red line, mean value of the respective samples. *P 0.05.


( h ) Oil Red O staining of striatal tissue from brain of two different unaffected control individuals ( top ) and two individuals with HD ( bottom ) . Nuclei are highlighted with hematoxylin . Lipid droplets are visible as red puncta . Subject identifiers are shown in each panel .

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(h) Oil Red O staining of striatal tissue from brain of two different unaffected control individuals (top) and two individuals with HD (bottom). Nuclei are highlighted with hematoxylin. Lipid droplets are visible as red puncta. Subject identifiers are shown in each panel.


( i ) The percentage of total cellular area occupied by lipid droplets ( left ) and the average number of lipid droplets per cell ( right ) was calculated for each of the samples by quantification of eight or nine different fields . * P 0 . 001 ; analysis of variance , P 0 . 01 for each person with HD versus each control individual by Tukey post hoc test . Subject identifiers are as in h .

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(i) The percentage of total cellular area occupied by lipid droplets (left) and the average number of lipid droplets per cell (right) was calculated for each of the samples by quantification of eight or nine different fields. *P 0.001; analysis of variance, P 0.01 for each person with HD versus each control individual by Tukey post hoc test. Subject identifiers are as in h.


( a ) Top : immunofluorescence for the mitochondrial marker COX IV in 18Q - htt and 111Q - htt MEFs maintained in the presence or absence of serum . Bottom : number of mitochondria per cell . Mean + s . d . of 10 - 20 cells in three different experiments .

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(a) Top: immunofluorescence for the mitochondrial marker COX IV in 18Q-htt and 111Q-htt MEFs maintained in the presence or absence of serum. Bottom: number of mitochondria per cell. Mean + s.d. of 10-20 cells in three different experiments.


( b ) Electron micrographs of striatal neurons from wild - type ( Ctr ) and HD94 mice grown over a wild - type ratastrocyte monolayer . Arrows , abnormally short ( black ) or abnormally long ( green ) mitochondria . Right : number of mitochondria per cell profile . Mean + s . d . of ten cells per group in triplicates .

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(b) Electron micrographs of striatal neurons from wild-type (Ctr) and HD94 mice grown over a wild-type ratastrocyte monolayer. Arrows, abnormally short (black) or abnormally long (green) mitochondria. Right: number of mitochondria per cell profile. Mean + s.d. of ten cells per group in triplicates.


( c ) Left : electron micrographs of lymphoblasts from controls ( UC ) or people with HD . Arrows as in b . Right : percentage of cellular area occupied by mitochondria in different individuals . Line indicates population mean .

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(c) Left: electron micrographs of lymphoblasts from controls (UC) or people with HD. Arrows as in b. Right: percentage of cellular area occupied by mitochondria in different individuals. Line indicates population mean.


( d , e ) Striatal cells from 7Q - htt and 111Q - htt mice ( d ) stained with Mitotracker and Mito - ROS . Right , merged images . Percentage of colocalization is indicated at the bottom in

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(d,e) Striatal cells from 7Q-htt and 111Q-htt mice (d) stained with Mitotracker and Mito-ROS. Right, merged images. Percentage of colocalization is indicated at the bottom in d


MEFs from 18Q - htt and 111Q - httmice ( e ) stained with Mitotracker and Mito - ROS . Right , merged images . Percentage of colocalization is indicated at the bottom in d and is displayed in the graph at the bottom in e . CCCP was added to control cells in e as a positive control for depolarization . * P 0 . 05 .

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MEFs from 18Q-htt and 111Q-httmice (e) stained with Mitotracker and Mito-ROS. Right, merged images. Percentage of colocalization is indicated at the bottom in d and is displayed in the graph at the bottom in e. CCCP was added to control cells in e as a positive control for depolarization. *P 0.05.


( f , g ) Striatal cells from 7Q - htt and 111Q - htt mice untreated or treated with vinblastine were co - stained for LC3 and Mitotracker ( f ) . Arrows , colocalization events . Extended study in Supplementary Figure 16 .

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(f,g) Striatal cells from 7Q-htt and 111Q-htt mice untreated or treated with vinblastine were co-stained for LC3 and Mitotracker (f). Arrows, colocalization events. Extended study in Supplementary Figure 16.


( f , g ) Striatal cells from 7Q - htt and 111Q - httmice untreated or treated with vinblastine were co - stained for LC3 and BODIPY 493 / 503 ( g ) . Arrows , colocalization events . Extended study in Supplementary Figure 16 .

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(f,g) Striatal cells from 7Q-htt and 111Q-httmice untreated or treated with vinblastine were co-stained for LC3 and BODIPY 493/503 (g). Arrows, colocalization events. Extended study in Supplementary Figure 16.


HeLa cells overexpressing the HA - tagged MICU1 WT , MICU1 S124D or MICU1 S124A mutants were stained for HA or HSP60 ( mitochondrial marker ) . Merged images are indicated ( merge ) . Scale bar 10

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HeLa cells overexpressing the HA-tagged MICU1 WT, MICU1 S124D or MICU1 S124A mutants were stained for HA or HSP60 (mitochondrial marker). Merged images are indicated (merge). Scale bar 10 μm


Representative images of the 2mt - GCaMP6m 474 / 410 ratio of ShMICU1 HeLa stable cells expressing an empty vector ( ctrl ) or the MICU1 WT , MICU1 SD , and MICU1 SA . Scale bar 10

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Representative images of the 2mt-GCaMP6m 474/410 ratio of ShMICU1 HeLa stable cells expressing an empty vector (ctrl) or the MICU1 WT, MICU1 SD, and MICU1 SA. Scale bar 10 μm


Resting mitochondrial calcium levels , evaluated through ratiometric imaging of the mitochondrial targeted GCaMP6m , in ShRNA control ( plko ) or ShRNA MICU1 HeLa stable clone cells transfected with the indicated constructs ( n = 5 independent experiments ; 55 - 67cells )

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Resting mitochondrial calcium levels, evaluated through ratiometric imaging of the mitochondrial targeted GCaMP6m, in ShRNA control (plko) or ShRNA MICU1 HeLa stable clone cells transfected with the indicated constructs (n=5 independent experiments; 55-67cells)


Representative kinetics ( E ) and analysis ( F ) of aequorin - based [ Ca2 + ] m measurements in ShRNA MICU1 HeLa stable clone cells transfected with the indicated constructs and challenged with 20 μM 2 , 5 - di - tert - butylhydroquinone ( TBHQ ) in the absence of extracellular Ca2 + ( n = 3 independent experiments )

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Representative kinetics (E) and analysis (F) of aequorin-based [Ca2+]m measurements in ShRNA MICU1 HeLa stable clone cells transfected with the indicated constructs and challenged with 20 μM 2,5-di-tert-butylhydroquinone (TBHQ) in the absence of extracellular Ca2+ (n=3 independent experiments)