( D ) Real - time imaging of undergoing biochemical fractionation . sNASP is tagged with mCherry , whilst LamnA / C is tagged with EGFP . left column of panels represents a maximum projection . time course on right represents a plane in z dimension reconstructed from 20 z - stacks penetrating 20 μm into medium . Scale bars represent 5

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(D) Real-time imaging of cells undergoing biochemical fractionation. sNASP is tagged with mCherry, whilst LamnA/C is tagged with EGFP. The left column of panels represents a maximum intensity projection. The time course on the right represents a plane in the z dimension reconstructed from 20 z-stacks penetrating 20 μm into the medium. Scale bars represent 5 μm


( A ) HeLa lines stably expressing eGFP - sNASP , eGFP - ASF1 eGFP - HAT1 . Scale bars represent 10 μm

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(A) HeLa cell lines stably expressing eGFP-sNASP, eGFP-ASF1 and eGFP-HAT1. Scale bars represent 10 μm


( B ) Histone chaperone immunoprecipitations separated by 15 % SDS - PAGE stained with Coomassie . Major bands that could be confidently identified by mass spectrometry are indicated . Due to high of acrylamide used , many of higher molecular bands are not resolved . Asterisks represent partial degradation . Background - corrected densitometry profiles are shown for portions of gels covering H3 H4 . Gel filtration purified recombinant H3 - H4 dimer is shown on

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

(B) Histone chaperone immunoprecipitations separated by 15% SDS-PAGE and stained with Coomassie. Major bands that could be confidently identified by mass spectrometry are indicated. Due to the high percentage of acrylamide used, many of the higher molecular weight bands are not resolved. Asterisks represent partial degradation. Background-corrected densitometry profiles are shown for the portions of the gels covering H3 and H4. Gel filtration purified recombinant H3-H4 dimer is shown on the right


Western blot of Fxr1 during ( A ) , TTX ( 48h treatment ) inducedupscaling ( n = 6 in each condition ) of postnatal cortical cultures . Student ' s T - * p < 0 . 05 , * * * p < 0 . 001 .

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Western blot analysis of Fxr1 during (A), TTX (48h treatment) inducedupscaling (n=6 in each condition) of primary postnatal cortical cultures. Student's T-test *p<0.05, ***p<0.001.


Western blot of Fxr1 during ( B ) , BIC ( 48h treatment ) induced downscaling ( n = 8 in each condition ) of postnatal cortical cultures . Student ' s T - * p < 0 . 05 , * * * p < 0 . 001 .

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Western blot analysis of Fxr1 during (B), BIC (48h treatment) induced downscaling (n=8 in each condition) of primary postnatal cortical cultures. Student's T-test *p<0.05, ***p<0.001.


C , D , Western blot of ( C ) , Fxr2 ( Veh n = 8 , TTX n = 8 ) ( D ) , Fmrp ( Veh n = 10 , TTX n = 12 ) during upscaling , Student ' s T - * p < 0 . 05 .

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C, D, Western blot analysis of (C), Fxr2 (Veh n=8, TTX n=8) and (D), Fmrp (Veh n=10, TTX n=12) during upscaling, Student's T-test *p<0.05.


E - G , RT - qPCR measurement of mRNA for ( E ) , Fxr1 , ( F ) , Fxr2 , ( G ) , Fmr1 during upscaling . n = 4 in each condition , Student ' s T - * p < 0 . 05 .

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E-G, RT-qPCR measurement of mRNA for (E), Fxr1, (F), Fxr2, (G), Fmr1 during upscaling. n=4 in each condition, Student's T-test *p<0.05.


H , Western blot for Fxr1 , pGsk3α / β , Gsk3α / β GAPDH in neuronal cultures treated with 1µM TTX ( TTX ) or 1mM LiCI ( Li ) or 1mM NaCI ( Ctrl ) for 48h .

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H, Western blot analysis for Fxr1, pGsk3α/β, Gsk3α/β and GAPDH in neuronal cultures treated with 1µM TTX (TTX) or 1mM LiCI (Li) or 1mM NaCI (Ctrl) for 48h.


I , Expression of Fxr1 in TTX ( n = 5 ) Li ( n = 5 ) conditions relative to Ctrl ( n = 6 ) condition . One way ANOVA with Dunnett ' s Multiple Comparison * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

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I, Expression of Fxr1 protein in TTX (n=5) and Li (n=5) conditions relative to Ctrl (n=6) condition. One way ANOVA with Dunnett's Multiple Comparison Test *p<0.05, **p<0.01, ***p<0.001.


J , Expression of pGsk3β / Gsk3β or pGsk3α / Gsk3α in TTX ( n = 5 ) Li ( n = 5 ) conditions relative to Ctrl ( n = 6 ) condition . One way ANOVA with Dunnett ' s Multiple Comparison * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

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J, Expression of pGsk3β/Gsk3β or pGsk3α/Gsk3α in TTX (n=5) and Li (n=5) conditions relative to Ctrl (n=6) condition. One way ANOVA with Dunnett's Multiple Comparison Test *p<0.05, **p<0.01, ***p<0.001.


B , Western blot of total GluA1 expression in Ctrl or Fxr1 overexpression ( Fxr1 ) condition during upscaling ( Ctrl / Veh n = 5 , Ctrl / TTX n = 5 , Fxr1 / Veh n = 6 , Fxr1 / Veh n = 6 ) . One way Anova with Dunnett ' s Multiple Comparison * * * p < 0 . 001 .

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B, Western blot analysis of total GluA1 expression in Ctrl or Fxr1 overexpression (Fxr1) condition during upscaling (Ctrl/Veh n=5, Ctrl/TTX n=5, Fxr1/Veh n=6, Fxr1/Veh n=6). One way Anova with Dunnett's Multiple Comparison Test ***p<0.001.


C , Western blot of total GluA1 expression in Ctrl or Fxr1over condition during downscaling ( Ctrl / Veh n = 4 , Ctrl / TTX n = 4 , Fxr1 / Veh n = 4 , Fxr1 / Veh n = 4 ) .

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C, Western blot analysis of total GluA1 expression in Ctrl or Fxr1over condition during downscaling (Ctrl/Veh n=4, Ctrl/TTX n=4, Fxr1/Veh n=4, Fxr1/Veh n=4).


D , RT - qPCR measurement of Gria1 mRNA in Ctrl or Fxr1over condition during upscaling ( Ctrl / Veh n = 3 , Ctrl / TTX n = 3 , Fxr1 / Veh n = 3 , Fxr1 / Veh n = 3 ) . One way Anova with Bonferroni ' s Multiple Comparison * * * p < 0 . 001 .

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D, RT-qPCR measurement of Gria1 mRNA in Ctrl or Fxr1over condition during upscaling (Ctrl/Veh n=3, Ctrl/TTX n=3, Fxr1/Veh n=3, Fxr1/Veh n=3). One way Anova with Bonferroni's Multiple Comparison Test ***p<0.001.


F , Measurement of relative luciferase signal after co - transfection of different tagged luciferase constructs along with GFP ( Ctrl ) or GFP - Fxr1 ( Fxr1 ) plasmids . n = 5 in each condition , Student ' s T - * p < 0 . 05 , * * * p < 0 . 001 .

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F, Measurement of relative luciferase signal after co-transfection of different tagged luciferase constructs along with GFP (Ctrl) or GFP-Fxr1 (Fxr1) plasmids. n=5 in each condition, Student's T-test *p<0.05, ***p<0.001.


G , Immunostaining for surface GluA1 in GFP ( Ctrl ) or GFP - Fxr1 ( Fxr1 over ) infected cultures after treatment with Veh , TTX or BIC for 48h .

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G, Immunostaining for surface GluA1 in GFP (Ctrl) or GFP-Fxr1 (Fxr1 over) infected cultures after treatment with Veh, TTX or BIC for 48h.


H , of surface GluA1 relative to mean of Ctrl / Veh condition ( Ctrl condition : Veh n = 63 , TTX n = 34 , BIC n = 29 , Fxr1 condition : Veh n = 56 , TTX n = 30 , BIC n = 29 ) . One way Anova with Bonferroni ' s Multiple Comparison * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

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H, Percentage of surface GluA1 relative to the mean of Ctrl/Veh condition (Ctrl condition: Veh n=63, TTX n=34, BIC n=29, Fxr1 condition: Veh n=56, TTX n=30, BIC n=29 ). One way Anova with Bonferroni's Multiple Comparison Test *p<0.05, **p<0.01, ***p<0.001.


A , Cumulative probability plots of mEPSCs amplitude ( 500 events per ) representative examples of mEPSCs ( left panel ) recorded from cultured cortical control neurons after 48 hours of 1 µM TTX or Veh exposure ( Ctrl / Veh n = 16 Ctrl / TTX n = 17 ) . B , A linear fit of Ctrl / TTX Ctrl / Veh amplitudes . C , degrees of overlap between Ctrl / TTX Ctrl / Veh data were assessed using various scaling factors . largest nonsignificant p - value was obtained with 1 . 47 scaling factor . D , Cumulative probability plots of mEPSCs amplitude of Ctrl / Veh , Ctrl / TTX Ctrl / TTX divided by scaling factor 1 . 47 , which yielded maximum overlap with Ctrl / Veh data .

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A, Cumulative probability plots of mEPSCs amplitude (500 events per cell) and representative examples of mEPSCs (left panel) recorded from cultured cortical control neurons after 48 hours of 1 µM TTX or Veh exposure (Ctrl/Veh n=16 and Ctrl/TTX n=17). B, A linear fit of Ctrl/TTX and Ctrl/Veh amplitudes. C, The degrees of overlap between Ctrl/TTX and Ctrl/Veh data were assessed using various scaling factors. The largest nonsignificant p-value was obtained with 1.47 scaling factor. D, Cumulative probability plots of the mEPSCs amplitude of Ctrl/Veh, Ctrl/TTX and Ctrl/TTX divided by scaling factor 1.47, which yielded the maximum overlap with Ctrl/Veh data.


Cumulative probability plots of mEPSCs amplitude ( 500 events per ) representative examples of mEPSCs ( left panel ) recorded from cultured cortical neurons after 48 hours of 1 µM TTX or Veh exposure ( E ) Fxr1overexpressing neurons ( Fxr1 / Veh n = 10 Fxr1 / TTX n = 8 )

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Cumulative probability plots of mEPSCs amplitude (500 events per cell) and representative examples of mEPSCs (left panel) recorded from cultured cortical neurons after 48 hours of 1 µM TTX or Veh exposure (E) Fxr1overexpressing neurons (Fxr1/Veh n=10 and Fxr1/TTX n=8)


Cumulative probability plots of mEPSCs amplitude ( 500 events per ) representative examples of mEPSCs ( left panel ) recorded from cultured cortical neurons after 48 hours of 1 µM TTX or Veh exposure ( F ) Gsk3 KO neurons ( Gsk3KO / Veh n = 10 Gsk3KO / TTX n = 11 )

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Cumulative probability plots of mEPSCs amplitude (500 events per cell) and representative examples of mEPSCs (left panel) recorded from cultured cortical neurons after 48 hours of 1 µM TTX or Veh exposure (F) Gsk3 KO neurons (Gsk3KO/Veh n=10 and Gsk3KO/TTX n=11)


Cumulative probability plots of mEPSCs amplitude ( 500 events per ) representative examples of mEPSCs ( left panel ) recorded from cultured cortical neurons after 48 hours of 1 µM TTX or Veh exposure ( G ) Fxr1 KO neurons ( Fxr1KO / Veh n = 16 Fxr1KO / TTX n = 8 ) . H , A linear fit of Fxr1 KO / Veh Ctrl / Veh amplitudes . I , degrees of overlap between Fxr1 KO / Veh Ctrl / Veh data were assessed using various scaling factors . largest non - significant p - value was obtained with 1 . 27 scaling factor . J , Cumulative probability plots of mEPSCs amplitude of Ctrl / Veh , Fxr1 KO / Veh Fxr1 KO / Veh divided by scaling factor 1 . 27 , which yielded maximum overlap with Ctrl / Veh data .

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Cumulative probability plots of mEPSCs amplitude (500 events per cell) and representative examples of mEPSCs (left panel) recorded from cultured cortical neurons after 48 hours of 1 µM TTX or Veh exposure (G) Fxr1 KO neurons (Fxr1KO/Veh n=16 and Fxr1KO/TTX n=8). H, A linear fit of Fxr1 KO/Veh and Ctrl/Veh amplitudes. I, The degrees of overlap between Fxr1 KO/Veh and Ctrl/Veh data were assessed using various scaling factors. The largest non-significant p-value was obtained with 1.27 scaling factor. J, Cumulative probability plots of the mEPSCs amplitude of Ctrl/Veh, Fxr1 KO/Veh and Fxr1 KO/Veh divided by scaling factor 1.27, which yielded the maximum overlap with Ctrl/Veh data.


K , Cumulative probability plots of mEPSCs amplitude ( 500 events per ) representative examples of mEPSCs ( left panel ) recorded from cultured cortical Gsk3 Fxr1 KO neurons after 48 hours of 1 µM TTX or Veh exposure ( Gsk3 / Fxr1KO / Veh n = 8 Gsk3 / Fxr1KO / TTX n = 11 ) . L , A linear fit of Gsk3 / Fxr1 KO / Veh Ctrl / Veh amplitudes . M , degrees of overlap between Gsk3 / Fxr1 KO / Veh Ctrl / Veh data were assessed using various scaling factors . largest non - significant p - value was obtained with 1 . 68 scaling factor . N , Cumulative probability plots of mEPSCs amplitude of Ctrl / Veh , Gsk3 / Fxr1 KO / Veh Gsk3 / Fxr1 KO / Veh divided by scaling factor 1 . 68 , which yielded maximum overlap with Ctrl / Veh data .

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

K, Cumulative probability plots of mEPSCs amplitude (500 events per cell) and representative examples of mEPSCs (left panel) recorded from cultured cortical Gsk3 and Fxr1 KO neurons after 48 hours of 1 µM TTX or Veh exposure (Gsk3/Fxr1KO/Veh n=8 and Gsk3/Fxr1KO/TTX n=11). L, A linear fit of Gsk3/Fxr1 KO/Veh and Ctrl/Veh amplitudes. M, The degrees of overlap between Gsk3/Fxr1 KO/Veh and Ctrl/Veh data were assessed using various scaling factors. The largest non-significant p-value was obtained with 1.68 scaling factor. N, Cumulative probability plots of the mEPSCs amplitude of Ctrl/Veh, Gsk3/Fxr1 KO/Veh and Gsk3/Fxr1 KO/Veh divided by scaling factor 1.68, which yielded the maximum overlap with Ctrl/Veh data.


O , mEPSC mean amplitude of cultured cortical neurons after 48 hours of 1 µM TTX or Veh exposure . One way Anova with Bonferroni ' s Multiple Comparison * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

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O, mEPSC mean amplitude of cultured cortical neurons after 48 hours of 1 µM TTX or Veh exposure. One way Anova with Bonferroni's Multiple Comparison Test *p<0.05, **p<0.01, ***p<0.001.


P , mEPSC frequency of cultured cortical neurons after 48 hours of 1 µM TTX or Veh exposure .

P, mEPSC frequency of cultured cortical neurons after 48 hours of 1 µM TTX or Veh exposure.


A , Hourly distribution of wakefulness ( WAKE ) , slow - wave sleep ( SWS ) paradoxical sleep ( PS ) during a 24 - h baseline ( BL ) recording a second 24 - h starting with 6 - h sleep deprivation ( SD ) ( recovery : REC ) in Ctrl Fxr1 overexpressing ( Fxr1 ) mice . Significant Group - by - Hour interactions were found for wakefulness during BL ( F23 , 230 = 1 . 67 , p = 0 . 046 ) REC ( F23 , 230 = 2 . 83 , p = 0 . 0029 ) , for SWS during BL ( F23 , 230 = 1 . 66 , p = 0 . 045 ) REC ( F23 , 230 = 2 . 89 , p = 0 . 0028 ) , for PS during REC ( F16 , 160 = 2 . 54 , p = 0 . 0018 , Two - way ANOVA Huynh - Feldt corrected , * p < 0 . 05 ) .

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A, Hourly distribution of wakefulness (WAKE), slow-wave sleep (SWS) and paradoxical sleep (PS) during a 24-h baseline (BL) recording and a second 24-h starting with 6-h sleep deprivation (SD) (recovery: REC) in Ctrl and Fxr1 overexpressing (Fxr1) mice. Significant Group-by-Hour interactions were found for wakefulness during BL (F23,230 = 1.67, p = 0.046) and REC (F23,230 = 2.83, p = 0.0029), for SWS during BL (F23,230 = 1.66, p = 0.045) and REC (F23,230 = 2.89, p=0.0028), and for PS during REC (F16,160 = 2.54, p=0.0018, Two-way ANOVA Huynh-Feldt corrected, *p<0.05).


B , Power spectra for WAKE , SWS , PS in Ctrl Fxr1 mice computed between 0 . 75 50 Hz per 0 . 25 - Hz for full 24 - h of BL REC .

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B, Power spectra for WAKE, SWS, and PS in Ctrl and Fxr1 mice computed between 0.75 and 50 Hz per 0.25-Hz for the full 24-h of BL and REC.


C , D , spectral activity of Fxr1 mice expressed relative to that of Ctrl mice for WAKE , SWS PS during ( C ) 24 - h BL ( D ) 24 - h REC . A significant difference between groups was found for frequency band 8 to 11 . 75 Hz during wakefulness ( t = 2 . 30 , p = 0 . 044 Student ' s T - * p < 0 . 05 ) .

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C, D, The spectral activity of Fxr1 mice expressed relative to that of Ctrl mice for WAKE, SWS and PS during (C) the 24-h BL and (D) the 24-h REC. A significant difference between groups was found for the frequency band 8 to 11.75 Hz during wakefulness (t = 2.30, p=0.044 Student's T-test *p<0.05).


E , Time course of wakefulness spectral activity between low alpha ( 8 . 5 - 10 . 5 Hz ) low theta ( 4 - 6 Hz ) during BL REC in Ctrl Fxr1 mice . A significant Group - by - Interval interaction was found for REC ( F22 , 220 = 1 . 96 , Two - way ANOVA Huynh - Feldt corrected ; * p < 0 . 05 ) , also when using BL REC intervals of periods ( F16 , 160 = 3 . 11 , p < 0 . 01 ) , .

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E, Time course of wakefulness spectral activity ratio between low alpha (8.5-10.5 Hz) and low theta (4-6 Hz) during BL and REC in Ctrl and Fxr1 mice. A significant Group-by-Interval interaction was found for REC (F22,220 = 1.96, Two-way ANOVA Huynh-Feldt corrected; *p < 0.05), and also when using BL and REC intervals of the light periods (F16,160 = 3.11, p < 0.01),.


F , Time course of SWS relative delta activity during BL REC in Ctrl Fxr1 mice . A significant Group - by - Interval interaction was found during REC ( F13 , 130 = 1 . 89 , Two - way ANOVA Huynh - Feldt corrected ; * p < 0 . 05 ) .

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F, Time course of SWS relative delta activity during BL and REC in Ctrl and Fxr1 mice. A significant Group-by-Interval interaction was found during REC (F13,130 = 1.89, Two-way ANOVA Huynh-Feldt corrected; *p < 0.05).


Western blot of ( B ) p845GluA1 GluA1 expression ( S n = 9 , SD n = 10 ) in prefrontal cortex of sleeping sleep deprived mice . Student ' s t - * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

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Western blot analysis of (B) p845GluA1 and GluA1 expression (S n=9, SD n=10) in the prefrontal cortex of sleeping and sleep deprived mice. Student's t-test *p < 0.05, **p < 0.01, ***p < 0.001.


Western blot of , ( C ) p880GluA2 GluA2 expression ( S n = 9 , SD n = 8 ) in prefrontal cortex of sleeping sleep deprived mice . Student ' s t - * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

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Western blot analysis of , (C) p880GluA2 and GluA2 expression (S n=9, SD n=8) in the prefrontal cortex of sleeping and sleep deprived mice. Student's t-test *p < 0.05, **p < 0.01, ***p < 0.001.


Western blot of ( D ) Fxr1 expression ( S n = 11 , SD n = 10 ) in prefrontal cortex of sleeping sleep deprived mice . Student ' s t - * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

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Western blot analysis of (D) Fxr1 expression (S n=11, SD n=10) in the prefrontal cortex of sleeping and sleep deprived mice. Student's t-test *p < 0.05, **p < 0.01, ***p < 0.001.


Western blot of ( E ) Fmrp expression ( S n = 11 , SD n = 10 ) in prefrontal cortex of sleeping sleep deprived mice . Student ' s t - * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

Western blot analysis of (E) Fmrp expression (S n=11, SD n=10) in the prefrontal cortex of sleeping and sleep deprived mice. Student's t-test *p < 0.05, **p < 0.01, ***p < 0.001.


F - H , RNAseq measurement of mRNA for ( F ) Fxr1 , ( G ) Fxr2 , ( H ) Fmr1 during SD . n = 3 in each condition , Student ' s T - * p < 0 . 05 .

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F-H, RNAseq measurement of mRNA for (F) Fxr1, (G) Fxr2, (H) Fmr1 during SD. n=3 in each condition, Student's T-test *p<0.05.


Cumulative probability plots of mEPSCs amplitude ( 500 events per ) representative examples of mEPSCs ( left panel ) recorded from brain slices of S SD mice . ( B ) Control neurons ( Ctrl / S n = 8 / 4 mice , Ctrl / SD n = 8 / 4 mice )

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Cumulative probability plots of mEPSCs amplitude (500 events per cell) and representative examples of mEPSCs (left panel) recorded from brain slices of S and SD mice. (B) Control neurons (Ctrl/S n=8 cells/4 mice, Ctrl/SD n=8 cells/4 mice)


Cumulative probability plots of mEPSCs amplitude ( 500 events per ) representative examples of mEPSCs ( left panel ) recorded from brain slices of S SD mice . ( C ) Fxr1 overexpressing ( Fxr1 / S n = 7 / 5 mice , Fxr1 over / SD n = 9 / 5 mice )

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Cumulative probability plots of mEPSCs amplitude (500 events per cell) and representative examples of mEPSCs (left panel) recorded from brain slices of S and SD mice. (C) Fxr1 overexpressing (Fxr1/S n=7 cells/5 mice, Fxr1 over/SD n=9 cells/5 mice)


Cumulative probability plots of mEPSCs amplitude ( 500 events per ) representative examples of mEPSCs ( left panel ) recorded from brain slices of S SD mice . ( D ) Gsk3 sKO ( Gsk3sKO / S n = 6 / 3 mice , Gsk3sKO / SD n = 7 / 4 mice ) .

Cumulative probability plots of mEPSCs amplitude (500 events per cell) and representative examples of mEPSCs (left panel) recorded from brain slices of S and SD mice. (D) Gsk3 sKO (Gsk3sKO/S n=6 cells/3 mice, Gsk3sKO/SD n=7 cells/4 mice).


mEPSC ( E ) mean amplitude of cortical neurons of S or SD mice . Control neurons ( Ctrl / S n = 8 / 4 mice , Ctrl / SD n = 8 / 4 mice ) , Fxr1 overexpressing ( Fxr1 / S n = 7 / 5 mice , Fxr1 over / SD n = 9 / 5 mice ) , Gsk3 sKO ( Gsk3sKO / S n = 6 / 3 mice , Gsk3sKO / SD n = 7 / 4 mice ) . Student ' s T - * p < 0 . 05 .

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mEPSC (E) mean amplitude of cortical neurons of S or SD mice. Control neurons (Ctrl/S n=8 cells/4 mice, Ctrl/SD n=8 cells/4 mice), Fxr1 overexpressing (Fxr1/S n=7 cells/5 mice, Fxr1 over/SD n=9 cells/5 mice), and Gsk3 sKO (Gsk3sKO/S n=6 cells/3 mice, Gsk3sKO/SD n=7 cells/4 mice). Student's T-test *p<0.05.


F , mEPSC ( F ) frequency of cortical neurons of S or SD mice . Control neurons ( Ctrl / S n = 8 / 4 mice , Ctrl / SD n = 8 / 4 mice ) , Fxr1 overexpressing ( Fxr1 / S n = 7 / 5 mice , Fxr1 over / SD n = 9 / 5 mice ) , Gsk3 sKO ( Gsk3sKO / S n = 6 / 3 mice , Gsk3sKO / SD n = 7 / 4 mice ) . Student ' s T - * p < 0 . 05 .

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F, mEPSC (F) frequency of cortical neurons of S or SD mice. Control neurons (Ctrl/S n=8 cells/4 mice, Ctrl/SD n=8 cells/4 mice), Fxr1 overexpressing (Fxr1/S n=7 cells/5 mice, Fxr1 over/SD n=9 cells/5 mice), and Gsk3 sKO (Gsk3sKO/S n=6 cells/3 mice, Gsk3sKO/SD n=7 cells/4 mice). Student's T-test *p<0.05.


G , Representative examples of current - voltage relationship of evoked EPSC amplitude recorded from S ( top panel ) SD ( bottom panel ) mice .

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G, Representative examples of the current-voltage relationship of evoked EPSC amplitude recorded from S (top panel) and SD (bottom panel) mice.


H , Summary bar graphs showing rectification index of control ( S n = 12 / 5 mice , SD n = 10 / 5 mice ) , Fxr1P overexpressing ( S n = 9 / 4 mice , SD n = 14 / 4 mice ) Gsk3 sKO ( S n = 9 / 3 mice , SD n = 9 / 3 mice ) neurons . One way ANOVA with Bonferroni ' s Multiple Comparison * p < 0 . 05 .

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H, Summary bar graphs showing rectification index of control (S n=12 cells/5 mice, and SD n=10 cells/5 mice), Fxr1P overexpressing (S n=9 cells/4 mice, and SD n=14 cells/4 mice) and Gsk3 sKO (S n=9 cells/3 mice, and SD n=9 cells/3 mice) neurons. One way ANOVA with Bonferroni's Multiple Comparison *p < 0.05.


B , Immunostaining for GFP HA in Ctrl Fxr1 mouse brain slices . Arrows indicate presence arrowheads indicate an absence of GFP tagged Fxr1 granules .

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B, Immunostaining for GFP and HA in Ctrl and Fxr1 mouse brain slices. Arrows indicate presence and arrowheads indicate an absence of GFP tagged Fxr1 granules.


C , of colocalization of GFP HA labeled neurons ( GFP + HA + 71 . 3 % ± 3 . 7 , n = 3 , GFP - Fxr1 + HA + 79 . 7 % ± 2 . 8 , n = 3 ) .

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C, Quantification of colocalization of GFP and HA labeled neurons (GFP+HA+ 71.3%±3.7, n=3, GFP-Fxr1+HA+ 79.7%±2.8, n=3).


G , Heat map showing transcripts that have bidirectional expression changes between Ctrl / S , Ctrl / SD , Fxr1 / SD conditions .

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G, Heat map showing transcripts that have bidirectional expression changes between Ctrl/S, Ctrl/SD, and Fxr1/SD conditions.


( B ) Electropherograms of sequencing , family pedigrees , audiograms of two unrelated . Electropherograms show a heterozygous c . 3610C > T substitution resulting in a stop codon ( R1204X ) . Arrows in family pedigrees indicate identified in present study . In audiograms : red blue indicate right left ; respectively , solid dashed lines square brackets show air conduction hearing bone conduction hearing ; respectively . bold ( at of 48 years ) thin ( at of 50 years ) in audiogram of 1 indicate progressive hearing loss ( green arrow ) .

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(B) Electropherograms of DNA sequencing, family pedigrees, and audiograms of two unrelated patients. Electropherograms show a heterozygous c.3610C>T substitution resulting in a stop codon (R1204X). Arrows in family pedigrees indicate the patients identified in the present study. In audiograms: red and blue indicate right and left; respectively, solid and dashed lines and square brackets show air conduction hearing and bone conduction hearing; respectively. The bold line (at the age of 48 years) and thin line (at the age of 50 years) in the audiogram of patient 1 indicate progressive hearing loss (green arrow).


GFP - tagged DIA1 , DIA1 ( M1190D ) , DIA1 ( R1204X ) plasmids were transfected into MDCKmCherry - ESPIN1 cultured on inserts . Twenty - four hours after transfection , a reconstructed lateral view of fixed was generated using a confocal microscope . Arrows indicate elongated microvilli visualized by mCherry - ESPIN1 with GFP - hDIA1 ( R1204X ) expression . Scale bars : 5 μm . Representative of 3 experiments .

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GFP-tagged DIA1, DIA1(M1190D), and DIA1(R1204X) plasmids were transfected into MDCKmCherry-ESPIN1 cells cultured on membrane inserts. Twenty-four hours after transfection, a reconstructed lateral view of fixed cells was generated using a confocal laser fluorescence microscope. Arrows indicate the elongated microvilli visualized by mCherry-ESPIN1 with GFP-hDIA1(R1204X) expression. Scale bars: 5 μm. Representative of 3 experiments.


Various GFP - tagged DIA1 plasmids were transfected into HeLa , fixed 24 h after transfection . Fixed were stained with Alexa568 - conjugated phalloidin DAPI ( A , B : representative of 5 experiments ) . ( A ) Stress fiber formation was observed with a confocal microscope . Note induction of stress by GFP - DIA1 ( M1190D ) ( asterisks ) . Arrow arrowheads indicate a plasma ( PM ) localization of GFP - DIA1 ( M1190D ) a dot - like localization of GFP - DIA1 ( R1204X ) adjacent to PM , respectively . Inset in top panel of DIA1 ( R1204X ) shows magnified image of region indicated by rectangle . Scale bars : 10 μm . .

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Various GFP-tagged DIA1 plasmids were transfected into HeLa cells, and fixed 24 h after transfection. Fixed cells were stained with Alexa568-conjugated phalloidin and DAPI (A, B: representative of 5 experiments).(A) Stress fiber formation was observed with a confocal laser microscope. Note the induction of stress fibers by GFP-DIA1(M1190D) (asterisks). Arrow and arrowheads indicate a plasma membrane (PM) localization of GFP-DIA1(M1190D) and a dot-like localization of GFP-DIA1(R1204X) adjacent to the PM, respectively. Inset in the top panel of DIA1 (R1204X) shows the magnified image of the region indicated by rectangle. Scale bars: 10 μm..


Various GFP - tagged DIA1 plasmids were transfected into HeLa , fixed 24 h after transfection . Fixed were stained with Alexa568 - conjugated phalloidin DAPI ( A , B : representative of 5 experiments ) . ( B ) Microvilli with or without GFP - DIA1 ( R1204X ) expression were observed under a confocal microscope . Note elongated thick microvilli ( arrowheads ) in GFP - DIA1 ( R1204X ) - transfected , but not in non - transfected ( circles ) . 3D movies are available in Movie EV1 ( R1204X ) , Movie EV2 ( WT ) , Movie EV3 ( ttaa ) . Scale bar : 10 μm .

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Various GFP-tagged DIA1 plasmids were transfected into HeLa cells, and fixed 24 h after transfection. Fixed cells were stained with Alexa568-conjugated phalloidin and DAPI (A, B: representative of 5 experiments).(B) Microvilli with or without GFP-DIA1(R1204X) expression were observed under a confocal laser microscope. Note elongated and thick microvilli (arrowheads) in GFP-DIA1(R1204X)- transfected cells, but not in non-transfected cells (circles). 3D movies are available in Movie EV1 (R1204X), Movie EV2 (WT), and Movie EV3 (ttaa). Scale bar: 10 μm.


( B ) Purified GST GST - tagged DID ( 50 nM ) were mixed with biotin - labeled DADs ( WT ; R1204X ; M1190D , 50 nM ) in binding buffer . After rotation , streptavidin - coupled magnetic beads were added to solution , mixture was agitated . material absorbed to beads was eluted in Laemmli ' s sample buffer , were subjected to SDS - PAGE , followed by immunoblotting using an HRP - conjugated GST Ab . Comparable of input ( GSTproteins ) are confirmed in right panel . Representative of 5 experiments .

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(B) Purified GST and GST-tagged DID proteins (50 nM) were mixed with biotin-labeled DADs (WT; R1204X; and M1190D, 50 nM) in binding buffer. After rotation, streptavidin-coupled magnetic beads were added to the solution, and the mixture was agitated. The material absorbed to the beads was eluted in Laemmli's sample buffer, and were subjected to SDS-PAGE, followed by immunoblotting using an HRP-conjugated GST Ab. Comparable levels of input (GSTproteins) are confirmed in the right panel. Representative of 5 experiments.


GFP - tagged DIA1 ( WT ) , DIA1 ( R1204X ) , DIA1 ( M1190D ) were transfected into XTC . XTC were spread on PLL - coated coverslips time - lapse were acquired every 300 ms , illuminating restricted areas near edges , by using a single - molecule speckle microscope . Representative are shown for DIA1 ( WT ) ( A , Movie EV5 ) , DIA1 ( R1204X ) ( B , Movie EV6 ) , DIA1 ( M1190D ) ( C , Movie EV7 ) . Circles indicate speckles showing directional movement over a of several micrometers . Trajectories of these speckles are also illustrated , crosses were plotted at end of directional movement . Speckles showing directional movement were frequently observed in expressing GFP - DIA1 ( R1204X ) ( B ) , whereas such speckles were scarce in expressing GFP - DIA1 ( WT ) ( A ) . However , density of these speckles in GFP - DIA1 ( R1204X ) - expressing appeared lower than that in GFP - DIA1 ( M1190D ) - expressing , which is considered a fully active mutant ( C ) . To evaluate frequency of speckles indicating directional movement , we performed a quantitative ( D ) . number of speckles showing directional movement during 3 continuous planes was counted were normalized by of each , which corresponds to expression level of GFP - tagged . * P = 0 . 0288 * * P = 0 . 0002 Bonferroni ' s post hoc following one - way ANOVA ( WT ; n = 5 , R1204X ; n = 6 , M1190D ; n = 6 ) . Time is in seconds . Scale bars : 5 µm .

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GFP-tagged DIA1(WT), DIA1(R1204X), and DIA1(M1190D) were transfected into XTC cells. XTC cells were spread on the PLL-coated coverslips and time-lapse images were acquired every 300 ms, illuminating the restricted areas near the cell edges, by using a single-molecule speckle microscope. Representative images are shown for DIA1(WT) (A, Movie EV5), DIA1(R1204X) (B, Movie EV6), and DIA1(M1190D) (C, Movie EV7). Circles indicate speckles showing directional movement over a distance of several micrometers. Trajectories of these speckles are also illustrated, and crosses were plotted at the end of the directional movement. Speckles showing directional movement were frequently observed in cells expressing GFP-DIA1(R1204X) (B), whereas such speckles were scarce in cells expressing GFP-DIA1(WT) (A). However, the density of these speckles in GFP-DIA1(R1204X)-expressing cells appeared lower than that in GFP-DIA1(M1190D)-expressing cells, which is considered a fully active mutant (C). To evaluate the frequency of speckles indicating directional movement, we performed a quantitative analysis (D). The number of speckles showing directional movement during 3 continuous planes was counted and were normalized by the fluorescent intensity of each cell, which corresponds to the expression level of the GFP-tagged protein. *P= 0.0288 and **P= 0.0002 Bonferroni's post hoc test following one-way ANOVA (WT; n = 5, R1204X; n = 6, M1190D; n = 6). Time is in seconds. Scale bars: 5 µm.


Cochleae from control , heterozygous Dia1 - KO ( Dia1 - / + ) homozygous Dia1 - KO ( Dia1 - / - ) mice were fixed at 5 of ( A , B ; representative of n = 6 ) . ( A ) Paraffin - embedded 4 μm sections of cochlea were stained with hematoxylin eosin . No hair ( HC ) loss ( either or OHC ) is observed at basal turn of Dia1 - / + or Dia1 - / - cochleae . No obvious differences are observed in spiral ganglion ( SV ) , spiral ligament ( SL ) , stria vascularis ( SV ) , Reissner ' s ( RM ) in Dia1 - / + , or Dia1 - / - mice compared with control mice . Lower panels ( Scale bars : 20 µm ) are magnified of region indicated rectangle in upper panels ( Scale bars : 100 µm ) .

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Cochleae from control, heterozygous Dia1-KO (Dia1-/+) and homozygous Dia1-KO (Dia1-/-) mice were fixed at 5 weeks of age (A, B; representative of n=6).(A) Paraffin-embedded 4 μm sections of cochlea were stained with hematoxylin and eosin. No hair cell (HC) loss (either IHC or OHC) is observed at the basal turn of the Dia1-/+ or Dia1-/-cochleae. No obvious differences are observed in the spiral ganglion (SV), spiral ligament (SL), stria vascularis (SV), and Reissner's membrane (RM) in Dia1-/+, or Dia1-/-mice compared with control mice. Lower panels (Scale bars: 20 µm) are magnified images of the region indicated rectangle in upper panels (Scale bars: 100 µm).


Cochleae from control , heterozygous Dia1 - KO ( Dia1 - / + ) homozygous Dia1 - KO ( Dia1 - / - ) mice were fixed at 5 of ( A , B ; representative of n = 6 ) . ( B ) Basal turns of dissected cochleae were immunostained using a Myo7a Ab followed by an Alexa488 secondary Ab with DAPI as a counterstain . No HC loss ( either or OHC ) is observed in Dia1 - / - mice . Scale bars : 20 µm .

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Cochleae from control, heterozygous Dia1-KO (Dia1-/+) and homozygous Dia1-KO (Dia1-/-) mice were fixed at 5 weeks of age (A, B; representative of n=6).(B) Basal turns of the dissected cochleae were immunostained using a Myo7a Ab followed by an Alexa488 secondary Ab with DAPI as a nuclear counterstain. No HC loss (either IHC or OHC) is observed in Dia1-/-mice. Scale bars: 20 µm.


( C ) ABR thresholds ( 10 , 20 , 40 kHz ) in control Dia1 - / - mice at 5 of ( dB SPL , mean SE ) . No hearing loss was observed in Dia1 - / - mice ( n = 8 ) compared with control mice ( n = 4 ) .

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(C) ABR thresholds (10, 20, 40 kHz) in control and Dia1-/-mice at 5 weeks of age (dB SPL, mean SE). No hearing loss was observed in Dia1-/-mice (n = 8) compared with control mice (n = 4).


( B ) Expression of FLAG - tagged DIA1 ( R1204X ) in inner ear heart of TG , but not WT , mice is confirmed by immunoblotting using a FLAG Ab . Representative of 3 experiments .

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(B) Expression of FLAG-tagged DIA1(R1204X) in the inner ear and heart of TG, but not WT, mice is confirmed by immunoblotting using a FLAG Ab. Representative of 3 experiments.


( C ) Expression of FLAG - tagged DIA1 ( R1204X ) relative to endogenous Dia1 in inner ear , thymus , heart were determined by immunoblotting using a Dia1 / DIA1 Ab ( inner ear ; n = 4 , thymus heart ; n = 3 ) , quantified in inner ear thymus ( * P = 0 . 0226 * * P = 0 . 0016 by two - Student ' s t - ) . Lysates of HEK293 overexpressing equal amounts of GFP - Dia1 GFP - DIA1 ( determined using a GFP Ab : right end ) are detected with equal affinity by Dia1 / DIA1 antibody . Comparable loading of is confirmed using an α − tubulin or GAPDH Ab .

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(C) Expression levels of FLAG-tagged DIA1(R1204X) relative to endogenous Dia1 in the inner ear, thymus, and heart were determined by immunoblotting using a Dia1/DIA1 Ab (inner ear; n = 4, thymus and heart; n = 3), and quantified in the inner ear and thymus (*P= 0.0226 and **P= 0.0016 by two-tailed Student's t-test). Lysates of HEK293 cells overexpressing equal amounts of GFP-Dia1 and GFP-DIA1 (determined using a GFP Ab: right end) are detected with equal affinity by the Dia1/DIA1 antibody. Comparable loading of proteins is confirmed using an α−tubulin or GAPDH Ab.


( A ) , - related click pure tone - burst ( 8 , 16 , 24 , 32 kHz ) ABR thresholds ( dB SPL , mean SE ) in DIA1 ( R1204X ) - TG ( 5 , n = 6 ; 10 , n = 16 ; 25 , n = 42 ) control mice ( 5 , n = 6 ; 10 , n = 16 ; 25 , n = 33 ) . Note progressive hearing loss of DIA1 ( R1204X ) - TG mice starting at high frequency sounds from 10 . * * P = 0 . 0039 ( 24 kHz ) * * P < 0 . 0001 ( 32 kHz ) at 10 * * P < 0 . 0001 ( click ) , * * P < 0 . 0001 ( 8 kHz ) , * * P = 0 . 0001 ( 16 kHz ) , * * P < 0 . 0001 ( 24 kHz ) , * * P < 0 . 0001 ( 32 kHz ) at 25 by Bonferroni ' s post hoc following two - way ANOVA .

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(A), Age-related click and pure tone-burst (8, 16, 24, 32 kHz) ABR thresholds (dB SPL, mean SE) in DIA1(R1204X)-TG (5 weeks, n = 6; 10 weeks, n = 16; 25 weeks, n = 42) and control mice (5 weeks, n = 6; 10 weeks, n = 16; 25 weeks, n = 33). Note the progressive hearing loss of DIA1(R1204X)-TG mice starting at high frequency sounds from 10 weeks. **P= 0.0039 (24 kHz) and **P< 0.0001 (32 kHz) at 10 weeks and **P< 0.0001 (click), **P< 0.0001 (8 kHz), **P= 0.0001 (16 kHz), **P< 0.0001 (24 kHz), and **P< 0.0001 (32 kHz) at 25 weeks by Bonferroni's post hoc test following two-way ANOVA.


( B ) Representative of organ of Corti at basal turn obtained from DIA1 ( R1204X ) - TG control mice at 5 , 10 , 25 of . red circles show OHC losses , respectively . Note OHC - dominant HC loss . Scale bars : 5 µm .

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(B) Representative SEM images of the organ of Corti at the basal turn obtained from DIA1(R1204X)-TG and control mice at 5, 10, and 25 weeks of age. White and red circles show IHC and OHC losses, respectively. Note OHC-dominant HC loss. Scale bars: 5 µm.


( C ) percentages of remaining OHC in each turn in DIA1 ( R1204X ) - TG control mice at 25 of ( n = 5 ) . Schematic illustration shows regions of cochlea divided into three parts : Ap ; apical turn , Md ; middle turn , Bs ; basal turn . * * P = 0 . 0017 ( OHC at Md ) , * * P < 0 . 0001 ( OHC at Bs ) , * P = 0 . 0117 ( at Ap ) , * P = 0 . 0330 ( at Md ) , * * P = 0 . 0003 ( at Bs ) by Bonferroni ' s post hoc following two - way ANOVA .

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(C) The percentages of remaining OHC and IHC in each turn in DIA1(R1204X)-TG and control mice at 25 weeks of age (n = 5). Schematic illustration shows the regions of the cochlea divided into three parts: Ap; apical turn, Md; middle turn, Bs; basal turn. **P= 0.0017 (OHC at Md), **P< 0.0001 (OHC at Bs), *P= 0.0117 (IHC at Ap), *P= 0.0330 (IHC at Md), and **P= 0.0003 (IHC at Bs) by Bonferroni's post hoc test following two-way ANOVA.


( D ) DPOAE ( f2 frequency at 8 , 12 , 16 , 20 kHz ) amplitude ( dB SPL , mean SE ) in DIA1 ( R1204X ) - TG ( n = 37 ) control mice ( n = 30 ) at 25 of . * P = 0 . 0127 , * * P = 0 . 0029 ( at 12 kHz ) , * * P = 0 . 0013 ( at 16 kHz ) by Bonferroni ' s post hoc following two - way ANOVA .

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(D) DPOAE (f2 frequency at 8, 12, 16, 20 kHz) amplitude (dB SPL, mean SE) in DIA1(R1204X)-TG (n = 37) and control mice (n = 30) at 25 weeks of age. *P= 0.0127, **P= 0.0029 (at 12 kHz), and **P= 0.0013 (at 16 kHz) by Bonferroni's post hoc test following two-way ANOVA.


( E ) High magnification of OHCs IHCs at basal turn of cochlea obtained from DIA1 ( R1204X ) - TG control mice at 25 of . Note sparse short ( arrow ) stereocilia of OHC , sparse fused ( arrowheads ) stereocilia of in DIA1 ( R1204X ) - TG mice . Scale bars : 1 µm in OHCs 5 µm in IHCs .

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(E) High magnification SEM images of OHCs and IHCs at the basal turn of cochlea obtained from DIA1(R1204X)-TG and control mice at 25 weeks of age. Note the sparse and short (arrow) stereocilia of OHC, and sparse and fused (arrowheads) stereocilia of IHC in DIA1(R1204X)-TG mice. Scale bars: 1 µm in OHCs and 5 µm in IHCs.


A MMS - induced checkpoint activation is not altered in irc5 - Δ1 . level of Rad53 phosphorylation was analyzed in wild type ( W303 - 1a ) irc5 - Δ1 ( IL012 ) by Western blot with an anti - Rad53 antibody

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A MMS-induced checkpoint activation is not altered in irc5-Δ1 cells. The level of Rad53 phosphorylation was analyzed in wild type (W303-1a) and irc5-Δ1 (IL012) cells by Western blot with an anti-Rad53 antibody


B Prolonged checkpoint activation in irc5 - Δ1 during recovery from MMS treatment . Rad53 phosphorylation status was analyzed in wild type ( W303 - 1a ) irc5 - Δ1 ( IL012 ) by Western blot with an anti - Rad53 antibody ; M , MMS

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B Prolonged checkpoint activation in irc5-Δ1 cells during recovery from MMS treatment. Rad53 phosphorylation status was analyzed in wild type (W303-1a) and irc5-Δ1 (IL012) cells by Western blot with an anti-Rad53 antibody; M, MMS


C Lack of Irc5 results in an elevated number of with Rad52 - YFP foci . At indicated time points samples of wild type ( W3749 - 14C ) irc5 - Δ1 ( TB039 ) were collected , washed processed for microscopic . Error bars represent mean value ± standard deviations of mean ( n = 5 ) . Unpaired t - was used to calculate P - value . Representative microscopic photos of are shown . Scale bars : 5 μm

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C Lack of Irc5 results in an elevated number of cells with Rad52-YFP foci. At indicated time points samples of wild type (W3749-14C) and irc5-Δ1 (TB039) cells were collected, washed and processed for microscopic analysis. Error bars represent mean value ± standard deviations of mean (n=5). Unpaired t-test was used to calculate the P-value. Representative microscopic photos of cells are shown. Scale bars: 5 μm


D Disruption of IRC5 leads to accumulation of ssDNA - containing lesions . ChEC of wild type ( BYR52MN ) irc5 - Δ1 ( TB040 ) cultured in presence or absence of 0 . 03 % MMS for 2 hours ( n = 3 )

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D Disruption of IRC5 leads to accumulation of ssDNA-containing lesions. ChEC analysis of wild type (BYR52MN) and irc5-Δ1 (TB040) cells cultured in the presence or absence of 0.03% MMS for 2 hours (n=3)


F Increased of bright Rfa1 - YFP foci in lacking Irc5 . At indicated time points culture samples of wild type ( W3775 - 12C ) irc5 - Δ1 ( TB041 ) were collected , washed processed for microscopic . Error bars represent mean value ± standard deviations of mean ( n = 5 ) . Unpaired t - was used to calculate P - value . Representative microscopic photos of are shown . Scale bars : 5

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F Increased levels of bright Rfa1-YFP foci in cells lacking Irc5. At indicated time points culture samples of wild type (W3775-12C) and irc5-Δ1 (TB041) cells were collected, washed and processed for microscopic analysis. Error bars represent mean value ± standard deviations of mean (n=5). Unpaired t-test was used to calculate the P-value. Representative microscopic photos of cells are shown. Scale bars: 5 μm


A irc5 - Δ1 mutant exhibits S - phase delay in presence of MMS . content of wild type ( W303 - 1a ) irc5 - Δ1 ( IL012 ) was measured by FACS plotted as histograms

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A The irc5-Δ1 mutant exhibits S-phase delay in the presence of MMS. DNA content of wild type (W303-1a) and irc5-Δ1 (IL012) cells was measured by FACS and plotted as histograms


B Completion of replication is delayed in irc5 - Δ1 mutant after MMS treatment . Intact chromosomes were isolated from wild type ( W303 - 1a ) irc5 - Δ1 ( IL012 ) processed for PFGE . M , 0 . 03 % MMS

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B Completion of DNA replication is delayed in the irc5-Δ1 mutant after MMS treatment. Intact chromosomes were isolated from wild type (W303-1a) and irc5-Δ1 (IL012) cells and processed for PFGE. M, 0.03% MMS


D Samples used for PFGE in B were processed for FACS analysis


A Lack of Irc5 translocase activity results in elevated number of with Rfa1 - YFP foci . At indicated time points samples of wild type ( TB042 ) irc5DAEA ( TB043 ) were collected , washed processed for microscopic . Error bars represent mean value ± standard deviations of mean ( n = 5 ) . Unpaired t - was used to calculate P -

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A Lack of Irc5 translocase activity results in elevated number of cells with Rfa1-YFP foci. At indicated time points samples of wild type (TB042) and irc5DAEA (TB043) cells were collected, washed and processed for microscopic analysis. Error bars represent mean value ± standard deviations of mean (n=5). Unpaired t-test was used to calculate the P-value


B Increased of bright Rad52 - YFP foci in expressing Irc5DAEA lacking ATPase activity . At indicated time points samples of wild type ( TB044 ) irc5DAEA ( TB045 ) were collected , washed processed for microscopic . Error bars represent mean value ± standard deviations of mean ( n = 5 ) . Unpaired t - was used to calculate P -

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B Increased levels of bright Rad52-YFP foci in cells expressing Irc5DAEA lacking ATPase activity. At indicated time points samples of wild type (TB044) and irc5DAEA (TB045) cells were collected, washed and processed for microscopic analysis. Error bars represent mean value ± standard deviations of mean (n=5). Unpaired t-test was used to calculate the P-value


C Translocase activity of Irc5 is important for completion of replication after MMS treatment . PFGE was performed as in Fig 2B using wild type ( EMD09 ) irc5DAEA ( EMD10 )

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C Translocase activity of Irc5 is important for completion of DNA replication after MMS treatment. PFGE was performed as in Fig 2B using wild type (EMD09) and irc5DAEA (EMD10) strains


E Samples used for PFGE in C were processed for FACS analysis


A , B ChIP - qPCR of Scc1 association with early replication origin ARS305 or ARS607 . PK - tagged Scc1 interaction with was monitored in wild type ( JC1513 ) irc5Δ - 1 ( TB064 ) . Error bars represent mean value ± standard deviations of mean ( n = 3 ) . ARS305 + 7 kb locus was used as a control for cohesin - free chromosomal region . POA1 was used as a control locus for chromosomal region that is bound by cohesin but is located ~ 30 . 000 bp from nearest replication origin

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A, B ChIP-qPCR analysis of Scc1 association with early replication origin ARS305 or ARS607. PK-tagged Scc1 interaction with chromatin was monitored in wild type (JC1513) and irc5Δ-1 (TB064) cells. Error bars represent mean value ± standard deviations of mean (n=3). ARS305 + 7 kb locus was used as a control for cohesin-free chromosomal region. POA1 was used as a control locus for the chromosomal region that is bound by cohesin but is located ~ 30.000 bp from the nearest replication origin


C , D ChIP - qPCR of Pol2 association with early replication origin ARS305 or ARS607 as well as late replication origin ARS501 . HA - tagged Pol2 interaction with was monitored in wild type ( TB065 ) irc5Δ - 1 ( TB066 ) . Error bars represent mean value ± standard deviations of mean ( n = 3 )

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C, D ChIP-qPCR analysis of Pol2 association with early replication origin ARS305 or ARS607 as well as late replication origin ARS501. HA-tagged Pol2 interaction with chromatin was monitored in wild type (TB065) and irc5Δ-1 (TB066) cells. Error bars represent mean value ± standard deviations of mean (n=3)


A , B ChIP - qPCR of Scc1 association with early replication origin ARS305 or ARS607 . HA - tagged Scc1 interaction with was monitored in wild type ( Y39 ) scc2 - 4 ( Y4366 ) . Error bars represent mean value ± standard deviations of mean ( n = 3 )

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A, B ChIP-qPCR analysis of Scc1 association with early replication origin ARS305 or ARS607. HA-tagged Scc1 interaction with chromatin was monitored in wild type (Y39) and scc2-4 (Y4366) cells. Error bars represent mean value ± standard deviations of mean (n=3)


C , D ChIP - qPCR of Pol2 association with early replication origin ARS305 or ARS607 . HA - tagged Pol2 interaction with was monitored in wild type ( TB067 ) scc2 - 4 ( TB068 ) . Error bars represent mean value ± standard deviations of mean ( n = 3 )

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C, D ChIP-qPCR analysis of Pol2 association with early replication origin ARS305 or ARS607. HA-tagged Pol2 interaction with chromatin was monitored in wild type (TB067) and scc2-4 (TB068) cells. Error bars represent mean value ± standard deviations of mean (n=3)


E Cohesin loading is required for completion of chromosome replication after MMS treatment . Intact chromosomes were isolated from wild type ( Y39 ) scc2 - 4 ( Y4366 ) cultivated at permissive ( 25 ° C ) processed for PFGE . M ,

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E Cohesin loading complex is required for completion of chromosome replication after MMS treatment. Intact chromosomes were isolated from wild type (Y39) and scc2-4 (Y4366) cells cultivated at permissive temperature (25°C) and processed for PFGE. M, MMS


A Sensitivity of scc2 - 4 scc2 - 4 irc5 - Δ1 mutants to damaging agents at permissive 25 ° C . Logarithmically growing cultures of wild type ( W303 - 1a ) , irc5 - Δ1 ( IL012 ) , scc2 - 4 ( TB069 ) irc5 - Δ1 scc2 - 4 ( TB070 ) strains were 10 - fold serially diluted plated onto solid YPD containing indicated concentrations of damaging

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A Sensitivity of scc2-4 and scc2-4 irc5-Δ1 mutants to DNA damaging agents at permissive temperature 25°C. Logarithmically growing cultures of wild type (W303-1a), irc5-Δ1 (IL012), scc2-4 (TB069) and irc5-Δ1 scc2-4 (TB070) strains were 10-fold serially diluted and plated onto solid YPD containing indicated concentrations of DNA damaging agents


B , C of repair foci in scc2 - 4 scc2 - 4 irc5 - Δ1 mutants . Logarithmically growing cultures of scc2 - 4 ( TB071 or TB073 ) scc2 - 4 irc5 - Δ1 ( TB072 or TB074 ) were treated with MMS for 1 hour at 25 ° C . Next , were washed processed for microscopic . Error bars represent mean value ± standard deviations of mean ( n = 3 )

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B, C Levels of DNA repair foci in scc2-4 and scc2-4 irc5-Δ1 mutants. Logarithmically growing cultures of scc2-4 (TB071 or TB073) and scc2-4 irc5-Δ1 (TB072 or TB074) were treated with MMS for 1 hour at 25°C. Next, cells were washed and processed for microscopic analysis. Error bars represent mean value ± standard deviations of mean (n=3)


D Sensitivity of scc1 - 73 scc1 - 73 irc5 - Δ1 mutants to damaging agents at permissive 25 ° C or semi - permissive 30 ° C . Logarithmically growing cultures of wild type ( W303 - 1a ) , irc5 - Δ1 ( IL012 ) , scc1 - 73 ( JC1339 ) irc5 - Δ1 scc1 - 73 ( TB075 ) strains were 10 - fold serially diluted plated onto solid YPD containing indicated concentrations of damaging

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D Sensitivity of scc1-73 and scc1-73 irc5-Δ1 mutants to DNA damaging agents at permissive temperature 25°C or semi-permissive temperature 30°C. Logarithmically growing cultures of wild type (W303-1a), irc5-Δ1 (IL012), scc1-73 (JC1339) and irc5-Δ1 scc1-73 (TB075) strains were 10-fold serially diluted and plated onto solid YPD containing indicated concentrations of DNA damaging agents


E , F of Rfa1 - YFP Rad52 - YFP foci in scc1 - 73 irc5 - Δ1 scc1 - 73 mutants . Logarithmically growing cultures of scc1 - 73 ( TB076 or TB078 ) irc5 - Δ1 scc1 - 73 ( TB077 or TB079 ) were treated with MMS for 1 hour at 25 ° C . Next , were washed processed for microscopic . Error bars represent mean value ± standard deviations of mean ( n = 3 )

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E, F Levels of Rfa1-YFP and Rad52-YFP foci in scc1-73 and irc5-Δ1 scc1-73 mutants. Logarithmically growing cultures of scc1-73 (TB076 or TB078) and irc5-Δ1 scc1-73 (TB077 or TB079) were treated with MMS for 1 hour at 25°C. Next, cells were washed and processed for microscopic analysis. Error bars represent mean value ± standard deviations of mean (n=3)


G Replication completion defect of scc1 - 73 is not exacerbated by IRC5 disruption . Intact chromosomes were isolated from scc1 - 73 ( JC1339 ) irc5 - Δ1 scc1 - 73 ( TB075 ) , cultured at 25 ° C , processed for PFGE . M , 0 . 03 %

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G Replication completion defect of scc1-73 is not exacerbated by IRC5 disruption. Intact chromosomes were isolated from scc1-73 (JC1339) and irc5-Δ1 scc1-73 cells (TB075), cultured at 25°C, and processed for PFGE. M, 0.03% MMS


A ) Ventral nests expansion at 0 min ( onset ) ; 245 min , when ventral spiracle nests fuse ; 515 min , when intersegment nests fuse ; 750 min , when most LECs are gone . We employed for these analyses following stock : w ; If / CyO ; ZCL2207 : : hh - DsRed / MKRS . green signal corresponds to ZCL2207 ( Septate junction GFP marker ) outlining apical membranes red signal to nuclei from posterior compartment expressing RFP under control of hedgehog promoter . Arrows point to spiracular ventral nests on top panel to junction between these two nests in second panel . of confocal sections have been rotated to maintain consistent alignment of dorso - ventral axis ( dorsal is up ) . outlines of confocal are marked with a yellow dotted . Scale bar = 40 µm .

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A) Ventral nests expansion at 0 min (onset); 245 min, when ventral and spiracle nests fuse; 515 min, when intersegment nests fuse; and 750 min, when most LECs are gone. We employed for these analyses the following stock: w; If/CyO; ZCL2207::hh-DsRed/MKRS. The green signal corresponds to ZCL2207 (Septate junction GFP marker) outlining all apical cell membranes and the red signal to nuclei from the posterior compartment expressing RFP under the control of the hedgehog promoter. Arrows point to the spiracular and ventral nests on the top panel and to the junction between these two nests in the second panel. Images of confocal sections have been rotated to maintain consistent alignment of the dorso-ventral axis (dorsal is up). The outlines of the confocal images are marked with a yellow dotted line. Scale bar = 40 µm.


B ) Comparison of manual vs . PIV surface kinetics [ evolution of nests ( red - manual , black - PIV ) LECs ( cyan - manual , blue - PIV ) ]

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B) Comparison of manual vs. PIV surface area kinetics [evolution of nests (red - manual, black - PIV) and LECs (cyan - manual, blue -PIV)]


C ) Nests rate of growth from a representative single nest . Note similarity in behavior ( red - manual , black - PIV ) in three sequential phases : Phase 1 ( Ph 1 ) , accelerated growth ; Phase 2 ( Ph 2 ) , sustained growth ; Phase 3 ( Ph 3 ) decelerated growth ) .

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C) Nests rate of growth from a representative single nest. Note the similarity in behavior (red - manual, black - PIV) in three sequential phases: Phase 1 (Ph 1), accelerated growth; Phase 2 (Ph 2), sustained growth; and Phase 3 (Ph 3) decelerated growth).


D ) Progression of elongation ( red - manual , black - PIV ) of nests Note initial fast decay on elongated shape of nests .

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D) Progression of the elongation ratio (red - manual, black - PIV) of nests Note the initial fast decay on the elongated shape of the nests.


E ) Nest orientation evolution ( red - manual , black - PIV ) . Anterior is π ( 3 . 1415 rads , dorso ventral is π / 2 ( 1 . 57 rads ) posterior is 0 .

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E) Nest orientation evolution (red - manual, black - PIV). Anterior is π (3.1415 rads, dorso ventral is π/2 (1.57 rads) and posterior is 0.


A ) Snapshots of divergence of velocity field from a representative movie . Note areas of positive divergence in nests , areas of positive negative divergence in LECs , an increase of magnitude on differences between them over time . Scale bar = 40 µm .

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A) Snapshots of the divergence of the velocity field from a representative movie. Note the areas of positive divergence in the nests, areas of positive and negative divergence in the LECs, and an increase of magnitude on the differences between them over time. Scale bar = 40 µm.


B ) Frequency distribution of divergence magnitude in nests LECs in different growth phases . Nests are always shown in red LECs in blue .

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B) Frequency distribution of the divergence magnitude in the nests and LECs in the different growth phases. Nests are always shown in red and LECs in blue.


C ) Color coded radial kymographs of divergence for a representative movie . Low divergences are blue , high are red ( see scales ) . Note overall positive divergence in nests negative in LECs .

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C) Color coded radial kymographs of divergence for a representative movie. Low divergences are blue, high are red (see scales). Note the overall positive divergence in the nests and negative in the LECs.


A ) numbers in nests ( blue - left scale ) LECs territories ( red - right scale ) ( profiles are averaged from three segmented movies ) .

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A) Cell numbers in nests (blue - left scale) and LECs territories (red - right scale) (profiles are averaged from three segmented movies).


B ) Mean evolution of histoblasts ( blue - left scale ) LECs ( red - right scale ) . Note progressive increase in mean histoblast ( profiles are average from three segmented movies ) .

B) Mean cell area evolution of histoblasts (blue - left scale) and LECs (red - right scale). Note the progressive increase in mean histoblast area (profiles are average from three segmented movies).


C ) Net change rate in number variation for nests ( red ) versus nests growth ( black ) . Note temporal correlation in phases , but reduced magnitude in numbers ( profiles are collected from single nests normalized ; control black lines are retrieved from Figure 1 ) .

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C) Net change rate in cell number variation for nests (red line) versus nests growth (black line). Note the temporal correlation in phases, but the reduced magnitude in cell numbers (profiles are collected from single nests and normalized; control black lines are retrieved from Figure 1).


D ) Net change rate in number variation for LECs ( blue ) versus tissue growth ( black ) . Note full pattern correlation oscillations in LECs net rate numbers ( profiles are collected from single nests normalized ; control black lines are retrieved from Figure 1 ) .

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D) Net change rate in cell number variation for LECs (blue line) versus tissue growth (black line). Note the full pattern correlation and the oscillations in LECs net rate numbers (profiles are collected from single nests and normalized; control black lines are retrieved from Figure 1).


E ) Evolution of net change rate of histoblasts MCA ( red ) versus net change rate of nest growth ( black ) . Note delay in reaching sustained growth phase reduced magnitude of MCA rate ( profiles are collected from single nests normalized ; control black lines are retrieved from Figure 1 ) .

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E) Evolution of the net change rate of histoblasts MCA (red line) versus net change rate of nest growth (black line). Note the delay in reaching the sustained growth phase and the reduced magnitude of the MCA rate (profiles are collected from single nests and normalized; control black lines are retrieved from Figure 1).


F ) Evolution of net change rate of LECs MCA ( blue ) versus net change rate of LECs tissue growth ( black ) . Note oscillatory MCA rate around 0 tendency toward negative values ( MCA decrease ) ( profiles are collected from single nests normalized ; control black lines are retrieved from Figure 1 ) .

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F) Evolution of the net change rate of LECs MCA (blue line) versus net change rate of LECs tissue growth (black line). Note the oscillatory MCA rate around 0 and the tendency toward negative values (MCA decrease) (profiles are collected from single nests and normalized; control black lines are retrieved from Figure 1).


A ) Histoblasts Mean ( MCA ) increases over time for ( black - whole nest ) , central ( blue ) peripheral histoblasts ( red ) . B ) Snapshots at specific time points of segmented movies depicting histoblasts with color - coded areas . Color scale on right . Scale bar = 40 µm . Note progressive increase of histoblasts , specially peripheral ones . Data information : In ( B ) top panel represents central histoblasts bottom panel peripheral . We employed an arbitrary annotation consider peripheral those present in 2 outermost rows of throughout perimeter of nest at earliest recorded time point .

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A) Histoblasts Mean Cell Area (MCA) increases over time for all (black - the whole nest), central (blue) and peripheral histoblasts (red). B) Snapshots at specific time points of segmented movies depicting histoblasts with color-coded cell areas. Color scale on the right. Scale bar = 40 µm. Note the progressive increase of the histoblasts size, specially the peripheral ones. Data information: In (B) the top panel represents the central histoblasts and the bottom panel the peripheral. We employed an arbitrary annotation and consider peripheral those cells present in the 2 outermost rows of cells all throughout the perimeter of the nest at the earliest recorded time point.


C ) Histoblasts Elongation decreases over time for ( black - whole nest ) , central ( blue ) peripheral histoblasts ( red ) . D ) Color coded snapshots of elongation at different time points . Color scale on right . Scale bar = 40 µm . Data information : In ( D ) , top panel represents central histoblasts bottom panel peripheral . We employed an arbitrary annotation consider peripheral those present in 2 outermost rows of throughout perimeter of nest at earliest recorded time point .

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C) Histoblasts Elongation Ratio decreases over time for all (black - the whole nest), central (blue) and peripheral histoblasts (red). D) Color coded snapshots of cell elongation at different time points. Color scale on the right. Scale bar = 40 µm. Data information: In (D), the top panel represents the central histoblasts and the bottom panel the peripheral. We employed an arbitrary annotation and consider peripheral those cells present in the 2 outermost rows of cells all throughout the perimeter of the nest at the earliest recorded time point.


A ) Density Power radial kymograph of a representative time - lapse . Each horizontal displays for each time point ( left scale ) mean of power value at every ( bottom scale ) to nest - LECs border ( vertical black ) . Each phase is represented in a colored background with three different densities of purple . Values scale is on right . Graph representing mean ( ) standard deviation ( shadow ) of power density radial kymographs ( left scale ) at different distances ( bottom scale ) by phases ( in coded densities of purple ) for samples .

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A) Density Power radial kymograph of a representative time-lapse. Each horizontal line displays for each time point (left scale) the mean of the power value at every distance (bottom scale) to the nest-LECs border (vertical black line). Each phase is represented in a colored background with three different densities of purple. Values scale is on the right. Graph representing the mean (line) and standard deviation (shadow) of the power density radial kymographs (left scale) at different distances (bottom scale) by phases (in coded densities of purple) for all samples.


Surface Stress radial kymograph of a representative time - lapse Phases are represented in different densities of brown . Graph representing mean standard deviation of surface stress radial kymographs Values color coded in different densities of brown .

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Surface Stress radial kymograph of a representative time-lapse Phases are represented in different densities of brown. Graph representing the mean and standard deviation of the surface stress radial kymographs Values color coded in different densities of brown.


A ) Density power radial kymograph of a representative time - lapse movie of nests expansion process after blocking histoblasts proliferation by overexpressing Dacapo . Each horizontal displays at each time point ( left scale ) mean of power value at every ( bottom scale ) to nest - LECs border ( vertical black ) . Each phase is represented in a colored background with three different densities of purple . Power values color scale is on right .

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

A) Density power radial kymograph of a representative time-lapse movie of the nests expansion process after blocking histoblasts proliferation by overexpressing Dacapo. Each horizontal line displays at each time point (left scale) the mean of the power value at every distance (bottom scale) to the nest-LECs border (vertical black line). Each phase is represented in a colored background with three different densities of purple. Power values color scale is on the right.


B ) Comparison of means standard deviations of density power radial kymographs in phase 3 for Dacapo overexpressing nests ( green ) wild type ( purple ) .

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B) Comparison of the means and standard deviations of the density power radial kymographs in phase 3 for Dacapo overexpressing nests (green) and wild type (purple).


C ) Surface Stress radial kymograph of a representative time - lapse movie Phases are represented in different densities of brown . Stress values color scale is on right .

O O O O O O 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) Surface Stress radial kymograph of a representative time-lapse movie Phases are represented in different densities of brown. Stress values color scale is on the right.


D ) Comparison of means standard deviations of surface stress radial kymographs in phase 3 for Dacapo overexpressing nests ( green ) wild type ( brown ) .

D) Comparison of the means and standard deviations of the surface stress radial kymographs in phase 3 for Dacapo overexpressing nests (green) and wild type (brown).


E ) Density power radial kymograph of a representative time - lapse movie after blocking LECs delamination by overexpressing P35 . Power values color scale is on right .

E) Density power radial kymograph of a representative time-lapse movie after blocking LECs delamination by overexpressing P35. Power values color scale is on the right.


F ) Comparison of means standard deviations of density power radial kymographs in phase 3 for P35 overexpressing LECs ( green ) wild type ( purple ) .

F) Comparison of the means and standard deviations of the density power radial kymographs in phase 3 for P35 overexpressing LECs (green) and wild type (purple).


G ) Surface Stress radial kymograph of a representative time - lapse movie Phases are represented in different densities of brown . Power values color scale is on right .

G) Surface Stress radial kymograph of a representative time-lapse movie Phases are represented in different densities of brown. Power values color scale is on the right.


H ) Comparison of means standard deviations of surface stress radial kymograph in phase 3 for P35 overexpressing LECs ( green ) wild type ( brown ) .

H) Comparison of the means and standard deviations of the surface stress radial kymograph in phase 3 for P35 overexpressing LECs (green) and wild type (brown).


A ) microsurgery was performed at different sides of ventral nests during phase 2 ( 300 minutes ) in parallel cuts [ in LEC tissue at 40 µm of nests / LEC border ( cyan ) within nests at 10 µm ( red ) 40 µm ( green ) of edge ] . Myo - GFP transgenic pupae were used to visualize cortex displacements . This example illustrates at anterior edge cuts positions . Scale bar = 15 µm .

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A) Laser microsurgery was performed at the different sides of the ventral nests during phase 2 (300 minutes) in parallel cuts [in the LEC tissue at 40 µm of the nests/LEC border (cyan) and within the nests at 10 µm (red) and 40 µm (green) of the edge]. Myo-GFP transgenic pupae were used to visualize the cortex displacements. This example illustrates at the anterior edge the laser cuts positions. Scale bar = 15 µm.


B ) Cortex recoil velocity ( pixels per second ) at different time points after cutting ( + 10 sec ) for a single experiment . Note exponentially decaying speed .

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B) Cortex recoil velocity (pixels per second) at different time points after laser cutting (+ 10 sec) for a single experiment. Note the exponentially decaying speed.


D ) Snapshots of a cut in nest . PIV arrows are shown in red . Scale bar = 5 µm .

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D) Snapshots of a laser cut in the nest area. PIV arrows are shown in red. Scale bar = 5 µm.


E ) Comparison of mean recoil velocities in phase 3 for Dacapo overexpressing nests ( green ) with wild type ( blue ) at different distances ( - 40 - n = 16 , - 10 - n = 22 , + 40 - n = 14 ) Error bars represent Standard Deviations .

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E) Comparison of the mean laser recoil velocities in phase 3 for Dacapo overexpressing nests (green) with wild type (blue) at different distances (-40 - n = 16, -10 - n =22, +40 - n = 14) Error bars represent Standard Deviations.


F ) Comparison of mean recoil velocities in phase 3 for P35 overexpressing nests ( green ) with wild type ( blue ) at different distances ( - 40 - n = 17 , - 10 - n = 20 , + 40 - n = 15 ) Error bars represent Standard Deviations .

F) Comparison of the mean laser recoil velocities in phase 3 for P35 overexpressing nests (green) with wild type (blue) at different distances (-40 - n = 17, -10 - n = 20, +40 - n = 15) Error bars represent Standard Deviations.


A . Serum of 15 - keto - dihydro - PGF2α in type 2 diabetic without diabetic retinopathy ( no DR ) , with non - proliferative diabetic retinopathy ( NPDR ) , with proliferative diabetic retinopathy ( PDR ) ( n = 20 - 24 ) . Data information : n . s . stands for " not significant . " Data were analyzed by Kruskal - Wallis with Dunn ' s multiple comparisons ( A ) , Data are represented as mean ± .

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A. Serum levels of 15-keto-dihydro-PGF2α in type 2 diabetic patients without diabetic retinopathy (no DR), with non-proliferative diabetic retinopathy (NPDR), and with proliferative diabetic retinopathy (PDR) (n=20-24). Data information: n.s. stands for "not significant." Data were analyzed by Kruskal-Wallis test with Dunn's multiple comparisons test (A), Data are represented as mean ± SEM.


C . Retinal PGF2α production in OIR mice during postnatal days 12 - 17 ( n = 4 ) . Data information : n . s . stands for " not significant . " Data were analyzed by , two - way ANOVA with Tukey ' s multiple comparisons ( C , Data are represented as mean ± .

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C. Retinal PGF2α production in OIR mice during postnatal days 12-17 (n=4). Data information: n.s. stands for "not significant." Data were analyzed by , two-way ANOVA with Tukey's multiple comparisons test (C, Data are represented as mean ± SEM.


D . mRNA expression of PGF2α synthases in retinas of OIR mice during postnatal days 12 - 17 ( n = E . mRNA expression of Ptgfr in retinas of OIR mice during postnatal days 12 - 17 ( n = 6 - 8 ) . Data information : n . s . stands for " not significant . " Data were analyzed by , two - way ANOVA with Tukey ' s multiple comparisons D , E , Data are represented as mean ± .

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D. mRNA expression of PGF2α synthases in the retinas of OIR mice during postnatal days 12-17 (n= E. mRNA expression of Ptgfr in the retinas of OIR mice during postnatal days 12-17 (n=6-8). Data information: n.s. stands for "not significant." Data were analyzed by , two-way ANOVA with Tukey's multiple comparisons test D, E, Data are represented as mean ± SEM.


F . mRNA expression of Ptgfr in each retinal layer from normoxic OIR mice on postnatal day 16 ( n = 6 ) . left - image displays representative cross - sections from OIR normoxic retinas , with isolectin B4 - stained vessels in green , DAPI - stained nuclei in blue , LCM isolation locations circled by dashed circles . INL , inner layer ; ONL , outer layer ; RGC , retinal ganglion . Data information : n . s . stands for " not significant . " Data were analyzed by two - way ANOVA with Tukey ' s multiple comparisons F ) Scale bar : 20 μm . Data are represented as mean ± .

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F. mRNA expression of Ptgfr in each retinal layer from normoxic and OIR mice on postnatal day 16 (n=6). The left-hand image displays representative cross-sections from OIR and normoxic retinas, with isolectin B4-stained vessels in green, DAPI-stained nuclei in blue, and LCM isolation locations circled by white dashed circles. INL, inner nuclear layer; ONL, outer nuclear layer; RGC, retinal ganglion cells. Data information: n.s. stands for "not significant." Data were analyzed by two-way ANOVA with Tukey's multiple comparisons test F) Scale bar: 20 μm. Data are represented as mean ± SEM.


G . Ptgfr mRNA expression in retinal endothelial ( CD31 + CD45 - ) sorted by MACS from normoxic OIR mice on postnatal day 16 ( n = 5 ) . Data information : n . s . stands for " not significant . " Data were analyzed Mann - Whitney ( G ) . Scale bar : 20 μm . Data are represented as mean ± .

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G. Ptgfr mRNA expression in retinal endothelial cells (CD31+CD45-) sorted by MACS from normoxic and OIR mice on postnatal day 16 (n=5). Data information: n.s. stands for "not significant." Data were analyzed Mann-Whitney test (G). Scale bar: 20 μm. Data are represented as mean ± SEM.


B . Ptgfr mRNA in retinal microvessels of CKO - T control mice ( n = 6 ) . INL , inner layer ; ONL , outer layer ; RGC , retinal ganglion . Data information : n . s . stands for " not significant . " Data were analyzed by Mann - Whitney ( B , Data are represented as mean ± .

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B. Ptgfr mRNA levels in retinal microvessels of CKO-T and control mice (n=6). INL, inner nuclear layer; ONL, outer nuclear layer; RGC, retinal ganglion cells. Data information: n.s. stands for "not significant." Data were analyzed by Mann-Whitney test (B, Data are represented as mean ± SEM.


C . Representative of OIR retinas from CKO - T control mice on postnatal day 17 . green color shows isolectin B4 - stained vessels . second - row panels are enlarged of boxes from first - row panels . third - row show neovascular tufts ( NV , ) fourth - row show vaso - obliteration ( VO , ) . D . Quantitation of oxygen - induced retinal neovascularization in CKO - T control mice ( n = 12 ) . E . Quantitation of retinal vaso - obliteration in CKO - T control mice ( n = 12 ) . Data information : n . s . stands for " not significant . " Data were analyzed by Mann - Whitney D , E ) . Scale bars : 500 μm ( unmagnified image ) , 150 μm ( magnified image ) . Data are represented as mean ± .

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C. Representative images of OIR retinas from CKO-T and control mice on postnatal day 17. The green color shows the isolectin B4-stained vessels. The second-row panels are the enlarged images of white boxes from the first-row panels. The third-row images show neovascular tufts (NV, white) and the fourth-row images show the vaso-obliteration (VO, white) area. D. Quantitation of oxygen-induced retinal neovascularization in CKO-T and control mice (n=12). E. Quantitation of retinal vaso-obliteration in CKO-T and control mice (n=12). Data information: n.s. stands for "not significant." Data were analyzed by Mann-Whitney test D, E). Scale bars: 500 μm (unmagnified image), 150 μm (magnified image). Data are represented as mean ± SEM.


A . Representative of scratch - induced migration of PGF2α - treated HRMECs . Yellow solid lines represent scratch locations , dashed lines represent post - migration locations , arrows indicate migration direction . B . Quantitation of migration in A ( n = 4 ) . C . Effect of PGF2α treatment on HRMEC proliferation in a dose - dependent manner . ( n = 6 ) . Data information : One data point represented mean value of three technical replicates from one independent biological replicate . Data were analyzed by Mann - Whitney ( B , Kruskal - Wallis with Dunn ' s multiple comparisons ( C ) . Scale bars : 100 μm ( A ) Data are represented as mean ± .

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A. Representative images of scratch-induced migration of PGF2α-treated HRMECs. Yellow solid lines represent scratch locations, dashed lines represent post-migration locations, and arrows indicate migration direction. B. Quantitation of the cell migration distance in A (n=4). C. Effect of PGF2α treatment on HRMEC proliferation in a dose-dependent manner. (n=6). Data information: One data point represented the mean value of three technical replicates from one independent biological replicate. Data were analyzed by Mann-Whitney test (B, Kruskal-Wallis test with Dunn's multiple comparisons test (C). Scale bars: 100 μm (A) Data are represented as mean ± SEM.


D . Representative of for BrdU incorporation in PGF2α - treated HRMECs . E . of BrdU + in D ( n = 4 ) . Data information : One data point represented mean value of three technical replicates from one independent biological replicate . Data were analyzed by Mann - Whitney Data are represented as mean ± .

D. Representative images of staining for BrdU incorporation in PGF2α-treated HRMECs. E. Quantification of BrdU+ cells in D (n=4). Data information: One data point represented the mean value of three technical replicates from one independent biological replicate. Data were analyzed by Mann-Whitney test Data are represented as mean ± SEM.


F . Representative tube formation of PGF2α - or vehicle ( DMSO ) - treated HRMECs . G - J . Quantitation of total tubule , number of junctions , number of meshes , of mesh ( n = 4 ) . Data information : One data point represented mean value of three technical replicates from one independent biological replicate . Data were analyzed by Mann - Whitney G , H , I , J , Scale bars : 200 μm ( F , Data are represented as mean ± .

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F. Representative tube formation images of PGF2α-or vehicle (DMSO)-treated HRMECs. G-J. Quantitation of total tubule length, number of junctions, number of meshes, and percentage of mesh area (n=4). Data information: One data point represented the mean value of three technical replicates from one independent biological replicate. Data were analyzed by Mann-Whitney test G, H, I, J, Scale bars: 200 μm (F, Data are represented as mean ± SEM.


K . Representative of endothelial sprouting of PGF2α - treated mouse aortic rings . Yellow lines indicate EC sprouting . L . of EC sprouting ( n = 6 ) . Data information : One data point represented mean value of three technical replicates from one independent biological replicate . Data were analyzed by Mann - Whitney L ) Scale bars : 200 μm K ) . Data are represented as mean ± .

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K. Representative images of endothelial cell sprouting of PGF2α-treated mouse aortic rings. Yellow lines indicate the EC sprouting area. L. Quantification of the EC sprouting area (n=6). Data information: One data point represented the mean value of three technical replicates from one independent biological replicate. Data were analyzed by Mann-Whitney test L) Scale bars: 200 μm K). Data are represented as mean ± SEM.


A . Heat map of upregulated genes in PGF2α - treated HRMECs ( n = 3 samples ) .

A. Heat map of upregulated genes in PGF2α-treated HRMECs (n=3 samples).


C . Effect of PGF2α treatment on ELR + CXC chemokine expression in HRMECs ( n = 3 ) . Data information : n . s . stands for " not significant . " Data were analyzed by unpaired Student ' s t - ( C ) Data in C .

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C. Effect of PGF2α treatment on ELR+ CXC chemokine expression in HRMECs (n=3). Data information: n.s. stands for "not significant." Data were analyzed by unpaired Student's t-test (C) Data in C.


D - G . Relative expression of enriched ELR + CXC chemokines in retinal microvascular ECs from with PDR ( n = 4 ) compared with those from normal subjects ( n = 7 ) ( Gene Expression Omnibus Database dataset GSE94019 ) . TPM stands for " Transcripts per kilobase million . " Data information : n . s . stands for " not significant . " Data were analyzed by Mann - Whitney ( D , E , F , G ) , Data are presented as mean + SD .

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D-G. Relative expression levels of enriched ELR+ CXC chemokines in retinal microvascular ECs from patients with PDR (n=4) compared with those from normal subjects (n=7) (Gene Expression Omnibus Database dataset GSE94019). TPM stands for "Transcripts per kilobase million." Data information: n.s. stands for "not significant." Data were analyzed by Mann-Whitney test (D, E, F, G), Data are presented as the mean + SD.


H . Effect of CXCR2 inhibitor SB265610 ( 1 μM ) on PGF2α - stimulated HRMEC migration . yellow solid indicates original position of scratch , dashed indicates position after migration , arrows indicate migratory direction . I . Quantitation of scratch - induced HRMEC migration in H ( n = 4 ) . J . Effect of SB265610 ( 1 μM ) on PGF2α - stimulated HRMEC proliferation ( n = 4 ) . Data information : n . s . stands for " not significant . " Data were analyzed by two - way ANOVA with Tukey ' s multiple comparisons ( I , J , Data in I . J . are represented as mean ± . Scale bar : 100 μm ( H )

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H. Effect of CXCR2 inhibitor SB265610 (1 μM) on PGF2α-stimulated HRMEC migration. The yellow solid line indicates the original position of the scratch, the dashed line indicates the position after cell migration, and the arrows indicate the migratory direction. I. Quantitation of scratch-induced HRMEC migration in H (n=4). J. Effect of SB265610 (1 μM) on PGF2α-stimulated HRMEC proliferation (n=4). Data information: n.s. stands for "not significant." Data were analyzed by two-way ANOVA with Tukey's multiple comparisons test (I, J, Data in I. J. are represented as mean ± SEM. Scale bar: 100 μm (H)