( 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 ) .

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(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 .

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(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 .

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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 .

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(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 .

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(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 ) .

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(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 .

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(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 ) .

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(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 .

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(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 .

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(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 .

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(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 .

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(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 .

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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 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

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(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 .

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(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 .

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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 .

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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 .

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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 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 .

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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 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 .

O O O O B-GENEPROD B-EXP_ASSAY O O O O O B-GENEPROD O B-GENEPROD I-GENEPROD O O B-TISSUE O O O O O O B-EXP_ASSAY O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O B-EXP_ASSAY O O O O O O O B-EXP_ASSAY O O O O O O B-EXP_ASSAY 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 of WT 11 RBP - KO ES lines after LIF withdrawal . Scale bar , 200μm .

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

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

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

B-TISSUE O O O O B-GENEPROD O O B-CELL_TYPE O O O B-CELL_TYPE O O B-TISSUE O O O O O O O O O O O B-GENEPROD O O B-CELL_TYPE O O O B-TISSUE 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 .

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

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

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

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 ) .

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

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

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

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

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 B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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 ) .

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

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 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

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 .

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

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

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

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

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 .

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

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 .

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

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 .

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

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 .

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

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 .

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

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 .

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

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 .

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

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 .

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

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 .

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

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 ) .

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

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 .

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

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 ) .

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

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 .

B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O B-GENEPROD O O O B-GENEPROD O O O B-CELL_TYPE O O O O B-TISSUE O O O B-TISSUE O O O B-TISSUE O O O O O O O

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 .

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

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 .

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

(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 ) .

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

(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 .

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

(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 .

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

(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 ) .

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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 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 .

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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 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 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 ) .

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 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 B-GENEPROD O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD I-GENEPROD I-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 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 ) 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).


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

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

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 of EGFP in spine heads relative to that in 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 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 of PSD - 95 were comparable under basal conditions for WT , E17R , 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 , were comparable under basal conditions for neurons expressing WT , E17R , 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 indicated CaM - IRES - ECFP plasmids infected at 14 DIV with lentivirus expressing shRNA against PSD - 95 ( sh95 ) 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 of intrinsic ECFP EGFP , surface GluA1 immunostaining at low ( top panels ; scale bar : 20 µm ) high enlargement ( bottom panels ; scale bar : 5 µm ) .

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

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 ) of surface GluA1 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 ; one - way ANOVA followed by Bonferroni ' s post hoc ) .

O O O O O B-GENEPROD O O O O O O O O O B-CELL_TYPE O B-GENEPROD I-GENEPROD I-GENEPROD 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 O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O 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) 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 indicated CaM - IRES - ECFP plasmids infected at 12 - 13 DIV with lentivirus expressing shRNA against PSD - 95 ( sh95 ) 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 ) averages of mEPSC events from single representative neurons ( right ) drawn to scale ( top ) normalized to peak ( bottom ) .

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

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

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

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

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(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 of transfected in ( B ) immunostained with anti - FLAG M2 antibody . Corresponding mean flourescence values are shown .

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(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 lysates derived from 293T transfected in ( B ) followed by immunoblotting with anti - FLAG M2 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 transfected with pQCXIP - FLAG - IFITM3 variants , HIV - 1 pNL4 - 3 , Vpr - Blam plasmid followed by immunoblotting with anti - FLAG M2 anti - p24 Gag ( 183 - H12 - 5C ) .

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

(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 for 4 hours - fusion was scored by flow cytometry . Mean + SD of 5 - 10 experiments ( each performed with produced from an independent transfection ) is shown . ( G ) used in ( E ) were used to infect SupT1 productive infection was scored at 48 - 60 hours by immunostaining with KC57 antibody flow cytometry . Expression of pCMV - MxA served as a negative control . mean + SD of 5 - 10 experiments is shown .

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(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 microscopy of 293T transfected with 0 . 5 μg pQCXIP - FLAG - IFITM3 variants immunostained with anti - FLAG M2

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(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 lysates derived from 293T transfected in ( H ) followed by immunoblotting with anti - FLAG M2 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 transfected with pQCXIP - FLAG - IFITM3 constructs , HIV - 1 pNL4 - 3 , Vpr - Blam plasmid followed by immunoblotting with anti - FLAG M2 anti - p24 Gag . Scale bar , 5 μm .

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(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 for 4 hours - fusion was scored by flow cytometry . 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 microscopy of SupT1 T 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 .

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(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 lines were treated or not with 500 ng / mL doxycycline overnight induction of IFITM3 was assessed anti - FLAG M2 immunostaining 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 lines were productively infected with NL4 - 3 VSV - G then treated with 500 ng / mL doxycycline overnight to produce from IFITM3 - IFITM3 + . 25 ng p24 equivalents of purified were used to infect fresh Tet - ON SupT1 targets , which were previously treated with 500 ng / mL doxycycline or not . Infection was scored at 72h post - infection by immunostaining with KC57 flow cytometry . Mean + SD of 3 experiments are shown .

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(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 used in ( C ) was subjected to SDS - PAGE . Immunoblotting was performed using anti - FLAG M2 , anti - p24 Gag , anti - gp120 Env ( NIH # 288 ) antibodies .

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

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

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(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 following transfection of 0 . 5 μg of pQCXIP - FLAG - IFITM3 variants 0 . 5 μg of pCI - HA - NEDD4 or pCI - HA - NEDD4 C867A . Immunoblotting was performed with anti - FLAG M2 , anti - IFITM3 ( Abcam , EPR5242 ) , an anti - ubiquitin antibody ( Enzo Life Sciences , recognizing K29 - , K48 - , K63 - linked mono - polyubiquitinylated ) . ' L ' indicates presence of chain antibody derived from FLAG antibody - coupled beads used for immunoprecipitation . * , * * , * * * indicate mono - , di - , tri - ubiquitinated forms of IFITM3 .

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(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 lysate input fractions from 293T used in ( B ) . Immunoblotting was performed with anti - FLAG M2 , anti - NEDD4 , anti - actin .

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(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 were transfected with 1 . 0 μg of pNL4 - 3 0 . 5 μg of pQCXIP - FLAG - IFITM3 variants . - containing supernatants were harvested 10 ng p24 equivalents were used to reinfect fresh SupT1 T . Productive infection was scored by immunostaining with KC57 antibody flow cytometry at 48 hours post - infection . 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 were seeded challenged with 10 ng of HIV - 1NL4 - 3 in presence of 2 μg / mL DEAE - Dextran . Productive infection was scored by immunostaining with KC57 flow cytometry at 48 hours post - infection . ' Colobus native ' represents full IFITM3 identified in colobus monkey . ( F ) As in ( E ) , except were challenged with Influenza A H1N1 PR / 8 / 34 , ( Charles River Laboratories ) , at a dose that resulted in ˜ 50 % infection of control ( Empty ) ( equivalent to 103 TCID50 / 0 . 2 mL ) . Infection was scored by immunostaining with an anti - IAV NP antibody flow cytometry at 18 hours post - infection . Results are presented in a logarithmic scale . Means + SD of 3 - 5 experiments are shown . Unpaired t - , * p < 0 . 05 , * * p < 0 . 005 .

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(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 , AID from siControl - siPhf5a - treated samples . 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 IgH / c - Myc translocation using genomic extracted from CIT - stimulated CH12F3 - 2A treated with siRNAs shown at bottom of each panel . Numbers on top indicate gel lanes . ( C ) Frequency of IgH / c - Myc chromosomal translocations derived from number of translocations detected in total number of PCR reactions per sample ( Boboila et al . , 2012b ) . values are presented as mean ± sd ( n = 3 ) . Statistical significance was assessed by two unpaired Student ' s t - ( * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 ) .

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(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 double - strand breaks by LM - PCR in CH12F3 - 2A transfected with indicated siRNA combinations . 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 GFP - positive obtained by FACS after co - transfection of I - SceI expression plasmid indicated siRNAs into a harboring NHEJ reporter construct . values are presented as mean ± sd ( n = 3 ) . Statistical significance was evaluated against corresponding siControl by two - unpaired Student ' s t - ( * * * p ≤ 0 . 001 ) .

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

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(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 of whole extract from CIT - stimulated CH12F3 - 2A transfected with indicated siRNAs . Expression of various including AID , variant H2As Ku80 were not decresed by Phf5a KD . ( ns , non - specific )

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

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(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 exogenously introduced Phf5a .

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


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

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(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 . ( A B ) In wild - type , SQST - 1 : : GFP is weakly expressed diffusely localized in cytoplasm . ( A ) DIC image of shown in B . ( C ) Elevated expression level accumulation of many SQST - 1 : : GFP in epg - 7 mutant .

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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 epg - 2 mutants , of SQST - 1 are dramatically elevated in atg - 3 epg - 7 mutant in an immunoblotting .

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(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 ( E F ) , endogenous SQST - 1 shows dramatically elevated accumulates into a large number of in epg - 7 mutant ( G ) . ∼ 200 - stage are shown in E - G . ( E ) DAPI image of shown in F .

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(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 per focal plane in wild type , atg - 3 mutants , epg - 7 mutants .

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


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

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(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 .

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


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

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(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 ( O - P ) sqst - 1 mutants ( R ) , but forms a large number of in epg - 7 mutants ( Q ) . ( O ) DIC image of shown in P .

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(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 , epg - 7 mutants . This experiment was completed once ( wild type : n = 2 , 964 ; epg - 7 : n = 602 ; epg - 1 : n = 215 ) . Statistical of L1 survival shows that epg - 7 mutants exhibit significant difference to wild type also to epg - 1 mutants ( log - rank , 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 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 B-GENEPROD I-GENEPROD I-GENEPROD 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

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

O O O B-EXP_ASSAY 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 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

(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 . ( In 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 ) .

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 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

(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 at 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 , 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 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

(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 accumulate in epg - 7 mutant 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 B-GENEPROD I-GENEPROD I-GENEPROD O 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 F ) No SQST - 1 are detected in epg - 7 mutants carrying an epg - 7 : : gfp reporter . ( E ) DAPI image of shown in F .

O O O O O B-GENEPROD I-GENEPROD I-GENEPROD 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 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 diffusely localized in cytoplasm in wild - type .

O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD O O O O O O B-SUBCELLULAR O O O O 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 ( H , arrow ) in 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 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.