Flowering time phenotype for Col - 0 , sec - 5 35S : : ATX1 - HA transgenic lines Experiments were repeated three times , for each , a total of 62 was counted for statistical of rosette leaf numbers Data are mean ± s . d . , ( * * ) , Statistical significance ( two - t - ) with P < 0 . 01 . n . s . , not significant .

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Flowering time phenotype analysis for Col-0, sec-5 and 35S::ATX1-HA transgenic lines Experiments were repeated three times, for each line, a total of 62 plants was counted for statistical analysis of rosette leaf numbers Data are mean ± s.d., (**), Statistical significance (two-tailed t-test) with P < 0.01. n.s., not significant.


Flowering time phenotype for Col - 0 , sec - 5 35S : : ATX1 - HA transgenic lines percentages of with visible buds bolting rates were calculated on day 21 day 24 after germination , respectively . Experiments were repeated three times , for each , a total of 62 was counted for statistical of visible buds bolting rates . Data are mean ± s . d . , ( * * ) , Statistical significance ( two - t - ) with P < 0 . 01 . n . s . , not significant .

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Flowering time phenotype analysis for Col-0, sec-5 and 35S::ATX1-HA transgenic lines The percentages of plants with visible buds and the bolting rates were calculated on day 21 and day 24 after plant germination, respectively. Experiments were repeated three times, for each line, a total of 62 plants was counted for statistical analysis of visible buds percent and bolting rates. Data are mean ± s.d., (**), Statistical significance (two-tailed t-test) with P < 0.01. n.s., not significant.


A Detection conformation of O - GlcNAc modification of His - ATX1ΔN catalyzed by His - SECΔN in vitro . His - ATX1ΔN ( expressing residues 592 - 952 of C - terminus ) His - SECΔN ( expressing residues 801 - 1062 of C - terminus ) were recombinantly expressed affinity purified separately . O - GlcNAcylation of His - ATX1ΔN was detected by anti - CTD110 . 6 antibody , further conformed by β - elimination .

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A Detection and conformation of O-GlcNAc modification of His-ATX1ΔN catalyzed by His-SECΔN in vitro. His-ATX1ΔN (expressing residues 592-952 of the C-terminus) and His-SECΔN (expressing residues 801-1062 of the C-terminus) were recombinantly expressed and affinity purified separately. O-GlcNAcylation of His-ATX1ΔN was detected by anti-CTD110.6 antibody, and further conformed by β-elimination analysis.


B His - SECΔN activates His - ATX1ΔN in vitro . Histone methyltransferase activity of ATX1ΔN was detected with or without recombinant SECΔN . H3K4A : mutated H3 in which fourth acid , a lysine ( K ) , was replaced with alanine ( A ) .

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B His-SECΔN activates His-ATX1ΔN in vitro. Histone methyltransferase activity of ATX1ΔN was detected with or without recombinant SECΔN. H3K4A: mutated H3 in which the fourth amino acid, a lysine (K), was replaced with alanine (A).


C of ATX1 O - GlcNAc modification in wild - type sec - 5 . Total soluble extracts from 12 - d - old seedlings were subjected to SDS - PAGE followed by immunoblotting using indicated antibodies . CBB : Coomassie brilliant blue , showing relative loading amount .

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C Analysis of ATX1 O-GlcNAc modification in wild-type and sec-5 plants. Total soluble protein extracts from 12-d-old seedlings were subjected to SDS-PAGE followed by immunoblotting using the indicated antibodies. CBB: Coomassie brilliant blue staining, showing relative protein loading amount.


D Loss of SEC function reduced ATX1 activity in Arabidopsis . were extracted ATX1 was immunoprecipitated with anti - ATX1 antibody from wild - type sec - 5 mutant , respectively , then used for histone H3K4 methyltransferase activity with recombinant H3 as catalyzing substrate . Band intensities were quantified with Image J . H3 signal was first normalized by input signal then was used for H3K4me3 signal normalization .

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D Loss of SEC function reduced ATX1 activity in Arabidopsis. Nuclear proteins were extracted and ATX1 was immunoprecipitated with anti-ATX1 antibody from wild-type and sec-5 mutant plants, respectively, and then used for histone H3K4 methyltransferase activity analysis with recombinant H3 as catalyzing substrate. Band intensities were quantified with Image J. The H3 signal was first normalized by input signal and then was used for H3K4me3 signal normalization.


E Comparison of ATX1 in Col - 0 sec - 5 at stages before after flowering . Seedlings were collected for extraction at 21 days ( before flowering ) 35 days ( after flowering ) after seeds were planted on plates . Anti - ATX1 antibody was used for immunoblot .

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E Comparison of ATX1 protein levels in Col-0 and sec-5 plants at stages before and after flowering. Seedlings were collected for protein extraction at 21 days (before flowering) and 35 days (after flowering) after seeds were planted on plates. Anti-ATX1 antibody was used for immunoblot assay.


A Mutation of either S947 alone or 12 serine threonine residues in SET domain reduced O - GlcNAc modification level of ATX1ΔN .

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A Mutation of either S947 alone or all 12 serine and threonine residues in the SET domain reduced O-GlcNAc modification level of ATX1ΔN.


B Site mutation of S947 or 12 serine threonine residues in ATX1 SET domain inhibited activation of ATX1 by SEC ; moreover , mutation of five conserved in SEC functional domain inhibited SEC activity .

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B Site mutation of S947 or all 12 serine and threonine residues in the ATX1 SET domain inhibited the activation of ATX1 by SEC; moreover, mutation of five conserved amino acids in the SEC functional domain inhibited SEC activity.


C Quantitative of HKMT activity of ATX1ΔN , ATX1ΔN - 12m , ATX1ΔN - m effect of SECΔN ΔSEC5m in activating those . Experiments were repeated three times . Error bars are SD . His - ΔSEC5m His - SECΔN were used as control .

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C Quantitative analysis of HKMT activity of ATX1ΔN, ATX1ΔN-12m, and ATX1ΔN-m and the effect of SECΔN and ΔSEC5m in activating those proteins. Experiments were repeated three times. Error bars are SD. His-ΔSEC5m and His-SECΔN were used as the control.


A Flowering of Col - 0 , atx1 - 2 , atx1 - 2 35S : : ATX1 - FLAG , atx1 - 2 35S : : ATX1m - FLAG ( with Ser947 replaced with alanine ) under LD conditions . Scale bar : 1 cm . Two independent lines are shown for each transformation .

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A Flowering phenotypes of Col-0, atx1-2, atx1-2 35S::ATX1-FLAG, and atx1-2 35S::ATX1m-FLAG (with Ser947 replaced with alanine) plants under LD conditions. Scale bar: 1 cm. Two independent lines are shown for each transformation.


B Quantitative real - time PCR of ATX1 FLC transcript in Col - 0 , atx1 - 2 , atx1 - 2 35S : : ATX1 - FLAG , atx1 - 2 35S : : ATX1m - FLAG . transcript were normalized to that of TUBULIN . Data are mean ± s . d . of three independent biological replicates , n = 3 .

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B Quantitative real-time PCR analysis of ATX1 and FLC transcript levels in Col-0, atx1-2, atx1-2 35S::ATX1-FLAG, and atx1-2 35S::ATX1m-FLAG plants. The transcript levels were normalized to that of TUBULIN. Data are mean ± s.d. of three independent biological replicates, n=3.


Flowering time phenotype of lines Three independent biological repeats were conducted , data from a total of 71 were used for statistical . Data are mean ± s . d . , ( * * ) , Statistical significance ( two - t - ) with P < 0 . 01 .

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Flowering time phenotype analysis of lines Three independent biological repeats were conducted, and data from a total of 71 plants were used for statistical analysis. Data are mean ± s.d., (**), Statistical significance (two-tailed t-test) with P < 0.01.


Flowering time phenotype of lines percentages of with visible buds bolting rates were calculated on day 21 day 24 after germination , respectively . Three independent biological repeats were conducted , data from a total of 71 were used for statistical . Data are mean ± s . d . , ( * * ) , Statistical significance ( two - t - ) with P < 0 . 01 .

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Flowering time phenotype analysis of lines The percentages of plants with visible buds and the bolting rates were calculated on day 21 and day 24 after plant germination, respectively. Three independent biological repeats were conducted, and data from a total of 71 plants were used for statistical analysis. Data are mean ± s.d., (**), Statistical significance (two-tailed t-test) with P < 0.01.


B : U2 - OS were transfected with indicated siRNAs . indicated were treated with etoposide ( 50 µM , 90 min . before ) . Whole - extracts were immunoblotted with indicated antibodies . result is representative of three biological replicates .

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B: U2-OS cells were transfected with the indicated siRNAs. The indicated cells were treated with etoposide (50 µM, 90 min. before analysis). Whole-cell extracts were immunoblotted with the indicated antibodies. The result is representative of three biological replicates.


D : U2 - OS were transfected with non - targeting ( mock ) or TSG101 - targeting siRNAs irradiated ( 20 Gy ) 90 min . before . Lysates were analyzed by EMSA results of fold changes in densitometric measurements of three independent experiments were compared with an ordinary one - way ANOVA ( * p < 0 . 05 ) in right panel .

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D: U2-OS cells were transfected with non-targeting (mock) or TSG101-targeting siRNAs and irradiated (20 Gy) 90 min. before analysis. Lysates were analyzed by EMSA and results of fold changes in densitometric measurements of the three independent experiments were compared with an ordinary one-way ANOVA (*p < 0.05) in the right panel.


A : - fractionation of U2 - OS . Indicated samples were transfected with siRNAs against TSG101 . PARylation was inhibited by olaparib ( 10 µM , 24 hours before ) . export of p - ATM was induced by etoposide ( 50 µM , 45 minutes before ) . fractions were immunoblotted with indicated antibodies . fractionation efficiency was controlled by respective subcellular marker ( PARP1 LDH - A ) . PARylation was detected using Pan ADPr reagent . Note that PARylation is activated by shearing forces during extract preparation ( lane 1 )

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A: Nuclear-cytoplasmic fractionation of U2-OS cells. Indicated samples were transfected with siRNAs against TSG101. PARylation was inhibited by olaparib (10 µM, 24 hours before analysis). Cytoplasmic export of p-ATM was induced by etoposide (50 µM, 45 minutes before analysis). Nuclear and cytoplasmic fractions were immunoblotted with the indicated antibodies. The fractionation efficiency was controlled by the respective subcellular marker proteins (PARP1 and LDH-A). PARylation was detected using the Pan ADPr reagent. Note that PARylation is activated by shearing forces during extract preparation (lane 1)


B : U2 - OS were transfected with non - targeting ( - ) or TSG101 - targeting ( + ) siRNAs were irradiated ( + ) or not ( - ) as indicated ( 20 Gy ) 10 min . before . Whole - extracts were immunoblotted with 10H PAR monoclonal antibody .

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B: U2-OS cells were transfected with non-targeting (-) or TSG101-targeting (+) siRNAs and cells were irradiated (+) or not (-) as indicated (20 Gy) 10 min. before analysis. Whole-cell extracts were immunoblotted with the 10H PAR monoclonal antibody.


C : siRNA - transfected U2 - OS as in B were etoposide - treated or not , as indicated . Whole - extracts were immunoblotted with Pan ADPr reagent that recognizes both poly - mono - ( ADP - ribose ) . Note that PARylation also occurred during lysis in untreated .

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C: siRNA-transfected U2-OS cells as in B were etoposide-treated or not, as indicated. Whole-cell extracts were immunoblotted with the Pan ADPr reagent that recognizes both poly- and mono-(ADP-ribose). Note that PARylation also occurred during cell lysis in untreated cells.


D : U2 - OS were transfected with non - targeting or VPS28 - targeting siRNAs . Whole - extracts were immunoblotted with indicated antibodies .

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D: U2-OS cells were transfected with non-targeting or VPS28-targeting siRNAs. Whole-cell extracts were immunoblotted with indicated antibodies.


F : were irradiated ( 20 Gy ) as indicated 10 min before . Whole - extracts were immunoblotted with indicated antibodies .

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F: Cells were irradiated (20 Gy) as indicated 10 min before analysis. Whole-cell extracts were immunoblotted with the indicated antibodies.


A : Proximity ligation ( PLA ) with unstimulated , etoposide ( Eto ) - treated ( 50 µM , 10 min . before ) , or etoposide plus olaparib - ( Olap ) co - treated ( 50 µM of etoposide , 10 min . before 10 µM of olaparib 24 hours before ) U2 - OS . red dots represent PARP1 - TSG101 interaction . nucleus is stained with DAPI ( blue ) . Scale bar : 10 µm . As negative controls , PLA was performed without PARP1 or TSG101 antibodies . As an additional control , PLA was performed in PARP1 depleted to show specificity B : of PLA dots per nuclei in A . Blind counting was performed from 30 replicates three biologically independent experiments . conditions were compared with an ordinary one - way ANOVA ( ns , p > 0 . 05 ; * p < 0 . 05 ; * * * p < 0 . 001 ; * * * * p < 0 . 0001 ) .

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A: Proximity ligation assay (PLA) assay with unstimulated, etoposide (Eto)-treated (50 µM, 10 min. before analysis), or etoposide plus olaparib-(Olap) co-treated (50 µM of etoposide, 10 min. before analysis and 10 µM of olaparib 24 hours before analysis) U2-OS cells. The red dots represent PARP1-TSG101 interaction. The nucleus is stained with DAPI (blue). Scale bar: 10 µm. As negative controls, PLA was performed without primary PARP1 or TSG101 antibodies. As an additional control, PLA was performed in PARP1 depleted cells to show specificity B: Quantification of PLA dots per nuclei in A. Blind counting was performed from 30 replicates three biologically independent experiments. The conditions were compared with an ordinary one-way ANOVA (ns, p > 0.05; *p < 0.05; ***p < 0.001; ****p < 0.0001).


C : U2 - OS were cotransfected with indicated deletion constructs of mCherry - tagged TSG101 EGFP - tagged PARP1 . Immunoprecipitation of GFP was performed using whole - extracts .

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C: U2-OS cells were cotransfected with the indicated deletion constructs of mCherry-tagged TSG101 and EGFP-tagged PARP1. Immunoprecipitation of GFP was performed using whole-cell extracts.


F : In - vitro PARylation reactions were carried out with indicated recombinant purified , including 8 ng PARP1 in presence of sssDNA ( sheared salmon sperm ) MgCl2 . 100 ng of TSG101 was added to reaction , as indicated . PARylation reaction was also carried out using 100 ng of BSA or RNF113A as negative controls .

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F: In-vitro PARylation reactions were carried out with the indicated recombinant and purified proteins, including 8 ng PARP1 in the presence of sssDNA (sheared salmon sperm DNA) and MgCl2. 100 ng of TSG101 was added to the reaction, as indicated. The PARylation reaction was also carried out using 100 ng of BSA or RNF113A as negative controls.


B : Representative of PARP1 - GFP association with - microirradiation sites in untreated ( mock ) , olaparib - treated ( 10 µM , 24 hours before ) or siTSG101 - transfected U2 - OS at indicated times post - irradiation . Scale bar : 7 . 5 µm . image is representative of nine replicates from three biologically independent experiments . Go to Movies EV1 - 3 for further illustration .

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B: Representative images of PARP1-GFP association with laser-microirradiation sites in untreated (mock), olaparib-treated (10 µM, 24 hours before analysis) or siTSG101-transfected U2-OS cells at indicated times post-irradiation. Scale bar: 7.5 µm. The image is representative of nine replicates from three biologically independent experiments. Go to Movies EV1-3 for further illustration.


C : Kinetics of PARP1 - GFP recruitment dissociation from lesions were measured at times indicated . Nine nuclei were analyzed for each indicated condition from three biologically independent experiments . data are shown as mean GFP in microirradiated ± , normalized to mean GFP of corresponding whole nuclei . D : Relative PARP1 - GFP recruitment to lesions for each indicated condition at 12 min . post - irradiation .

C: Kinetics of PARP1-GFP recruitment and dissociation from DNA lesions were measured at times indicated. Nine nuclei were analyzed for each indicated condition from three biologically independent experiments. The data are shown as mean GFP intensity in the microirradiated area ± SEM, normalized to the mean GFP intensity of corresponding whole nuclei. D: Relative PARP1-GFP recruitment to DNA lesions for each indicated condition at 12 min. post-irradiation.


C : were transfected with non - targeting or TSG101 - targeting siRNAs . Immunofluorescence was performed with indicated antibodies ( red : caspase 3 ; green : γH2AX ) . Nuclei were stained with DAPI ( blue ) . image is a representative of 3 biologically independent experiments . Scale bar : 30 µm . See Fig EV6D for of γH2AX positive as an evidence of IR - treatment . D : of caspase 3 was determined by blind counting of approximately 100 for each condition from 3 biologically independent experiments . Conditions were compared with an unpaired t - ( * p < 0 . 05 , * * p < 0 . 01 ) .

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C: Cells were transfected with non-targeting or TSG101-targeting siRNAs. Immunofluorescence staining was performed with the indicated antibodies (red: cleaved caspase 3; green: γH2AX). Nuclei were stained with DAPI (blue). The image is a representative of 3 biologically independent experiments. Scale bar: 30 µm. See Fig EV6D for percentage of γH2AX positive cells as an evidence of IR-treatment. D: Percentage of cleaved caspase 3 was determined by blind counting of approximately 100 cells for each condition from 3 biologically independent experiments. Conditions were compared with an unpaired t-test (*p < 0.05, **p < 0.01).


E : γH2AX of TSG101 - targeting or non - targeting siRNA transfected U2 - OS reveals - like foci at 2 or 7 days after irradiation .

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E: γH2AX staining of TSG101-targeting or non-targeting siRNA transfected U2-OS cells reveals puncta-like foci at 2 or 7 days after irradiation.


F : number of - like γH2AX foci per was determined by blind counting of approximately 100 for each condition from 3 biologically independent experiments .

F: The number of puncta-like γH2AX foci per cell was determined by blind counting of approximately 100 cells for each condition from 3 biologically independent experiments.


Representative of seed segregation in 420 / + + in wild type ( Col ) hcr2 . Scatterplots to right show red ( dsRed ) green ( eGFP ) values in 420 / + + Col ( top ) hcr2 ( bottom ) . Scale bars : 2 mm .

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Representative images of seed fluorescence segregation in 420/++ in wild type (Col) and hcr2. Scatterplots to the right show red (dsRed) and green (eGFP) fluorescence values in 420/++ Col (top) and hcr2 (bottom). Scale bars: 2 mm.


End - point RT - PCR of HSBP in Col , hsbp - 3 , hsbp - 2 hsbp - 1 . Hash asterisk indicate aberrant long short splicing variants of HSBP in hsbp - 3 , respectively . Image J was used to measure relative PCR band for hsbp - 3 ( 53 % ) , hsbp - 2 ( 9 % ) hsbp - 1 ( 70 % ) . TUB2 was used as an internal control . As in E , but showing immunoblot of HSBP . hsbp - 3 , hsbp - 2 hsbp - 1 accumulate about 58 % , 17 % 77 % of HSBP , respectively . Coomassie - stained was used as a loading control .

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End-point RT-PCR analysis of HSBP in Col, hsbp-3, hsbp-2 and hsbp-1. Hash and asterisk indicate aberrant long and short splicing variants of HSBP in hsbp-3, respectively. Image J was used to measure relative PCR band intensity for hsbp-3 (53%), hsbp-2 (9%) and hsbp-1 (70%). TUB2 was used as an internal control. As in E, but showing immunoblot analysis of HSBP. hsbp-3, hsbp-2 and hsbp-1 accumulate about 58%, 17% and 77% of HSBP levels, respectively. Coomassie-stained membrane was used as a loading control.


Representative of ASY1 ( green ) RAD51 ( red ) immunostaining in Col hsbp - 3 . Nuclei spreads were stained with DAPI ( blue ) . Scale bars : 10 μm . of RAD51 foci numbers per in Col ( blue ) hsbp - 3 ( red ) . n = 20 of biological replicates .

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Representative images of ASY1 (green) and RAD51 (red) immunostaining in Col and hsbp-3. Nuclei spreads were stained with DAPI (blue). Scale bars: 10 μm. Quantification of RAD51 foci numbers per cell in Col (blue) and hsbp-3 (red). n = 20 cells of biological replicates.


Representative of MLH1 ( red ) immunostaining in Col , hsbp - 3 , hsbp - 2 , Col / Ler , meiMIGS - HSBP Col / Ler . was stained with DAPI . Scale bar : 5 μm . of number of MLH1 foci per shown in ( G ) . n ≥ 32 of biological replicates .

Representative images of MLH1 (red) immunostaining in Col, hsbp-3, hsbp-2, Col/Ler, and meiMIGS-HSBP Col/Ler. Nuclear DNA was stained with DAPI. Scale bar: 5 μm. Quantification of the number of MLH1 foci per cell shown in (G). n ≥ 32 cells of biological replicates.


Representative of HEI10 ( red ) ASY1 ( green ) immunostaining in Col hsbp - 3 . Scale bar : 2 μm .

Representative images of HEI10 (red) and ASY1 (green) immunostaining in Col and hsbp-3. Scale bar: 2 μm.


of number of HEI10 foci per shown in ( I ) . n ≥ 21 of biological replicates .

Quantification of the number of HEI10 foci per cell shown in (I). n ≥ 21 cells of biological replicates.


Immunoblot of HEI10 HEI10 - Myc in Col , hsbp - 3 , HEI10 - myc HEI10 - myc . Experiments were performed at least three times .

Immunoblot analysis of HEI10 and HEI10-Myc in Col, hsbp-3, HEI10-myc and HEI10-myc plants. Experiments were performed at least three times.


Figure 1 . DENND2B binds ITSN Purified GST - SH3 - containing were incubated with lysates of HEK - 293T expressing Flag - DENND2B . Total bound were detected by Ponceau S Western blot , respectively .

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Figure 1. DENND2B binds ITSN  Purified GST-SH3-containing proteins were incubated with lysates of HEK-293T cells expressing Flag-DENND2B. Total proteins and bound proteins were detected by Ponceau S staining and Western blot, respectively.


Purified GST - SH3 domains of ITSN were incubated with lysates of HEK - 293T expressing Flag - DENND2B . Total bound were detected by Ponceau S Western blot , respectively .

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Purified GST-SH3 domains of ITSN were incubated with lysates of HEK-293T cells expressing Flag-DENND2B. Total proteins and bound proteins were detected by Ponceau S staining and Western blot, respectively.


Lysates from HEK - 293T expressing Flag - ITSN - s GFP or GFP - DENND2B were incubated with A beads or A beads coupled to anti - GFP . Bound were detected by Western blot .

Lysates from HEK-293T cells expressing Flag-ITSN-s and GFP or GFP-DENND2B were incubated with protein A beads or protein A beads coupled to anti-GFP. Bound proteins were detected by Western blot.


D , E HeLa expressing indicated constructs . Flag - ITSN - s was visualized with anti - Flag AlexaFluor561 or AlexaFluor488 in D E respectively . Scale bar : 5 µm . Boxed regions are magnified on bottom . arrows red arrows point to colocalizing non - colocalizing structures , respectively .

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D,E HeLa cells expressing the indicated constructs. Flag-ITSN-s was visualized with anti-Flag and AlexaFluor561 or AlexaFluor488 in D and E respectively. Scale bar: 5 µm. Boxed regions are magnified on the bottom. White arrows and red arrows point to colocalizing and non-colocalizing structures, respectively.


Figure 2 . DENND2B phosphorylation by PKD disrupts ITSN interaction A HEK - 293T were treated with DMSO or okadaic acid ( OA ) , lysed analyzed by Western blot .

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

Figure 2. DENND2B phosphorylation by PKD disrupts ITSN interaction A HEK-293T cells were treated with DMSO or okadaic acid (OA), lysed and analyzed by Western blot.


B HEK - 293T expressing Flag - DENND2B were treated with DMSO or 250 nM OA , lysates were incubated with GST - . Total bound were detected by Ponceau S Western blot , respectively .

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

B HEK-293T cells expressing Flag-DENND2B were treated with DMSO or 250 nM OA, and cell lysates were incubated with GST-proteins. Total proteins and bound proteins were detected by Ponceau S staining and Western blot, respectively.


C of phosphorylated peptides following DMSO or OA treatment as determined by mass spectrometry . Low abundance peptides not included . Refer to Table EV1 for a complete peptide list .

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

C Percentage of phosphorylated peptides following DMSO or OA treatment as determined by mass spectrometry. Low abundance peptides not included. Refer to Table EV1 for a complete peptide list.


E HEK - 293T expressing Flag - DENND2B were treated with DMSO , 250 nM OA or with increasing concentrations of PKD inhibitor CID755673 . lysates were analyzed by Western blot .

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 B-CONTROLLED_VAR O O O O O O O O

E HEK-293T cells expressing Flag-DENND2B were treated with DMSO, 250 nM OA or with increasing concentrations of PKD inhibitor CID755673. Cell lysates were analyzed by Western blot.


F HEK - 293T expressing Flag - DENND2B or control were treated with 250 nM OA or DMSO , incubated with G beads coupled to anti - Flag analyzed by Western blot .

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

F HEK-293T cells expressing Flag-DENND2B or control cells were treated with 250 nM OA or DMSO, incubated with protein G beads coupled to anti-Flag and analyzed by Western blot.


G Lysates from HEK - 293T expressing Flag - DENND2B PKD - dominant negative ( DN ) were incubated with G beads or G beads coupled to anti - Flag . Bound were detected by Western blot .

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

G Lysates from HEK-293T cells expressing Flag-DENND2B and PKD-dominant negative (DN) were incubated with protein G beads or protein G beads coupled to anti-Flag. Bound proteins were detected by Western blot.


H HEK - 293T co - expressing Flag - DENND2B with pcDNA3 or PKD - constitutively active ( CA ) , were incubated with G beads or G beads coupled to anti - Flag bound were detected by Western blot .

O O O O O O O O O O O O O O O O O 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 HEK-293T cells co-expressing Flag-DENND2B with pcDNA3 or PKD-constitutively active (CA), were incubated with protein G beads or protein G beads coupled to anti-Flag and bound proteins were detected by Western blot.


I of H where PKD - CA treatment is normalized to pcDNA3 control . Mean + / - SD . Welch ' s t - * p = 0 . 016 , n = 6 for both treatment groups from 3 independent experiments .

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

I Quantification of H where PKD-CA treatment is normalized to pcDNA3 control. Mean +/- SD. Welch's t-test *p=0.016, n=6 for both treatment groups from 3 independent experiments.


Figure 3 . 14 - 3 - 3 outcompetes ITSN for binding to phosphorylated DENND2B A Purified GST - 14 - 3 - 3 WT or K50E were incubated with lysates of HEK - 293T expressing GFP - DENND2B WT or S30A . Total bound were detected by Ponceau S Western blot , respectively .

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

Figure 3. 14-3-3 outcompetes ITSN for binding to phosphorylated DENND2B A Purified GST-14-3-3 WT or K50E were incubated with lysates of HEK-293T cells expressing GFP-DENND2B WT or S30A. Total proteins and bound proteins were detected by Ponceau S staining and Western blot, respectively.


B HEK - 293T expressing Flag - DENND2B were treated with DMSO or 250 nM OA lysates were incubated with GST - 14 - 3 - 3 . Total bound were detected by Ponceau S Western blot , respectively .

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

B HEK-293T cells expressing Flag-DENND2B were treated with DMSO or 250 nM OA and cell lysates were incubated with GST-14-3-3. Total proteins and bound proteins were detected by Ponceau S staining and Western blot, respectively.


C of B . where bound DENND2B is normalized to SM . Mean + / - SD . Welch ' s t - * * p = 0 . 006 n = 8 for both treatment groups from 5 independent

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

C Quantification of B. where bound DENND2B is normalized to the SM. Mean +/- SD. Welch's t-test **p=0.006 n=8 for both treatment groups from 5 independent experiments


D HEK - 293T expressing Flag - DENND2B were treated with 250 nM OA alone or with 50 µm PKD inhibitor CID755673 lysates were incubated with GST - 14 - 3 - 3 . Total bound were detected by Ponceau S Western blot , respectively .

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

D HEK-293T cells expressing Flag-DENND2B were treated with 250 nM OA alone or with 50 µm PKD inhibitor CID755673 and cell lysates were incubated with GST-14-3-3. Total proteins and bound proteins were detected by Ponceau S staining and Western blot, respectively.


E of D . where bound DENND2B is normalized to SM . Mean + / - SD . Welch ' s t - * p = 0 . 040 . n = 5 for both treatment groups from 3 independent

E Quantification of D. where bound DENND2B is normalized to the SM. Mean +/- SD. Welch's t-test *p=0.040. n=5 for both treatment groups from 3 independent experiments


F GST - SH3A was incubated with lysates of HEK - 293T expressing GFP - DENND2B WT or S30A . Total bound were detected by Ponceau S Western blot , respectively .

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

F GST-SH3A was incubated with lysates of HEK-293T cells expressing GFP-DENND2B WT or S30A. Total proteins and bound proteins were detected by Ponceau S staining and Western blot, respectively.


G Lysates of HEK - 293T expressing Flag - DENND2B were pre - incubated with increasing concentrations of purified 14 - 3 - 3 WT subsequently incubated with GST - SH3A ( top panel ) or GST - Grb2 ( bottom panel ) . Total bound were detected by Ponceau S Western blot , respectively .

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

G Lysates of HEK-293T cells expressing Flag-DENND2B were pre-incubated with increasing concentrations of purified 14-3-3 WT and subsequently incubated with GST-SH3A (top panel) or GST-Grb2 (bottom panel). Total proteins and bound proteins were detected by Ponceau S staining and Western blot, respectively.


Figure 4 . DENND2B promotes EGFR recycling A , B HeLa expressing indicated constructs . Boxed regions are magnified on bottom . Scale bars : 5 µm in top panel 1 µm bottom panel . arrows arrowheads point to colocalization on vesicles plasma , respectively .

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

Figure 4. DENND2B promotes EGFR recycling A,B HeLa cells expressing the indicated constructs. Boxed regions are magnified on the bottom. Scale bars: 5 µm in top panel and 1 µm bottom panel. White arrows and arrowheads point to colocalization on vesicles and plasma membrane, respectively.


C Transduced MCF10A were quantified by real - time PCR . Mean + / - SD , ANOVA with Dunnett post - hoc * * * p = 0 . 000 for both comparisons , n = 3 from 3 independent experiments .

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

C Transduced MCF10A cells were quantified by real-time PCR. Mean +/- SD, ANOVA with Dunnett post-hoc test ***p=0.000 for both comparisons, n=3 from 3 independent experiments.


D Transduced HEK - 293T expressing Flag - DENND2B were analyzed by Western blot .

D Transduced HEK-293T cells expressing Flag-DENND2B were analyzed by Western blot.


E Transduced MCF10A , were biotinylated lysates were incubated with streptavidin beads . Bound were detected by Western blot .

E Transduced MCF10A cells, were biotinylated and cell lysates were incubated with streptavidin beads. Bound proteins were detected by Western blot.


F of E . where surface EGFR is normalized to total EGFR . Mean + / - SD , Anova with Dunnett post - hoc * p = 0 . 013 * * * p = 0 . 000 . n = 6 for treatment groups from 4 independent

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

F Quantification of E. where surface EGFR is normalized to total EGFR. Mean +/- SD, Anova with Dunnett post-hoc test *p=0.013 and ***p=0.000. n=6 for all treatment groups from 4 independent experiments


G of E where total EGFR is normalized to Hsc70 . Mean + / - SD n = 3 for treatment groups from 3 independent

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

G Quantification of E where total EGFR is normalized to Hsc70. Mean +/- SD n=3 for all treatment groups from 3 independent experiments


I MCF10A expressing GFP - DENND2B were labelled for surface EGFR on ice , incubated at 16 ° C to allow internalization , acid washed to remove remaining labelled - EGFR processed for immunofluorescence to visualize internal pool of EGFR ( post - endocytosis , top panel ) or incubated at 37 ° C to allow recycling , acid washed again processed for immunofluorescence to visualize remaining internal pool of EGFR ( post - recycling , bottom panel ) .

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

I MCF10A cells expressing GFP-DENND2B were labelled for surface EGFR on ice, incubated at 16°C to allow internalization, acid washed to remove remaining labelled-EGFR and processed for immunofluorescence to visualize the internal pool of EGFR (post-endocytosis, top panel) or incubated at 37°C to allow recycling, acid washed again and processed for immunofluorescence to visualize the remaining internal pool of EGFR (post-recycling, bottom panel).


J of I top panel , internal EGFR post - endocytosis . Mean per + / - SD , n = 13 for GFP - DENND2B n = 21 for control pooled from 2 independent experiments .

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

J Quantification of I top panel, internal EGFR post-endocytosis. Mean fluorescence per cell +/- SD, n=13 for GFP-DENND2B and n=21 for control cells pooled from 2 independent experiments.


K of I bottom panel , internal EGFR post - recycling . Mean per + / - SD , n = 10 for GFP - DENND2B n = 26 for control pooled from 2 independent experiments .

K Quantification of I bottom panel, internal EGFR post-recycling. Mean fluorescence per cell +/- SD, n=10 for GFP-DENND2B and n=26 for control cells pooled from 2 independent experiments.


Figure 5 . EGF treatment dissociates DENND2B from ITSN A HEK - 293T were serum starved , treated with 100 ng / mL EGF for indicated times lysates were analyzed by Western blot .

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

Figure 5. EGF treatment dissociates DENND2B from ITSN A HEK-293T cells were serum starved, treated with 100 ng/mL EGF for the indicated times and cell lysates were analyzed by Western blot.


B of A where P - PKD is normalized to Hsc70 . Mean + / - SD . ANOVA with Dunnett post - * p = 0 . 015 * * p = 0 . 003 n = 3 from 3 independent experiments .

B Quantification of A where P-PKD is normalized to Hsc70. Mean +/- SD. ANOVA with Dunnett post-test *p=0.015 and ** p=0.003 n=3 from 3 independent experiments.


C HEK - 293T were serum starved , treated with 100 ng / mL EGF for 5 min lysates were incubated with G beads or G beads coupled to anti - Flag . Bound were detected by Western blot .

C HEK-293T cells were serum starved, treated with 100 ng/mL EGF for 5 min and cell lysates were incubated with protein G beads or protein G beads coupled to anti-Flag. Bound proteins were detected by Western blot.


D of B where P - PKD substrate is normalized to Flag - DENND2B . Mean + / - SD Welch ' s t - * p = 0 . 036 , n = 4 from 3 independent experiments .

D Quantification of B where P-PKD substrate is normalized to Flag-DENND2B. Mean +/- SD Welch's t-test *p=0.036, n=4 from 3 independent experiments.


E HEK - 293T were serum starved , treated with 100 ng / mL EGF for 5 min lysates were incubated with GST - SH3A . Total bound were detected by Ponceau S Western blot , respectively .

E HEK-293T cells were serum starved, treated with 100 ng/mL EGF for 5 min and cell lysates were incubated with GST-SH3A. Total proteins and bound proteins were detected by Ponceau S staining and Western blot, respectively.


F of E where bound Flag - DENND2B is normalized to SM . Mean + / - SD , Welch ' s t - * p = 0 . 012 , n = 6 from 3 independent experiments .

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

F Quantification of E where bound Flag-DENND2B is normalized to SM. Mean +/- SD, Welch's t-test *p=0.012, n=6 from 3 independent experiments.


( A , B ) In situ cryo - electron tomogram displaying proximal region of a mature mother centriole ( A ) procentriole ( B ) . Mature centriole , MC ; procentriole , PC ; mitochondria , mito ; vacuole , vac ; dashed , lamella edge . Scale bars , 100 nm .

(A, B) In situ cryo-electron tomogram displaying the proximal region of a mature mother centriole (A) and procentriole (B). Mature centriole, MC; procentriole, PC; mitochondria, mito; vacuole, vac; white dashed line, lamella edge. Scale bars, 100 nm.


( C ) Side view z - projection of cartwheels containing central hub several CIDs from a mature centriole . Central hub , CH ; cartwheel inner densities , CID . Scale bar , 20 nm .

(C) Side view z-projection of cartwheels containing the central hub and several CIDs from a mature centriole. Central hub, CH; cartwheel inner densities, CID. Scale bar, 20 nm.


( D ) Cross section of cartwheel - containing regions from a mature centriole . Microtubule triplet , MTT ; spokes , SP . Scale bar , 200 nm .

O O O O O O 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) Cross section of the cartwheel-containing regions from a mature centriole. Microtubule triplet, MTT; spokes, SP. Scale bar, 200 nm.


( E ) Side view z - projection of cartwheels containing central hub several CIDs from a procentriole . Scale bar , 20 nm .

(E) Side view z-projection of cartwheels containing the central hub and several CIDs from a procentriole. Scale bar, 20 nm.


( F ) Cross section of cartwheel - containing regions from a procentriole . Scale bar , 200 nm .

(F) Cross section of the cartwheel-containing regions from a procentriole. Scale bar, 200 nm.


( G ) Nine - fold symmetrized cross sections of cartwheel - containing region from a mature centriole ( left side ) a procentriole ( right side ) . Dashed circle , central hub . Scale bars , 100 nm .

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

(G) Nine-fold symmetrized cross sections of the cartwheel-containing region from a mature centriole (left side) and a procentriole (right side). Dashed white circle, central hub. Scale bars, 100 nm.


( H ) Longitudinal periodicity measurements of central hub CIDs . Central hub , blue ; CID , red . Mean values are displayed above data range . Blue data points are measured between units of central hub , red data points are measured distances between units of cartwheel inner density . Mature centriole , central hub , n = 30 , mean = 4 . 1 + / - 0 . 67 ( S . D ) ; mature centriole , cartwheel inner density , n = 18 , mean = 8 . 7 + / - 1 . 6 ( S . D ) ; procentriole , central hub , n = 10 , mean = 4 . 0 + / - 0 . 66 ( S . D ) ; procentriole , cartwheel inner density , n = 5 , mean = 8 . 6 + / - 1 . 2 ( S . D ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Longitudinal periodicity measurements of the central hub and CIDs. Central hub, blue; CID, red. Mean values are displayed above the data range. Blue data points are measured distance between individual units of the central hub, red data points are measured distances between individual units of the cartwheel inner density. Mature centriole, central hub, n = 30, mean = 4.1 +/- 0.67 (S.D); mature centriole, cartwheel inner density, n = 18, mean = 8.7 +/- 1.6 (S.D); procentriole, central hub, n = 10, mean = 4.0 +/- 0.66 (S.D); procentriole, cartwheel inner density, n = 5, mean = 8.6 +/- 1.2 (S.D).


( I , J ) Nine - fold symmetrized central hub z - projections , starting at proximal end of cartwheel continuing distally along cartwheel by 5 . 4 nm steps in a mature centriole ( I ) a procentriole ( J ) . Red arrow , CID ; blue arrow ; central hub . Scale bar : 20 nm .

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

(I, J) Nine-fold symmetrized central hub z-projections, starting at the proximal end of the cartwheel and continuing distally along the cartwheel by 5.4 nm steps in a mature centriole (I) and a procentriole (J). Red arrow, CID; blue arrow; central hub. Scale bar: 20 nm.


( A , B , C ) Cryo - electron tomograms of proximal regions of a P . tetraurelia centriole ( A ) , a N . gruberi centriole ( B ) , a H . sapiens procentriole ( C ) . arrows denote a broken cartwheel ; procentriole , PC ; mature centriole , MC ; Scale bar , 100 nm . Note that most N . gruberi H . sapiens centrioles were heavily compressed during cryo - EM preparation , as previously described ( Guichard et al . , 2010 ) . displayed N . gruberi centriole illustrates damage caused by compression . periodicities of N . gruberi H . sapiens cartwheels were measured only on regions that were not

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

(A, B, C) Cryo-electron tomograms of the proximal regions of a P. tetraurelia centriole (A), a N. gruberi centriole (B), and a H. sapiens procentriole (C). White arrows denote a broken cartwheel; procentriole, PC; mature centriole, MC; Scale bar, 100 nm. Note that most N. gruberi and H. sapiens centrioles were heavily compressed during the cryo-EM preparation, as previously described (Guichard et al., 2010). The displayed N. gruberi centriole illustrates the damage caused by compression. The periodicities of N. gruberi and H. sapiens cartwheels were measured only on regions that were not damaged


( D ) Cross sections from cartwheel - containing region of P . tetraurelia centrioles . Scale bar , 50 nm . ( E ) Zoomed side view of cartwheel from P . tetraurelia , displaying central hub ( CH ) several cartwheel inner densities ( CIDs ) , arrow . Scale bar , 25 nm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Cross sections from cartwheel-containing region of P. tetraurelia centrioles. Scale bar, 50 nm. (E) Zoomed side view of cartwheel from P. tetraurelia, displaying the central hub (CH) and several cartwheel inner densities (CIDs), white arrow. Scale bar, 25 nm.


( F ) Cross section from cartwheel - containing region of N . gruberi , ( G ) Zoomed side view of cartwheel from N . gruberi displaying central hub ( CH ) several cartwheel inner densities ( CIDs ) , arrow .

(F) Cross section from cartwheel-containing region of N. gruberi, (G) Zoomed side view of cartwheel from N. gruberi displaying the central hub (CH) and several cartwheel inner densities (CIDs), white arrow.


( H ) Cross section from cartwheel - containing region of H . sapiens centriole . ( I ) Zoomed side view of cartwheel from H . sapiens , displaying central hub ( CH ) several cartwheel inner densities ( CIDs ) , arrow .

O O O O O O O O O O O O O O O O O O O O O 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) Cross section from cartwheel-containing region of H. sapiens centriole. (I) Zoomed side view of cartwheel from H. sapiens, displaying the central hub (CH) and several cartwheel inner densities (CIDs), white arrow.


( J ) Longitudinal periodicity of central hub CIDs in P . tetraurelia , N . gruberi , H . sapiens . Mean values are displayed above data range . Black lines indicate mean standard deviation . Blue data points are measured between units of central hub , red data points are measured distances between units of cartwheel inner density . P . tetraurelia , central hub , n = 10 , mean = 4 . 3 + / - 0 . 38 ( S . D ) ; N . gruberi , central hub , n = 10 , mean = 4 . 4 + / - 0 . 53 ( S . D ) ; H . sapiens , central hub , n = 10 , mean = 4 . 2 + / - 0 . 68 ( S . D ) ; P . tetraurelia , cartwheel inner density , n = 8 , mean = 8 . 4 + / - 1 . 3 ( S . D ) ; N . gruberi , cartwheel inner density , n = 8 , mean = 8 . 3 + / - 1 . 8 ( S . D ) ; H . sapiens , cartwheel inner density , n = 8 , mean = 8 . 1 + / - 2 . 5 ( S . D ) .

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

(J) Longitudinal periodicity of the central hub and CIDs in P. tetraurelia, N. gruberi, and H. sapiens. Mean values are displayed above data range. Black lines indicate the mean and the standard deviation. Blue data points are measured distance between individual units of the central hub, red data points are measured distances between individual units of the cartwheel inner density. P. tetraurelia, central hub, n = 10, mean = 4.3 +/- 0.38 (S.D); N. gruberi, central hub, n = 10, mean = 4.4 +/- 0.53 (S.D); H. sapiens, central hub, n = 10, mean = 4.2 +/- 0.68 (S.D); P. tetraurelia, cartwheel inner density, n = 8, mean = 8.4 +/- 1.3 (S.D); N. gruberi, cartwheel inner density, n = 8, mean = 8.3 +/- 1.8 (S.D); H. sapiens, cartwheel inner density, n = 8, mean = 8.1 +/- 2.5 (S.D).


( K Proximal protrusion of cartwheel beyond microtubule triplets in C . reinhardtii , P . tetraurelia , N . gruberi , H . sapiens . Internal cartwheel inside microtubule barrel , blue ( INT ) ; external cartwheel beyond microtubule wall , blue ( EXT ) . Mean values are displayed above each bar plot with black lines indicating standard deviation ( K ) . Mature C . reinhardtii , n = 4 , external cartwheel = 37 . 6 + / - 3 . 4 , internal cartwheel 74 . 2 + / - 16 . 6 ; C . reinhardtii procentriole , n = 2 , external cartwheel mean = 52 . 8 + / - 22 . 0 , internal cartwheel 26 . 5 + / - 29 . 7 ; P . tetraurelia , n = 23 , external cartwheel mean = 11 . 5 + / - 9 . 2 , internal cartwheel 66 . 6 + / - 15 . 7 ; N . gruberi , n = 19 , external cartwheel mean = 41 . 0 + / - 22 . 5 , internal cartwheel 259 . 0 + / - 87 . 2 ; human procentriole , n = 7 , external cartwheel 23 . 1 + / - 10 . 3 , internal cartwheel 154 . 7 + / - 47 . 7 . Error reported is standard deviation . Scale bar , 50 nm .

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

(K Proximal protrusion length of the cartwheel beyond the microtubule triplets in C. reinhardtii, P. tetraurelia, N. gruberi, and H. sapiens. Internal cartwheel inside the microtubule barrel, dark blue (INT); external cartwheel beyond the microtubule wall, light blue (EXT). Mean values are displayed above each bar plot with the black lines indicating the standard deviation (K). Mature C. reinhardtii, n = 4, external cartwheel length = 37.6 +/- 3.4, internal cartwheel length 74.2 +/- 16.6; C. reinhardtii procentriole, n = 2, external cartwheel mean length = 52.8 +/- 22.0, internal cartwheel length 26.5 +/- 29.7; P. tetraurelia, n = 23, external cartwheel mean length = 11.5 +/- 9.2, internal cartwheel length 66.6 +/- 15.7; N. gruberi, n = 19, external cartwheel mean length = 41.0 +/- 22.5, internal cartwheel length 259.0 +/- 87.2; human procentriole, n = 7, external cartwheel length 23.1 +/- 10.3, internal cartwheel length 154.7 +/- 47.7. Error reported is standard deviation. Scale bar, 50 nm.


start of each microtubule wall is delineated by a dashed ( L ) . Mature C . reinhardtii , n = 4 , external cartwheel = 37 . 6 + / - 3 . 4 , internal cartwheel 74 . 2 + / - 16 . 6 ; C . reinhardtii procentriole , n = 2 , external cartwheel mean = 52 . 8 + / - 22 . 0 , internal cartwheel 26 . 5 + / - 29 . 7 ; P . tetraurelia , n = 23 , external cartwheel mean = 11 . 5 + / - 9 . 2 , internal cartwheel 66 . 6 + / - 15 . 7 ; N . gruberi , n = 19 , external cartwheel mean = 41 . 0 + / - 22 . 5 , internal cartwheel 259 . 0 + / - 87 . 2 ; human procentriole , n = 7 , external cartwheel 23 . 1 + / - 10 . 3 , internal cartwheel 154 . 7 + / - 47 . 7 . Error reported is standard deviation . Scale bar , 50 nm .

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

The start of each microtubule wall is delineated by a dashed white line (L). Mature C. reinhardtii, n = 4, external cartwheel length = 37.6 +/- 3.4, internal cartwheel length 74.2 +/- 16.6; C. reinhardtii procentriole, n = 2, external cartwheel mean length = 52.8 +/- 22.0, internal cartwheel length 26.5 +/- 29.7; P. tetraurelia, n = 23, external cartwheel mean length = 11.5 +/- 9.2, internal cartwheel length 66.6 +/- 15.7; N. gruberi, n = 19, external cartwheel mean length = 41.0 +/- 22.5, internal cartwheel length 259.0 +/- 87.2; human procentriole, n = 7, external cartwheel length 23.1 +/- 10.3, internal cartwheel length 154.7 +/- 47.7. Error reported is standard deviation. Scale bar, 50 nm.


( A ) Top view of cartwheel reconstruction from P . tetraurelia . Scale bar , 50 nm . orange arrow marks D2 - rod . Purple arrow marks part of pinhead . Dotted yellow box with arrows denotes central hub - focused reslice shown in panel B ; dashed box with arrows denotes spoke - focused reslice shown in panel D .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Top view of cartwheel reconstruction from P. tetraurelia. Scale bar, 50 nm. Dark orange arrow marks the D2-rod. Purple arrow marks part of the pinhead. Dotted yellow box with arrows denotes the central hub-focused reslice shown in panel B; dashed white box with arrows denotes the spoke-focused reslice shown in panel D.


( B ) Reslice of central hub - containing region with spokes in P . tetraurelia . Scale bar , 50 nm . Dashed yellow denotes zoomed view shown in panels C , black with arrows indicates ~ 25 nm repeat between D1 densities formed by merged spokes . orange arrow marks D2 - rod .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Reslice of central hub-containing region with spokes in P. tetraurelia. Scale bar, 50 nm. Dashed light yellow line denotes the zoomed view shown in panels C, black line with arrows indicates the ~25 nm repeat distance between D1 densities formed by merged spokes. Dark orange arrow marks the D2-rod.


( C ) Zoomed view displaying periodic repeats of central hub ( CH ) several cartwheel inner densities ( CIDs ) in P . tetraurelia . CIDs , red arrowheads red plot profile ; CH , blue arrowheads blue plot profile . Overlay between CID CH peaks is plotted on right in red blue . Mean between CID peaks , red 8 . 6 + / - . 8 ( n = 5 ) . Distances between CH peaks split into two distinct populations : smaller ( within a ring - pair ) , green 3 . 1 + / - 1 . 3 ( n = 6 ) ; larger ( between ring - pairs ) , blue 5 . 5 + / - . 7 ( n = 5 ) ; average periodicity , black 4 . 2 + / - 1 . 3 ( n = 11 ) .

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

(C) Zoomed view displaying periodic repeats of the central hub (CH) and several cartwheel inner densities (CIDs) in P. tetraurelia. CIDs, red arrowheads and red plot profile; CH, blue arrowheads and blue plot profile. Overlay between CID and CH peaks is plotted on the right in red and blue. Mean distance between CID peaks, red 8.6 +/- .8 (n= 5). Distances between CH peaks split into two distinct populations: smaller (within a ring-pair), green 3.1 +/- 1.3 (n= 6); larger (between ring-pairs), blue 5.5 +/- .7 (n= 5); average periodicity, black 4.2 +/- 1.3 (n= 11).


( D ) Serial z - projections of ~ 4 nm thickness from one cartwheel repeat unit of P . tetraurelia . Left - most z - projections display central hub , right - most projection shows microtubule wall . Blue circles delineate one ring - pair . arrows mark emanating spokes . arrowheads mark fused spokes . dashed follows tilt of spokes . Black arrow indicates final merged spoke ( D1 density ) . orange arrow marks D2 - rod . Scale bar , 50 nm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Serial z-projections of ~4 nm thickness from one cartwheel repeat unit of P. tetraurelia. Left-most z-projections display the central hub, right-most projection shows the microtubule wall. Blue circles delineate one ring-pair. White arrows mark individual emanating spokes. White arrowheads mark fused spokes. White dashed line follows the tilt of the spokes. Black arrow indicates the final merged spoke (D1 density). Dark orange arrow marks the D2-rod. Scale bar, 50 nm.


( E ) Top view of cartwheel reconstruction from C . reinhardtii . Scale bar , 50 nm . orange arrow marks D2 - rod . Purple arrow marks part of pinhead . Dotted yellow box with arrows denotes central hub - focused reslice shown in panel F ; dashed box with arrows denotes spoke - focused reslice shown in panel H .

(E) Top view of cartwheel reconstruction from C. reinhardtii. Scale bar, 50 nm. Dark orange arrow marks the D2-rod. Purple arrow marks part of the pinhead. Dotted yellow box with arrows denotes the central hub-focused reslice shown in panel F; dashed white box with arrows denotes the spoke-focused reslice shown in panel H.


( F ) Reslice of central hub - containing region with spokes in C . reinhardtii . Scale bar , 50 nm . Dashed yellow denotes zoomed view shown in panel G , black with arrows indicates ~ 25 nm repeat between D1 densities formed by merged spokes . orange arrow marks D2 - rod .

(F) Reslice of central hub-containing region with spokes in C. reinhardtii. Scale bar, 50 nm. Dashed light yellow line denotes the zoomed view shown in panel G, black line with arrows indicates the ~25 nm repeat distance between D1 densities formed by merged spokes. Dark orange arrow marks the D2-rod.


( G ) Zoomed view displaying periodic repeats of central hub ( CH ) several cartwheel inner densities ( CIDs ) in C . reinhardtii . CIDs , red arrowheads red plot profile ; CH , blue arrowheads blue plot profile . Overlay between CID CH peaks is plotted on right in red blue . Mean between CID peaks , red 8 . 8 + / - 1 . 0 ( n = 5 ) . Distances between CH peaks split into two distinct populations : smaller ( within a ring - pair ) , green 3 . 5 + / - 1 . 3 ( n = 6 ) ; larger ( between ring - pairs ) , blue 5 . 1 + / - . 4 ( n = 5 ) ; average periodicity , black 4 . 2 + / - 1 . 3 ( n = 11 ) .

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

(G) Zoomed view displaying periodic repeats of the central hub (CH) and several cartwheel inner densities (CIDs) in C. reinhardtii. CIDs, red arrowheads and red plot profile; CH, blue arrowheads and blue plot profile. Overlay between CID and CH peaks is plotted on the right in red and blue. Mean distance between CID peaks, red 8.8 +/- 1.0 (n= 5). Distances between CH peaks split into two distinct populations: smaller (within a ring-pair), green 3.5 +/- 1.3 (n= 6); larger (between ring-pairs), blue 5.1 +/- .4 (n= 5); average periodicity, black 4.2 +/- 1.3 (n= 11).


( H ) Serial z - projections of ~ 4 nm thickness from one cartwheel repeat unit of C . reinhardtii . Left - most z - projections display central hub , right - most projection shows microtubule wall . Blue circles delineate one ring - pair . arrows mark emanating spokes . arrowheads mark fused spokes . dashed follows tilt of spokes . Black arrow indicates final merged spoke ( D1 density ) . orange arrow marks D2 - rod . Scale bar , 50 nm .

(H) Serial z-projections of ~4 nm thickness from one cartwheel repeat unit of C. reinhardtii. Left-most z-projections display the central hub, right-most projection shows the microtubule wall. Blue circles delineate one ring-pair. White arrows mark individual emanating spokes. White arrowheads mark fused spokes. White dashed line follows the tilt of the spokes. Black arrow indicates the final merged spoke (D1 density). Dark orange arrow marks the D2-rod. Scale bar, 50 nm.


( A ) Cryo - electron tomogram of P . tetraurelia centriole . Blue arrow denotes cartwheel , orange arrow denotes inner scaffold . dashed box delimits inset represented in ( D , E ) . Red lines arrows indicate position direction of cross - sections made in ( B ) . Scale bar , 100 nm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Cryo-electron tomogram of P. tetraurelia centriole. Blue arrow denotes cartwheel, orange arrow denotes inner scaffold. White dashed box delimits the inset represented in (D, E). Red lines and arrows indicate the position and the direction of the cross-sections made in (B). Scale bar, 100 nm.


( B ) Nine - fold symmetrizations of serial cross - sections taken along proximal to distal axis in P . tetraurelia . Each section is a z - projection of 20 . 7 nm . dashed circles delineate structures highlighted in C . Scale bar , 60 nm .

O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Nine-fold symmetrizations of serial cross-sections taken along the proximal to distal axis in P. tetraurelia. Each section is a z-projection of 20.7 nm. White dashed circles delineate the structures highlighted in C. Scale bar, 60 nm.


( C ) Zoomed of proximal centriole substructures from nine - fold symmetrizations of P . tetraurelia along proximal - distal axis . Each panel corresponds to above image from panel B . Purple arrow , pinhead ; green arrow , triplet base ; turquoise arrow , A - C linker ; orange arrow , inner scaffold .

(C) Zoomed images of proximal centriole substructures from nine-fold symmetrizations of P. tetraurelia along the proximal-distal axis. Each panel corresponds to the above image from panel B. Purple arrow, pinhead; light green arrow, triplet base; turquoise arrow, A-C linker; orange arrow, inner scaffold.


( D ) Side view showing transition from pinhead to inner scaffold in P . tetraurelia .

(D) Side view showing the transition from pinhead to inner scaffold in P. tetraurelia.


( G ) Cryo - electron tomogram of C . reinhardtii centriole . Blue arrow denotes cartwheel , orange arrow denotes inner scaffold . dashed box delimits inset represented in ( J , K ) . Red lines arrows indicate position direction of cross - sections made in ( H ) . Scale bar , 100 nm .

(G) Cryo-electron tomogram of C. reinhardtii centriole. Blue arrow denotes cartwheel, orange arrow denotes inner scaffold. White dashed box delimits the inset represented in (J, K). Red lines and arrows indicate the position and the direction of the cross-sections made in (H). Scale bar, 100 nm.


( H ) Nine - fold symmetrizations of serial cross - sections taken along proximal to distal axis in C . reinhardtii . Each section is a z - projection of 20 . 7 nm . dashed circles delineate structures highlighted in I . Scale bar , 60 nm .

(H) Nine-fold symmetrizations of serial cross-sections taken along the proximal to distal axis in C. reinhardtii. Each section is a z-projection of 20.7 nm. White dashed circles delineate the structures highlighted in I. Scale bar, 60 nm.


( I ) Zoomed of proximal centriole substructures from nine - fold symmetrizations of C . reinhardtii along proximal - distal axis . Each panel corresponds to above image from panel H . Purple arrow , pinhead ; green arrow , triplet base ; turquoise arrow , A - C linker ; orange arrow , inner scaffold .

(I) Zoomed images of proximal centriole substructures from nine-fold symmetrizations of C. reinhardtii along the proximal-distal axis. Each panel corresponds to the above image from panel H. Purple arrow, pinhead; light green arrow, triplet base; turquoise arrow, A-C linker; orange arrow, inner scaffold.


( J , K ) Side views showing transition from pinhead to inner scaffold in C . reinhardtii from two consecutive sections from a tomogram .

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

(J, K) Side views showing the transition from pinhead to inner scaffold in C. reinhardtii from two consecutive sections from a tomogram.


( M , N ) Positioning of different structures along proximal from representative P . tetraurelia ( M ) C . reinhardtii ( N ) centrioles . between ends of pinhead cartwheel regions is denoted by zone 1 ( for , see Figure EV4E ) . between end of pinhead region start of inner scaffold region is denoted by zone

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

(M, N) Positioning of the different structures along the proximal length from representative P. tetraurelia (M) and C. reinhardtii (N) centrioles. Distance between the ends of the pinhead and cartwheel regions is denoted by zone 1 (for quantification, see Figure EV4E). Distance between end of the pinhead region and start of the inner scaffold region is denoted by zone 2


( O ) Cartwheel A - C linker in C . reinhardtii ( n = 5 centrioles ) , P . tetraurelia ( n = 16 centrioles ) , N . gruberi ( n = 11 centrioles ) , H . sapiens ( n = 5 centrioles ) . Means standard deviations of mean are displayed above range . A - C linker , turquoise ; cartwheel , blue ; microtubule triplets , grey .

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

(O) Cartwheel and A-C linker length in C. reinhardtii (n= 5 centrioles), P. tetraurelia (n= 16 centrioles), N. gruberi (n= 11 centrioles), and H. sapiens (n= 5 centrioles). Means and standard deviations of the mean are displayed above the range. A-C linker, turquoise; cartwheel, blue; microtubule triplets, grey.


differentially expressed RBPs during embryoid ( EB ) differentiation , neural differentiation cardiac differentiation , which are classified as different clusters . right plots show dynamic expression pattern of each cluster , gray lines represent expression of genes in cluster , while red represents average expression level . number on graph indicates number of RBPs in cluster . expression value was firstly z - score scaled before plotting . EBD4 : embryoid at day 4 ; EBD8 : embryoid at day8 ; NSC : Neural ; NPC : Neural Progenitor ; MES : mesodermal ; CP : Cardiac Precursors ; CM : Cardiomyocytes .

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

The differentially expressed RBPs during embryoid body (EB) differentiation, neural differentiation and cardiac differentiation, which are classified as different clusters. The right line plots show the dynamic expression pattern of each cluster, gray lines represent the expression of all genes in individual cluster, while the red line represents the average expression level. The number on the graph indicates the number of RBPs in the cluster. The expression value was firstly z-score scaled before plotting. EBD4: embryoid body at day 4; EBD8: embryoid body at day8; NSC: Neural Stem Cells; NPC: Neural Progenitor Cell; MES: mesodermal cells; CP: Cardiac Precursors; CM: Cardiomyocytes.


showing expression pattern of 29 RBPs during EB differentiation .

Heatmap showing the expression pattern of the 29 RBPs during EB differentiation.


AP of 11 RBP - KO ES lines maintained under ES culture conditions . Scale bar , 200 μm .

AP staining of 11 RBP-KO ES cell lines maintained under ES cells culture conditions. Scale bar, 200 μm.


EB formation from WT RBP - KO ES lines . WT ES - derived EBs formed cystic structures by day 8 of differentiation ; most of RBP - KO ES - derived EBs were more compact . Scale bar , 600 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

EB formation from WT and RBP-KO ES cell lines. WT ES cell-derived EBs formed cystic structures by day 8 of differentiation; most of RBP-KO ES cell-derived EBs were more compact. Scale bar, 600 μm.


Time - course of Oct4 expression three lineage differentiation markers ( Hand1 , Brachyury FGF5 ) during EB formation . It was quantified by qPCR , relative expression of genes was calculated by double delta Ct ( ΔΔCt ) method , normalized to expression value at day 0 . relative expression data was represented as .

Time-course analysis of Oct4 expression and three lineage differentiation markers (Hand1, Brachyury and FGF5) during EB formation. It was quantified by qPCR, the relative expression of genes was calculated by the double delta Ct (ΔΔCt) method, normalized to the expression value at day 0. The relative expression data was represented as the heatmap.


AP of WT 11 RBP - KO ES lines after LIF withdrawal . Scale bar , 200μm .

AP staining of WT and 11 RBP-KO ES cell lines after LIF withdrawal. Scale bar, 200μm.


RBP ranking on ES abilities to self - renewal ( determined by colony formation ( ± LIF ) Oct4 expression ) exit - from - pluripotency ( determined by AP , EB formation marker genes expression ) in context of RBP KO . expression value of Oct4 , FGF5 , Hand1 Brachyury was represented as relative level of these genes compared to their expressions in WT ES . quantitation of other , such as colony formation , AP EB formation was manually defined .

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

RBP ranking based on ES cell abilities to self-renewal (determined by colony formation (±LIF) and Oct4 expression) and exit-from-pluripotency (determined by AP staining, EB formation and marker genes expression) in the context of RBP KO. The expression value of Oct4, FGF5, Hand1 and Brachyury was represented as the relative level of these genes compared to their expressions in WT ES cells. The quantitation of other phenotypes, such as colony formation, AP staining and EB formation was manually defined.


Immunofluorescence of Oct4 hnRNPLL in WT hnRNPLL - KO ES from two clones ( H8 G6 ) . hnRNPLL was not expressed in either of hnRNPLL - KO clones . DAPI stained nuclei . Scale bar , 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

Immunofluorescence staining of Oct4 and hnRNPLL in WT and hnRNPLL-KO ES cells from two clones (H8 and G6). hnRNPLL was not expressed in either of the hnRNPLL-KO clones. DAPI stained the nuclei. Scale bar, 100 μm.


EB formation from WT two hnRNPLL - KO ES clones . WT ES - derived EBs formed cystic structures by day 8 of differentiation ; While both hnRNPLL - KO ES clones - derived EBs were more compact . Scale bar , 200 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

EB formation from WT and the two hnRNPLL-KO ES cell clones. WT ES cell-derived EBs formed cystic structures by day 8 of differentiation; While both hnRNPLL-KO ES cell clones-derived EBs were more compact. Scale bar, 200 μm.


AP of WT both hnRNPLL - KO ES clones after LIF withdrawal . Scale bar , 200 μm .

AP staining of WT and both hnRNPLL-KO ES cell clones after LIF withdrawal. Scale bar, 200 μm.


Cumulative distribution showing relative expression of WT - differentially expressed genes in hnRNPLL - KO ES during EB differentiation . Red lines green lines represent up - down - regulated genes during EB differentiation of WT ES , while black stands for expressed genes in WT hnRNPLL - KO ES . Kolmogorov - Smirnov was used to determine significance . relative expression was log2 - transformed before plotting .

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

Cumulative distribution showing the relative expression of WT-differentially expressed genes in hnRNPLL-KO ES cells during EB differentiation. Red lines and green lines represent the up- and down-regulated genes during EB differentiation of WT ES cells, while the black line stands for all expressed genes in WT and hnRNPLL-KO ES cells. Kolmogorov-Smirnov test was used to determine the significance. The relative expression ratio was log2-transformed before plotting.


of key pluripotency differentiation associated gene expression during EB formation from WT hnRNPLL - KO clones ( H8 G6 ) .

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

Heatmap of key pluripotency and differentiation associated gene expression during EB formation from WT and hnRNPLL-KO clones (H8 and G6).


Hematoxylin eosin labeling Oct4 immunohistochemical of teratomas derived from WT hnRNPLL - KO ES ( left ) . Representative Oct4 - positive regions are shown . of Oct4 positive region ( % ) is plotted ( right ) . data represent means ± SD , ( n = 3 , from biological repeats ) . * P ＜ 0 . 05 ; * * * P ＜ 0 . 001 , T - was used to determine significance . Scale bar , 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 O O O O O O O O O O O O O O O O O O O O O O O O O O

Hematoxylin and eosin labeling and Oct4 immunohistochemical staining of teratomas derived from WT and hnRNPLL-KO ES cells (left). Representative Oct4-positive regions are shown. The percentage of Oct4 positive region (%) is plotted (right). The data represent the means ± SD, (n=3, from biological repeats). *P＜0.05; ***P＜0.001, T-test was used to determine the significance. Scale bar, 100 μm.


AP of WT hnRNPLL - overexpression ES lines maintained under ES culture conditions colony formation . Scale bar , 200 μm . data represent means ± SD ( n = 3 biological repeats ) .

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

AP staining of WT and hnRNPLL-overexpression ES cell lines maintained under ES cells culture conditions and colony formation assay. Scale bar, 200 μm. The data represent the means ± SD (n=3 biological repeats).


Relative expression of Oct4 Sox2 after hnRNPLL overexpression . data represent means ± SD ( n = 3 , from biological repeats ) . * * * P ＜ 0 . 001 . T - was used to determine significance .

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

Relative expression of Oct4 and Sox2 after hnRNPLL overexpression. The data represent the means ± SD (n=3, from biological repeats). ***P＜0.001. T-test was used to determine the significance.


Birth rate of different hnRNPLL genotypes , birth rate of hnRNPLL - / - mice was significantly lower than that of WT hnRNPLL + / + mice . data represent means ± SD . T - was used to determine significance .

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

Birth rate of the different hnRNPLL genotypes, the birth rate of hnRNPLL-/- mice was significantly lower than that of WT hnRNPLL+/+ mice. The data represent the means ± SD. T-test was used to determine the significance.


Representative of hnRNPLL + / + hnRNPLL - / - at 3 .

Representative images of hnRNPLL+/+ and hnRNPLL-/- littermates at 3 weeks.


growth curve of mice at different postnatal days . data represent means ± SD ( n = 5 - 10 ) . * * * P ＜ 0 . 001 . T - was used to determine significance .

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

Body weight growth curve of mice at different postnatal days. The data represent the means ± SD (n=5-10). ***P＜0.001. T-test was used to determine the significance.


Survival curve for hnRNPLL + / + , hnRNPLL + / - hnRNPLL - / - mice ( ten mice in hnRNPLL + / + / + / - group seven mice in hnRNPLL - / - group ) .

Survival curve analysis for hnRNPLL+/+, hnRNPLL+/-and hnRNPLL-/-mice (ten mice in hnRNPLL+/+/+/-group and seven mice in hnRNPLL-/-group).


Genotypes representative of hnRNPLL + / + retarded hnRNPLL - / - at E11 . 5 which generated from hnRNPLL + / - intercrosses . Scale bar , 1mm .

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

Genotypes and representative images of hnRNPLL+/+ embryo and retarded hnRNPLL-/- embryos at E11.5 which generated from hnRNPLL+/- intercrosses. Scale bar, 1mm.


Silver nitrate - stained NuPAGE gel of hnRNPLL interacting . Red star indicate hnRNPLL specific interacting compared to IgG .

Silver nitrate-stained NuPAGE gel of hnRNPLL interacting proteins. Red star indicate hnRNPLL specific interacting proteins compared to IgG.


Mass spectrum identification of hnRNPLL interactome in ES . splicing associated are well enriched by hnRNPLL IP . enrichment score was log2 - transformed before plotting .

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

Mass spectrum identification of the hnRNPLL interactome in ES cells. RNA splicing associated proteins are well enriched by hnRNPLL IP. The enrichment score was log2-transformed before plotting.


Western blot validation interactions between hnRNPLL indicated splicing associated .

Western blot validation the interactions between hnRNPLL and the indicated splicing associated proteins.


Cumulative distribution of PSI with exon skipping events between WT hnRNPLL - KO ES . Kolmogorov - Smirnov was used to determine significance .

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

Cumulative distribution of PSI with exon skipping events between WT and hnRNPLL-KO ES cells. Kolmogorov-Smirnov test was used to determine the significance.


Volcano plot of differential exon skipping events between WT hnRNPLL - KO ES . Indicated AS events were labeled in purple ( up - regulated ) green ( down - regulated ) with gene names .

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

Volcano plot of differential exon skipping events between WT and hnRNPLL-KO ES cells. Indicated AS events were labeled in purple (up-regulated) and green (down-regulated) with gene names.


Western blot of hnRNPLL during EB formation of WT ES at indicated days .

Western blot analysis of hnRNPLL protein during EB formation of WT ES cells at indicated days.


of 38 25 divergent exon skipping events in Figure 5I . * indicates different exon skipping events of same gene .

Heatmap of the 38 and 25 divergent exon skipping events in Figure 5I. * indicates the different exon skipping events of the same gene.


RT - PCR validation of partial divergent exon skipping events shown in Figure 5J . Red boxes represent ESC preferred exons , blue boxes indicate EB preferred exons .

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RT-PCR validation of partial divergent exon skipping events shown in Figure 5J. Red boxes represent ESC preferred exons, blue boxes indicate EB preferred exons.


RIP - PCR validation of transcripts that interact with hnRNPLL . left panel shows results of western blots verification of hnRNPLL Immunoprecipitation efficiency . data represent means ± SD ( n = 3 , from biological repeats ) . * * * P ＜ 0 . 001 . T - was used to determine significance .

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RIP-PCR validation of the transcripts that interact with hnRNPLL. The left panel shows the results of western blots verification of hnRNPLL Immunoprecipitation efficiency. The data represent the means ± SD (n=3, from biological repeats). ***P＜0.001. T-test was used to determine the significance.


Purification of hnRNPLL - detection of targets in by PCR . left panel shows position labeled as yellow square which was cut to extract hnRNPLL - . bands around 70KD indicate hnRNPLL , band around 50KD indicate heavy chain of antibodies used in Immunoprecipitation . right plot indicates enrichment of these targets in hnRNPLL - . data represent means ± SD ( n = 3 , from biological repeats ) . * P ＜ 0 . 05 ; * * * P ＜ 0 . 001 . T - was used to determine significance .

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Purification of hnRNPLL-RNA complex and detection of the RNA targets in the complex by PCR. The left panel shows the membrane position labeled as the yellow square which was cut to extract the hnRNPLL-RNA complex. The bands around 70KD indicate hnRNPLL proteins, the band around 50KD indicate the heavy chain of antibodies used in Immunoprecipitation. The right plot indicates the enrichment of these RNA targets in the hnRNPLL-RNA complex. The data represent the means ± SD (n=3, from biological repeats). *P＜0.05; ***P＜0.001. T-test was used to determine the significance.


Detection of Bptf Tbx3 exon skipping events during EB formation in WT hnRNPLL - KO ES ( left ) in hnRNPLL - overexpression ES under ES culture conditions ( right ) by RT - PCR . OE , hnRNPLL overexpression .

Detection of Bptf and Tbx3 exon skipping events during EB formation in WT and hnRNPLL-KO ES cells (left) and in hnRNPLL-overexpression ES cells under ES cells culture conditions (right) by RT-PCR.OE, hnRNPLL overexpression.


RT - PCR validation of Bptf exon24a Tbx3 exon2a deletion in WT ES .

RT-PCR validation of Bptf exon24a and Tbx3 exon2a deletion in WT ES cells.


AP of Bptf E24a KO or Tbx3 E2a KO ES maintained under normal ES culture conditions ( upper ) . Scale bar , 200 μm . Colony formation of Bptf E24a KO or Tbx3 E2a KO ES under ES culture condition ( lower left ) . data represent means ± SD ( n = 3 , from biological repeats ) . * * * P ＜ 0 . 001 . Colony diameter of Tbx3 E2a KO ES ( lower right ) . data represent means ± SD ( n ＞ 80 ) . * * * P ＜ 0 . 001 . T - was used to determine significance .

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AP staining of Bptf E24a KO or Tbx3 E2a KO ES cells maintained under normal ES cells culture conditions (upper). Scale bar, 200 μm. Colony formation assay of Bptf E24a KO or Tbx3 E2a KO ES cells under ES cells culture condition (lower left). The data represent the means ± SD (n=3, from biological repeats). ***P＜0.001. Colony diameter of Tbx3 E2a KO ES cells (lower right). The data represent the means ± SD (n＞80). ***P＜0.001. T-test was used to determine the significance.


Relative expression of Oct4 Rex1 , determined by qRT - PCR . data represent means ± SD ( n = 3 , from biological repeats ) . * * * P ＜ 0 . 001 . T - was used to determine significance .

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Relative expression of Oct4 and Rex1, determined by qRT-PCR. The data represent the means ± SD (n=3, from biological repeats). ***P＜0.001. T-test was used to determine the significance.


RT - PCR validation of Bptf Tbx3 alternative exon deletion , separately in hnRNPLL - KO H8 ( upper ) simultaneously in hnRNPLL - KO H8 ( lower ) .

RT-PCR validation of Bptf and Tbx3 alternative exon deletion, separately in hnRNPLL-KO H8 cells (upper) and simultaneously in hnRNPLL-KO H8 cells (lower).


AP of H8 - Bptf or H8 - Tbx3 exon - KO ES after LIF withdrawal . hnRNPLL - H8 , hnRNPLL - KO ES ; H8 - Bptf KO , alternative Bptf exon was deleted in hnRNPLL - KO H8 ; H8 - Tbx3 KO , alternative Tbx3 exon was deleted in hnRNPLL - KO H8 ; H8 - Bptf / Tbx3 KO , alternative Bptf or Tbx3 exons were simultaneously deleted in hnRNPLL - KO H8 . Scale bar , 200 μm .

AP staining of H8-Bptf or H8-Tbx3 exon-KO ES cells after LIF withdrawal. hnRNPLL-H8, hnRNPLL-KO ES cells; H8-Bptf KO, alternative Bptf exon was deleted in hnRNPLL-KO H8 cells; H8-Tbx3 KO, alternative Tbx3 exon was deleted in hnRNPLL-KO H8 cells; H8-Bptf/Tbx3 KO, alternative Bptf or Tbx3 exons were simultaneously deleted in hnRNPLL-KO H8 cells. Scale bar, 200 μm.


Colony formation of H8 - Bptf or H8 - Tbx3 exon - KO ES after LIF withdrawal . data represent means ± SD ( n = 3 , from biological repeats ) .

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Colony formation assay of H8-Bptf or H8-Tbx3 exon-KO ES cells after LIF withdrawal. The data represent the means± SD (n=3, from biological repeats).


Hematoxylin eosin labeling of teratomas derived from WT H8 - Bptf or H8 - Tbx3 exon - KO ES . blue triangle represent ectoderm , one represent mesoderm green one represent endoderm . Scale bar , 100 μm .

Hematoxylin and eosin labeling of teratomas derived from WT and H8-Bptf or H8-Tbx3 exon-KO ES cells. The blue triangle represent ectoderm, the dark one represent mesoderm and the green one represent endoderm. Scale bar, 100 μm.


Oct4 immunohistochemical of teratomas derived from WT H8 - Bptf or H8 - Tbx3 exon - KO ES . Representative Oct4 - positive regions are shown ( left ) . Scale bar , 100 µm . Oct4 - positive region ( % ) is shown ( right ) . data represent means ± SD ( n = 3 biological repeats ) . * * P ＜ 0 . 01 ; ns , not significant . T - was used to determine significance .

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Oct4 immunohistochemical staining of teratomas derived from WT and H8-Bptf or H8-Tbx3 exon-KO ES cells. Representative Oct4-positive regions are shown (left). Scale bar, 100 µm. The percentage Oct4-positive region (%) is shown (right). The data represent the means± SD (n=3 biological repeats). **P＜0.01; ns, not significant. T-test was used to determine the significance.


( A ) Overlay of 15N - 1H HSQC spectra of 15N - labeled Ca2 + / CaM by itself ( red peaks ) after addition of saturating , unlabeled PSD - 95 ( 1 - 21 ) ( blue peaks ) or saturating , unlabeled PSD - 95 _ pT19 ( green peaks ) . NMR experiments were recorded at 600 MHz 1H frequency .

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(A) Overlay of 15N-1H HSQC spectra of 15N-labeled Ca2+/CaM by itself (red peaks) and after addition of saturating, unlabeled PSD-95 (1-21) (blue peaks) or saturating, unlabeled PSD-95_pT19 (green peaks). All NMR experiments were recorded at 600 MHz 1H frequency.


( B ) Chemical shift perturbation of CaM amide resonances in absence vs . presence of PSD - 95 _ pT19 . Chemical shift difference ( CSD ) plotted on vertical axis was defined as CSD = { ( HNA - HNB ) 2 + ( 15NA - 15NB ) 2 } 1 / 2 , where " A " " B " designate free bound states of CaM . HN 15N represent amide 1H 15N chemical shifts , respectively . Perturbations greater than average were used as active ambiguous restraints in Haddock docking calculation ( see Methods ) .

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(B) Chemical shift perturbation of CaM amide resonances in the absence vs. presence of PSD-95_pT19. Chemical shift difference (CSD) plotted on the vertical axis was defined as CSD = {(HNA - HNB)2 + (15NA - 15NB)2}1/2 , where "A" and "B" designate free and bound states of CaM. HN and 15N represent amide 1H and 15N chemical shifts, respectively. Perturbations greater than the average were used as active ambiguous restraints in the Haddock docking calculation (see Methods).


( C ) Representative data from IPAP [ 15N - 1H ] - HSQC spectra of 15N - labeled Ca2 + / CaM bound to PSD - 95 _ pT19 in absence ( blue ) presence of 17 mg / ml filamentous bacteriophage Pf1 ( red ) . Residual dipolar couplings ( RDCs ) for backbone HN resonances are calculated ( in Hertz ) as difference in splitting for 15N - { 1H } doublet components ( red peaks ) relative to isotropic 1JNH coupling ( blue peaks ) . RDC values are calculated to be + 40 , + 30 , + 20 , + 20 , - 20 Hz for I100 , I27 , N137 , D64 , I130 , respectively .

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(C) Representative data from IPAP [15N-1H]-HSQC spectra of 15N-labeled Ca2+/CaM bound to PSD-95_pT19 in the absence (blue) and presence of 17 mg/ml filamentous bacteriophage Pf1 (red). Residual dipolar couplings (RDCs) for backbone HN resonances are calculated (in Hertz) as the difference in splitting for the 15N-{1H} doublet components (red peaks) relative to the isotropic 1JNH coupling (blue peaks). RDC values are calculated to be +40, +30, +20, +20, and -20 Hz for I100, I27, N137, D64, and I130, respectively.


( D ) Correlation between observed RDCs ( panel C ) versus RDCs calculated from final NMR derived structure of Ca2 + / CaM bound to PSD - 95 _ pT19 . correlation coefficient ( R ) equals 0 . 99 Q is 0 . 11 .

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(D) Correlation between observed RDCs (panel C) versus RDCs calculated from the final NMR derived structure of Ca2+/CaM bound to PSD-95_pT19. The correlation coefficient (R) equals 0.99 and Q is 0.11.


( A ) NMR structure of Ca2 + / CaM ( grey ) bound to unphosphorylated PSD - 95 . PSD - 95 residues D2 ( depicted as starting point ) to R13 are resolved form an - helix ( cyan ) ( Zhang et al . , 2014 ) . Residues 14 - 19 of unphosphorylated PSD - 95 are unstructured not shown . Ca2 + ions bound to CaM are depicted by orange spheres .

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(A) NMR structure of Ca2+/CaM (grey) bound to unphosphorylated PSD-95. PSD-95 residues D2 (depicted as starting point) to R13 are resolved and form an -helix (cyan) (Zhang et al., 2014). Residues 14-19 of unphosphorylated PSD-95 are unstructured and not shown. Ca2+ ions bound to CaM are depicted by orange spheres.


( B ) NMR structure of Ca2 + / CaM bound to PSD - 95 phosphorylated at T19 ( PDB accession no . 5J7J ) . Residues 14 - 19 of phosphorylated PSD - 95 adopt a hook - like structure ( highlighted in red ) not seen in unphosphorylated PSD - 95 . This hook - like structure is stabilized by PSD - 95 residues E17 pT19 forming salt bridges with R126 K115 , respectively . Side - chain atoms are depicted by a stick representation for PSD - 95 residues Q15 , E17 T19 ( red ) CaM residues K115 , T117 , R126 ( blue ) .

(B) NMR structure of Ca2+/CaM bound to PSD-95 phosphorylated at T19 (PDB accession no. 5J7J). Residues 14-19 of phosphorylated PSD-95 adopt a hook-like structure (highlighted in red) not seen in unphosphorylated PSD-95. This hook-like structure is stabilized by PSD-95 residues E17 and pT19 forming salt bridges with R126 and K115, respectively. Side-chain atoms are depicted by a stick representation for PSD-95 residues Q15, E17 and T19 (red) and CaM residues K115, T117, and R126 (blue).


( A ) FP titrations showing binding of wild - type ( WT ) CaM to PSD - 95 ( 1 - 21 ) peptides , either WT ( blue ) , Q15A ( salmon ) , E17R ( black ) , T19E ( green ) , or T19K ( yellow ) . solid lines represent optimal theoretical curves calculated using a one - site binding model described in Methods . calculated dissociation constants in each case are given in Table 1 .

(A) FP titrations showing the binding of wild-type (WT) CaM to PSD-95 (1-21) peptides, either WT (blue), Q15A (salmon), E17R (black), T19E (green), or T19K (yellow). The solid lines represent optimal theoretical curves calculated using a one-site binding model described in Methods. The calculated dissociation constants in each case are given in Table 1.


( B ) FP titrations showing binding of R126E CaM to PSD - 95 peptides , either WT ( blue ) or E17R ( black ) .

(B) FP titrations showing the binding of R126E CaM to PSD-95 peptides, either WT (blue) or E17R (black).


( C ) FP titrations showing binding of K115E CaM to PSD - 95 peptides T19K ( yellow ) or T19E ( green ) .

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(C) FP titrations showing the binding of K115E CaM to PSD-95 peptides T19K (yellow) or T19E (green).


Cultured cortical neurons were treated with BIC ( 50 μM ) at 15 DIV for 48h or at 17 DIV for 2 h or left untreated before harvesting , extraction , of palmitoylation by biotin switch method pull down with NeutrAvidin - agarose beads . ( A ) Representative immunoblots of pull down samples ( top two panels ) total lysate ( bottom two panels ) for PSD - 95 , as control , trimeric G subunit G - i3 . Omission of NH2OH before biotinylation resulted in no NeutrAvidin pull down as negative control for non - specific pull down .

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Cultured cortical neurons were treated with BIC (50 μM) at 15 DIV for 48h or at 17 DIV for 2 h or left untreated before harvesting, extraction, and analysis of palmitoylation by the biotin switch method and pull down with NeutrAvidin-agarose beads. (A) Representative immunoblots of pull down samples (top two panels) and total lysate (bottom two panels) for PSD-95 and, as control, the trimeric G protein subunit G-i3. Omission of NH2OH before biotinylation resulted in no NeutrAvidin pull down as negative control for non-specific pull down.


( B ) of PSD - 95 palmitoylation . Seven samples from three independent sets of cultures were analyzed per condition ( * * p < 0 . 01 ; t - ) .

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(B) Quantification of PSD-95 palmitoylation. Seven samples from three independent sets of cultures were analyzed per condition (**p<0.01; t-test).


Cultured hippocampal neurons were infected at 14 DIV with lentivirus for expression of both shRNA against PSD - 95 ( sh95 ) sh - resistant PSD - 95 - EGFP . Thus endogenous PSD - 95 was replaced with either wild - type ( WT ) , E17R , or T19K PSD - 95 . Cultures were treated with or without BIC ( 50 μM ) at 17 DIV for 24 - 48 h or left untreated before fixation for surface GluA1 . ( A ) Representative confocal microscopic of PSD - 95 - EGFP ( green ) surface GluA1 immunostaining ( red ) at low ( top panels ; scale bar : 20 µm ) high enlargement ( bottom panels ; scale bar : 5 µm ) .

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Cultured hippocampal neurons were infected at 14 DIV with lentivirus for expression of both shRNA against PSD-95 (sh95) and sh-resistant PSD-95-EGFP. Thus endogenous PSD-95 was replaced with either wild-type (WT), E17R, or T19K PSD-95. Cultures were treated with or without BIC (50 μM) at 17 DIV for 24-48 h or left untreated before fixation and staining for surface GluA1. (A) Representative confocal microscopic images of PSD-95-EGFP fluorescence (green) and surface GluA1 immunostaining (red) at low (top panels; scale bar: 20 µm) and high enlargement (bottom panels; scale bar: 5 µm).


( B ) of surface GluA1 from ( A ) showed reduction in AMPAR surface during scaling down in neurons expressing WT PSD - 95 but not in those expressing either E17R or T19K mutant . Three dendritic segments from each of 31 - 42 neurons from three independent sets of cultures were analyzed per condition ( * * p < 0 . 01 ; n . s . , not significant ; one - way ANOVA followed by Bonferroni ' s post hoc ) .

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(B) Quantification of surface GluA1 intensity from (A) showed reduction in AMPAR surface levels during scaling down in neurons expressing WT PSD-95 but not in those expressing either E17R or T19K mutant. Three dendritic segments from each of 31-42 neurons from three independent sets of cultures were analyzed per condition (**p<0.01; n.s., not significant; one-way ANOVA followed by Bonferroni's post hoc test).