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

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(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 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

O O O O O O O O O O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM O O O O B-ORGANISM I-ORGANISM I-ORGANISM O O O B-SUBCELLULAR O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O 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 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 .

O O O B-SUBCELLULAR O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O B-ORGANISM I-ORGANISM I-ORGANISM O O O O B-SUBCELLULAR O O B-ORGANISM I-ORGANISM I-ORGANISM O O O O B-SUBCELLULAR O O B-ORGANISM I-ORGANISM I-ORGANISM O O O O B-SUBCELLULAR O O O B-ORGANISM I-ORGANISM I-ORGANISM O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O O B-SUBCELLULAR O O O B-SUBCELLULAR 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.


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 .

O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O B-TISSUE O B-TISSUE I-TISSUE O O O O B-TISSUE O B-TISSUE I-TISSUE O O O B-CELL_TYPE O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O O O O O O O B-CELL_TYPE O B-CELL_TYPE I-CELL_TYPE O O O O O O B-CELL_TYPE O B-CELL_TYPE 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.


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

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


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

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


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 .

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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 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 .

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

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

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


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 .

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

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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 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 .

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

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


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 .

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


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

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


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 .

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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 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 ) .

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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 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 .

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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 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 .

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

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


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 .

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

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


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

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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 proteins . Red star indicate hnRNPLL specific interacting proteins compared to IgG .

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


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 .

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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 the interactions between hnRNPLL and the indicated splicing associated proteins .

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


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

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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 and hnRNPLL - KO ES cells . Indicated AS events were labeled in purple ( up - regulated ) and green ( down - regulated ) with gene names .

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

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


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

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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 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 .

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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 - 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 .

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

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 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 .

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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 and Tbx3 exon2a deletion in WT ES cells .

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


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 .

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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 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 .

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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 and Tbx3 alternative exon deletion , separately in hnRNPLL - KO H8 cells ( upper ) and simultaneously in hnRNPLL - KO H8 cells ( lower ) .

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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 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 .

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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 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 ) .

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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 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 .

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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 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 .

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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 ) 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 .

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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 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 ) .

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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 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 .

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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 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 .

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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 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 .

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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 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 ) .

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(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 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 .

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

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


( C ) FP titrations showing the 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 , 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 .

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

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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 ) 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 ) .

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

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 ) 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 ) .

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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).


Cultured hippocampal neurons were co - transfected with mCherry and EGFP - tagged PSD - 95 wild - type ( WT ) , E17R , or T19K at 10 - 11 DIV and treated with BIC ( 50 μM ) or left untreated at 17 DIV for 24 - 48 h before fixation . ( A ) Representative confocal microscopic images of PSD - 95 - EGFP ( green ) and mCherry ( red ) used as " fill " to visualize the dendrite with spine heads ( scale bar : 2 µm ) after control ( top ) and BIC treatment ( bottom ) . Individual channels are shown in grayscale .

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Cultured hippocampal neurons were co-transfected with mCherry and EGFP-tagged PSD-95 wild-type (WT), E17R, or T19K at 10-11 DIV and treated with BIC (50 μM) or left untreated at 17 DIV for 24-48 h before fixation. (A) Representative confocal microscopic images of PSD-95-EGFP (green) and mCherry (red) used as "fill" to visualize the dendrite with spine heads (scale bar: 2 µm) after control (top) and BIC treatment (bottom). Individual channels are shown in grayscale.


( B ) BIC treatment reduced spine enrichment of WT but not E17R or T19K PSD - 95 as quantified as the ratio of EGFP intensity in spine heads relative to that in the adjacent dendritic shaft . Values were normalized to control treatments , which were set to equal 100 % for each PSD - 95 construct .

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(B) BIC treatment reduced spine enrichment of WT but not E17R or T19K PSD-95 as quantified as the ratio of EGFP intensity in spine heads relative to that in the adjacent dendritic shaft. Values were normalized to control treatments, which were set to equal 100% for each PSD-95 construct.


( C ) mCherry showed no enrichment in spines and BIC had no effect on its localization .

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(C) mCherry showed no enrichment in spines and BIC had no effect on its localization.


( D - F ) Spine enrichment and puncta size and intensity of PSD - 95 were comparable under basal conditions for WT , E17R , and T19K PSD - 95 ( A . U . , arbitrary unit ) .

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(D-F) Spine enrichment and puncta size and intensity of PSD-95 were comparable under basal conditions for WT, E17R, and T19K PSD-95 (A.U., arbitrary unit).


( G - I ) Spine density , head diameter , and length were comparable under basal conditions for neurons expressing WT , E17R , and T19K PSD - 95 .

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(G-I) Spine density, head diameter, and length were comparable under basal conditions for neurons expressing WT, E17R, and T19K PSD-95.


Cultured hippocampal neurons were transfected at 10 - 11 DIV with the indicated CaM - IRES - ECFP plasmids and infected at 14 DIV with lentivirus expressing shRNA against PSD - 95 ( sh95 ) and sh - resistant wild - type ( WT ) or E17R PSD - 95 - EGFP . Cultures were treated with BIC ( 50 μM ) or left untreated at 17 DIV for 24 - 48 h before fixation . ( A ) Representative confocal microscopic images of intrinsic ECFP and EGFP fluorescence , and surface GluA1 immunostaining at low ( top panels ; scale bar : 20 µm ) and high enlargement ( bottom panels ; scale bar : 5 µm ) .

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

Cultured hippocampal neurons were transfected at 10-11 DIV with the indicated CaM-IRES-ECFP plasmids and infected at 14 DIV with lentivirus expressing shRNA against PSD-95 (sh95) and sh-resistant wild-type (WT) or E17R PSD-95-EGFP. Cultures were treated with BIC (50 μM) or left untreated at 17 DIV for 24-48 h before fixation. (A) Representative confocal microscopic images of intrinsic ECFP and EGFP fluorescence, and surface GluA1 immunostaining at low (top panels; scale bar: 20 µm) and high enlargement (bottom panels; scale bar: 5 µm).


( B ) Quantification of surface GluA1 intensity from ( A ) showed impaired AMPAR downscaling in neurons expressing PSD - 95 - E17R in presence of wild - type CaM ( CaM - WT ) that was rescued by coexpression of CaM - R126E . Three dendritic segments from each of 18 - 26 neurons from three independent sets of cultures were analyzed per condition ( * * p < 0 . 01 , * p < 0 . 05 vs . untreated basal levels ; one - way ANOVA followed by Bonferroni ' s post hoc test ) .

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(B) Quantification of surface GluA1 intensity from (A) showed impaired AMPAR downscaling in neurons expressing PSD-95-E17R in presence of wild-type CaM (CaM-WT) that was rescued by coexpression of CaM-R126E. Three dendritic segments from each of 18-26 neurons from three independent sets of cultures were analyzed per condition (**p<0.01,*p<0.05 vs. untreated basal levels; one-way ANOVA followed by Bonferroni's post hoc test).


Cultured hippocampal neurons were co - transfected at 10 - 11 DIV with RFP and the indicated CaM - IRES - ECFP plasmids and infected at 12 - 13 DIV with lentivirus expressing shRNA against PSD - 95 ( sh95 ) and sh - resistant wild - type ( WT ) or E17R PSD - 95 - EGFP . mEPSC recordings were performed at DIV 17 - 19 , following 48 h of control or BIC ( 50 μM ) treatment . ( A ) Representative traces of AMPAR mEPSC recordings ( left ) and averages of all mEPSC events from single representative neurons ( right ) drawn to scale ( top ) and normalized to peak ( bottom ) .

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Cultured hippocampal neurons were co-transfected at 10-11 DIV with RFP and the indicated CaM-IRES-ECFP plasmids and infected at 12-13 DIV with lentivirus expressing shRNA against PSD-95 (sh95) and sh-resistant wild-type (WT) or E17R PSD-95-EGFP. mEPSC recordings were performed at DIV 17-19, following 48 h of control or BIC (50 μM) treatment. (A) Representative traces of AMPAR mEPSC recordings (left) and averages of all mEPSC events from single representative neurons (right) drawn to scale (top) and normalized to peak (bottom).


( B ) Quantification of mEPSC amplitude revealed downscaling by BIC in neurons expressing PSD - 95WT / CaMWT or PSD - 95E17R / CaMR126E but not PSD - 95WT / CaMR126E ( left ) over the full range of amplitude distributions ( right ) .

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

(B) Quantification of mEPSC amplitude revealed downscaling by BIC in neurons expressing PSD-95WT / CaMWT or PSD-95E17R / CaMR126E but not PSD-95WT / CaMR126E (left) over the full range of amplitude distributions (right).


( C , D ) Mini - EPSC frequency ( C ) and decay time ( D ) were comparable among all conditions ( n = 11 - 25 neurons per condition from 8 - 14 independent sets of cultures prepared from the embryos of 8 - 14 dams ; * p < 0 . 05 ; two - way ANOVA followed by Bonferroni ' s test ) .

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(C,D) Mini-EPSC frequency (C) and decay time (D) were comparable among all conditions (n=11-25 neurons per condition from 8-14 independent sets of cultures prepared from the embryos of 8-14 dams;* p<0.05; two-way ANOVA followed by Bonferroni's test).


( B ) Confocal fluorescence microscopy of 293T cells transfected with 0 . 5 μg pQCXIP encoding IFITM3 variants containing N - terminal FLAG ( pQCXIP - FLAG - IFITM3 ) and immunostained with anti - FLAG M2 antibody . Scale bar , 5 μm .

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

(B) Confocal fluorescence microscopy of 293T cells transfected with 0.5 μg pQCXIP encoding IFITM3 variants containing N-terminal FLAG (pQCXIP-FLAG-IFITM3) and immunostained with anti-FLAG M2 antibody. Scale bar, 5 μm.


( C ) Flow cytometry analysis of transfected cells in ( B ) immunostained with anti - FLAG M2 antibody . Corresponding mean flourescence intensity values are shown .

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

(C) Flow cytometry analysis of transfected cells in (B) immunostained with anti-FLAG M2 antibody. Corresponding mean flourescence intensity values are shown.


( D ) SDS - PAGE of cell lysates derived from 293T cells transfected in ( B ) followed by immunoblotting with anti - FLAG M2 and anti - actin antibodies .

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(D) SDS-PAGE of cell lysates derived from 293T cells transfected in (B) followed by immunoblotting with anti-FLAG M2 and anti-actin antibodies.


( E ) SDS - PAGE of Opti - prep purified supernatants ( 25 ng p24 equivalent ) derived from 293T cells transfected with pQCXIP - FLAG - IFITM3 variants , HIV - 1 pNL4 - 3 , and Vpr - Blam plasmid followed by immunoblotting with anti - FLAG M2 and anti - p24 Gag ( 183 - H12 - 5C ) .

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(E) SDS-PAGE of Opti-prep purified supernatants (25 ng p24 equivalent) derived from 293T cells transfected with pQCXIP-FLAG-IFITM3 variants, HIV-1 pNL4-3, and Vpr-Blam plasmid followed by immunoblotting with anti-FLAG M2 and anti-p24 Gag (183-H12-5C).


( F ) Opti - prep purified supernatants from ( E ) were incubated with SupT1 target cells for 4 hours and virus - cell fusion was scored by flow cytometry . Mean + SD of 5 - 10 experiments ( each performed with virus produced from an independent transfection ) is shown . ( G ) Virus used in ( E ) were used to infect SupT1 cells and productive infection was scored at 48 - 60 hours by immunostaining with the KC57 antibody and flow cytometry . Expression of pCMV - MxA served as a negative control . The mean + SD of 5 - 10 experiments is shown .

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

(F) Opti-prep purified supernatants from (E) were incubated with SupT1 target cells for 4 hours and virus-cell fusion was scored by flow cytometry. Mean + SD of 5-10 experiments (each performed with virus produced from an independent transfection) is shown. (G) Virus used in (E) were used to infect SupT1 cells and productive infection was scored at 48-60 hours by immunostaining with the KC57 antibody and flow cytometry. Expression of pCMV-MxA served as a negative control. The mean + SD of 5-10 experiments is shown.


( H ) Confocal fluorescence microscopy of 293T cells transfected with 0 . 5 μg pQCXIP - FLAG - IFITM3 variants and immunostained with anti - FLAG M2

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

(H) Confocal fluorescence microscopy of 293T cells transfected with 0.5 μg pQCXIP-FLAG-IFITM3 variants and immunostained with anti-FLAG M2 antibody


( I ) SDS - PAGE of cell lysates derived from 293T cells transfected in ( H ) followed by immunoblotting with anti - FLAG M2 and anti - actin antibodies .

(I) SDS-PAGE of cell lysates derived from 293T cells transfected in (H) followed by immunoblotting with anti-FLAG M2 and anti-actin antibodies.


( J ) SDS - PAGE of Opti - prep purified supernatants ( 25 ng p24 equivalent ) derived from 293T cells transfected with pQCXIP - FLAG - IFITM3 constructs , HIV - 1 pNL4 - 3 , and Vpr - Blam plasmid followed by immunoblotting with anti - FLAG M2 and anti - p24 Gag . Scale bar , 5 μm .

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

(J) SDS-PAGE of Opti-prep purified supernatants (25 ng p24 equivalent) derived from 293T cells transfected with pQCXIP-FLAG-IFITM3 constructs, HIV-1 pNL4-3, and Vpr-Blam plasmid followed by immunoblotting with anti-FLAG M2 and anti-p24 Gag. Scale bar, 5 μm.


( K ) Opti - prep purified supernatants from ( J ) were incubated with SupT1 target cells for 4 hours and virus - cell fusion was scored by flow cytometry . The mean + SD of 3 experiments is shown .

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(K) Opti-prep purified supernatants from (J) were incubated with SupT1 target cells for 4 hours and virus-cell fusion was scored by flow cytometry. The mean + SD of 3 experiments is shown.


( A ) Confocal fluorescent microscopy of SupT1 T cells transduced with Tet - inducible pQCXIP - FLAG - IFITM3 or pQCXIP - FLAG - IFITM3 Δ1 - 21 ( Tet - ON ) immunostained with anti - FLAG M2 antibody following overnight treatment with 500 ng / mL doxycycline . Scale bar , 10 μm .

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

(A) Confocal fluorescent microscopy of SupT1 T cells transduced with Tet-inducible pQCXIP-FLAG-IFITM3 or pQCXIP-FLAG-IFITM3 Δ1-21 (Tet-ON) immunostained with anti-FLAG M2 antibody following overnight treatment with 500 ng/mL doxycycline. Scale bar, 10 μm.


( B ) Tet - ON SupT1 cell lines were treated or not with 500 ng / mL doxycycline overnight and induction of IFITM3 protein was assessed anti - FLAG M2 immunostaining and flow cytometry .

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(B) Tet-ON SupT1 cell lines were treated or not with 500 ng/mL doxycycline overnight and induction of IFITM3 protein was assessed anti-FLAG M2 immunostaining and flow cytometry.


( C ) Tet - ON SupT1 cell lines were productively infected with NL4 - 3 VSV - G and then treated with 500 ng / mL doxycycline overnight to produce virus from IFITM3 - and IFITM3 + cells . 25 ng p24 equivalents of purified were used to infect fresh Tet - ON SupT1 cells cell targets , which were previously treated with 500 ng / mL doxycycline or not . Infection was scored at 72h post - infection by immunostaining with KC57 and flow cytometry . Mean + SD of 3 experiments are shown .

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

(C) Tet-ON SupT1 cell lines were productively infected with NL4-3 VSV-G and then treated with 500 ng/mL doxycycline overnight to produce virus from IFITM3- and IFITM3+ cells. 25 ng p24 equivalents of purified were used to infect fresh Tet-ON SupT1 cells cell targets, which were previously treated with 500 ng/mL doxycycline or not. Infection was scored at 72h post-infection by immunostaining with KC57 and flow cytometry. Mean + SD of 3 experiments are shown.


( D ) 25 ng p24 equivalents of virus used in ( C ) was subjected to SDS - PAGE . Immunoblotting was performed using the anti - FLAG M2 , anti - p24 Gag , and anti - gp120 Env ( NIH # 288 ) antibodies .

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

(D) 25 ng p24 equivalents of virus used in (C) was subjected to SDS-PAGE. Immunoblotting was performed using the anti-FLAG M2, anti-p24 Gag, and anti-gp120 Env (NIH #288) antibodies.


( E ) 293T cells were transfected with the indicated amount of pQCXIP - FLAG - IFITM3 or pQCXIP - FLAG - Δ1 - 21 or pCMV - MxA and 1 . 0 μg of HIV - 1 pNL4 - 3 . SDS - PAGE was performed on whole cell lysates followed by immunoblotting with anti - FLAG M2 , anti - p24 Gag , anti - Env gp120 ( NIH # 288 ) , and anti - tubulin antibodies . Western blot images are representative of 3 independent experiments . Unpaired t - test , * p < 0 . 05 , ♯ p = 0 . 12 , non statistically significant . Comparisons were made between the condition indicated and the correponding condition in Tet - ON IFITM3 cells .

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

(E) 293T cells were transfected with the indicated amount of pQCXIP-FLAG-IFITM3 or pQCXIP-FLAG-Δ1-21 or pCMV-MxA and 1.0 μg of HIV-1 pNL4-3. SDS-PAGE was performed on whole cell lysates followed by immunoblotting with anti-FLAG M2, anti-p24 Gag, anti-Env gp120 (NIH #288), and anti-tubulin antibodies. Western blot images are representative of 3 independent experiments. Unpaired t-test, *p<0.05, ♯p=0.12, non statistically significant. Comparisons were made between the condition indicated and the correponding condition in Tet-ON IFITM3 cells.


( A ) Confocal fluorescence microscopy of 293T cells transfected with 0 . 5 μg of pQCXIP - FLAG - Human IFITM3 mutated at indicated residues and immunostained with anti - FLAG M2 antibody . Scale bars , 5 μm . The P17L , P18H , Δ22 - 24 , P18L / L23P and P18H / Δ19 - 23 mutations were introduced into the human IFITM3 background .

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

(A) Confocal fluorescence microscopy of 293T cells transfected with 0.5 μg of pQCXIP-FLAG-Human IFITM3 mutated at indicated residues and immunostained with anti-FLAG M2 antibody. Scale bars, 5 μm. The P17L, P18H, Δ22-24, P18L/L23P and P18H/Δ19-23 mutations were introduced into the human IFITM3 background.


( B ) SDS - PAGE of FLAG - immunoprecipitated fractions from 293T cells following transfection of 0 . 5 μg of pQCXIP - FLAG - IFITM3 variants and 0 . 5 μg of pCI - HA - NEDD4 or pCI - HA - NEDD4 C867A . Immunoblotting was performed with anti - FLAG M2 , anti - IFITM3 ( Abcam , EPR5242 ) , and an anti - ubiquitin antibody ( Enzo Life Sciences , recognizing K29 - , K48 - , and K63 - linked mono - and polyubiquitinylated proteins ) . ' L ' indicates the presence of light chain antibody derived from the FLAG antibody - coupled beads used for immunoprecipitation . * , * * , and * * * indicate mono - , di - , and tri - ubiquitinated forms of IFITM3 .

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

(B) SDS-PAGE of FLAG-immunoprecipitated fractions from 293T cells following transfection of 0.5 μg of pQCXIP-FLAG-IFITM3 variants and 0.5 μg of pCI-HA-NEDD4 or pCI-HA-NEDD4 C867A. Immunoblotting was performed with anti-FLAG M2, anti-IFITM3 (Abcam, EPR5242), and an anti-ubiquitin antibody (Enzo Life Sciences, recognizing K29-, K48-, and K63-linked mono- and polyubiquitinylated proteins). 'L' indicates the presence of light chain antibody derived from the FLAG antibody-coupled beads used for immunoprecipitation. *, **, and *** indicate mono-, di-, and tri-ubiquitinated forms of IFITM3.


( C ) SDS - PAGE of whole cell lysate input fractions from 293T cells used in ( B ) . Immunoblotting was performed with anti - FLAG M2 , anti - NEDD4 , and anti - actin .

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

(C) SDS-PAGE of whole cell lysate input fractions from 293T cells used in (B). Immunoblotting was performed with anti-FLAG M2, anti-NEDD4, and anti-actin.


( D ) 293T cells were transfected with 1 . 0 μg of pNL4 - 3 and 0 . 5 μg of pQCXIP - FLAG - IFITM3 variants . Virus - containing supernatants were harvested and 10 ng p24 equivalents were used to reinfect fresh SupT1 T cells . Productive infection was scored by immunostaining with the KC57 antibody and flow cytometry at 48 hours post - infection . The means + SD of 3 experiments are shown .

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(D) 293T cells were transfected with 1.0 μg of pNL4-3 and 0.5 μg of pQCXIP-FLAG-IFITM3 variants. Virus-containing supernatants were harvested and 10 ng p24 equivalents were used to reinfect fresh SupT1 T cells. Productive infection was scored by immunostaining with the KC57 antibody and flow cytometry at 48 hours post-infection. The means + SD of 3 experiments are shown.


( E ) 2 . 0 x 10ˆ5 cells were seeded and challenged with 10 ng of HIV - 1NL4 - 3 in the presence of 2 μg / mL DEAE - Dextran . Productive infection was scored by immunostaining with KC57 and flow cytometry at 48 hours post - infection . The ' Colobus native ' sequence represents the full IFITM3 sequence identified in the colobus monkey . ( F ) As in ( E ) , except cells were challenged with Influenza A virus H1N1 PR / 8 / 34 , ( Charles River Laboratories ) , at a dose that resulted in ˜ 50 % infection of control ( Empty ) cells ( equivalent to 103 TCID50 / 0 . 2 mL ) . Infection was scored by immunostaining with an anti - IAV NP antibody and flow cytometry at 18 hours post - infection . Results are presented in a logarithmic scale . Means + SD of 3 - 5 experiments are shown . Unpaired t - test , * p < 0 . 05 , * * p < 0 . 005 .

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

(E) 2.0 x 10ˆ5 cells were seeded and challenged with 10 ng of HIV-1NL4-3 in the presence of 2 μg/mL DEAE-Dextran. Productive infection was scored by immunostaining with KC57 and flow cytometry at 48 hours post-infection. The 'Colobus native' sequence represents the full IFITM3 sequence identified in the colobus monkey. (F) As in (E), except cells were challenged with Influenza A virus H1N1 PR/8/34, (Charles River Laboratories), at a dose that resulted in ˜50% infection of control (Empty) cells (equivalent to 103 TCID50 / 0.2 mL). Infection was scored by immunostaining with an anti-IAV NP antibody and flow cytometry at 18 hours post-infection. Results are presented in a logarithmic scale. Means + SD of 3-5 experiments are shown. Unpaired t-test, *p<0.05, **p<0.005.


( E ) Quantitative RT - PCR ( qRT - PCR ) of μGLT , αGLT , and AID from siControl - and siPhf5a - treated samples . The values are presented as mean ± sd ( n = 3 ) .

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(E) Quantitative RT-PCR (qRT-PCR) of μGLT, αGLT, and AID from siControl- and siPhf5a-treated samples. The values are presented as mean ± sd (n=3).


( B ) Southern blots of the IgH / c - Myc translocation assay using genomic DNA extracted from CIT - stimulated CH12F3 - 2A cells treated with the siRNAs shown at the bottom of each panel . Numbers on the top indicate gel lanes . ( C ) Frequency of IgH / c - Myc chromosomal translocations derived from the number of translocations detected in the total number of PCR reactions per sample ( Boboila et al . , 2012b ) . The values are presented as mean ± sd ( n = 3 ) . Statistical significance was assessed by two tailed unpaired Student ' s t - test ( * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 ) .

O O O B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY O O O O O O B-SMALL_MOLECULE O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD B-SUBCELLULAR B-EXP_ASSAY O O O O O B-EXP_ASSAY O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(B) Southern blots of the IgH/c-Myc translocation assay using genomic DNA extracted from CIT-stimulated CH12F3-2A cells treated with the siRNAs shown at the bottom of each panel. Numbers on the top indicate gel lanes. (C) Frequency of IgH/c-Myc chromosomal translocations derived from the number of translocations detected in the total number of PCR reactions per sample (Boboila et al., 2012b). The values are presented as mean ± sd (n=3). Statistical significance was assessed by two tailed unpaired Student's t-test (** p ≤ 0.01, *** p ≤ 0.001).


( D - E ) Detection of Sμ DNA double - strand breaks by LM - PCR in CH12F3 - 2A cells transfected with the indicated siRNA combinations . The DNA samples were either left untreated ( none ) or treated with indicated end processing enzymes prior to linker ligation .

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(D-E) Detection of Sμ DNA double-strand breaks by LM-PCR in CH12F3-2A cells transfected with the indicated siRNA combinations. The DNA samples were either left untreated (none) or treated with indicated end processing enzymes prior to linker ligation.


( B ) Effect of Phf5a knockdown on I - SceI - induced NHEJ . Plot represents the GFP - positive cells obtained by FACS analysis after co - transfection of the I - SceI expression plasmid and the indicated siRNAs into a cell line harboring the NHEJ reporter construct . The values are presented as mean ± sd ( n = 3 ) . Statistical significance was evaluated against corresponding siControl by two - tailed unpaired Student ' s t - test ( * * * p ≤ 0 . 001 ) .

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

(B) Effect of Phf5a knockdown on I-SceI-induced NHEJ. Plot represents the GFP-positive cells obtained by FACS analysis after co-transfection of the I-SceI expression plasmid and the indicated siRNAs into a cell line harboring the NHEJ reporter construct. The values are presented as mean ± sd (n=3). Statistical significance was evaluated against corresponding siControl by two-tailed unpaired Student's t-test (*** p ≤0.001).


( C ) Top : schematic diagram of the position of the ChIP assay PCR products specific to the S region . Bottom : ChIP assay of the indicated DNA repair proteins using CIT - stimulated CH12F3 - 2A cells transfected with siControl or siPhf5a . The values are presented as mean ± sd ( n = 3 ) . Statistical significance was evaluated in reference to siControl by two - tailed unpaired Student ' s t - test ( * p ≤ 0 . 05 , * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 ) .

O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-SMALL_MOLECULE O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Top: schematic diagram of the position of the ChIP assay PCR products specific to the S region. Bottom: ChIP assay of the indicated DNA repair proteins using CIT-stimulated CH12F3-2A cells transfected with siControl or siPhf5a. The values are presented as mean ± sd (n=3). Statistical significance was evaluated in reference to siControl by two-tailed unpaired Student's t-test (*p ≤ 0.05, ** p ≤0.01, *** p ≤0.001).


( C ) Western blot analysis of the whole cell extract from CIT - stimulated CH12F3 - 2A cells transfected with the indicated siRNAs . Expression of various proteins including AID , variant H2As and Ku80 were not decresed by Phf5a KD . ( ns , non - specific )

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

(C) Western blot analysis of the whole cell extract from CIT-stimulated CH12F3-2A cells transfected with the indicated siRNAs. Expression of various proteins including AID, variant H2As and Ku80 were not decresed by Phf5a KD. (ns, non-specific)


( C ) CH12F3 - 2A cells were transfected with WT Phf5a or its mutants ( Phf5aR - MF ) along with siRNA against Phf5a . An anti - Flag antibody was used to immunoprecipitate and immunodetect Phf5aR - MF . An anti - Phf5a immunoblot confirmed the knockdown efficiency of the endogenous Phf5a . The antibodies used to detect the co - associated proteins are indicated next to the respective blot .

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

(C) CH12F3-2A cells were transfected with WT Phf5a or its mutants (Phf5aR-MF) along with siRNA against Phf5a. An anti-Flag antibody was used to immunoprecipitate and immunodetect Phf5aR-MF. An anti-Phf5a immunoblot confirmed the knockdown efficiency of the endogenous Phf5a. The antibodies used to detect the co-associated proteins are indicated next to the respective blot.


( B ) Western blot showing expression of endogenous and exogenously introduced Phf5a .

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

(B) Western blot showing expression of endogenous and exogenously introduced Phf5a.


( C ) H2A . Z ChIP assay was performed using CH12F3 - 2A cells transfected with indicated siRNA and the Phf5a expression constructs . The data are compiled from 2 experiments and presented relative to siControl treated sample , which was set as 100 . The error bars represent the mean ± sd .

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

(C) H2A.Z ChIP assay was performed using CH12F3-2A cells transfected with indicated siRNA and the Phf5a expression constructs. The data are compiled from 2 experiments and presented relative to siControl treated sample, which was set as 100. The error bars represent the mean ± sd.


Mutations in epg - 7 cause defective degradation of SQST - 1 aggregates . ( A and B ) In wild - type embryos , SQST - 1 : : GFP is weakly expressed and diffusely localized in the cytoplasm . ( A ) DIC image of the embryo shown in B . ( C ) Elevated expression level and accumulation of many SQST - 1 : : GFP aggregates in epg - 7 mutant embryos .

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

Mutations in epg-7 cause defective degradation of SQST-1 aggregates. (A and B) In wild-type embryos, SQST-1::GFP is weakly expressed and diffusely localized in the cytoplasm. (A) DIC image of the embryo shown in B. (C) Elevated expression level and accumulation of many SQST-1::GFP aggregates in epg-7 mutant embryos.


( D ) Compared with wild type and epg - 2 mutants , levels of SQST - 1 are dramatically elevated in atg - 3 and epg - 7 mutant embryos in an immunoblotting assay .

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

(D) Compared with wild type and epg-2 mutants, levels of SQST-1 are dramatically elevated in atg-3 and epg-7 mutant embryos in an immunoblotting assay.


( E - G ) Compared with wild - type embryos ( E and F ) , endogenous SQST - 1 shows dramatically elevated levels and accumulates into a large number of aggregates in epg - 7 mutant embryos ( G ) . ∼ 200 - cell stage embryos are shown in E - G . ( E ) DAPI image of the embryo shown in F .

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

(E-G) Compared with wild-type embryos (E and F), endogenous SQST-1 shows dramatically elevated levels and accumulates into a large number of aggregates in epg-7 mutant embryos (G). ∼200-cell stage embryos are shown in E-G. (E) DAPI image of the embryo shown in F.


( H ) Number of SQST - 1 aggregates per focal plane in wild type , atg - 3 mutants , and epg - 7 mutants .

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

(H) Number of SQST-1 aggregates per focal plane in wild type, atg-3 mutants, and epg-7 mutants.


( I - L ) At the ∼ 100 - cell stage , far fewer SQST - 1 aggregates are formed in epg - 7 mutants than in atg - 3 mutants . ( I and K ) DAPI images of the embryos shown in J and L , respectively .

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

(I-L) At the ∼100-cell stage, far fewer SQST-1 aggregates are formed in epg-7 mutants than in atg-3 mutants. (I and K) DAPI images of the embryos shown in J and L, respectively.


( M ) SQST - 1 directly interacts with ZK1053 . 4 in a GST pull - down assay .

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

(M) SQST-1 directly interacts with ZK1053.4 in a GST pull-down assay.


( N ) Compared with wild - type animals , levels of ZK1053 . 4 : : GFP are dramatically elevated in atg - 3 and epg - 7 mutants .

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

(N) Compared with wild-type animals, levels of ZK1053.4::GFP are dramatically elevated in atg-3 and epg-7 mutants.


( O - R ) ZK1053 . 4 : : GFP is weakly expressed in wild - type embryos ( O - P ) and sqst - 1 mutants ( R ) , but forms a large number of aggregates in epg - 7 mutants ( Q ) . ( O ) DIC image of the embryo shown in P .

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

(O-R) ZK1053.4::GFP is weakly expressed in wild-type embryos (O-P) and sqst-1 mutants (R), but forms a large number of aggregates in epg-7 mutants (Q). (O) DIC image of the embryo shown in P.


( S ) Survival curve of L1 larvae under food depletion conditions in wild type , epg - 1 mutants , and epg - 7 mutants . This experiment was completed once ( wild type : n = 2 , 964 ; epg - 7 : n = 602 ; epg - 1 : n = 215 ) . Statistical analysis of L1 survival shows that epg - 7 mutants exhibit significant difference to wild type and also to epg - 1 mutants ( log - rank test , P = 0 . 000 ) .

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

(S) Survival curve of L1 larvae under food depletion conditions in wild type, epg-1 mutants, and epg-7 mutants. This experiment was completed once (wild type: n = 2,964; epg-7: n = 602; epg-1: n = 215). Statistical analysis of L1 survival shows that epg-7 mutants exhibit significant difference to wild type and also to epg-1 mutants (log-rank test, P = 0.000).


( T ) Loss of function of epg - 7 does not ameliorate the degeneration of mec - 4 : : gfp - labeled touch neurons caused by mec - 4 ( u231 ) .

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

(T) Loss of function of epg-7 does not ameliorate the degeneration of mec-4::gfp-labeled touch neurons caused by mec-4(u231).


( U ) Survival curves of wild type and various mutants . sqst - 1 ; epg - 7 mutants have a shortened mean and maximum life span . ( P = 0 . 000 for epg - 7 or wild type vs . sqst - 1 ; epg - 7 in all three repeats ; log - rank test ) .

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

(U) Survival curves of wild type and various mutants. sqst-1;epg-7 mutants have a shortened mean and maximum life span. (P = 0.000 for epg-7 or wild type vs. sqst-1;epg-7 in all three repeats; log-rank test).


( V ) Survival of hydrogen peroxide - treated animals . ( In all three repeats , P = 0 . 000 for epg - 7 vs . sqst - 1 ; epg - 7 ; P > 0 . 2 for wild type vs . sqst - 1 ; epg - 7 ; log - rank test ) .

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

(V) Survival of hydrogen peroxide-treated animals. (In all three repeats, P = 0.000 for epg-7 vs. sqst-1;epg-7; P > 0.2 for wild type vs. sqst-1;epg-7; log-rank test).


( W ) Survival of animals at the indicated time after heat shock treatment . ( P < 0 . 05 for wild type vs . epg - 7 or epg - 7 vs . sqst - 1 ; epg - 7 at 5 , 6 , and 7 h ) .

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

(W) Survival of animals at the indicated time after heat shock treatment. (P < 0.05 for wild type vs. epg-7 or epg-7 vs. sqst-1;epg-7 at 5, 6, and 7 h).


( X ) Dauer formation of daf - 2 ( e1370 ) mutants in different backgrounds .

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

(X) Dauer formation of daf-2(e1370) mutants in different backgrounds.


( D ) A large number of SQST - 1 : : GFP aggregates accumulate in epg - 7 mutant embryos carrying an epg - 7 ( del Atg11 ) transgene .

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

(D) A large number of SQST-1::GFP aggregates accumulate in epg-7 mutant embryos carrying an epg-7(del Atg11) transgene.


( E and F ) No SQST - 1 aggregates are detected in epg - 7 mutants carrying an epg - 7 : : gfp reporter . ( E ) DAPI image of the embryo shown in F .

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

(E and F) No SQST-1 aggregates are detected in epg-7 mutants carrying an epg-7::gfp reporter. (E) DAPI image of the embryo shown in F.


( G ) EPG - 7 : : GFP is weakly expressed and diffusely localized in the cytoplasm in wild - type embryos .

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

(G) EPG-7::GFP is weakly expressed and diffusely localized in the cytoplasm in wild-type embryos.


( H - I ) EPG - 7 : : GFP is expressed in pharyngeal muscles ( H , arrow ) and cells in the tail region ( I ) at larval stages .

O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD O O O B-TISSUE I-TISSUE O O O O O O O O O B-TISSUE O O O O O O O O

(H-I) EPG-7::GFP is expressed in pharyngeal muscles (H, arrow) and cells in the tail region (I) at larval stages.


( J ) Compared with wild - type animals , levels of EPG - 7 : : GFP are dramatically elevated in epg - 4 mutants , but remain unchanged in sqst - 1 mutants .

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

(J) Compared with wild-type animals, levels of EPG-7::GFP are dramatically elevated in epg-4 mutants, but remain unchanged in sqst-1 mutants.


( K ) The expression level of EPG - 7 : : GFP is dramatically elevated and many EPG - 7 : : GFP aggregates are formed in lgg - 1 mutant embryos .

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

(K) The expression level of EPG-7::GFP is dramatically elevated and many EPG-7::GFP aggregates are formed in lgg-1 mutant embryos.


( L and M ) No EPG - 7 aggregates , detected by anti - EPG - 7 antibody , are formed in wild - type embryos . ( L ) DAPI image of the embryo shown in M . ( N ) Levels of EPG - 7 are dramatically elevated and a large number of EPG - 7 aggregates are formed in atg - 3 mutants . ( O ) No EPG - 7 aggregates are formed in sqst - 1 mutant embryos .

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

(L and M) No EPG-7 aggregates, detected by anti-EPG-7 antibody, are formed in wild-type embryos. (L) DAPI image of the embryo shown in M. (N) Levels of EPG-7 are dramatically elevated and a large number of EPG-7 aggregates are formed in atg-3 mutants. (O) No EPG-7 aggregates are formed in sqst-1 mutant embryos.


( A ) EPG - 7 colocalizes with SQST - 1 aggregates in epg - 8 mutant embryos . Colocalization was defined as two aggregates showing > 70 % overlap in their fluorescence signals . Insets show a magnified view .

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

(A) EPG-7 colocalizes with SQST-1 aggregates in epg-8 mutant embryos. Colocalization was defined as two aggregates showing >70% overlap in their fluorescence signals. Insets show a magnified view.


( B ) Percentage of EPG - 7 aggregates colocalized with SQST - 1 in indicated autophagy mutants .

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

(B) Percentage of EPG-7 aggregates colocalized with SQST-1 in indicated autophagy mutants.


( C ) A coimmunoprecipitation assay reveals that EPG - 7 associates with SQST - 1 .

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

(C) A coimmunoprecipitation assay reveals that EPG-7 associates with SQST-1.


( D ) EPG - 7 interacts with SQST - 1 in a pull - down assay .

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

(D) EPG-7 interacts with SQST-1 in a pull-down assay.


( E ) EPG - 7 self - associates in a pull - down assay .

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

(E) EPG-7 self-associates in a pull-down assay.


( F ) SDS - PAGE and blue - native PAGE of purified EPG - 7 ( 520 - 714 ) proteins . Lane M shows molecular weight marker . Lanes 1 , 2 , 3 , and 4 contain 10 , 5 , 2 . 5 , and 1 . 25 µg of proteins , respectively .

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

(F) SDS-PAGE and blue-native PAGE of purified EPG-7(520-714) proteins. Lane M shows molecular weight marker. Lanes 1, 2, 3, and 4 contain 10, 5, 2.5, and 1.25 µg of proteins, respectively.


( G ) An EPG - 7 fragment containing amino acids 531 - 631 self - interacts .

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

(G) An EPG-7 fragment containing amino acids 531-631 self-interacts.


( H and I ) SQST - 1 aggregates accumulate in epg - 7 mutant embryos carrying an epg - 7 ( del 531 - 631 ) : : gfp transgene . ( H ) DAPI image of the embryo shown in I .

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

(H and I) SQST-1 aggregates accumulate in epg-7 mutant embryos carrying an epg-7(del 531-631)::gfp transgene. (H) DAPI image of the embryo shown in I.


( J and K ) EPG - 7 ( del 531 - 631 ) : : GFP is weakly expressed and diffusely localized in epg - 7 ( tm2508 ) embryos . Its expression is not elevated in lgg - 1 ( RNAi ) animals . H - K show embryos carrying the same transgene . Images in J and K were taken using the same exposure time .

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

(J and K) EPG-7(del 531-631)::GFP is weakly expressed and diffusely localized in epg-7(tm2508) embryos. Its expression is not elevated in lgg-1(RNAi) animals. H-K show embryos carrying the same transgene. Images in J and K were taken using the same exposure time.


( L and M ) SQST - 1 self - interacts ( L ) and the self - interaction is mediated by a fragment containing amino acids 26 - 181 ( M ) .

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

(L and M) SQST-1 self-interacts (L) and the self-interaction is mediated by a fragment containing amino acids 26-181 (M).


( N ) Levels of SQST - 1 ( del 26 - 181 ) : : GFP are dramatically elevated in epg - 7 and atg - 3 mutants .

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

(N) Levels of SQST-1(del 26-181)::GFP are dramatically elevated in epg-7 and atg-3 mutants.


( O and P ) SQST - 1 ( del 26 - 181 ) : : GFP is weakly expressed and diffusely localized in sqst - 1 ( ok2892 ) mutant embryos . ( O ) DIC image of the embryo shown in P .

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

(O and P) SQST-1(del 26-181)::GFP is weakly expressed and diffusely localized in sqst-1(ok2892) mutant embryos. (O) DIC image of the embryo shown in P.


( Q and R ) SQST - 1 ( del 26 - 181 ) : : GFP is expressed at dramatically elevated levels and accumulates into a large number of aggregates in lgg - 1 ( Q ) and epg - 7 ( R ) mutants .

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

(Q and R) SQST-1(del 26-181)::GFP is expressed at dramatically elevated levels and accumulates into a large number of aggregates in lgg-1 (Q) and epg-7 (R) mutants.


( A - D ) Expression of LGG - 1 in wild - type embryos at the ∼ 100 - cell ( A and B ) , the ∼ 200 - cell ( C ) , and the fourfold ( D ) stages . ( A ) DAPI image of the embryo shown in B .

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(A-D) Expression of LGG-1 in wild-type embryos at the ∼100-cell (A and B), the ∼200-cell (C), and the fourfold (D) stages. (A) DAPI image of the embryo shown in B.


( E ) Loss of function of epg - 7 does not increase levels of LGG - 1 and also does not suppress accumulation of LGG - 1 in epg - 3 mutant embryos .

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(E) Loss of function of epg-7 does not increase levels of LGG-1 and also does not suppress accumulation of LGG-1 in epg-3 mutant embryos.


( F - H ) Expression of LGG - 1 in epg - 7 mutant embryos before the ∼ 100 - cell stage ( F ) , at the ∼ 200 - cell stage ( G ) , and at the fourfold stage ( H ) . Images in B - D and F - H were taken using the same exposure time .

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(F-H) Expression of LGG-1 in epg-7 mutant embryos before the ∼100-cell stage (F), at the ∼200-cell stage (G), and at the fourfold stage (H). Images in B-D and F-H were taken using the same exposure time.


( I ) SQST - 1 aggregates are separable from LGG - 1 puncta in epg - 7 mutant embryos . Insets show a magnified view . ( J ) In epg - 6 mutant embryos , LGG - 1 puncta and SQST - 1 aggregates are enlarged and colocalized . ( K ) LGG - 1 and SQST - 1 aggregates are smaller in size and largely separable in epg - 6 ; epg - 7 double mutants . ( I - K ) DAPI images of the embryos shown in the same row .

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(I) SQST-1 aggregates are separable from LGG-1 puncta in epg-7 mutant embryos. Insets show a magnified view. (J) In epg-6 mutant embryos, LGG-1 puncta and SQST-1 aggregates are enlarged and colocalized. (K) LGG-1 and SQST-1 aggregates are smaller in size and largely separable in epg-6;epg-7 double mutants. (I-K) DAPI images of the embryos shown in the same row.


( L - O ) Enlarged LGG - 1 and SQST - 1 aggregates are colocalized in epg - 4 mutants ( L and M ) . Simultaneously depleting epg - 7 reduces the size of LGG - 1 puncta and SQST - 1 aggregates , which become largely separable ( N and O ) . ( L and N ) DAPI images of the embryos shown in M and O , respectively .

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(L-O) Enlarged LGG-1 and SQST-1 aggregates are colocalized in epg-4 mutants (L and M). Simultaneously depleting epg-7 reduces the size of LGG-1 puncta and SQST-1 aggregates, which become largely separable (N and O). (L and N) DAPI images of the embryos shown in M and O, respectively.


( P ) In atg - 9 mutant embryos , LGG - 1 puncta largely colocalize with SQST - 1 aggregates at the comma stage . Notably , there are more SQST - 1 aggregates than LGG - 1 puncta . ( Q ) SQST - 1 aggregates are separable from LGG - 1 puncta in atg - 9 ; epg - 7 mutants at the comma stage .

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(P) In atg-9 mutant embryos, LGG-1 puncta largely colocalize with SQST-1 aggregates at the comma stage. Notably, there are more SQST-1 aggregates than LGG-1 puncta. (Q) SQST-1 aggregates are separable from LGG-1 puncta in atg-9;epg-7 mutants at the comma stage.


( R ) Percentage of LGG - 1 puncta colocalized with SQST - 1 aggregates in indicated autophagy mutants . Comma - stage embryos were examined for atg - 9 and atg - 9 ; epg - 7 mutants . ∼ 200 - cell stage embryos were examined for other autophagy mutants .

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(R) Percentage of LGG-1 puncta colocalized with SQST-1 aggregates in indicated autophagy mutants. Comma-stage embryos were examined for atg-9 and atg-9;epg-7 mutants. ∼200-cell stage embryos were examined for other autophagy mutants.


( A ) EPG - 7 coimmunoprecipitates with ATG - 9 : : HA .

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(A) EPG-7 coimmunoprecipitates with ATG-9::HA.


( B ) EPG - 7 directly interacts with ATG - 9 in a GST pull - down assay .

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(B) EPG-7 directly interacts with ATG-9 in a GST pull-down assay.


( C and D ) Coimmunoprecipitation reveals that EPG - 7 associates with LGG - 3 ( C )

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(C and D) Coimmunoprecipitation reveals that EPG-7 associates with LGG-3 (C)


( C and D ) Coimmunoprecipitation reveals that EPG - 7 associates with LGG - 1 ( D ) .

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(C and D) Coimmunoprecipitation reveals that EPG-7 associates with LGG-1 (D).


( E ) EPG - 7 directly interacts with LGG - 3 .

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(E) EPG-7 directly interacts with LGG-3.


( F ) EPG - 7 fragments containing amino acids 852 - 1003 or 1004 - 1142 , but not 762 - 851 , interact with LGG - 1 .

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(F) EPG-7 fragments containing amino acids 852-1003 or 1004-1142, but not 762-851, interact with LGG-1.


( G ) Fragments of EPG - 7 containing amino acids 762 - 851 or 852 - 1003 directly interact with ATG - 18 .

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(G) Fragments of EPG-7 containing amino acids 762-851 or 852-1003 directly interact with ATG-18.