Figure 1 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 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 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 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 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

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1 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 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 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 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 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


Figure 2 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 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

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

Figure 2 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 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


Figure 3 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 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 - valueC 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 ) strainsE Samples used for PFGE in C were processed for FACS

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O 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-PANEL_START O O O O O O O O O O

Figure 3 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 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-valueC 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) strainsE Samples used for PFGE in C were processed for FACS analysis


Figure 5 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 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 )

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

Figure 5 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 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)


Figure 6 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 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 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 ,

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6 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 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 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


Figure 7A 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 agentsB , 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 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 agentsE , 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 ) and irc5 - Δ1 scc1 - 73 cells ( TB075 ) , cultured at 25 ° C , and processed for PFGE . M , 0 . 03 %

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7A 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 agentsB, 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 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 agentsE, 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) and irc5-Δ1 scc1-73 cells (TB075), cultured at 25°C, and processed for PFGE. M, 0.03% MMS


Figure 1A ) 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 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 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 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 ) and posterior is 0 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1A) 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 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 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 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) and posterior is 0.


Figure 2A ) 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 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 the overall positive divergence in the nests and negative in the LECs .

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Figure 2A) 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 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 the overall positive divergence in the nests and negative in the LECs.


Figure 3A ) Cell numbers in nests ( blue - left scale ) and LECs territories ( red - right scale ) ( profiles are averaged 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 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 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 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 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 ) .

O B-PANEL_START O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3A) Cell numbers in nests (blue - left scale) and LECs territories (red - right scale) (profiles are averaged 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 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 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 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 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).


Figure 4A ) 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 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 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4A) 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 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.


Figure 6A ) 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 the mean and standard deviation of the surface stress radial kymographs Values color coded in different densities of brown .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6A) 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 the mean and standard deviation of the surface stress radial kymographs Values color coded in different densities of brown.


Figure 7A ) 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 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 the right . 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 the right . 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 the right . 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 ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O 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-PANEL_START O O O O O O 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-PANEL_START O O O O O O O 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-PANEL_START O O O O O 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-PANEL_START O O O O O O O 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-PANEL_START O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7A) 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 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 the right.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 the right.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 the right.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).


Figure 8A ) 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 laser cutting ( + 10 sec ) for a single experiment . Note the exponentially decaying speed . D ) Snapshots of a laser cut in the nest area . PIV arrows are shown in red . Scale bar = 5 µm . 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 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 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O 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-PANEL_START 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 8A) 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 laser cutting (+ 10 sec) for a single experiment. Note the exponentially decaying speed.D) Snapshots of a laser cut in the nest area. PIV arrows are shown in red. Scale bar = 5 µm.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 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.


Figure 1A . 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 test ( C , Data are represented as mean ± SEM . 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 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 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 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1A. 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 test (C, Data are represented as mean ± SEM.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 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 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.


Figure 2B . 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 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 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2B. 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 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.


Figure 3A . 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 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 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 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 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3A. 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 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 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 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.


Figure 4A . 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 - test ( C ) Data in C . 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 . 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 ) K . Effect of CXCR2 inhibitor SB265610 ( 1μM ) on tube formation in PGF2α - treated HRMECs . L . Statistical graph of tube formation experiments in K , comparing the total tubule length , number of junctions , number of meshes , and percentage of mesh area ( n = 4 ) . Data information : n . s . stands for " not significant . " Data were analyzed by two - way ANOVA with Tukey ' s multiple comparisons test L ) . Data in L are represented as mean ± SEM . Scale bar : 200 μm ( K ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4A. 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-test (C) Data in C.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. 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)K. Effect of CXCR2 inhibitor SB265610 (1μM) on tube formation in PGF2α-treated HRMECs. L. Statistical graph of tube formation experiments in K, comparing the total tubule length, number of junctions, number of meshes, and percentage of mesh area (n=4). Data information: n.s. stands for "not significant." Data were analyzed by two-way ANOVA with Tukey's multiple comparisons test L). Data in L are represented as mean ± SEM. Scale bar: 200 μm (K).


Figure 5A . Effect of CXCR2 inhibitor SB265610 ( 1 μM ) on PGF2α - promoted sprouting of aortic rings . Yellow lines show the sprouting area . B . Quantitation of the sprouting areas in A ( n = 6 ) . Data information : n . s . stands for " not significant . " Data were analyzed by two - way ANOVA with Tukey ' s multiple comparisons test ( B , Scale bar : 200 μm ( A ) , image ) . Data are represented as mean ± SEM . C . mRNA expression of ELR + CXC chemokines in retinas from OIR mice during postnatal days 12 - 17 ( n = 4 - 6 ) . Data information : n . s . stands for " not significant . " Data were analyzed by two - way ANOVA with Tukey ' s multiple comparisons test image ) . Data are represented as mean ± SEM . D . Effect of PTGFR deficiency in ECs on ELR + CXC chemokine expression in retinas from OIR mice on postnatal day 16 ( n = 6 ) . Data information : n . s . stands for " not significant . " Data were analyzed by two - way ANOVA with Tukey ' s multiple comparisons test image ) . Data are represented as mean ± SEM . F . Retinal CXCL1 protein - expression levels in Cxcl1 - expressing lentivirus - injected CKO mice ( n = 3 ) . G . Quantitation of retinal CXCL1 protein in F ( n = 3 ) . Data information : n . s . stands for " not significant . " Data were analyzed by two - way ANOVA with Tukey ' s multiple comparisons test G , image ) . Data are represented as mean ± SEM . H . Representative images of oxygen - induced retinal angiogenesis in Cxcl1 - expressing lentivirus - injected CKO mice on postnatal day 17 . Green represents the isolectin B4 - stained vessels , the second - row panels display the enlarged images of white boxes in 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 . I . Quantitation of oxygen - induced retinal neovascularization in H ( n = 8 ) . J . Quantitation of retinal vaso - obliteration in H ( n = 8 ) . Data information : n . s . stands for " not significant . " Data were analyzed by two - way ANOVA with Tukey ' s multiple comparisons test , I , J ) . Scale bar : 500 μm ( H , unmagnified image ) , 150 μm ( H , magnified image ) . Data are represented as mean ± SEM .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5A. Effect of CXCR2 inhibitor SB265610 (1 μM) on PGF2α-promoted sprouting of aortic rings. Yellow lines show the sprouting area. B. Quantitation of the sprouting areas in A (n=6). Data information: n.s. stands for "not significant." Data were analyzed by two-way ANOVA with Tukey's multiple comparisons test (B, Scale bar: 200 μm (A), image).Data are represented as mean ± SEM.C. mRNA expression of ELR+ CXC chemokines in retinas from OIR mice during postnatal days 12-17 (n=4-6). Data information: n.s. stands for "not significant." Data were analyzed by two-way ANOVA with Tukey's multiple comparisons test image).Data are represented as mean ± SEM.D. Effect of PTGFR deficiency in ECs on ELR+ CXC chemokine expression in retinas from OIR mice on postnatal day 16 (n=6). Data information: n.s. stands for "not significant." Data were analyzed by two-way ANOVA with Tukey's multiple comparisons test image).Data are represented as mean ± SEM.F. Retinal CXCL1 protein-expression levels in Cxcl1-expressing lentivirus-injected CKO mice (n=3). G. Quantitation of retinal CXCL1 protein in F (n=3). Data information: n.s. stands for "not significant." Data were analyzed by two-way ANOVA with Tukey's multiple comparisons test G, image).Data are represented as mean ± SEM.H. Representative images of oxygen-induced retinal angiogenesis in Cxcl1-expressing lentivirus-injected CKO mice on postnatal day 17. Green represents the isolectin B4-stained vessels, the second-row panels display the enlarged images of white boxes in 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. I. Quantitation of oxygen-induced retinal neovascularization in H (n=8). J. Quantitation of retinal vaso-obliteration in H (n=8). Data information: n.s. stands for "not significant." Data were analyzed by two-way ANOVA with Tukey's multiple comparisons test , I, J). Scale bar: 500 μm (H, unmagnified image), 150 μm (H, magnified image).Data are represented as mean ± SEM.


Figure 6A . Heat map of differentially expressed transcription factors in HRMECs in response to 500 nM PGF2α ( n = 3 ) . B . Relative expression levels of FOS in retinal microvascular ECs from patients with PDR ( n = 4 ) compared with the corresponding expression levels 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 ( B , Data in B are expressed as the mean + SD . C . Effect of FOS knockdown on 500 nM PGF2α - induced CXCL8 expression in HRMECs ( n = 4 ) . D . Effect of FOS knockdown on 500 nM PGF2α - induced HRMEC CXCL8 secretion in the culture medium ( n = 4 ) . Data information : n . s . stands for " not significant . " Data were analyzed by Mann - Whitney test C , D , Data in C , D , are represented as mean ± SEM . E . and F . Effect of RA ( 1 μM , AP - 1 dimer inhibitor ) , SN - 50 ( 100 μg / mL , NFκB inhibitor ) , and Inca - 6 ( 2 . 5 μM , NFAT inhibitor ) on 500 nM PGF2α - induced CXCL8 expression in HRMECs and HRMEC CXCL8 secretion in the culture medium ( n = 4 ) . Data information : n . s . stands for " not significant . " Data were analyzed by Mann - Whitney test E , F , Data in E , F , are represented as mean ± SEM . G . Representative images of scratch - induced migration of 500 nM PGF2α - treated HRMECs with or without FOS knockdown . The yellow solid line indicates the original position of the scratch , the dashed line indicates the position of the cells after migration , and the arrows represent the migratory direction . H . Quantitation of the cell - migration distance in H ( n = 4 ) . I . Effect of FOS knockdown on 500 nM PGF2α - induced 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 ( H , I Scale bar : 100 μm ( G ) Data in H , I , are represented as mean ± SEM . J . Representative images of tube formation by 500 nM PGF2α - treated HRMECs with or without FOS knockdown . K . Quantitation of total tube length , number of junctions , number of meshes , and percentage of mesh area in J ( n = 4 ) . Data information : n . s . stands for " not significant . " Data were analyzed by two - way ANOVA with Tukey ' s multiple comparisons test K , Scale bar : 200 μm ( J , Data in K , are represented as mean ± SEM . L . Fos mRNA levels in retinas from OIR mice during postnatal days 12 - 17 ( n = 3 - 4 ) . Data information : n . s . stands for " not significant . " Data were analyzed by two - way ANOVA with Tukey ' s multiple comparisons test L , Data in , L , are represented as mean ± SEM . M . Effect of PTGFR deficiency in ECs on Fos expression in the retina from OIR mice on postnatal day 16 ( n = 6 ) . Data information : n . s . stands for " not significant . " Data were analyzed by Mann - Whitney test M , Data in M , are represented as mean ± SEM . N . Effect of FOS inhibitor SR11302 ( 1 μM ) on 500 nM PGF2α - promoted sprouting of mouse aortic rings . Data information : Scale bar : 200 μm N ) . O . Quantitation of sprouting areas in N ( n = 6 ) . Data information : n . s . stands for " not significant . " Data were analyzed by two - way ANOVA with Tukey ' s multiple comparisons test O ) . Data in O , are represented as mean ± SEM . P . Effect of FOS inhibitor SR11302 ( 1 μM ) on 500 nM PGF2α - promoted CXCL1 secretion in mouse aortic ring culture medium ( n = 6 ) . Data information : n . s . stands for " not significant . " Data were analyzed by Mann - Whitney test P ) Data in P are represented as mean ± SEM .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6A. Heat map of differentially expressed transcription factors in HRMECs in response to 500 nM PGF2α (n=3).B. Relative expression levels of FOS in retinal microvascular ECs from patients with PDR (n=4) compared with the corresponding expression levels 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 (B, Data in B are expressed as the mean + SD.C. Effect of FOS knockdown on 500 nM PGF2α-induced CXCL8 expression in HRMECs (n=4). D. Effect of FOS knockdown on 500 nM PGF2α-induced HRMEC CXCL8 secretion in the culture medium (n=4). Data information: n.s. stands for "not significant." Data were analyzed by Mann-Whitney test C, D, Data in C, D, are represented as mean ± SEM. E. and F. Effect of RA (1 μM, AP-1 dimer inhibitor), SN-50 (100 μg/mL, NFκB inhibitor), and Inca-6 (2.5 μM, NFAT inhibitor) on 500 nM PGF2α-induced CXCL8 expression in HRMECs and HRMEC CXCL8 secretion in the culture medium (n=4). Data information: n.s. stands for "not significant." Data were analyzed by Mann-Whitney test E, F, Data in E, F, are represented as mean ± SEM. G. Representative images of scratch-induced migration of 500 nM PGF2α-treated HRMECs with or without FOS knockdown. The yellow solid line indicates the original position of the scratch, the dashed line indicates the position of the cells after migration, and the arrows represent the migratory direction. H. Quantitation of the cell-migration distance in H (n=4). I. Effect of FOS knockdown on 500 nM PGF2α-induced 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 (H, I Scale bar: 100 μm (G) Data in H, I, are represented as mean ± SEM. J. Representative images of tube formation by 500 nM PGF2α-treated HRMECs with or without FOS knockdown. K. Quantitation of total tube length, number of junctions, number of meshes, and percentage of mesh area in J (n=4). Data information: n.s. stands for "not significant." Data were analyzed by two-way ANOVA with Tukey's multiple comparisons test K, Scale bar: 200 μm (J, Data in K, are represented as mean ± SEM. L. Fos mRNA levels in retinas from OIR mice during postnatal days 12-17 (n=3-4). Data information: n.s. stands for "not significant." Data were analyzed by two-way ANOVA with Tukey's multiple comparisons test L, Data in , L, are represented as mean ± SEM. M. Effect of PTGFR deficiency in ECs on Fos expression in the retina from OIR mice on postnatal day 16 (n=6). Data information: n.s. stands for "not significant." Data were analyzed by Mann-Whitney test M, Data in M, are represented as mean ± SEM. N. Effect of FOS inhibitor SR11302 (1 μM) on 500 nM PGF2α-promoted sprouting of mouse aortic rings. Data information: Scale bar: 200 μm N).O. Quantitation of sprouting areas in N (n=6). Data information: n.s. stands for "not significant." Data were analyzed by two-way ANOVA with Tukey's multiple comparisons test O). Data in O, are represented as mean ± SEM. P. Effect of FOS inhibitor SR11302 (1 μM) on 500 nM PGF2α-promoted CXCL1 secretion in mouse aortic ring culture medium (n=6). Data information: n.s. stands for "not significant." Data were analyzed by Mann-Whitney test P) Data in P are represented as mean ± SEM.


Figure 7B . Effect of U73122 ( 10 μM ) , BAPTA ( 50 μM ) , Y27632 ( 5 μM ) , and Ly294002 ( 15 μM ) treatment on 500 nM PGF2α - induced FOS gene expression in HRMECs ( n = 4 ) . C . Effect of KN93 ( 10 μM ) and RO - 318220 ( 250 nM ) treatment on 500 nM PGF2α - induced FOS expression in HRMECs ( n = 4 ) . Data information : n . s . stands for " not significant . " Data were analyzed by Mann - Whitney test ( B , C Data are represented as mean ± SEM . D . Effect of SB203580 ( 2 μM ) , PD98059 ( 20 μM ) , and SP600125 ( 60 nM ) treatment on 500 nM PGF2α - induced FOS gene expression in HRMECs ( n = 4 ) . Data information : n . s . stands for " not significant . " Data were analyzed by Mann - Whitney test Data are represented as mean ± SEM . E . Effect of U73122 ( 10 μM ) , BAPTA ( 50 μM ) , and KN93 ( 10 μM ) treatment on 500 nM PGF2α - induced p38 phosphorylation in HRMECs . F . Quantitation of the ratio of p38 phosphorylation to total p38 protein in E ( n = 3 ) . Data information : n . s . stands for " not significant . " Data were analyzed by unpaired student ' s t - test ( F , Data are represented as mean ± SEM . G . Effect of U73122 ( 10 μM ) , BAPTA ( 50 μM ) , and SB203580 ( 2 μM ) on the phosphorylation of CAMK2 isoforms in 500 nM PGF2α - treated HRMECs . H . Quantitation of the ratio of CAMK2G phosphorylation to total CAMK2G in G ( n = 3 ) . I . Quantitation of the ratio of phosphorylated CAMK2D to total CAMK2D in G ( n = 3 ) . Data information : n . s . stands for " not significant . " Data were analyzed by unpaired student ' s t - test H , I , Data are represented as mean ± SEM . J . Effect of CAMK2G or CAMK2D knockdown on 500 nM PGF2α - triggered p38 phosphorylation in HRMECs . K . Quantitation of the ratio of p38 phosphorylation to total p38 protein in J ( n = 3 ) . Data information : n . s . stands for " not significant . " Data were analyzed by unpaired student ' s t - test Data are represented as mean ± SEM . L . Effect of CAMK2G or CAMK2D knockdown on 500 nM PGF2α - induced FOS expression in HRMECs ( n = 4 ) . Data information : n . s . stands for " not significant . " Data were analyzed by Mann - Whitney test L ) Data are represented as mean ± SEM . M . Effect of U73122 ( 10 μM ) , BAPTA ( 50 μM ) , KN93 ( 10 μM ) , and SB203580 ( 2 μM ) on ELK - 1 phosphorylation in 500 nM PGF2α - treated HRMECs . N . Quantitation of the ratio of phosphorylated ELK - 1 to total ELK - 1 in M ( n = 3 ) . Data information : n . s . stands for " not significant . " Data were analyzed by unpaired student ' s t - test N ) . Data are represented as mean ± SEM .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7B. Effect of U73122 (10 μM), BAPTA (50 μM), Y27632 (5 μM), and Ly294002 (15 μM) treatment on 500 nM PGF2α-induced FOS gene expression in HRMECs (n=4). C. Effect of KN93 (10 μM) and RO-318220 (250 nM) treatment on 500 nM PGF2α-induced FOS expression in HRMECs (n=4). Data information: n.s. stands for "not significant." Data were analyzed by Mann-Whitney test (B, C Data are represented as mean ± SEM.D. Effect of SB203580 (2 μM), PD98059 (20 μM), and SP600125 (60 nM) treatment on 500 nM PGF2α-induced FOS gene expression in HRMECs (n=4). Data information: n.s. stands for "not significant." Data were analyzed by Mann-Whitney test Data are represented as mean ± SEM.E. Effect of U73122 (10 μM), BAPTA (50 μM), and KN93 (10 μM) treatment on 500 nM PGF2α-induced p38 phosphorylation in HRMECs. F. Quantitation of the ratio of p38 phosphorylation to total p38 protein in E (n=3). Data information: n.s. stands for "not significant." Data were analyzed by unpaired student's t-test (F, Data are represented as mean ± SEM.G. Effect of U73122 (10 μM), BAPTA (50 μM), and SB203580 (2 μM) on the phosphorylation of CAMK2 isoforms in 500 nM PGF2α-treated HRMECs. H. Quantitation of the ratio of CAMK2G phosphorylation to total CAMK2G in G (n=3). I. Quantitation of the ratio of phosphorylated CAMK2D to total CAMK2D in G (n=3). Data information: n.s. stands for "not significant." Data were analyzed by unpaired student's t-test H, I, Data are represented as mean ± SEM.J. Effect of CAMK2G or CAMK2D knockdown on 500 nM PGF2α-triggered p38 phosphorylation in HRMECs. K. Quantitation of the ratio of p38 phosphorylation to total p38 protein in J (n=3). Data information: n.s. stands for "not significant." Data were analyzed by unpaired student's t-test Data are represented as mean ± SEM.L. Effect of CAMK2G or CAMK2D knockdown on 500 nM PGF2α-induced FOS expression in HRMECs (n=4). Data information: n.s. stands for "not significant." Data were analyzed by Mann-Whitney test L) Data are represented as mean ± SEM.M. Effect of U73122 (10 μM), BAPTA (50 μM), KN93 (10 μM), and SB203580 (2 μM) on ELK-1 phosphorylation in 500 nM PGF2α-treated HRMECs. N. Quantitation of the ratio of phosphorylated ELK-1 to total ELK-1 in M (n=3). Data information: n.s. stands for "not significant." Data were analyzed by unpaired student's t-test N). Data are represented as mean ± SEM.


Figure 8B . Representative images of OIR retinas in WT and Cxcr2 - / - mice with or without AL8810 treatment . The green color shows the isolectin B4 - stained vessels , the second - row panels display the enlarged images of white boxes in 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 . C . Quantitation of oxygen - induced retinal neovascularization in WT and Cxcr2 - / - mice with or without AL8810 treatment ( n = 12 ) . D . Quantitation of retinal vaso - obliteration in WT and Cxcr2 - / - mice with or without AL8810 treatment ( n = 12 ) . Data information : n . s . stands for " not significant . " Data were analyzed by two - way ANOVA with Tukey ' s multiple comparisons test ( C , D ) Scale bar : 500 μm ( unmagnified image ) , 150 μm ( magnified image ) . Data are represented as mean ± SEM . E . Effect of AL8810 on retinal Fos expression in WT and Cxcr2 - / - OIR mice on postnatal day 16 ( n = 6 ) . F . Effect of AL8810 on retinal Cxcl1 expression in WT and Cxcr2 - / - OIR mice on postnatal day 16 ( n = 6 ) . Data information : n . s . stands for " not significant . " Data were analyzed by two - way ANOVA with Mann - Whitney test ( E , F ) . Data are represented as mean ± SEM .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 8B. Representative images of OIR retinas in WT and Cxcr2-/- mice with or without AL8810 treatment. The green color shows the isolectin B4-stained vessels, the second-row panels display the enlarged images of white boxes in 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. C. Quantitation of oxygen-induced retinal neovascularization in WT and Cxcr2-/- mice with or without AL8810 treatment (n=12). D. Quantitation of retinal vaso-obliteration in WT and Cxcr2-/- mice with or without AL8810 treatment (n=12). Data information: n.s. stands for "not significant." Data were analyzed by two-way ANOVA with Tukey's multiple comparisons test (C, D) Scale bar: 500 μm (unmagnified image), 150 μm (magnified image). Data are represented as mean ± SEM.E. Effect of AL8810 on retinal Fos expression in WT and Cxcr2-/- OIR mice on postnatal day 16 (n=6). F. Effect of AL8810 on retinal Cxcl1 expression in WT and Cxcr2-/- OIR mice on postnatal day 16 (n=6). Data information: n.s. stands for "not significant." Data were analyzed by two-way ANOVA with Mann-Whitney test (E, F). Data are represented as mean ± SEM.


Figure 1 ( D , E ) Representative immunofluorescence microscopy images of the distribution of septin 7 ( red ) or borg3 or borg4 ( green ) in young , aged and aged LT - HSCs treated with CASIN ( 5µM ) . Nuclei were stained with DAPI ( blue ) , scale bar = 5µm .

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Figure 1(D,E) Representative immunofluorescence microscopy images of the distribution of septin 7 (red) or borg3 or borg4 (green) in young, aged and aged LT-HSCs treated with CASIN (5µM). Nuclei were stained with DAPI (blue), scale bar= 5µm.


Figure 2 ( A ) Representative immunofluorescence confocal microscopy images of young , aged and aged + CASIN ( 5µM ) treated LT - HSCs for the proximity ligation assay ( red signal ) , testing the extent of close physical interaction of Cdc42 and borg4 . A red fluorescent signal indicates close proximity of the two proteins tested . Nuclei were stained with DAPI ( blue ) , scale bar = 5µm . ( B ) Quantification of the level of fluorescent signal in young , aged and aged + CASIN ( 5µM ) treated LT - HSCs to quantify the Cdc42 - borg4 proximity interaction . Data are normalized to the mean fluorescence signal of aged LT - HSCs . 3 biological repeats , n = at least 17 cells per condition , error bars indicate mean ± SEM , unpaired t - test , * * * * P < 0 . 0001 . ( C ) Representative immunofluorescence confocal microscopy images of young , aged and aged + CASIN ( 5µM ) treated LT - HSCs for the proximity ligation assay ( red signal ) , testing the extent of close physical interaction of borg4 and septin 7 . A red fluorescent signal indicates close proximity of the two proteins tested . Nuclei were stained with DAPI ( blue ) , scale bar = 5µm . ( D ) Quantification of the level of fluorescent signal in young , aged and aged + CASIN ( 5µM ) treated LT - HSCs to quantify the borg4 - septin 7 proximity interaction . Data are normalized to the mean fluorescence signal of aged LT - HSCs . 3 biological repeats , n = at least 17 cells per condition , error bars indicate mean ± SEM , unpaired t - test , * * * P < 0 . 001 , * * * * P < 0 . 0001 .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2(A) Representative immunofluorescence confocal microscopy images of young, aged and aged + CASIN (5µM) treated LT-HSCs for the proximity ligation assay (red signal), testing the extent of close physical interaction of Cdc42 and borg4. A red fluorescent signal indicates close proximity of the two proteins tested. Nuclei were stained with DAPI (blue), scale bar= 5µm. (B) Quantification of the level of fluorescent signal in young, aged and aged + CASIN (5µM) treated LT-HSCs to quantify the Cdc42-borg4 proximity interaction. Data are normalized to the mean fluorescence signal of aged LT-HSCs. 3 biological repeats, n=at least 17 cells per condition, error bars indicate mean±SEM, unpaired t-test, ****P<0.0001. (C) Representative immunofluorescence confocal microscopy images of young, aged and aged + CASIN (5µM) treated LT-HSCs for the proximity ligation assay (red signal), testing the extent of close physical interaction of borg4 and septin 7. A red fluorescent signal indicates close proximity of the two proteins tested. Nuclei were stained with DAPI (blue), scale bar= 5µm. (D) Quantification of the level of fluorescent signal in young, aged and aged + CASIN (5µM) treated LT-HSCs to quantify the borg4-septin 7 proximity interaction. Data are normalized to the mean fluorescence signal of aged LT-HSCs. 3 biological repeats, n=at least 17 cells per condition, error bars indicate mean±SEM, unpaired t-test, ***P<0.001, ****P<0.0001.


Figure 3 ( A , B ) Representative immunofluorescence microscopy images of the distribution of septin 7 ( red ) , Cdc42 ( red ) and tubulin ( green ) in LT - HSCs from borg4fl / fl or borg4 ∆ / ∆ mice . Nuclei were stained with DAPI ( blue ) , scale bar = 5 µm . ( C ) Percentage of cells with a polar distribution of septin 7 , Cdc42 or tubulin in borg4fl / fl or borg4 ∆ / ∆ LT - HSCs . At least 3 biological repeats , at least 50 cells were scored per sample . Error bars indicate mean ± SEM , Two - way ANOVA analysis , * * * P < 0 . 001 , * * P < 0 . 01 , * P < 0 . 05 . ( D , E ) Representative immunofluorescence microscopy images of the distribution of borg4 ( red ) , Cdc42 ( red ) or tubulin ( green ) in LT - HSCs from septin 7fl / fl or septin 7 ∆ / ∆ mice . Nuclei were stained with DAPI ( blue ) . scale bar = 5 µm . ( F ) Percentage of cells with a polar distribution of borg4 , Cdc42 or tubulin in septin 7fl / fl or septin 7 ∆ / ∆ LT - HSCs . At least 3 biological repeats , at least 30 to 50 cells were scored per sample . Error bars indicate mean ± SEM , Two - way ANOVA analysis , * * P < 0 . 01 , * P < 0 . 05 .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3(A,B) Representative immunofluorescence microscopy images of the distribution of septin 7 (red), Cdc42 (red) and tubulin (green) in LT-HSCs from borg4fl/fl or borg4∆/∆ mice. Nuclei were stained with DAPI (blue), scale bar= 5 µm. (C) Percentage of cells with a polar distribution of septin 7, Cdc42 or tubulin in borg4fl/fl or borg4∆/∆ LT-HSCs. At least 3 biological repeats, at least 50 cells were scored per sample. Error bars indicate mean±SEM, Two-way ANOVA analysis, ***P<0.001, **P<0.01, *P<0.05. (D,E) Representative immunofluorescence microscopy images of the distribution of borg4 (red), Cdc42 (red) or tubulin (green) in LT-HSCs from septin 7fl/fl or septin 7∆/∆ mice. Nuclei were stained with DAPI (blue). scale bar= 5 µm. (F) Percentage of cells with a polar distribution of borg4, Cdc42 or tubulin in septin 7fl/fl or septin 7∆/∆ LT-HSCs. At least 3 biological repeats, at least 30 to 50 cells were scored per sample. Error bars indicate mean±SEM, Two-way ANOVA analysis, **P<0.01, *P<0.05.


Figure 4 ( J - O ) Percentage of donor derived ( borg4fl / fl or borg4 ∆ / ∆ or septin 7fl / fl or septin 7 ∆ / ∆ ) B - cells ( B220 positive cells ) , T - cells ( CD3 positive cells ) and myeloid cells ( Mac - 1 + or Gr + positive cells ) among BM cells , percentage of donor derived LT - HSCs , ST - HSCs and LMPPs in LSK cells and percentage of donor derived CLPs in lin - Sca1C - kit low cells in animals at post - transplantation ( n = 12 ) . Error bars indicate mean ± SD , * * * * P < 0 . 0001 , * * P < 0 . 01 and * P < 0 . 05 , Two - way ANOVA analysis and 2 tailed unpaired t - test .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4(J-O) Percentage of donor derived (borg4fl/fl or borg4∆/∆ or septin 7fl/fl or septin 7∆/∆) B-cells (B220 positive cells), T-cells (CD3 positive cells) and myeloid cells (Mac-1+ or Gr+ positive cells) among BM cells, percentage of donor derived LT-HSCs, ST-HSCs and LMPPs in LSK cells and percentage of donor derived CLPs in lin-Sca1C-kit low cells in animals at post- transplantation (n=12). Error bars indicate mean±SD, ****P<0.0001, **P<0.01 and *P<0.05, Two-way ANOVA analysis and 2 tailed unpaired t-test.


Figure 5 ( A ) Percentage of single LT - HSCs from septin 7fl / fl of septin 7 ∆ / ∆ mice that completed first and second division up to 48 hours post initiation of culture . ( 192 sorted HSCs cells were analysed , error bars indicate mean ± SD ) .

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Figure 5(A) Percentage of single LT-HSCs from septin 7fl/fl of septin 7∆/∆ mice that completed first and second division up to 48 hours post initiation of culture. (192 sorted HSCs cells were analysed, error bars indicate mean±SD).


Figure 1C . Western blot for POT1 and DDR markers upon POT1 deletion induced with 0 . 5 μM 4 - OHT . Cl75 - parental cell line . gRNA1 - POT1 knock - out in the population by transient transfection with pSpCas9 ( BB ) - 2A - puro containing gRNA1 sequence . EV - corresponding empty vector . D . Time course of telomere length changes upon POT1 removal in clone 35 . d ( days ) TP0 - time point 0 , no 4OHT . E . Growth curve for POT1 WT and POT1 knockout cells . Population doublings ( PDL ) are represented as mean ± SD ( three biological replicates ) . F . Quantification for TIFs in EdU positive and EdU negative cells upon POT1 removal in clone 35 . The bars show percentage of cells containing more than 5 TIFs ± SD . Experiments were performed in triplicate . At least 120 cells were analyzed per condition per replicate . Significance was determined using two - way ANOVA . p - values are indicated on the graph .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O 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-PANEL_START O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1C. Western blot for POT1 and DDR markers upon POT1 deletion induced with 0.5 μM 4-OHT. Cl75 - parental cell line. gRNA1 - POT1 knock-out in the population by transient transfection with pSpCas9(BB)-2A-puro containing gRNA1 sequence. EV - corresponding empty vector.D. Time course of telomere length changes upon POT1 removal in clone 35. d (days) TP0 - time point 0, no 4OHT.E. Growth curve for POT1 WT and POT1 knockout cells. Population doublings (PDL) are represented as mean ± SD (three biological replicates).F. Quantification for TIFs in EdU positive and EdU negative cells upon POT1 removal in clone 35. The bars show percentage of cells containing more than 5 TIFs ± SD. Experiments were performed in triplicate. At least 120 cells were analyzed per condition per replicate. Significance was determined using two-way ANOVA. p-values are indicated on the graph.


Figure 2A . G - overhang assay for clone 35 upon POT1 removal . The bars show relative amounts of single stranded telomeric DNA ± SD . Experiments were performed in triplicate . B . 2D gels for telomeric DNA from clone 35 cells with POT1 ( NT ) and upon POT1 removal for 7 days ( 7d 4OHT ) . C . Examples of metaphase spreads for non - treated ( NT ) clone 35 cells and clone 35 treated with 4 - OHT for 4 and 7 days . White arrowheads indicate fragile telomeres , the red arrowhead indicates a sister - chromatid fusion . Scale bar equals 6 µm . D . Quantification of fragile telomeres ( left graph ) , sister chromatid fusions ( middle graph ) and chromosome fusions ( right graph ) as a percentage of events per metaphase . Experiments were performed in triplicate . At least 25 metaphases were examined per condition per replicate . The mean is displayed and error bars represent ± SD . Significance was determined using one - way ANOVA . p - values are indicated on the graph . E . Quantification and representative images of anaphases with bridges and micronuclei for non - treated ( NT ) clone 35 cells and clone 35 treated with 4 - OHT for 4 and 7 days . Scale bar equals 6 µm . Experiments were performed in triplicate . The bars show percentage of cells containing anaphases , anaphases with bridges or micronuclei + SD . At least 650 cells were examined per condition per replicate . Significance was determined using one - way ANOVA . p - values are indicated on the graph . In all experiments , POT1 deletion was induced with 0 . 5 μM 4 - OHT .

O B-PANEL_START O O O O O O O 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-PANEL_START O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2A. G-overhang assay for clone 35 upon POT1 removal. The bars show relative amounts of single stranded telomeric DNA ± SD. Experiments were performed in triplicate.B. 2D gels for telomeric DNA from clone 35 cells with POT1 (NT) and upon POT1 removal for 7 days (7d 4OHT).C. Examples of metaphase spreads for non-treated (NT) clone 35 cells and clone 35 treated with 4-OHT for 4 and 7 days. White arrowheads indicate fragile telomeres, the red arrowhead indicates a sister-chromatid fusion. Scale bar equals 6 µm. D. Quantification of fragile telomeres (left graph), sister chromatid fusions (middle graph) and chromosome fusions (right graph) as a percentage of events per metaphase. Experiments were performed in triplicate. At least 25 metaphases were examined per condition per replicate. The mean is displayed and error bars represent ± SD. Significance was determined using one-way ANOVA. p-values are indicated on the graph. E. Quantification and representative images of anaphases with bridges and micronuclei for non-treated (NT) clone 35 cells and clone 35 treated with 4-OHT for 4 and 7 days. Scale bar equals 6 µm. Experiments were performed in triplicate. The bars show percentage of cells containing anaphases, anaphases with bridges or micronuclei + SD. At least 650 cells were examined per condition per replicate. Significance was determined using one-way ANOVA. p-values are indicated on the graph. In all experiments, POT1 deletion was induced with 0.5 μM 4-OHT.


Figure 3A . Western blot for POT1 and DDR markers upon POT1 removal in clone 35 and overexpression of ectopic WT POT1 . B . Telomere length and G - overhang length analysis for clone 35 upon POT1 removal and overexpression of ectopic WT POT1 ( constant - field gel electrophoresis ) . D , E Telomere length and G - overhang length analysis for clone 35 upon POT1 removal and overexpression of ectopic POT1 variants carrying disease - associated mutations ( constant - field gel electrophoresis ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3A. Western blot for POT1 and DDR markers upon POT1 removal in clone 35 and overexpression of ectopic WT POT1.B. Telomere length and G-overhang length analysis for clone 35 upon POT1 removal and overexpression of ectopic WT POT1 (constant-field gel electrophoresis).D, E Telomere length and G-overhang length analysis for clone 35 upon POT1 removal and overexpression of ectopic POT1 variants carrying disease-associated mutations (constant-field gel electrophoresis).


Figure 4B . Quantification of precipitated telomeric DNA in 2 - step QTIP ( % of input ) and fold enrichment of precipitated telomeric DNA compared with precipitated Alu repeat DNA ( based on dot blot analyses ) . Bars represent data from three independent experiments ± SD . C . Scatter plots representing immunoprecipitation specificity ( 2 - step QTIP / IgG ratio ) versus difference between non treated cells ( NT ) and cells at day 4 upon 4 - OHT induction ( 4d 4OHT / NT ratio ) or difference between non treated cells ( NT ) and cells at day 7 upon 4 - OHT induction ( 7d 4OHT / NT ratio ) respectively ( data from Table EV1 ) . MRN complex in red , 9 - 1 - 1 complex in blue , shelterin complex in orange except for POT1 in black . Values are the average of the 3 biological replicates . D . Heat map representing changes in levels of selected proteins involved in DNA damage ( MRN complex in red , 9 - 1 - 1 complex in blue ) and HDR proteins ( purple ) after POT1 knockout induction . Proteins marked with * pass t - test , but not t - test with Benjamini - Hochberg correction ( p < 0 . 05 , q < 0 . 09 ) . Data information : 4 - OHT - 4 - hydroxytamoxifen , NT - non - treated cells .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4B. Quantification of precipitated telomeric DNA in 2-step QTIP (% of input) and fold enrichment of precipitated telomeric DNA compared with precipitated Alu repeat DNA (based on dot blot analyses). Bars represent data from three independent experiments ± SD.C. Scatter plots representing immunoprecipitation specificity (2-step QTIP/IgG ratio) versus difference between non treated cells (NT) and cells at day 4 upon 4-OHT induction (4d 4OHT/NT ratio) or difference between non treated cells (NT) and cells at day 7 upon 4-OHT induction (7d 4OHT/NT ratio) respectively (data from Table EV1). MRN complex in red, 9-1-1 complex in blue, shelterin complex in orange except for POT1 in black. Values are the average of the 3 biological replicates.D. Heat map representing changes in levels of selected proteins involved in DNA damage (MRN complex in red, 9-1-1 complex in blue) and HDR proteins (purple) after POT1 knockout induction. Proteins marked with * pass t-test, but not t-test with Benjamini-Hochberg correction (p < 0.05, q < 0.09). Data information: 4-OHT - 4-hydroxytamoxifen, NT - non-treated cells.