A Normalised cumulative nucleosome ( 150 ± 30 bp size class ) position frequency profiles for 4013 S . pombe genes aligned at the transcription start site ( TSS ) plotted from low MNase ( biorep1 ) and high MNase ( biorep 2 ) datasets . P - values ( calculated using a two - tailed unpaired t - test ) for the difference in means between random n = 100 subsets of the frequency values at the points indicated by the dotted lines ( " - 4 " nucleosome and " + 4 " nucleosome ) are shown .

O O O B-SUBCELLULAR O O O O O O O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O O O O O O B-GENEPROD O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O 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-SUBCELLULAR O O O O O B-SUBCELLULAR O O O O

A Normalised cumulative nucleosome (150 ± 30 bp size class) position frequency profiles for 4013 S. pombe genes aligned at the transcription start site (TSS) plotted from low MNase (biorep1) and high MNase (biorep 2) datasets. P-values (calculated using a two-tailed unpaired t-test) for the difference in means between random n = 100 subsets of the frequency values at the points indicated by the dotted lines ("-4" nucleosome and "+4" nucleosome) are shown.


B Nucleosome position frequency values for the coding regions of 4013 S . pombe genes were k - means clustered ( k = 9 ) using the abo1Δ data from biorep 1 ( low MNase ) and displayed with positive values coloured yellow and other values coloured blue ( left - hand panel ) . The cluster order was then used to display the equivalent wild type frequency values in the right - hand panel .

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B Nucleosome position frequency values for the coding regions of 4013 S. pombe genes were k-means clustered (k = 9) using the abo1Δ data from biorep 1 (low MNase) and displayed with positive values coloured yellow and other values coloured blue (left-hand panel). The cluster order was then used to display the equivalent wild type frequency values in the right-hand panel.


C Level of histone gene mRNAs was determined by qRT - PCR . Data are the mean of four independent biological repeats and error bars are ± SEM . Two - tailed unpaired t - tests showed no significant differences ( P > 0 . 05 ) between wild type and abo1Δ cells .

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C Level of histone gene mRNAs was determined by qRT-PCR. Data are the mean of four independent biological repeats and error bars are ±SEM. Two-tailed unpaired t-tests showed no significant differences (P > 0.05) between wild type and abo1Δ cells.


D Whole cell extracts were subjected to western blotting with histone H3 ( Abcam ) , histone H2A ( Abcam ) and tubulin antibodies . Examples of the primary data are shown ( left ) along with a quantification of histone H2A and H3 levels normalized to tubulin ( right ) . Data are the mean of at least three independent repeats and error bars represent ± SEM . P - values were calculated using a two - tailed unpaired t - test .

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D Whole cell extracts were subjected to western blotting with histone H3 (Abcam), histone H2A (Abcam) and tubulin antibodies. Examples of the primary data are shown (left) along with a quantification of histone H2A and H3 levels normalized to tubulin (right). Data are the mean of at least three independent repeats and error bars represent ±SEM. P-values were calculated using a two-tailed unpaired t-test.


A Whole cell extracts ( WCE ) were prepared from the indicated strains , immunoprecipitated ( IP ) with anti - GFP antibody and subjected to western blotting with anti - V5 - Pk ( Serotec ) and anti - GFP ( Life Technologies ) antibodies . Data are representative of three independent biological repeats . B As for A .

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A Whole cell extracts (WCE) were prepared from the indicated strains, immunoprecipitated (IP) with anti-GFP antibody and subjected to western blotting with anti-V5-Pk (Serotec) and anti-GFP (Life Technologies) antibodies. Data are representative of three independent biological repeats.B As for A.


C RNA purified from wild type , abo1Δ and spt16 - 18 cells was analysed by northern blotting using a probe to the 3 ′ end of spbc19c7 . 11 ( top panel ) . RNA ( 5 µg ) used for northern blotting was analysed on an ethidium bromide stained 1 % TAE agarose gel ( bottom panel ) . Data are representative of two independent biological repeats .

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C RNA purified from wild type, abo1Δ and spt16-18 cells was analysed by northern blotting using a probe to the 3′ end of spbc19c7.11 (top panel). RNA (5 µg) used for northern blotting was analysed on an ethidium bromide stained 1% TAE agarose gel (bottom panel). Data are representative of two independent biological repeats.


D RNA purified from wild type , and abo1Δ cells was analysed by strand specific RT - PCR . RNA from hip1Δ cells was analysed as a control . One primer , complementary to either the forward or reverse transcripts , was included during the reverse transcription step the second primer was then added during PCR amplification . Control reactions omitting the reverse transcription step ( - RT ) were included to demonstrate the absence of contaminating genomic DNA . Data are representative of two independent biological repeats .

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D RNA purified from wild type, and abo1Δ cells was analysed by strand specific RT-PCR. RNA from hip1Δ cells was analysed as a control. One primer, complementary to either the forward or reverse transcripts, was included during the reverse transcription step the second primer was then added during PCR amplification. Control reactions omitting the reverse transcription step (-RT) were included to demonstrate the absence of contaminating genomic DNA. Data are representative of two independent biological repeats.


E The indicated strains were subjected to ChIP analysis with anti - GFP antibodies and the resulting DNA was analysed by qPCR for the indicated locus . Data are the mean of three independent biological repeats and error bars represent ± SEM . P - values were calculated using a two - tailed unpaired t - test . Significant ( P < 0 . 05 ) values are indicated .

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E The indicated strains were subjected to ChIP analysis with anti-GFP antibodies and the resulting DNA was analysed by qPCR for the indicated locus. Data are the mean of three independent biological repeats and error bars represent ±SEM. P-values were calculated using a two-tailed unpaired t-test. Significant (P < 0.05) values are indicated.


F ChIP analysis of histone H3 levels at act1 + was determined by qPCR . Data are the mean of three independent biological repeats and error bars represent ± SEM . P - value was calculated using a two - tailed unpaired t - test .

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F ChIP analysis of histone H3 levels at act1+ was determined by qPCR. Data are the mean of three independent biological repeats and error bars represent ±SEM. P-value was calculated using a two-tailed unpaired t-test.


G The indicated strains were subjected to ChIP analysis as described for E .

G The indicated strains were subjected to ChIP analysis as described for E.


H Log phase cells were subjected to five - fold serial dilution and spotted onto rich ( YE5S ) agar or agar supplemented with MMS ( 0 . 006 % ) . Plates were incubated for 3 - 5 days at 30 ° C or 7 days at 20 ° C . Images are representative of three independent biological repeats .

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H Log phase cells were subjected to five-fold serial dilution and spotted onto rich (YE5S) agar or agar supplemented with MMS (0.006%). Plates were incubated for 3-5 days at 30°C or 7 days at 20°C. Images are representative of three independent biological repeats.


A The position of ura4 + reporter alleles in centromere 1 is shown in the top panel . Strains containing the imr : : ura4 + allele were grown to log phase in YE5S medium , subjected to 5 - fold serial dilutions and spotted onto YE5S agar or YE5S agar supplemented with 5 - FOA ( 1 mg / ml ) . Data are representative of three independent biological repeats .

O O O O B-GENEPROD O O O O B-SUBCELLULAR O O O O O O O O O O O B-SMALL_MOLECULE O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O

A The position of ura4+ reporter alleles in centromere 1 is shown in the top panel. Strains containing the imr::ura4+ allele were grown to log phase in YE5S medium, subjected to 5-fold serial dilutions and spotted onto YE5S agar or YE5S agar supplemented with 5-FOA (1 mg/ml). Data are representative of three independent biological repeats.


B Strains carrying the otr : : ura4 + were analysed as described for A .

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B Strains carrying the otr::ura4+ were analysed as described for A.


C RNA was purified from the indicated strains and the level of centromeric transcripts determined by qRT - PCR . Data is the mean of three independent repeats and error bars indicate ± SEM . P - value was calculated using a two - tailed unpaired t - test .

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C RNA was purified from the indicated strains and the level of centromeric transcripts determined by qRT-PCR. Data is the mean of three independent repeats and error bars indicate ± SEM. P-value was calculated using a two-tailed unpaired t-test.


D RNA was purified from the indicated strains and subjected to qRT - PCR for the subtelomeric gene , tlh1 + . Data is the mean of three biological repeats and error bars indicate ± SEM . P - value was calculated using a two - tailed unpaired Student ' s t - test .

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D RNA was purified from the indicated strains and subjected to qRT-PCR for the subtelomeric gene, tlh1+. Data is the mean of three biological repeats and error bars indicate ± SEM. P-value was calculated using a two-tailed unpaired Student's t-test.


E The position of insertion of ade6 + reporter allele in the silent mat locus is shown in the top panel . Log phase cells containing the mat3 - M : : ade6 + allele were subjected to five - fold serial dilution and spotted onto YE5S agar plates lacking adenine ( Low Ade ) were incubated for 4 days at 30 ° C . Data are representative of at least three independent biological repeats . Note , microarray analysis indicates that deletion of abo1 + does not influence the expression of ade6 + ( or ura4 + ) when these genes are present at their normal genomic loci .

O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O O O O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O O O O O O O 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-GENEPROD O O O O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O O

E The position of insertion of ade6+ reporter allele in the silent mat locus is shown in the top panel. Log phase cells containing the mat3-M::ade6+ allele were subjected to five-fold serial dilution and spotted onto YE5S agar plates lacking adenine (Low Ade) were incubated for 4 days at 30°C. Data are representative of at least three independent biological repeats. Note, microarray analysis indicates that deletion of abo1+ does not influence the expression of ade6+ (or ura4+) when these genes are present at their normal genomic loci.


A ChIP analysis was performed on wild type ( untagged ) and abo1 - GFP cells and the resulting DNA analysed by qPCR for centromeric ( dh , dg and imr ) repeat sequences . Data are the mean of four independent biological repeats and error bars represent ± SEM . P - values calculated using a two - tailed unpaired t - test indicates that all loci are significantly enriched ( P < 0 . 05 ) relative the untagged control .

O B-EXP_ASSAY O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O B-SMALL_MOLECULE O O B-EXP_ASSAY O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 ChIP analysis was performed on wild type (untagged) and abo1-GFP cells and the resulting DNA analysed by qPCR for centromeric (dh, dg and imr) repeat sequences. Data are the mean of four independent biological repeats and error bars represent ±SEM. P-values calculated using a two-tailed unpaired t-test indicates that all loci are significantly enriched (P < 0.05) relative the untagged control.


B The indicated strains expressing GFP - Swi6 were subjected to ChIP analysis . The level of centromeric ( dg ) repeat sequences relative to the euchromatic control locus , adh1 + was determined by qPCR and scaled to a clr4Δ ( - H3 K9me control ) mutant . Data are the mean of two independent ChIP experiments and error bars represent the range of the data .

O O O O O B-GENEPROD O B-GENEPROD O O O B-EXP_ASSAY O O O O O O O B-SMALL_MOLECULE O O O O O O B-SUBCELLULAR O O O B-GENEPROD O O O O B-EXP_ASSAY O O O O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O

B The indicated strains expressing GFP-Swi6 were subjected to ChIP analysis. The level of centromeric (dg) repeat sequences relative to the euchromatic control locus, adh1+ was determined by qPCR and scaled to a clr4Δ (- H3 K9me control) mutant. Data are the mean of two independent ChIP experiments and error bars represent the range of the data.


C ChIP analysis of histone H3 levels at centromeric ( dg ) repeat sequences was determined by qPCR . Data are the mean of three independent ChIP experiments and error bars are ± SEM . P - values were calculated using a two - tailed unpaired t - test .

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C ChIP analysis of histone H3 levels at centromeric (dg) repeat sequences was determined by qPCR. Data are the mean of three independent ChIP experiments and error bars are ±SEM. P-values were calculated using a two-tailed unpaired t-test.


D . ChIP analysis of Abo1 - GFP at centromeric ( dh and imr ) repeat sequences regions in wild type and pob3Δ cells . Data are the mean of three independent experiments and error bars represent ± SEM . P - values , calculated using a two - tailed unpaired t - test , indicated no significant difference ( P > 0 . 05 ) for wild type and pob3Δ cells .

O O B-EXP_ASSAY O O B-GENEPROD O B-GENEPROD O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O 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-GENEPROD O O

D. ChIP analysis of Abo1-GFP at centromeric (dh and imr) repeat sequences regions in wild type and pob3Δ cells. Data are the mean of three independent experiments and error bars represent ±SEM. P-values, calculated using a two-tailed unpaired t-test, indicated no significant difference (P > 0.05) for wild type and pob3Δ cells.


E ChIP analysis of Abo1 - GFP at centromeric ( dh and imr ) repeat sequences in wild type and pob3Δ cells . Data are the mean of duplicate experiments and error bars represent the range of the data .

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E ChIP analysis of Abo1-GFP at centromeric (dh and imr) repeat sequences in wild type and pob3Δ cells. Data are the mean of duplicate experiments and error bars represent the range of the data.


F ChIP analysis of Abo1 - GFP over the dh and imr regions in wild type and swi6Δ cells . Data are the mean of three independent experiments and error bars represent ± SEM . P - values , calculated using a two - tailed unpaired t - test , indicated no significant differences ( P > 0 . 05 ) for wild type and swi6Δ cells .

O B-EXP_ASSAY O O B-GENEPROD O B-GENEPROD O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O 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-GENEPROD O O

F ChIP analysis of Abo1-GFP over the dh and imr regions in wild type and swi6Δ cells. Data are the mean of three independent experiments and error bars represent ±SEM. P-values, calculated using a two-tailed unpaired t-test, indicated no significant differences (P > 0.05) for wild type and swi6Δ cells.


A RNA was extracted from mid log phase cells and Tf2 mRNA levels were determined by qRT - PCR , normalised to act1 + mRNA and scaled relative to the wild type level . Data are the mean of three independent biological repeats and error bars represent ± SEM .

O O O O O O O O O O B-GENEPROD O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD O O O 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 RNA was extracted from mid log phase cells and Tf2 mRNA levels were determined by qRT-PCR, normalised to act1+ mRNA and scaled relative to the wild type level. Data are the mean of three independent biological repeats and error bars represent ±SEM.


B Mid log phase cells with the indicated integrated lacZ reporter were subjected to quantitative β - galactosidase assays . Data are the mean of three independent biological repeats and error bars represent ± SEM . C As for

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B Mid log phase cells with the indicated integrated lacZ reporter were subjected to quantitative β-galactosidase assays. Data are the mean of three independent biological repeats and error bars represent ±SEM.C As for B


D ChIP DNA samples from wild type ( untagged ) and abo1 - GFP cells were analysed by qPCR for Tf2 LTR . Data are the mean of four independent biological repeats and error bars represent ± SEM .

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D ChIP DNA samples from wild type (untagged) and abo1-GFP cells were analysed by qPCR for Tf2 LTR. Data are the mean of four independent biological repeats and error bars represent ±SEM.


E Normalised cumulative nucleosome ( 150 ± 30 bp ) position frequency profiles for Tf2 LTR retrotransposons aligned at the ATG plotted from low MNase ( biorep1 ) and high MNase ( biorep 2 ) datasets . P - values were calculated using a two - tailed unpaired t - test .

O O O B-SUBCELLULAR O O O O O O O O O O B-GENEPROD B-SUBCELLULAR O O O O O O O O B-GENEPROD O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O

E Normalised cumulative nucleosome (150 ± 30 bp) position frequency profiles for Tf2 LTR retrotransposons aligned at the ATG plotted from low MNase (biorep1) and high MNase (biorep 2) datasets.P-values were calculated using a two-tailed unpaired t-test.


A Log phase cells were subjected to five - fold serial dilution and spotted onto rich ( YE5S ) agar or agar supplemented with TBZ ( 15 μg / ml ) and incubated for 3 - 5 days 30 ° C . Data are representative of at three independent biological repeats .

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A Log phase cells were subjected to five-fold serial dilution and spotted onto rich (YE5S) agar or agar supplemented with TBZ (15 μg/ml) and incubated for 3-5 days 30°C. Data are representative of at three independent biological repeats.


B The frequency of Ch16 minichromosome loss was assayed by plating cells onto adenine limiting agar and determining the frequency of half sectored ( red - white ) colonies . At least two independently derived strain isolates were used for each genotype and the total number of colonies counted for each genotype is indicated in parenthesis .

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B The frequency of Ch16 minichromosome loss was assayed by plating cells onto adenine limiting agar and determining the frequency of half sectored (red-white) colonies. At least two independently derived strain isolates were used for each genotype and the total number of colonies counted for each genotype is indicated in parenthesis.


C Heterothallic ( h90 ) strains were cultured on nitrogen limiting ( EMMG ) medium for 3 days at 25 ° C to induce mating and meiosis . The resulting asci were stained with DAPI and visualised by fluorescence microscopy . Data are representative of three biological

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C Heterothallic (h90) strains were cultured on nitrogen limiting (EMMG) medium for 3 days at 25°C to induce mating and meiosis. The resulting asci were stained with DAPI and visualised by fluorescence microscopy. Data are representative of three biological repeats


D Spore viability was measured by dissecting spores onto YE5S agar followed by incubation for 4 - 5 days at 30 ° C . Data are the mean of three independent biological repeats and error bars are ± SEM . P - value was calculated using a two - tailed unpaired t - test .

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D Spore viability was measured by dissecting spores onto YE5S agar followed by incubation for 4-5 days at 30°C. Data are the mean of three independent biological repeats and error bars are ±SEM. P-value was calculated using a two-tailed unpaired t-test.


H & amp ; amp ; E staining of adult head sections of ( A ) repo > GFP . nls and ( B ) repo > Shits1 at 29 ° C for 3 days . Lamina degeneration was identified as vacuoles in ( B ) . Cryosection of adult ( C ) repo > H2B - RFP exhibited the expression of the nuclear red fluorescent protein ( RFP ) in the glia ( epithelialglialnucleus : arrowhead ) and ( D ) repo > Shits1 at 28 ° C for 12 days . Vacuoles were identified in the lamina neuropile ( D ) . Epithelial ( arrowhead ) ( E ) , marginal ( arrow ) and distal satellite ( arrowhead ) glianuclei ( F ) are labeled by HisCl - GAL4 and NP2109 - GAL4 , respectively . Note that HisCl - GAL4 is not expressed in all epithelialglia . ( G , H ) Weak lamina vacuolization was identified in HisCl > Shits1 ( G , 0 . 92 % ) and NP2109 > Shits1 ( H , 1 . 46 % ) at 29 ° C for 14 days . ( I , J ) A single MARCMglia clone ( GFP , green ) expressed Shits1 at 21 ° C ( I ) and 29 ° C ( J ) . ( J ) A vacuole occurred within a glial clone . DAPI ( white ) stains the nuclei ( C , D , and G - J ) . ( K ) The percentage of the vacuole area in the lamina of the repo > Shits1flies at 28 ° C progressively increased . n indicated in each column . P - values were calculated using one - way ANOVA with Bonferroni ' s post - test . ( L ) When repo > Shits1flies were shifted to 28 ° C for 12 days and then shifted to 17 ° C for 9 additional days , the vacuolization was not alleviated . P - values were calculated using one - way ANOVA with Tukey ' s post - test . n indicated in each column .

B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-TISSUE O O O O O O O B-GENEPROD O O O O O O O O B-GENEPROD O O O O O O O O B-TISSUE O O O O B-SUBCELLULAR O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O B-SUBCELLULAR O O O O B-GENEPROD O O O B-CELL_TYPE O B-CELL_TYPE O O O O O O O O O B-GENEPROD O O O O O O O O B-SUBCELLULAR O O O O B-TISSUE I-TISSUE O O O O B-TISSUE O O O O O O O O O O O O O B-CELL_TYPE O O O B-CELL_TYPE O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O O O O B-CELL_TYPE O O O O O O O B-TISSUE O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O B-EXP_ASSAY O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O B-TISSUE O O O O B-GENEPROD O O O O O O O O O O O O O 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-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

H&amp;E staining of adult head sections of (A) repo>GFP.nls and (B) repo>Shits1 at 29°C for 3 days. Lamina degeneration was identified as vacuoles in (B). Cryosection of adult (C) repo>H2B-RFP exhibited the expression of the nuclear red fluorescent protein (RFP) in the glia (epithelialglialnucleus: arrowhead) and (D) repo>Shits1 at 28°C for 12 days. Vacuoles were identified in the lamina neuropile (D). Epithelial (arrowhead) (E), marginal (arrow) and distal satellite (arrowhead) glianuclei (F) are labeled by HisCl-GAL4 and NP2109-GAL4, respectively. Note that HisCl-GAL4 is not expressed in all epithelialglia. (G, H) Weak lamina vacuolization was identified in HisCl>Shits1 (G, 0.92%) and NP2109>Shits1 (H, 1.46%) at 29°C for 14 days. (I, J) A single MARCMglia clone (GFP, green) expressed Shits1 at 21°C (I) and 29°C (J). (J) A vacuole occurred within a glial clone. DAPI (white) stains the nuclei (C, D, and G-J). (K) The percentage of the vacuole area in the lamina of the repo>Shits1flies at 28°C progressively increased. n indicated in each column. P-values were calculated using one-way ANOVA with Bonferroni's post-test. (L) When repo>Shits1flies were shifted to 28°C for 12 days and then shifted to 17°C for 9 additional days, the vacuolization was not alleviated. P-values were calculated using one-way ANOVA with Tukey's post-test. n indicated in each column.


( A - E ) Horizontal sections of the adult head lamina cartridge . ( A ) In repo > GFP . nls flies , two lamina neurons L1 , L2 axons ( L ) and R1 - 6 axons ( blue area ) were surrounded by the electron - dense cytoplasm of the epithelial glia ( G ) . The section was examined at three different depths , and the size of a single cartridge is not significantly different at different depths . ( B - D ) repo > Shits1 adults maintained at 29 ° C for 4 days . Axons were enlarged but intact . A large number of small and medium vacuoles were identified in the epithelial glia . ( C ) Small vacuoles in the glia that contained a double - membrane structure ( arrowhead ) . ( C ' ) Higher magnification of the boxed area in ( C ) . ( D ) A double - membrane autophagosome - like structure ( arrowhead ) in the glialcytoplasm . ( E ) In repots > DERDN adults maintained at 29 ° C for 2 days , large vacuoles were identified in between the lamina cartridges . N : glialnuclei . ( E ' ) Higher magnification of the boxed area in ( E ) , which shows autophagosome - like vesicles ( arrowhead ) in the gliacytoplasm .

O O O O O O O O O O B-TISSUE I-TISSUE O O O O O O O O B-GENEPROD O O B-ORGANISM O O B-TISSUE I-TISSUE O O O B-TISSUE O O O O O O O B-TISSUE O O O O O O O O O O O B-SUBCELLULAR O O B-CELL_TYPE I-CELL_TYPE O O O O O O O O O O O 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-GENEPROD O O O O O O O O O O B-TISSUE O O O O O O O O O O O O B-SUBCELLULAR O O O O B-CELL_TYPE I-CELL_TYPE O O O O O B-SUBCELLULAR O O B-CELL_TYPE O O O O O O O O O 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-SUBCELLULAR O O O O O O O O B-CELL_TYPE O O O O O O O B-GENEPROD O O O O O O O O O O O B-SUBCELLULAR O O O O O B-TISSUE O O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O B-SUBCELLULAR O O O O O B-CELL_TYPE O

(A-E) Horizontal sections of the adult head lamina cartridge. (A) In repo>GFP.nls flies, two lamina neurons L1, L2 axons (L) and R1-6 axons (blue area) were surrounded by the electron-dense cytoplasm of the epithelial glia (G). The section was examined at three different depths, and the size of a single cartridge is not significantly different at different depths. (B-D) repo>Shits1 adults maintained at 29°C for 4 days. Axons were enlarged but intact. A large number of small and medium vacuoles were identified in the epithelial glia. (C) Small vacuoles in the glia that contained a double-membrane structure (arrowhead). (C') Higher magnification of the boxed area in (C). (D) A double-membrane autophagosome-like structure (arrowhead) in the glialcytoplasm. (E) In repots>DERDN adults maintained at 29°C for 2 days, large vacuoles were identified in between the lamina cartridges. N: glialnuclei. (E') Higher magnification of the boxed area in (E), which shows autophagosome-like vesicles (arrowhead) in the gliacytoplasm.


( A ) Rh1 > lacZ + H2B - RFP exhibited nuclearRFP expression in the R1 - 6 photoreceptors ( red ) . ( B ) Rh1 > lacZ + Shits1 maintained at 29 ° C for 14 days resulted in lamina vacuolization . ( C ) Pan - Rh7 > H2B - RFP exhibited expression in R7 ( arrow ) and R8 ( arrowhead ) ( red ) . ( D ) Pan - Rh7 > Shits1 incubated at 29 ° C for 14 days did not exhibit lamina degeneration . ( E ) Lamina monopolar ( L ) neurons ( red ) were selectively labeled by the Ln - GAL4 driven H2B - RFP with repo - GAL80 . ( F ) Ln > Shits1 , repoGAL80 shifted to 29 ° C for 14 days did not exhibit lamina degeneration . ( G ) R1 - 6 photoreceptors were killed in Rh1ts > lacZ + Hid shifted to 29 ° C for 14 days . Rh1ts indicates tubGAL80ts ; Rh1 - GAL4 . Degeneration was induced in the lamina in addition to the retina . ( H ) rdgC306 / + mutant exposed to constant light for 14 days exhibited degeneration in the retina , as well as the lamina . DAPI : nuclei ( white in A - H ) . ( I , K ) Rh1 > lacZ + Shits1 at 29 ° C for 4 days exhibited vacuoles in the electron dense cytoplasm of epithelialglia or near the glianucleus . Arrow : vacuole with internal debris . Arrowhead : large vacuole . ( J ) Lamina cartridge of Rh1 > lacZ + H2B - RFP . N indicates glialnuclei . ( L ) The percentage of the vacuole area in the lamina at 29 ° C for 14 days was examined . All P - values were calculated using one - way ANOVA with Tukey ' s post - test . Scale bar : 20 μm .

O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-SUBCELLULAR O O O O O O B-TISSUE O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O B-TISSUE O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O B-TISSUE O O O O B-TISSUE O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O B-TISSUE O O O O O B-TISSUE O O O O B-CELL_TYPE O O O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O O O O O O B-GENEPROD O O O O O O O O O O O O O B-TISSUE O O O O O O O O B-TISSUE O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O O B-TISSUE O O O O B-TISSUE O O O O B-GENEPROD O O O O O O O O O O O O O O B-TISSUE O O O O O B-TISSUE O B-EXP_ASSAY O B-SUBCELLULAR O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O B-SUBCELLULAR O O O O B-SUBCELLULAR O B-CELL_TYPE O O O B-CELL_TYPE O O O B-SUBCELLULAR O O O O O O O B-SUBCELLULAR O O O O B-TISSUE O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O B-CELL_TYPE O O O O O O O O B-SUBCELLULAR O O O B-TISSUE O O O O O O O O O O O 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) Rh1>lacZ+H2B-RFP exhibited nuclearRFP expression in the R1-6 photoreceptors (red). (B) Rh1>lacZ+Shits1 maintained at 29°C for 14 days resulted in lamina vacuolization. (C) Pan-Rh7>H2B-RFP exhibited expression in R7 (arrow) and R8 (arrowhead) (red). (D) Pan-Rh7>Shits1 incubated at 29°C for 14 days did not exhibit lamina degeneration. (E) Lamina monopolar (L) neurons (red) were selectively labeled by the Ln-GAL4 driven H2B-RFP with repo-GAL80. (F) Ln>Shits1, repoGAL80 shifted to 29°C for 14 days did not exhibit lamina degeneration. (G) R1-6 photoreceptors were killed in Rh1ts>lacZ+Hid shifted to 29°C for 14 days. Rh1ts indicates tubGAL80ts; Rh1-GAL4. Degeneration was induced in the lamina in addition to the retina. (H) rdgC306/+ mutant exposed to constant light for 14 days exhibited degeneration in the retina, as well as the lamina. DAPI: nuclei (white in A-H). (I, K) Rh1>lacZ+Shits1 at 29°C for 4 days exhibited vacuoles in the electron dense cytoplasm of epithelialglia or near the glianucleus. Arrow: vacuole with internal debris. Arrowhead: large vacuole. (J) Lamina cartridge of Rh1>lacZ+H2B-RFP. N indicates glialnuclei. (L) The percentage of the vacuole area in the lamina at 29°C for 14 days was examined. All P-values were calculated using one-way ANOVA with Tukey's post-test. Scale bar: 20 μm.


( A ) repots > Shits1 + H2B - RFP , ( B ) repots > Shits1 + Egfrλtop and ( C ) repots > Shits1 + RlSem incubated for 12 days at 28 ° C . ( D ) repots > DERDN for 7 days at 28 ° C . ( E ) Epithelial glia MARCM clone and ( F ) Egfrco mutant glia MARCM clone ( labeled with Tomato , green ) . The penetrance is indicated as the number of samples with vacuoles over the total number of samples . DAPI : nuclei ( white in A - F ) . ( G ) Percentage of vacuole area in the lamina neuropile of ( A - D ) . The P - values were calculated using one - way ANOVA with Dunnett ' s post - test . ( H ) Percentage of vacuole area in the lamina of repots > Shits1 flies in the indicated genetic background . The adults were shifted to 28 ° C for 12 days . The P - values were calculated using one - way ANOVA with Tukey ' s post - test . Scale bar : 20 μm .

O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O B-GENEPROD O B-GENEPROD O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O B-CELL_TYPE I-CELL_TYPE B-EXP_ASSAY O O O O O B-GENEPROD O B-CELL_TYPE B-EXP_ASSAY O O O O B-GENEPROD O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O B-EXP_ASSAY O B-SUBCELLULAR O O O O O O O O O O O O O B-SUBCELLULAR O O O B-TISSUE I-TISSUE O O O O O O O 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-SUBCELLULAR O O O O O O O B-GENEPROD B-ORGANISM O O O O O O O O O O O O O O O O O O O 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) repots>Shits1+H2B-RFP, (B) repots>Shits1+Egfrλtop and (C) repots>Shits1+RlSem incubated for 12 days at 28°C. (D) repots>DERDN for 7 days at 28°C. (E) Epithelial glia MARCM clone and (F) Egfrco mutant glia MARCM clone (labeled with Tomato, green). The penetrance is indicated as the number of samples with vacuoles over the total number of samples. DAPI: nuclei (white in A-F). (G) Percentage of vacuole area in the lamina neuropile of (A-D). The P-values were calculated using one-way ANOVA with Dunnett's post-test. (H) Percentage of vacuole area in the lamina of repots>Shits1 flies in the indicated genetic background. The adults were shifted to 28°C for 12 days. The P-values were calculated using one-way ANOVA with Tukey's post-test. Scale bar: 20 μm.


( 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 .

O O O O O O O O B-GENEPROD O O O B-EXP_ASSAY O B-ORGANISM O B-TISSUE O B-GENEPROD O O O O O B-TISSUE O B-TISSUE O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O B-TISSUE O O O O B-ORGANISM O O O O O B-TISSUE O O O O O O B-TISSUE O O O O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O B-TISSUE O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O O O O O O O O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O O B-TISSUE O O O O O O O O O O O O O O O O O B-TISSUE O O O B-TISSUE B-GENEPROD O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O B-SUBCELLULAR O O O O O O O O O O B-TISSUE O O O O O 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-EXP_ASSAY O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O

(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 .

O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O B-CELL_TYPE O O O O B-GENEPROD O O O O O O O O O O O O O O O O B-GENEPROD O O O B-GENEPROD O O O B-GENEPROD O O O O O B-TISSUE O O O O O B-CELL_TYPE O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O B-CELL_TYPE O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O B-GENEPROD O O B-CELL_TYPE O O O O O O O O O O O O O O O O O B-GENEPROD O O O O B-TISSUE O O O O O O O O O O O B-GENEPROD O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O B-SUBCELLULAR O O O O O O O O O O O O O O O O

(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 .

O O O O O B-EXP_ASSAY O B-EXP_ASSAY O O O O O O O O O O O O O O O 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-GENEPROD O O O O O O O O O O B-GENEPROD O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O B-TISSUE O O O O O O O O O O O O O O O O O O O B-GENEPROD O O B-GENEPROD O O O O O O B-GENEPROD O O O B-CELL_TYPE O O O O O O B-GENEPROD O B-GENEPROD O O O O O O B-GENEPROD O B-GENEPROD O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O 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-CELL_TYPE O O O O O O O O O O O B-CELL_TYPE I-CELL_TYPE O O O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O B-SUBCELLULAR B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O

(A-C) 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 .

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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 .

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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 .

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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 .

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(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 .

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(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 .

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(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 .

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(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 .

O O O O O O O O B-ORGANISM B-TISSUE O O B-SMALL_MOLECULE O O O B-TISSUE I-TISSUE O O O B-SMALL_MOLECULE O O O O O O O O O O O O O B-TISSUE O O O O B-TISSUE O O O O B-TISSUE O O O O O B-SMALL_MOLECULE O O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O

(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 .

O O O B-SMALL_MOLECULE I-SMALL_MOLECULE B-EXP_ASSAY O O B-TISSUE I-TISSUE I-TISSUE O O O O O B-DISEASE O O O B-TISSUE O O O O O O B-EXP_ASSAY O O O O O B-TISSUE I-TISSUE B-EXP_ASSAY O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O B-EXP_ASSAY O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O

(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 B-SMALL_MOLECULE I-SMALL_MOLECULE B-EXP_ASSAY O O B-ORGANISM B-TISSUE I-TISSUE O O O B-GENEPROD O B-SMALL_MOLECULE O O O O O O O B-TISSUE I-TISSUE O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-SMALL_MOLECULE I-SMALL_MOLECULE B-EXP_ASSAY O O O O O B-GENEPROD O O O O B-GENEPROD O O O B-CELL_LINE O O O O O O B-EXP_ASSAY O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O 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-GENEPROD O O B-GENEPROD O O O B-CELL_TYPE O O O B-TISSUE O O O O B-SUBCELLULAR O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O B-EXP_ASSAY O B-GENEPROD O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O B-GENEPROD O O B-CELL_TYPE O O O O O O O O O O B-SMALL_MOLECULE O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O B-EXP_ASSAY 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 .

O O O O B-SMALL_MOLECULE O O B-GENEPROD O O O B-CELL_TYPE O O O O O B-SMALL_MOLECULE O O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O O O O B-SMALL_MOLECULE O O O O B-GENEPROD O B-GENEPROD O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O

(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 .

O O O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O O B-CELL_TYPE O O O B-GENEPROD O O O O O O O B-EXP_ASSAY O O O O O B-EXP_ASSAY O B-EXP_ASSAY O B-GENEPROD O O O O O O O B-EXP_ASSAY O O O B-GENEPROD O O O O O O O O O O B-GENEPROD O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O

(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-GENEPROD O O B-SUBCELLULAR O B-GENEPROD O O O B-CELL_TYPE O O O O O B-SMALL_MOLECULE O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O O B-SUBCELLULAR O B-GENEPROD 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-GENEPROD O O B-SUBCELLULAR O B-GENEPROD O O O B-CELL_TYPE O O O O B-GENEPROD O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O B-SUBCELLULAR 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-GENEPROD O O B-SUBCELLULAR O B-GENEPROD O O O B-CELL_TYPE O O B-EXP_ASSAY O B-SUBCELLULAR B-GENEPROD O O O O O O O O O O O O O O O O O O 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-SUBCELLULAR 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-GENEPROD B-SUBCELLULAR O O O O O O B-CELL_TYPE O O B-GENEPROD O O O B-ORGANISM O O O B-CELL_TYPE O B-TISSUE O O O O O O B-GENEPROD O O O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O B-SUBCELLULAR O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O O B-EXP_ASSAY O B-SUBCELLULAR B-GENEPROD O O O O O O O O B-GENEPROD O O B-SUBCELLULAR O O B-GENEPROD O O O O O O O O O O O O O O O O O O O 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-SUBCELLULAR 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 B-GENEPROD B-SUBCELLULAR O O O O O O B-CELL_TYPE O O B-GENEPROD O O O B-ORGANISM O O O B-CELL_TYPE O B-TISSUE O O O O O O B-GENEPROD O O O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O B-EXP_ASSAY O B-GENEPROD O O B-GENEPROD O O B-GENEPROD O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O B-GENEPROD O O O O O O O O O B-GENEPROD O B-GENEPROD O O B-EXP_ASSAY O O O O B-GENEPROD O O O B-GENEPROD O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O B-GENEPROD O O O O O O O O O O B-GENEPROD 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 .

O B-GENEPROD B-SUBCELLULAR O O B-CELL_TYPE O O B-GENEPROD O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O B-GENEPROD O O O B-ORGANISM O O O B-CELL_TYPE O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD O O O 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 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 .

O O O O O O O O B-SMALL_MOLECULE O O O O B-TISSUE O O B-SMALL_MOLECULE O O B-EXP_ASSAY O O O O O O O O O O O O B-EXP_ASSAY O B-SUBCELLULAR B-GENEPROD O O O O O O O O B-GENEPROD B-EXP_ASSAY O B-SUBCELLULAR O O B-GENEPROD O O O O O O O O O O O O O O O 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-SUBCELLULAR O O O O O O O O O

(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 .

O O O O O B-GENEPROD O O O B-CELL_TYPE O O O O O B-ORGANISM O O O B-GENEPROD O O O O O B-ORGANISM O O O B-GENEPROD O O O O O O B-GENEPROD O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O

(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 .

O O O B-EXP_ASSAY O O O O B-GENEPROD O O O B-GENEPROD O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O O O O O B-EXP_ASSAY O B-GENEPROD O O O O O O O O O O O O O O

(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 .

O O O B-EXP_ASSAY O O O O B-GENEPROD O O O B-GENEPROD O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O O O O O B-EXP_ASSAY O B-GENEPROD O O O O B-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD B-EXP_ASSAY O O O O O O O O O O O O B-EXP_ASSAY O O O O O B-GENEPROD O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O

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 .

O O O O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD O B-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O B-GENEPROD O O O B-CELL_TYPE O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(D) 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 .

O O O O O O O O O O B-GENEPROD O O O B-CELL_LINE O O O O O O B-ORGANISM O O O B-GENEPROD O O O O O B-ORGANISM O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O

(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 .

O O O O O O O O O O B-GENEPROD O O O B-CELL_LINE O O O O O O O O B-GENEPROD O O O O O O O B-GENEPROD O O O B-GENEPROD O O B-GENEPROD B-EXP_ASSAY O O B-GENEPROD O O O B-CELL_LINE O O O O O O O O O O O O O O O O O O O O

(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 .

O O B-GENEPROD B-EXP_ASSAY O B-CELL_TYPE O O O B-GENEPROD O B-SMALL_MOLECULE O O B-ORGANISM O O O B-CELL_TYPE O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O B-GENEPROD O O O O O O O B-GENEPROD B-EXP_ASSAY O O O O O O B-GENEPROD O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O

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 .

O O B-GENEPROD B-EXP_ASSAY O B-CELL_TYPE O O O B-GENEPROD O B-SMALL_MOLECULE O B-ORGANISM B-CELL_LINE O B-CELL_LINE O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O B-GENEPROD O O O O O O O B-GENEPROD B-EXP_ASSAY O O O O O O B-GENEPROD O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O

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 .

O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD O B-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O B-GENEPROD O O O B-CELL_LINE O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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 .

O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD O B-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O B-GENEPROD O O O B-CELL_LINE O O O O O B-GENEPROD O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-EXP_ASSAY O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O

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

B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O O O O O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O O O O O O B-GENEPROD O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O B-EXP_ASSAY O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O B-GENEPROD O O O O O O O O O O O O O

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