Confocal microscopy images of co - localization analysis of the BNIP3 - proficient ( shCntl , n = 27 cells ) or deficient ( shBNIP3 , n = 31 cells ) B16 - F10 cells transiently transfected with GFP - LC3 and immunostained for TOMM20 ( mitochondrial marker ) . Colocalization events of GFP - LC3 puncta ( green ) with TOMM20 ( red ) are highlighted with white arrows . The percentage of GFP - LC3 puncta colocalizing with TOMM20 was calculated in n cells from 3 biologically independent experiments and analyzed with Mann - Whitney ' s non - parametric test . Scale bars represent 10µm .

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Confocal microscopy images of co-localization analysis of the BNIP3-proficient (shCntl, n=27 cells) or deficient (shBNIP3, n=31 cells) B16-F10 cells transiently transfected with GFP-LC3 and immunostained for TOMM20 (mitochondrial marker). Colocalization events of GFP-LC3 puncta (green) with TOMM20 (red) are highlighted with white arrows. The percentage of GFP-LC3 puncta colocalizing with TOMM20 was calculated in n cells from 3 biologically independent experiments and analyzed with Mann-Whitney's non-parametric test. Scale bars represent 10µm.


Glut1 , Hk2 , Pkm1 , Pkm2 , Pdk1 , Mct1 , Ldha , Ldhb and Mct4 transcript levels measured by qPCR from the corresponding B16 - F10 cell lysates . Transcript expression is represented as the fold - change relative to their corresponding shCntl value . Heatmap shows the average values in each condition ( n = 4 ) and they were analyzed with a one - sample T - test only against shCntl , except for Glut1 shATG5 that was analyzed with a Wilcoxon rank test .

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Glut1, Hk2, Pkm1, Pkm2, Pdk1, Mct1, Ldha, Ldhb and Mct4 transcript levels measured by qPCR from the corresponding B16-F10 cell lysates. Transcript expression is represented as the fold-change relative to their corresponding shCntl value. Heatmap shows the average values in each condition (n=4) and they were analyzed with a one-sample T-test only against shCntl, except for Glut1 shATG5 that was analyzed with a Wilcoxon rank test.


Immunoblot detection of Hydroxylated HIF - 1α ( HIF - 1α - OH ) , total HIF - 1α , LDHA and ACTIN protein levels from lysates of B16 - F10 cells ( n = 4 ) or B16 - F10 tumors [ CntlKD ( n = 5 ) , BNIP3KD ( n = 5 ) and ATG5KD ( n = 7 ) ] ) . The HIF - 1α - OH / HIF - 1α ratio is shown in a bar graph below . Densitometric quantifications relative to ACTIN levels are shown below each corresponding band . Analysis was performed as indicated in the Data Information section , unless for HIF - 1α / Actin ratio ( in vivo ) , which was analyzed with Kruskal - Wallis non - parametric test with Dunn ' s multiple comparisons test , and LDHA / Actin ratio for shATG5 condition ( in vitro ) , which was analyzed with Wilcoxon ' s rank test .

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Immunoblot detection of Hydroxylated HIF-1α (HIF-1α-OH), total HIF-1α, LDHA and ACTIN protein levels from lysates of B16-F10 cells (n=4) or B16-F10 tumors [CntlKD (n=5), BNIP3KD (n=5) and ATG5KD (n=7)]). The HIF-1α-OH /HIF-1α ratio is shown in a bar graph below. Densitometric quantifications relative to ACTIN levels are shown below each corresponding band. Analysis was performed as indicated in the Data Information section, unless for HIF-1α/Actin ratio (in vivo), which was analyzed with Kruskal-Wallis non-parametric test with Dunn's multiple comparisons test, and LDHA/Actin ratio for shATG5 condition (in vitro), which was analyzed with Wilcoxon's rank test.


Immunohistochemical staining for HIF - 1α ( brown ) representative from CntlKD , BNIP3KD and ATG5KD , B16 - F10 tumor tissues . Higher magnification of the highlighted ( with a white square ) stained sections is shown below .

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Immunohistochemical staining for HIF-1α (brown) representative from CntlKD, BNIP3KD and ATG5KD, B16-F10 tumor tissues. Higher magnification of the highlighted (with a white square) stained sections is shown below.


Immunoblot detection of Hydroxylated HIF - 1α ( HIF - 1α - OH ) , total HIF - 1α , LDHA , PHD2 and ACTIN protein levels from normoxic lysates of B16 - F10 cells transfected in presence of non - targeting siRNA sequences ( Scr ) or siRNA against PHD2 .

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Immunoblot detection of Hydroxylated HIF-1α (HIF-1α-OH), total HIF-1α, LDHA, PHD2 and ACTIN protein levels from normoxic lysates of B16-F10 cells transfected in presence of non-targeting siRNA sequences (Scr) or siRNA against PHD2.


Relative intracellular Fe2 + levels in normoxic B16 - F10 cells ( n = 4 ) . Representative pictures of FerroOrange intensity are shown on the left and analyzed using a RM one - way ANOVA ( Geisser - Greenhouse correction ) with Holm - Sidak ' s multiple comparisons test . Scale bars represent 50µm .

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Relative intracellular Fe2+ levels in normoxic B16-F10 cells (n=4). Representative pictures of FerroOrange intensity are shown on the left and analyzed using a RM one-way ANOVA (Geisser-Greenhouse correction) with Holm-Sidak's multiple comparisons test. Scale bars represent 50µm.


Immunoblot detection of Hydroxylated HIF - 1α ( HIF - 1α - OH ) , total HIF - 1α , LDHA , NCOA4 and ACTIN levels from normoxic lysates of B16 - F10 cells transfected in presence of non - targeting siRNA sequences ( Scr ) or siRNA against NCOA4 ( n = 4 ) .

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Immunoblot detection of Hydroxylated HIF-1α (HIF-1α-OH), total HIF-1α, LDHA, NCOA4 and ACTIN levels from normoxic lysates of B16-F10 cells transfected in presence of non-targeting siRNA sequences (Scr) or siRNA against NCOA4 (n=4).


Growth curves of B16 - F10 tumors generated by shCntl or shBNIP3 cells expressing Luciferase ( Luc ) or an undegradable HIF - 1α ( HIF - 1α - AA ) construct ( n = 7 for CntlKD Luc / BNIP3KD HIF - 1α - AA , n = 8 CntlKD HIF - 1α - AA and n = 5 for BNIP3KD Luc ) and analyzed with a two - way ANOVA with Tukey ' s multiple comparisons test . Representative double immunostaining for CD31 and αSMA in B16 - F10 tumors ( n = 5 per condition ) . Scale bars represent 50µm .

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Growth curves of B16-F10 tumors generated by shCntl or shBNIP3 cells expressing Luciferase (Luc) or an undegradable HIF-1α (HIF-1α-AA) construct (n=7 for CntlKD Luc/BNIP3KD HIF-1α-AA, n=8 CntlKD HIF-1α-AA and n=5 for BNIP3KD Luc) and analyzed with a two-way ANOVA with Tukey's multiple comparisons test. Representative double immunostaining for CD31 and αSMA in B16-F10 tumors (n=5 per condition). Scale bars represent 50µm.


HEK293T cells were transfected with 200 nM siRNAs per well . After 24 h , the cells were infected with HIV - luc / 2019 - nCoV pseudoviruses . After washing and incubation with fresh medium for 48 h , the intracellular luciferase activity was measured . Ordinary one - way ANOVA test was used for this analysis . The results are shown as the mean and SEM . * P < 0 . 05 .

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HEK293T cells were transfected with 200 nM siRNAs per well. After 24 h, the cells were infected with HIV-luc/2019-nCoV pseudoviruses. After washing and incubation with fresh medium for 48 h, the intracellular luciferase activity was measured. Ordinary one-way ANOVA test was used for this analysis. The results are shown as the mean and SEM. *P< 0.05.


assay . B , Teicoplanin inhibit the entry of 2019 - nCoV and SARS - nCoV . Chemical structure of teicoplanin ( Left ) . A549 cells were seeded in a 96 - well plate , and 24 h later , the cells were infected with HIV - luc / 2019 - nCoV , HIV - luc / SARS - nCoV or HIV - luc / VSVG pseudoviruses . After washing and incubation with fresh medium for 48 h , the intracellular luciferase activity was measured . Ordinary one - way ANOVA test was used for this analysis . The results are shown as the mean and SEM . * P < 0 . 05 .

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assay.B, Teicoplanin inhibit the entry of 2019-nCoV and SARS-nCoV. Chemical structure of teicoplanin (Left). A549 cells were seeded in a 96-well plate, and 24 h later, the cells were infected with HIV-luc/2019-nCoV, HIV-luc/SARS-nCoV or HIV-luc/VSVG pseudoviruses. After washing and incubation with fresh medium for 48 h, the intracellular luciferase activity was measured. Ordinary one-way ANOVA test was used for this analysis. The results are shown as the mean and SEM. *P< 0.05.C


, A549 cells were infected with HIV - luc / 2019 - nCoV pseudoviruses and then incubated with teicoplanin at various concentrations . The intracellular luciferase activity was measured at 48 h post - infection . The IC50 was calculated using GraphPad Prism software .

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, A549 cells were infected with HIV-luc/2019-nCoV pseudoviruses and then incubated with teicoplanin at various concentrations. The intracellular luciferase activity was measured at 48 h post-infection. The IC50 was calculated using GraphPad Prism software.


types . A , HEK293T cells were infected with HIV - luc / 2019 - nCoV pseudoviruses and then incubated with teicoplanin at various concentrations . The intracellular luciferase activity was measured at 48 h post - infection . The IC50 was calculated using GraphPad Prism software .

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types.A, HEK293T cells were infected with HIV-luc/2019-nCoV pseudoviruses and then incubated with teicoplanin at various concentrations. The intracellular luciferase activity was measured at 48 h post-infection. The IC50 was calculated using GraphPad Prism software.B


software . B , Huh7 cells were infected with HIV - luc / 2019 - nCoV pseudoviruses and then incubated with teicoplanin at various concentrations . The intracellular luciferase activity was measured at 48 h post - infection . The IC50 was calculated using GraphPad Prism software .

software.B, Huh7 cells were infected with HIV-luc/2019-nCoV pseudoviruses and then incubated with teicoplanin at various concentrations. The intracellular luciferase activity was measured at 48 h post-infection. The IC50 was calculated using GraphPad Prism software.C


A549 cells were incubated with dalbavancin ( D ) at various concentrations and infected with HIV - luc / 2019 - nCoV pseudoviruses . The intracellular luciferase activity was measured at 48 h post - infection . Ordinary one - way ANOVA test was used for this analysis .

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A549 cells were incubated with dalbavancin (D) at various concentrations and infected with HIV-luc/2019-nCoV pseudoviruses. The intracellular luciferase activity was measured at 48 h post-infection. Ordinary one-way ANOVA test was used for this analysis. dalbavancin


D , A549 cells were incubated with vancomycin at various concentrations and infected with HIV - luc / 2019 - nCoV pseudoviruses . The intracellular luciferase activity was measured at 48 h post - infection . Ordinary one - way ANOVA test was used for this analysis . Left : Chemical structure of

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D, A549 cells were incubated with vancomycin at various concentrations and infected with HIV-luc/2019-nCoV pseudoviruses. The intracellular luciferase activity was measured at 48 h post-infection. Ordinary one-way ANOVA test was used for this analysis. Left: Chemical structure of vancomycin


( A ) NIH3T3 fibroblasts co - expressing DsRed - CA7 and EGFP - CA2 ( n = 4 independent replicates ) .

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(A) NIH3T3 fibroblasts co-expressing DsRed-CA7 and EGFP-CA2 (n = 4 independent replicates).


( B - E ) Co - localization of EGFP and the two CA isoforms with filamentous actin studied in fibroblasts expressing ( B ) EGFP , ( C ) EGFP - CA2 , or ( D , E ) EGFP - CA7 and stained with phalloidin - 594 to visualize F - actin ( n = 4 , 10 and 8 independent replicates , respectively ) . A magnification of the area marked with the yellow rectangle in ( C ) and ( D ) shows the localization of EGFP - CA2 and EGFP - CA7 compared to phalloidin - 594 . ( E ) EGFP - CA7 caused a prominent overexpression phenotype with thick and curvy cytosolic actin bundles ( arrow ) and plasmalemmal protrusions ( arrowhead ) .

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(B-E) Co-localization of EGFP and the two CA isoforms with filamentous actin studied in fibroblasts expressing (B) EGFP, (C) EGFP-CA2, or (D, E) EGFP-CA7 and stained with phalloidin-594 to visualize F-actin (n = 4, 10 and 8 independent replicates, respectively). A magnification of the area marked with the yellow rectangle in (C) and (D) shows the localization of EGFP-CA2 and EGFP-CA7 compared to phalloidin-594. (E) EGFP-CA7 caused a prominent overexpression phenotype with thick and curvy cytosolic actin bundles (arrow) and plasmalemmal protrusions (arrowhead).


( F - H ) The normalized fluorescence emission intensity profiles for F - actin ( red line ) and ( F ) EGFP , ( G ) EGFP - CA2 , or ( H ) EGFP - CA7 ( black line ) .

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(F-H) The normalized fluorescence emission intensity profiles for F-actin (red line) and (F) EGFP, (G) EGFP-CA2, or (H) EGFP-CA7 (black line).


( A ) Actin co - sedimentation assay shows that CA7 binds to F - actin . The binding is enhanced at more acidic pH ( 6 . 5 vs . 7 . 4 ) n = 3 independent replicates at each actin concentration , two - way ANOVA , P < 0 . 001 .

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(A) Actin co-sedimentation assay shows that CA7 binds to F-actin. The binding is enhanced at more acidic pH (6.5 vs. 7.4) n = 3 independent replicates at each actin concentration, two-way ANOVA, P < 0.001.


( B ) Fluorescence time ‐ lapse images of F - actin bundling in an in vitro bundling assay . A mixture of unlabeled and Rhodamine labelled non - muscle actin was polymerized in the absence ( PBS control , upper panel ) or presence of mCA7 ( lower panel ) . Numbers in images indicate the time after the onset of the experiment ( 0 , 5 and 23 min ) . Intensity based Fire - coloring ( Fiji ) was used to visualize intensity changes . Scale bar 10 µm . ( C ) Quantification of the mean increase in filament length ( n = 10 filaments at each time point ) and the mean relative fluorescence intensity values of cross - sections for individual filaments / bundles ( n = 30 - 31 ) in the absence and presence of mCA7 ( 1 . 12 µM ) . The data were analyzed using a general mixed model with time as a within - unit factor and the presence of CA7 as a between - unit factor . n = 3 independent repetitions , experiment repeats were included as a covariate and were non - significant .

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(B) Fluorescence time‐lapse images of F-actin bundling in an in vitro bundling assay. A mixture of unlabeled and Rhodamine labelled non-muscle actin was polymerized in the absence (PBS control, upper panel) or presence of mCA7 (lower panel). Numbers in images indicate the time after the onset of the experiment (0, 5 and 23 min). Intensity based Fire-coloring (Fiji) was used to visualize intensity changes. Scale bar 10 µm. (C) Quantification of the mean increase in filament length (n = 10 filaments at each time point) and the mean relative fluorescence intensity values of cross-sections for individual filaments /bundles (n = 30 - 31) in the absence and presence of mCA7 (1.12 µM). The data were analyzed using a general mixed model with time as a within-unit factor and the presence of CA7 as a between-unit factor. n = 3 independent repetitions, experiment repeats were included as a covariate and were non-significant.


( D ) Kymographs showing different time points in the line of interest ( line width 1 µm ) from the experiments analyzed in ( C ) . Kymographs were generated with Fiji Multi Kymograph function . Total time is 159 frames = 26 . 5 minutes . Scale bar 5 μm ( full height is 20 μm ) . F - actin bundles can be detected as clear lines in kymographs .

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(D) Kymographs showing different time points in the line of interest (line width 1 µm) from the experiments analyzed in (C). Kymographs were generated with Fiji Multi Kymograph function. Total time is 159 frames = 26.5 minutes. Scale bar 5 μm (full height is 20 μm). F-actin bundles can be detected as clear lines in kymographs.


( E ) Fluorescence time ‐ lapse images of F - actin bundling in an in vitro bundling assay . A mixture of unlabeled and Rhodamine labelled non - muscle actin was polymerized in the presence of mCA7 ( 0 . 11 µM ) . Numbers above images indicate the time after the onset of the experiment ( 22 min - 23 min 20 sec ) . Intensity based Fire - coloring ( Fiji ) was used to visualize intensity changes . White arrows highlight the bundling filaments . Scale bar 5 μm . This is a representative video from 8 similar experiments .

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(E) Fluorescence time‐lapse images of F-actin bundling in an in vitro bundling assay. A mixture of unlabeled and Rhodamine labelled non-muscle actin was polymerized in the presence of mCA7 (0.11 µM). Numbers above images indicate the time after the onset of the experiment (22 min -23 min 20 sec). Intensity based Fire-coloring (Fiji) was used to visualize intensity changes. White arrows highlight the bundling filaments. Scale bar 5 μm. This is a representative video from 8 similar experiments.


( A , B ) NIH3T3 cells transfected with DsRed ( A ) or DsRed - CA7 ( B ) were incubated in growth medium with 5 µM Latrunculin B for 0 , 2 , 5 , 10 , or 30 minutes , or in an equal amount of DMSO for 60 minutes . Analyses of experiments show that in cells transfected with DsRed - CA7 F - actin structures collapse more slowly ( 0 min : 83 % " normal " ; 2 min : 80 % , p = 0 . 44 ; 5 min : 74 % , p = 0 . 39 ; 10 min : 27 % , p = 0 . 08 ; 30 min : 16 % , p = 0 . 02 ; DMSO : 82 % , p = 0 . 99 ; tested against 0 min with two - way ANOVA , Dunnett ' s multiple comparison test ) than in the DsRed - transfected ones ( 0 min : 89 % " normal " ; 2 min : 48 % , p = 0 . 14 ; 5 min : 31 % , p = 0 . 04 ; 10 min : 2 % , p = 0 . 001 ; 30 min : 0 . 7 % , p = 0 . 001 ; DMSO : 88 % , p = 0 . 8 ; tested against 0 min with two - way ANOVA , Dunnett ' s multiple comparison test ) . For the analysis , cells were categorized to three groups as " normal " , " some shape / F - actin left " and " round " . The upper panel shows example images of the cells in all three categories for ( A ) DsRed - or ( B ) DsRed - CA7 transfected cells ( actin visualized with Phalloidin - 488 ) .

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(A,B) NIH3T3 cells transfected with DsRed (A) or DsRed-CA7 (B) were incubated in growth medium with 5 µM Latrunculin B for 0, 2, 5, 10, or 30 minutes, or in an equal amount of DMSO for 60 minutes. Analyses of experiments show that in cells transfected with DsRed-CA7 F-actin structures collapse more slowly (0 min: 83% "normal"; 2 min: 80%, p = 0.44; 5 min: 74%, p = 0.39; 10 min: 27%, p = 0.08; 30 min: 16%, p = 0.02; DMSO: 82%, p = 0.99; tested against 0 min with two-way ANOVA, Dunnett's multiple comparison test) than in the DsRed-transfected ones (0 min: 89% "normal"; 2 min: 48%, p = 0.14; 5 min: 31%, p = 0.04; 10 min: 2%, p = 0.001; 30 min: 0.7%, p = 0.001; DMSO: 88%, p = 0.8; tested against 0 min with two-way ANOVA, Dunnett's multiple comparison test) . For the analysis, cells were categorized to three groups as "normal", "some shape/F-actin left" and "round". The upper panel shows example images of the cells in all three categories for (A) DsRed- or (B) DsRed-CA7 transfected cells (actin visualized with Phalloidin-488).


( A - C ) NIH3T3 fibroblasts expressing EGFP - CA7 - mutant1 ( A ) , EGFP - CA7 - mutant2 ( B ) , and EGFP - CA7 - mutant3 ( C ) . F - actin is visualized with Phalloidin - 594 . In the right - most panel of ( A - C ) are the normalized fluorescence intensity profiles of the mutated CA7 EGFP signal ( black ) and actin ( red ) and the yellow line in left - most panels indicates the cross - section from which the pixel intensities were measured . Scale bars 20 µm .

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(A-C) NIH3T3 fibroblasts expressing EGFP-CA7-mutant1 (A), EGFP-CA7-mutant2 (B), and EGFP-CA7-mutant3 (C). F-actin is visualized with Phalloidin-594. In the right-most panel of (A-C) are the normalized fluorescence intensity profiles of the mutated CA7 EGFP signal (black) and actin (red) and the yellow line in left-most panels indicates the cross-section from which the pixel intensities were measured. Scale bars 20 µm.


( D ) Analysis of the mutated CA7 and F - actin co - localization in cultured fibroblasts . Scatterplots of fluorescent intensities per pixel ( EGFP vs . Phalloidin - 594 ) along a cross section through a representative cell . Pearson ' s correlation coefficient ( r ) for the analyzed cell is given in each panel .

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(D) Analysis of the mutated CA7 and F-actin co-localization in cultured fibroblasts. Scatterplots of fluorescent intensities per pixel (EGFP vs. Phalloidin-594) along a cross section through a representative cell. Pearson's correlation coefficient (r) for the analyzed cell is given in each panel.


( E ) Pearson ' s correlation coefficient values calculated for the depicted constructs and compared to CA7 . F - actin had a strong positive correlation coefficient with EGFP - CA7 ( r = 0 . 91 ± 0 . 02 , n = 26 cells ) . Neither EGFP alone ( r = 0 . 01 ± 0 . 03 , n = 56 ) nor EGFP - CA2 ( r = 0 . 09 ± 0 . 03 , n = 53 ) co - localized with F - actin ( P < 0 . 0001 for both constructs , when compared to CA7 ) . From the five mutated CA7 constructs , EGFP - CA7 - mutant1 ( r = 0 . 51 ± 0 . 04 , P < 0 . 0001 , n = 25 ) and EGFP - CA7 - mutant3 ( r = 0 . 72 ± 0 . 03 , P = 0 . 05 , n = 21 ) co - localized less with F - actin actin when compared to CA7 . The co - localization of the other four mutated CA7 constructs , EGFP - CA7 - mutant2 ( r = 0 . 92 ± 0 . 02 , P = 0 . 99 , n = 18 ) , EGFP - CA7 - R223E ( r = 0 . 86 ± 0 . 02 , P = 0 . 99 , n = 24 ) and EGFP - CA7 - H96 / 98C ( r = 0 . 79 ± 0 . 04 , P = 0 . 14 , n = 18 ) did not differ significantly from that of CA7 . Data are shown as mean ± SEM . Data did not pass Shapiro - Wilk test for normality , statistical comparison against CA7 was done with Kruskall - Wallis test corrected for multiple comparisons .

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(E) Pearson's correlation coefficient values calculated for the depicted constructs and compared to CA7. F-actin had a strong positive correlation coefficient with EGFP-CA7 (r = 0.91 ± 0.02, n = 26 cells). Neither EGFP alone (r = 0.01 ± 0.03, n = 56) nor EGFP-CA2 (r = 0.09 ± 0.03, n = 53) co-localized with F-actin (P < 0.0001 for both constructs, when compared to CA7). From the five mutated CA7 constructs, EGFP-CA7-mutant1 (r= 0.51 ± 0.04, P < 0.0001, n = 25) and EGFP-CA7-mutant3 (r = 0.72 ± 0.03, P = 0.05, n = 21) co-localized less with F-actin actin when compared to CA7. The co-localization of the other four mutated CA7 constructs, EGFP-CA7-mutant2 (r = 0.92 ± 0.02, P = 0.99, n = 18), EGFP-CA7-R223E (r = 0.86 ± 0.02, P = 0.99, n = 24) and EGFP-CA7-H96/98C (r = 0.79 ± 0.04, P = 0.14, n = 18) did not differ significantly from that of CA7. Data are shown as mean ± SEM. Data did not pass Shapiro-Wilk test for normality, statistical comparison against CA7 was done with Kruskall-Wallis test corrected for multiple comparisons.


( A ) Isoform - specific subcellular localization shown in cultured hippocampal neurons ( DIV14 ) co - expressing DsRed - CA7 ( left ) and EGFP - CA2 ( middle ) .

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(A) Isoform-specific subcellular localization shown in cultured hippocampal neurons (DIV14) co-expressing DsRed-CA7 (left) and EGFP-CA2 (middle).


( B , C ) Representative confocal images of precocious in vivo expression of ( B ) EGFP - CA2 and ( C ) EGFP - CA7 in P40 mouse cortical layer 2 / 3 pyramidal neurons . Neurons were transfected at E14 . 5 with EGFP - CA2 or EGFP - CA7 using in utero electroporation and images were taken from fixed slices . Right panels in ( B ) and ( C ) show higher magnification of the primary apical dendrite marked with a box . ( D ) The expression of EGFP - CA7 disrupted the normal spine morphology and induced the formation of thick , filopodia - like protrusions .

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(B,C) Representative confocal images of precocious in vivo expression of (B) EGFP-CA2 and (C) EGFP-CA7 in P40 mouse cortical layer 2/3 pyramidal neurons. Neurons were transfected at E14.5 with EGFP-CA2 or EGFP-CA7 using in utero electroporation and images were taken from fixed slices. Right panels in (B) and (C) show higher magnification of the primary apical dendrite marked with a box. (D) The expression of EGFP-CA7 disrupted the normal spine morphology and induced the formation of thick, filopodia-like protrusions.


( A ) Comparison of mEPSCs in cortical layer 2 / 3 pyramidal neurons from P30 - P40 WT and CA7 KO mice . Sample traces of mEPSC recordings from WT and CA7 KO neurons , low - pas filtered at 1 kHz ( left ) . The data are summarized in the bar diagrams ( right ) . mEPSC frequency ( P = 0 . 63 ) and amplitude ( P = 0 . 90 ) were not significantly different between WT and CA7 KO and neurons ( n = 7 and 5 neurons , respectively , Student ' s independent samples t - test ) .

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(A) Comparison of mEPSCs in cortical layer 2/3 pyramidal neurons from P30 - P40 WT and CA7 KO mice. Sample traces of mEPSC recordings from WT and CA7 KO neurons, low-pas filtered at 1 kHz (left). The data are summarized in the bar diagrams (right). mEPSC frequency (P = 0.63) and amplitude (P = 0.90) were not significantly different between WT and CA7 KO and neurons (n = 7 and 5 neurons, respectively, Student's independent samples t-test).


( B ) Representative confocal images of apical dendrites from Lucifer Yellow injected cortical layer 2 / 3 pyramidal neurons from WT and CA7 KO mice . The dendritic spine density and spine head size were examined in fixed slice preparations from P34 - P37 mice . Scale bar 2 µm .

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(B) Representative confocal images of apical dendrites from Lucifer Yellow injected cortical layer 2/3 pyramidal neurons from WT and CA7 KO mice. The dendritic spine density and spine head size were examined in fixed slice preparations from P34 - P37 mice. Scale bar 2 µm.


( C ) Summary of the spine density analysis done from the Lucifer Yellow injected neurons . Spine density was a higher in CA7 KO neurons both in apical and basal dendrites ( n = 28 neurons for both ) compared to WT ( n = 29 neurons for apical and n = 30 for basal dendrite analysis ) ( P = 0 . 000002 for apical dendrites , analyzed with Mann - Whitney test , and P = 6 , 8 x 10 - 8 for basal dendrites , Student ' s t - test with Welch - correction . ) A total of 8279 spines were analyzed from four CA7 KO mice and 8730 spines from two WT control mice .

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(C) Summary of the spine density analysis done from the Lucifer Yellow injected neurons. Spine density was a higher in CA7 KO neurons both in apical and basal dendrites (n = 28 neurons for both) compared to WT (n= 29 neurons for apical and n=30 for basal dendrite analysis) (P = 0.000002 for apical dendrites, analyzed with Mann-Whitney test, and P = 6,8 x 10-8 for basal dendrites, Student's t-test with Welch-correction.) A total of 8279 spines were analyzed from four CA7 KO mice and 8730 spines from two WT control mice.


( D ) The spine head width distribution7differed significantly between the genotypes ( n = 467 spines from WT and n = 421 spines from CA7 KO animals , 15 neurons analyzed from both genotypes , Wilcoxon rank sum test with continuity correction , W = 134540 , P < 0 . 001 ) .

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(D) The spine head width distribution7differed significantly between the genotypes (n= 467 spines from WT and n = 421 spines from CA7 KO animals, 15 neurons analyzed from both genotypes, Wilcoxon rank sum test with continuity correction, W = 134540, P < 0.001).


MARCH2 + / + ( n = 8 ) or MARCH2 - / - ( n = 8 ) mice were intravenously infected with VSV - GFP ( 2 × 108 PFU / mouse ) , and serum samples were collected at 12 hpi ( hours post - infection ) . ( A ) Virus titer was analyzed in a plaque assay .

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MARCH2+/+ (n = 8) or MARCH2-/- (n = 8) mice were intravenously infected with VSV-GFP (2 × 108 PFU/mouse), and serum samples were collected at 12 hpi (hours post-infection). (A) Virus titer was analyzed in a plaque assay.


MARCH2 + / + ( n = 8 ) or MARCH2 - / - ( n = 8 ) mice were intravenously infected with VSV - GFP ( 2 × 108 PFU / mouse ) , and serum samples were collected at 12 hpi ( hours post - infection ) . ( B ) Secretion of IFN - β , IL - 6 , IL - 12 , and TNFα was measured in specific ELISAs .

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MARCH2+/+ (n = 8) or MARCH2-/- (n = 8) mice were intravenously infected with VSV-GFP (2 × 108 PFU/mouse), and serum samples were collected at 12 hpi (hours post-infection). (B) Secretion of IFN-β, IL-6, IL-12, and TNFα was measured in specific ELISAs.


C MARCH2 + / + ( n = 6 ) or MARCH2 - / - ( n = 6 ) mice were intravenously injected with poly ( I : C ) ( 200 μg / mouse ) . Serum samples were collected at 2 hpi and secretion of IFN - β , IL - 6 , IL - 12 , and TNFα was measured in specific ELISAs .

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C MARCH2+/+ (n = 6) or MARCH2-/- (n = 6) mice were intravenously injected with poly(I:C) (200 μg/mouse). Serum samples were collected at 2 hpi and secretion of IFN-β, IL-6, IL-12, and TNFα was measured in specific ELISAs.


MARCH2 + / + ( n = 8 ) or MARCH2 - / - ( n = 8 ) mice were intraperitoneally challenged with 1 × 106 CFU of Listeria monocytogenes . ( D ) The percentage of surviving mice ( D , log - rank test , * * P < 0 . 01 )

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MARCH2+/+ (n = 8) or MARCH2-/- (n = 8) mice were intraperitoneally challenged with 1 × 106 CFU of Listeria monocytogenes. (D) The percentage of surviving mice (D, log-rank test, **P < 0.01)


MARCH2 + / + ( n = 8 ) or MARCH2 - / - ( n = 8 ) mice were intraperitoneally challenged with 1 × 106 CFU of Listeria monocytogenes . body weight changes for each group ( E ) are shown .

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MARCH2+/+ (n = 8) or MARCH2-/- (n = 8) mice were intraperitoneally challenged with 1 × 106 CFU of Listeria monocytogenes. body weight changes for each group (E) are shown.


MARCH2 + / + ( n = 8 ) or MARCH2 - / - ( n = 8 ) mice were intraperitoneally challenged with 1 × 106 CFU of Listeria monocytogenes . ( F , G ) At 3 dpc , the bacterial load in the spleen ( F ) and liver ( G ) was examined .

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MARCH2+/+ (n = 8) or MARCH2-/- (n = 8) mice were intraperitoneally challenged with 1 × 106 CFU of Listeria monocytogenes. (F, G) At 3 dpc, the bacterial load in the spleen (F) and liver (G) was examined.


MARCH2 + / + ( n = 8 ) or MARCH2 - / - ( n = 8 ) mice were intraperitoneally challenged with 1 × 106 CFU of Listeria monocytogenes . ( H - J ) Amount of IL - 6 , IL - 12 , CCL5 , and CXCL10 in serum ( H ) , spleen ( I ) , and liver ( J ) of mice in each group at 24 hpc .

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MARCH2+/+ (n = 8) or MARCH2-/- (n = 8) mice were intraperitoneally challenged with 1 × 106 CFU of Listeria monocytogenes. (H-J) Amount of IL-6, IL-12, CCL5, and CXCL10 in serum (H), spleen (I), and liver (J) of mice in each group at 24 hpc.


MARCH2 + / + ( n = 8 ) or MARCH2 - / - ( n = 8 ) mice were challenged intraperitoneally with LPS ( 24 mg / kg ) . Percentage of surviving mice ( A , log - rank test , * * P < 0 . 01 )

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MARCH2+/+ (n = 8) or MARCH2-/- (n = 8) mice were challenged intraperitoneally with LPS (24 mg/kg). Percentage of surviving mice (A, log-rank test, **P < 0.01)


MARCH2 + / + ( n = 8 ) or MARCH2 - / - ( n = 8 ) mice were challenged intraperitoneally with LPS ( 24 mg / kg ) . body weight changes ( B ) in each groups .

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MARCH2+/+ (n = 8) or MARCH2-/- (n = 8) mice were challenged intraperitoneally with LPS (24 mg/kg). body weight changes (B) in each groups.


C MARCH2 + / + ( n = 4 ) or MARCH2 - / - ( n = 4 ) mice were challenged intraperitoneally with LPS ( 24 mg / kg ) . Representing slides of H & E staining of spleen sections from each group . Scale bar , 6mm .

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C MARCH2+/+ (n = 4) or MARCH2-/- (n = 4) mice were challenged intraperitoneally with LPS (24 mg/kg). Representing slides of H&E staining of spleen sections from each group. Scale bar, 6mm.


D MARCH2 + / + ( n = 6 ) or MARCH2 - / - ( n = 6 ) mice were challenged intraperitoneally with LPS ( 24 mg / kg ) . Levels of IL - 6 , TNF - α , CXCL - 10 , CCL - 5 in serum from mice in each groups were measured at12 hpc by ELISA .

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D MARCH2+/+ (n = 6) or MARCH2-/- (n = 6) mice were challenged intraperitoneally with LPS (24 mg/kg). Levels of IL-6, TNF-α, CXCL-10, CCL-5 in serum from mice in each groups were measured at12 hpc by ELISA.


E , F MARCH2 + / + ( n = 5 ) or MARCH2 - / - ( n = 5 ) mice were challenged intraperitoneally with LPS ( 24 mg / kg ) . cDNA was prepared from total RNA extracted from spleen and liver of mice . Expression of mRNA encoding IL - 6 , TNF - α , CXCL10 , IL - 1β in spleen ( E ) and liver ( F ) from mice in each group was examined at 6 hpc by qPCR .

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E, F MARCH2+/+ (n = 5) or MARCH2-/- (n = 5) mice were challenged intraperitoneally with LPS (24 mg/kg). cDNA was prepared from total RNA extracted from spleen and liver of mice. Expression of mRNA encoding IL-6, TNF-α, CXCL10, IL-1β in spleen (E) and liver (F) from mice in each group was examined at 6 hpc by qPCR.


A - C BMDMs isolated from MARCH2 + / + or MARCH2 - / - mice were infected with PR8 - GFP ( A , MOI = 3 ) , VSV - GFP ( B , MOI = 5 ) , or HSV - GFP ( C , MOI = 3 ) , and virus titer was measured in a plaque assay .

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A-C BMDMs isolated from MARCH2+/+ or MARCH2-/- mice were infected with PR8-GFP (A, MOI=3), VSV-GFP (B, MOI = 5), or HSV-GFP (C, MOI = 3), and virus titer was measured in a plaque assay.


D , E BMDMs isolated from MARCH2 + / + or MARCH2 - / - mice were infected with viruses or treated with poly ( I : C ) ( 80 µg / ml ) or poly ( dA : dT ) ( 1 µg / ml ) . The concentration of secreted IFN - β ( D ) and IL - 6 ( E ) in the supernatants was measured in an ELISA .

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D, E BMDMs isolated from MARCH2+/+ or MARCH2-/- mice were infected with viruses or treated with poly(I:C) (80 µg/ml) or poly (dA:dT) (1 µg/ml). The concentration of secreted IFN-β (D) and IL-6 (E) in the supernatants was measured in an ELISA.


PBMCs isolated from whole peripheral blood from MARCH2 + / + or MARCH2 - / - mice were infected with VSV - GFP ( MOI = 1 ) . ( F ) The virus titer was measured in a plaque assay .

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PBMCs isolated from whole peripheral blood from MARCH2+/+ or MARCH2-/- mice were infected with VSV-GFP (MOI =1). (F) The virus titer was measured in a plaque assay.


PBMCs isolated from whole peripheral blood from MARCH2 + / + or MARCH2 - / - mice were infected with VSV - GFP ( MOI = 1 ) . ( G ) Concentration of IFN - β and IL - 6 in the supernatant was measured in an ELISA .

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PBMCs isolated from whole peripheral blood from MARCH2+/+ or MARCH2-/- mice were infected with VSV-GFP (MOI =1). (G) Concentration of IFN-β and IL-6 in the supernatant was measured in an ELISA.


PMs isolated from MARCH2 + / + or MARCH2 - / - mice infected with CVB - GFP ( MOI = 3 ) . ( H ) Virus titer was measured in a plaque assay .

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PMs isolated from MARCH2+/+ or MARCH2-/- mice infected with CVB-GFP (MOI = 3). (H) Virus titer was measured in a plaque assay.


PMs isolated from MARCH2 + / + or MARCH2 - / - mice infected with CVB - GFP ( MOI = 3 ) . ( I ) Concentration of IFN - β and IL - 6 in the supernatant was measured in an ELISA .

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PMs isolated from MARCH2+/+ or MARCH2-/- mice infected with CVB-GFP (MOI = 3). (I) Concentration of IFN-β and IL-6 in the supernatant was measured in an ELISA.


J , K BMDMs ( J ) or PMs ( K ) isolated from MARCH2 + / + or MARCH2 - / - mice were infected with Salmonella typhimurium or L . monocytogenes , or treated with LPS or zymosan . The concentration of IL - 6 and IL - 12 in supernatant was analyzed in an ELISA .

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J, K BMDMs (J) or PMs (K) isolated from MARCH2+/+ or MARCH2-/- mice were infected with Salmonella typhimurium or L. monocytogenes, or treated with LPS or zymosan. The concentration of IL-6 and IL-12 in supernatant was analyzed in an ELISA.


L , M RAW 264 . 7 cells transfected with control siRNA ( si - control ) or MARCH2 - specific siRNA ( si - MARCH2 ) were infected with PR8 - GFP ( MOI = 1 ) , VSV - GFP ( MOI = 0 . 5 ) , poly ( I : C ) ( 20 µg / ml ) or HSV - GFP ( MOI = 1 ) poly ( dA : dT ) ( 1 µg / ml ) and IFN - β ( L ) , and IL - 6 ( M ) levels in the supernatant were measured in an ELISA .

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L, M RAW 264.7 cells transfected with control siRNA (si-control) or MARCH2-specific siRNA (si-MARCH2) were infected with PR8-GFP (MOI = 1), VSV-GFP (MOI = 0.5), poly (I:C) (20 µg/ml) or HSV-GFP (MOI = 1) poly (dA:dT) (1 µg/ml) and IFN-β (L), and IL-6 (M) levels in the supernatant were measured in an ELISA.


N RAW 264 . 7 cells transfected with control siRNA ( si - control ) or MARCH2 - specific siRNA ( si - MARCH2 ) were infected with S . typhimurium , or L . monocytogenes , or treated with LPS ( 100 ng / ml ) or zymosan ( 100 µg / ml ) and IL - 6 secretion into the cell supernatant was measured in an

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N RAW 264.7 cells transfected with control siRNA (si-control) or MARCH2-specific siRNA (si-MARCH2) were infected with S. typhimurium, or L. monocytogenes, or treated with LPS (100 ng/ml) or zymosan (100 µg/ml) and IL-6 secretion into the cell supernatant was measured in an ELISA


HEK293T cells were transfected with a firefly luciferase reporter plasmid encoding the IFN - β promoter , plus a TK renilla plasmid and an increasing dose of flag - tagged MARCH2 plasmid ( 50 , 100 , 200 , 400 ng ) plus expression plasmids for RIG - I 2CARD , RIG - I , MDA - 5 , MAVS or stimulated with poly ( I : C ) , for 24 h . Results are expressed relative to those of renilla luciferase alone ( Internal control ) .

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HEK293T cells were transfected with a firefly luciferase reporter plasmid encoding the IFN-β promoter , plus a TK renilla plasmid and an increasing dose of flag-tagged MARCH2 plasmid (50, 100, 200, 400 ng) plus expression plasmids for RIG-I 2CARD, RIG-I, MDA-5, MAVS or stimulated with poly (I:C), for 24 h. Results are expressed relative to those of renilla luciferase alone (Internal control).


HEK293T cells were transfected with a firefly luciferase reporter plasmid encoding the IFN - β promoter plus a TK renilla plasmid and an increasing dose of flag - tagged MARCH2 plasmid ( 50 , 100 , 200 , 400 ng ) plus expression plasmids for TBK1 TRIF , for 24 h . Results are expressed relative to those of renilla luciferase alone ( Internal control ) .

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HEK293T cells were transfected with a firefly luciferase reporter plasmid encoding the IFN-β promoter plus a TK renilla plasmid and an increasing dose of flag-tagged MARCH2 plasmid (50, 100, 200, 400 ng) plus expression plasmids for TBK1 TRIF , for 24 h. Results are expressed relative to those of renilla luciferase alone (Internal control).


HEK293T cells were transfected with a firefly luciferase reporter plasmid encoding the the NF - κB promoter plus a TK renilla plasmid and an increasing dose of flag - tagged MARCH2 plasmid ( 50 , 100 , 200 , 400 ng ) plus expression plasmids for TRAF 6 , TRAF 2 , NEMO for 24 h . Results are expressed relative to those of renilla luciferase alone ( Internal control ) .

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HEK293T cells were transfected with a firefly luciferase reporter plasmid encoding the the NF-κB promoter plus a TK renilla plasmid and an increasing dose of flag-tagged MARCH2 plasmid (50, 100, 200, 400 ng) plus expression plasmids for TRAF 6, TRAF 2, NEMO for 24 h. Results are expressed relative to those of renilla luciferase alone (Internal control).


HEK293T cells were transfected with a firefly luciferase reporter plasmid encoding the NF - κB promoter plus a TK renilla plasmid and an increasing dose of flag - tagged MARCH2 plasmid ( 50 , 100 , 200 , 400 ng ) plus expression plasmids for TBK1 TRIF , for 24 h . Results are expressed relative to those of renilla luciferase alone ( Internal control ) .

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HEK293T cells were transfected with a firefly luciferase reporter plasmid encoding the NF-κB promoter plus a TK renilla plasmid and an increasing dose of flag-tagged MARCH2 plasmid (50, 100, 200, 400 ng) plus expression plasmids for TBK1 TRIF , for 24 h. Results are expressed relative to those of renilla luciferase alone (Internal control).


Interaction between MARCH2 and NEMO in response to viral infection . HEK293T cells ( E ) were infected with NDV - GFP in a time - dependent manner in the presence of MG132 ( proteasome inhibitor , 10 μM ) . Cell lysates were subjected to immunoprecipitation with an anti - NEMO antibody , followed by immunoblotting with an anti - MARCH2 antibody .

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Interaction between MARCH2 and NEMO in response to viral infection. HEK293T cells (E) were infected with NDV-GFP in a time-dependent manner in the presence of MG132 (proteasome inhibitor, 10 μM). Cell lysates were subjected to immunoprecipitation with an anti-NEMO antibody, followed by immunoblotting with an anti-MARCH2 antibody.


Interaction between MARCH2 and NEMO in response to viral infection . Raw264 . 7 cells ( F ) were infected with NDV - GFP in a time - dependent manner in the presence of MG132 ( proteasome inhibitor , 10 μM ) . Cell lysates were subjected to immunoprecipitation with an anti - NEMO antibody , followed by immunoblotting with an anti - MARCH2 antibody .

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Interaction between MARCH2 and NEMO in response to viral infection. Raw264.7 cells (F) were infected with NDV-GFP in a time-dependent manner in the presence of MG132 (proteasome inhibitor, 10 μM). Cell lysates were subjected to immunoprecipitation with an anti-NEMO antibody, followed by immunoblotting with an anti-MARCH2 antibody.


G Confocal microscopy was conducted to examine time - dependent colocalization of MARCH2 and NEMO in HeLa cells upon NDV infection ( MOI = 1 ) in the presence of MG132 ( 10 μM ) . Arrow indicates the co - localized NEMO and MARCH2 protein .

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G Confocal microscopy was conducted to examine time-dependent colocalization of MARCH2 and NEMO in HeLa cells upon NDV infection (MOI = 1) in the presence of MG132 (10 μM). Arrow indicates the co-localized NEMO and MARCH2 protein.


Interaction between MARCH2 and NEMO in response to bacterial infection . Raw264 . 7 cells were infected with L . monocytogenes ( H ) or S . typhimurium ( I ) in a time - dependent manner in the presence of MG132 . Cell lysates were subjected to immunoprecipitation with an anti - NEMO antibody , followed by immunoblotting with an anti - MARCH2 antibody .

Interaction between MARCH2 and NEMO in response to bacterial infection. Raw264.7 cells were infected with L. monocytogenes (H) or S. typhimurium (I) in a time-dependent manner in the presence of MG132. Cell lysates were subjected to immunoprecipitation with an anti-NEMO antibody, followed by immunoblotting with an anti-MARCH2 antibody.


J Confocal microscopy was conducted to examine time - dependent colocalization of MARCH2 and NEMO in HeLa cells expressing TLR2 upon zymosan treatment ( 100 μg / ml ) in the presence of MG132 ( 10 μM ) . Arrow indicates the co - localized NEMO and MARCH2 protein .

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J Confocal microscopy was conducted to examine time-dependent colocalization of MARCH2 and NEMO in HeLa cells expressing TLR2 upon zymosan treatment (100 μg/ml) in the presence of MG132 (10 μM). Arrow indicates the co-localized NEMO and MARCH2 protein.


NEMO degradation assay . ( A ) HEK293T cells were transfected with different doses of Strep - tagged MARCH2 in the presence HA - tagged NEMO plasmid .

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NEMO degradation assay. (A) HEK293T cells were transfected with different doses of Strep-tagged MARCH2 in the presence HA-tagged NEMO plasmid.


NEMO degradation assay . ( B ) MARCH2 + / + and MARCH2 - / - HEK293T cells were infected with PR8 - GFP ( MOI = 1 ) in time - dependent manner . Cells were harvested following infection , NEMO expression level , total and phosphorylated IRF - 3 , and TBK - 1 were measured by immunoblotting . β - actin was used to confirm equal loading of protein .

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NEMO degradation assay. (B) MARCH2+/+ and MARCH2-/- HEK293T cells were infected with PR8-GFP (MOI=1) in time-dependent manner. Cells were harvested following infection, NEMO expression level, total and phosphorylated IRF-3, and TBK-1 were measured by immunoblotting. β-actin was used to confirm equal loading of protein.


C HEK293T cells transfected with a Strep - tagged empty vector or MARCH2 together with GST - tagged NEMO were treated with MG132 ( 10 μM ) , chloroquine , NH4Cl , or Z - VAD for 6 h before harvest . Whole cell lysates were subjected to immunoblotting with indicated antibodies .

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C HEK293T cells transfected with a Strep-tagged empty vector or MARCH2 together with GST-tagged NEMO were treated with MG132 (10 μM), chloroquine, NH4Cl, or Z-VAD for 6 h before harvest. Whole cell lysates were subjected to immunoblotting with indicated antibodies.


D HEK293T cells were transfected with different doses of Strep - tagged MARCH2 together with and GST - tagged NEMO plasmid . MG132 ( 10 μM ) was added to cells for 6 h prior to harvest . Whole cell lysates were subjected to immunoblotting with the indicated antibodies .

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D HEK293T cells were transfected with different doses of Strep-tagged MARCH2 together with and GST-tagged NEMO plasmid. MG132 (10 μM) was added to cells for 6 h prior to harvest. Whole cell lysates were subjected to immunoblotting with the indicated antibodies.


E Confocal microscopy was used to examine colocalization of MARCH2 and NEMO in HeLa cells upon NDV infection ( MOI = 1 ) in the presence or absence of MG132 ( 10 μM ) . Scale bar , 10μm . Arrow indicates the co - localized NEMO and MARCH2 protein .

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E Confocal microscopy was used to examine colocalization of MARCH2 and NEMO in HeLa cells upon NDV infection (MOI = 1) in the presence or absence of MG132 (10 μM). Scale bar, 10μm. Arrow indicates the co-localized NEMO and MARCH2 protein.


F HEK293T cells transfected with Strep - tagged empty vector or MARCH2 with GST - tagged NEMO and each different HA - tagged ubiquitin mutants ( indicated lysine ( K ) only , other lysines ( K ) mutated to arginines ( R ) were treated with MG132 ( 10 μM ) for 6 h before harvest . Lysates were subjected to pull down with GST beads , followed by immunoblotting with an anti - HA antibody . Whole cell lysates were determined by immunoblotting with the indicated antibodies .

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F HEK293T cells transfected with Strep-tagged empty vector or MARCH2 with GST-tagged NEMO and each different HA-tagged ubiquitin mutants (indicated lysine (K) only, other lysines (K) mutated to arginines (R) were treated with MG132 (10 μM) for 6 h before harvest. Lysates were subjected to pull down with GST beads, followed by immunoblotting with an anti-HA antibody. Whole cell lysates were determined by immunoblotting with the indicated antibodies.


G NEMO ubiquitination assay . HEK293T cells were transfected with different doses of Strep - tagged MARCH2 in the presence of a HA - tagged NEMO plasmid . Lysates were immunoprecipitated with anti - HA antibody followed by immunoblotting with anti - K48 antibody .

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G NEMO ubiquitination assay. HEK293T cells were transfected with different doses of Strep-tagged MARCH2 in the presence of a HA-tagged NEMO plasmid. Lysates were immunoprecipitated with anti-HA antibody followed by immunoblotting with anti-K48 antibody.


MARCH2 + / + HEK293T cells harboring empty vector or MARCH2 - / - HEK293T cells harboring empty vector , Flag - tagged MARCH2 , or MARCH2CCH ( a mutant harboring mutations in residues within the catalytic domain : C64S , C67S , and H90Q ) were infected with VSV - GFP ( MOI = 0 . 05 ) . Viral replication was assessed under a fluorescence microscope ( H )

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

MARCH2+/+ HEK293T cells harboring empty vector or MARCH2-/- HEK293T cells harboring empty vector, Flag-tagged MARCH2, or MARCH2CCH (a mutant harboring mutations in residues within the catalytic domain: C64S, C67S, and H90Q) were infected with VSV-GFP (MOI = 0.05). Viral replication was assessed under a fluorescence microscope (H)


MARCH2 + / + HEK293T cells harboring empty vector or MARCH2 - / - HEK293T cells harboring empty vector , Flag - tagged MARCH2 , or MARCH2CCH ( a mutant harboring mutations in residues within the catalytic domain : C64S , C67S , and H90Q ) were infected with VSV - GFP ( MOI = 0 . 05 ) . by measuring GFP expression using a fluorescence

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MARCH2+/+ HEK293T cells harboring empty vector or MARCH2-/- HEK293T cells harboring empty vector, Flag-tagged MARCH2, or MARCH2CCH (a mutant harboring mutations in residues within the catalytic domain: C64S, C67S, and H90Q) were infected with VSV-GFP (MOI = 0.05). by measuring GFP expression using a fluorescence modulator


MARCH2 + / + HEK293T cells harboring empty vector or MARCH2 - / - HEK293T cells harboring empty vector , Flag - tagged MARCH2 , or MARCH2CCH ( a mutant harboring mutations in residues within the catalytic domain : C64S , C67S , and H90Q ) were infected with VSV - GFP ( MOI = 0 . 05 ) . ( J ) Levels of IFN - β ( top ) and IL - 6 ( bottom ) in cell supernatants were measured in an ELISA .

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MARCH2+/+ HEK293T cells harboring empty vector or MARCH2-/- HEK293T cells harboring empty vector, Flag-tagged MARCH2, or MARCH2CCH (a mutant harboring mutations in residues within the catalytic domain: C64S, C67S, and H90Q) were infected with VSV-GFP (MOI = 0.05). (J) Levels of IFN-β (top) and IL-6 (bottom) in cell supernatants were measured in an ELISA.


K In vitro ubiquitination assay for NEMO . HEK293T cells were transfected with GST - tagged NEMO , Strep - tagged MARCH2 or MARCH2CCH individually . Immunoprecipitates were incubated with a reaction mixture containing ubiquitin , E1 , and UbcH6 ( E2 ) , followed by immunoblotting with an anti - K48 - linkage specific polyubiquitin antibody .

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K In vitro ubiquitination assay for NEMO. HEK293T cells were transfected with GST-tagged NEMO, Strep-tagged MARCH2 or MARCH2CCH individually. Immunoprecipitates were incubated with a reaction mixture containing ubiquitin, E1, and UbcH6 (E2), followed by immunoblotting with an anti-K48-linkage specific polyubiquitin antibody.


( B ) HEK293T cells were transfected with Strep - tagged MARCH2 domain constructs together with GST - tagged NEMO . Whole cell lysates were subjected to a Strep - PD assay , followed by immunoblotting with an anti - GST antibody .

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(B) HEK293T cells were transfected with Strep-tagged MARCH2 domain constructs together with GST-tagged NEMO. Whole cell lysates were subjected to a Strep-PD assay, followed by immunoblotting with an anti-GST antibody.


( D ) HEK293T cells were transfected with GST - tagged NEMO domain constructs together with Strep - tagged MARCH2 - TM . Whole cell lysates were subjected to a Strep - PD assay , followed by immunoblotting with an anti - GST antibody .

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(D) HEK293T cells were transfected with GST-tagged NEMO domain constructs together with Strep-tagged MARCH2-TM. Whole cell lysates were subjected to a Strep-PD assay, followed by immunoblotting with an anti-GST antibody.


( F ) HEK293T cells were transfected with HA - tagged NEMO domain constructs together with GST - tagged MARCH2 . Whole cell lysates were subjected to a GST - PD assay , followed by immunoblotting with an anti - HA antibody .

(F) HEK293T cells were transfected with HA-tagged NEMO domain constructs together with GST-tagged MARCH2. Whole cell lysates were subjected to a GST-PD assay, followed by immunoblotting with an anti-HA antibody.


G HEK293T cells transfected with His - tagged ubiquitin and HA - tagged NEMO WT or NEMO lacking the CC1 domain together with Strep - tagged empty vector or MARCH2 were treated with MG132 ( 10 μM ) for 6 h before harvest . Lysates were subjected to immunoprecipitation with an anti - HA antibody , followed by immunoblotting with an anti - His antibody . WCL were subjected to immunoblotting with anti - HA , anti - Strep , or anti - β - actin antibody .

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G HEK293T cells transfected with His-tagged ubiquitin and HA-tagged NEMO WT or NEMO lacking the CC1 domain together with Strep-tagged empty vector or MARCH2 were treated with MG132 (10 μM) for 6 h before harvest. Lysates were subjected to immunoprecipitation with an anti-HA antibody, followed by immunoblotting with an anti-His antibody. WCL were subjected to immunoblotting with anti-HA, anti-Strep, or anti-β-actin antibody.


( I ) HEK293T cells transfected with GST - tagged NEMO mutants together with Strep - tagged empty vector or MARCH2 were subjected to immunoblotting with anti - GST , anti - Strep , or anti - β - actin antibody .

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(I) HEK293T cells transfected with GST-tagged NEMO mutants together with Strep-tagged empty vector or MARCH2 were subjected to immunoblotting with anti-GST, anti-Strep, or anti-β-actin antibody.


J HEK293T cells transfected with HA - tagged ubiquitin and GST - tagged NEMO mutants together with Strep - tagged empty vector or MARCH2 were subjected to pulldown with GST beads , followed by immunoblotting with anti - Lys48 ( K48 ) - linkage specific polyubiquitin ( K48 - ub ) , anti - Lys63 ( K63 ) - linkage specific polyubiquitin ( K63 - ub ) , or anti - Strep antibody . Whole cell lysates were determined by immunoblotting with the indicated antibodies .

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J HEK293T cells transfected with HA-tagged ubiquitin and GST-tagged NEMO mutants together with Strep-tagged empty vector or MARCH2 were subjected to pulldown with GST beads, followed by immunoblotting with anti-Lys48 (K48)-linkage specific polyubiquitin (K48-ub), anti-Lys63 (K63)-linkage specific polyubiquitin (K63-ub), or anti-Strep antibody. Whole cell lysates were determined by immunoblotting with the indicated antibodies.


A HEK293T cells were transfected with GST - tagged NEMO - WT and its point mutants with or without Strep - tagged MARCH2 . Whole cell lysates were subjected to immunoblotting with the indicated antibodies .

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A HEK293T cells were transfected with GST-tagged NEMO-WT and its point mutants with or without Strep-tagged MARCH2. Whole cell lysates were subjected to immunoblotting with the indicated antibodies.


B HEK293T cells were transfected with GST - tagged NEMO - WT and its point mutants together with Strep - tagged MARCH2 in the presence of MG132 . Whole cell lysates were subjected to a GST - PD assay , followed by immunoblotting with an anti - K48 linkage specific polyubiquitin antibody .

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B HEK293T cells were transfected with GST-tagged NEMO-WT and its point mutants together with Strep-tagged MARCH2 in the presence of MG132. Whole cell lysates were subjected to a GST-PD assay, followed by immunoblotting with an anti-K48 linkage specific polyubiquitin antibody.


C In vitro ubiquitination assay for NEMO - K326R . HEK293T cells were transfected with GST - tagged NEMO , GST - tagged NEMO - K326R or Strep - tagged MARCH2 individually . Immunoprecipitates were incubated with a reaction mixture containing ubiquitin , E1 , and UbcH6 ( E2 ) , followed by immunoblotting with an anti - K48 - linkage specific polyubiquitin antibody .

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C In vitro ubiquitination assay for NEMO-K326R. HEK293T cells were transfected with GST-tagged NEMO, GST-tagged NEMO-K326R or Strep-tagged MARCH2 individually. Immunoprecipitates were incubated with a reaction mixture containing ubiquitin, E1, and UbcH6 (E2), followed by immunoblotting with an anti-K48-linkage specific polyubiquitin antibody.


HEK293T cells transfected with GST - tagged empty vector , NEMO - WT , or NEMO - K326R , with or without Strep - tagged MARCH2 were infected with VSV - GFP . ( D - F ) Viral replication was determined by fluorescence microscopy ( D )

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HEK293T cells transfected with GST-tagged empty vector, NEMO-WT, or NEMO-K326R, with or without Strep-tagged MARCH2 were infected with VSV-GFP. (D-F) Viral replication was determined by fluorescence microscopy (D)


HEK293T cells transfected with GST - tagged empty vector , NEMO - WT , or NEMO - K326R , with or without Strep - tagged MARCH2 were infected with VSV - GFP . , fluorescence absorbance ( E )

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HEK293T cells transfected with GST-tagged empty vector, NEMO-WT, or NEMO-K326R, with or without Strep-tagged MARCH2 were infected with VSV-GFP. , fluorescence absorbance (E)


HEK293T cells transfected with GST - tagged empty vector , NEMO - WT , or NEMO - K326R , with or without Strep - tagged MARCH2 were infected with VSV - GFP . plaque assay ( F ) .

HEK293T cells transfected with GST-tagged empty vector, NEMO-WT, or NEMO-K326R, with or without Strep-tagged MARCH2 were infected with VSV-GFP. plaque assay (F).


HEK293T cells transfected with GST - tagged empty vector , NEMO - WT , or NEMO - K326R , with or without Strep - tagged MARCH2 were infected with VSV - GFP . ( G ) IFN - β and IL - 6 in the cell supernatants were measured in an ELISA .

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

HEK293T cells transfected with GST-tagged empty vector, NEMO-WT, or NEMO-K326R, with or without Strep-tagged MARCH2 were infected with VSV-GFP. (G) IFN-β and IL-6 in the cell supernatants were measured in an ELISA.


H , I IFN - β ( H ) and NF - κB ( I ) luciferase reporter assay .

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

H, I IFN-β (H) and NF-κB (I) luciferase reporter assay.


HUVECs transfected with control or CEP41 siRNAs were scratched ( 0 h ) to induce wounding and then incubated for 12 h to allow wound closure . The wound margins were observed every 4 h in the CEP41 # 1 and # 2 siRNA - transfected cells and compared to those of control cells . Representative images of cells subjected to the wound closure assay in ( A ) . Scale bars , 600 μm . Quantification of the extent of wound closure in ( B ) presented graphically by measuring the distance between the dotted lines at each time point .

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

HUVECs transfected with control or CEP41 siRNAs were scratched (0 h) to induce wounding and then incubated for 12 h to allow wound closure. The wound margins were observed every 4 h in the CEP41#1 and #2 siRNA-transfected cells and compared to those of control cells. Representative images of cells subjected to the wound closure assay in (A). Scale bars, 600 μm. Quantification of the extent of wound closure in (B) presented graphically by measuring the distance between the dotted lines at each time point.


The siRNA - transfected HUVECs were plated inside a transwell chamber and incubated with serum for 18 h . The cells that invaded were observed after staining with crystal violet ( CV ) solution . Scale bars , 600 μm . The numbers of cells that invaded in each field of view was counted with the ImageJ software in ( D ) .

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

The siRNA-transfected HUVECs were plated inside a transwell chamber and incubated with serum for 18 h. The cells that invaded were observed after staining with crystal violet (CV) solution. Scale bars, 600 μm. The numbers of cells that invaded in each field of view was counted with the ImageJ software in (D).


Tubulogenesis of control and CEP41 - knockdown cells for 18 h was compared via an in vitro angiogenesis assay . Scale bars , 600 μm . Quantification of tube node numbers in ( F ) and tube length in ( G ) from each field of view using the ImageJ angiogenesis analyzer at the indicated time points . The graph compares the relative length of control cells and CEP41 - knockdown cells . Data are shown as mean ± SD of five independent experiments with ≥ 5 tubulogenesis regions per condition .

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

Tubulogenesis of control and CEP41-knockdown cells for 18 h was compared via an in vitro angiogenesis assay. Scale bars, 600 μm. Quantification of tube node numbers in (F) and tube length in (G) from each field of view using the ImageJ angiogenesis analyzer at the indicated time points. The graph compares the relative length of control cells and CEP41-knockdown cells. Data are shown as mean ± SD of five independent experiments with ≥ 5 tubulogenesis regions per condition.


Blood vessels were observed in MOs ( control , cep41 AUG ( 2 . 5 ng ) , or cep41 SB ( 2 ng ) ) - injected or cep41 - mutated Tg ( kdrl : eGFP ) zebrafish at 40 hours post - fertilization ( hpf ) by fluorescent microscopy . Asterisks and arrowheads indicate impaired ISVs and DLAVs , respectively . The representative images for analysis of ISV lumen diameter are indicated by dotted rectangles . A , anterior ; P , posterior ; DLAV , dorsal longitudinal anastomotic vessel ; ISV , intersegmental vessel . Scale bars , 100 μm . Quantification of ISV lumen diameter in ( B ) , the numbers of defective ISVs in ( C ) , the numbers of embryos with aberrant DLAVs in ( D ) , and the numbers of ruptured DLAVs in ( E ) from data observed in equivalent fields of view ( within eight somites ) . The severity of blood vessel defects in cep41 - deficient zebrafish : B ( narrowed ISVs ) < C ( shorten , fused , and missing ISVs ) < D and E ( ruptured DLAVs ) . Data are shown as mean ± SD of three independent experiments with ≥ 20 embryos per condition .

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

Blood vessels were observed in MOs (control, cep41 AUG (2.5 ng), or cep41 SB (2 ng))-injected or cep41-mutated Tg(kdrl:eGFP) zebrafish at 40 hours post-fertilization (hpf) by fluorescent microscopy. Asterisks and arrowheads indicate impaired ISVs and DLAVs, respectively. The representative images for analysis of ISV lumen diameter are indicated by dotted rectangles. A, anterior; P, posterior; DLAV, dorsal longitudinal anastomotic vessel; ISV, intersegmental vessel. Scale bars, 100 μm. Quantification of ISV lumen diameter in (B), the numbers of defective ISVs in (C), the numbers of embryos with aberrant DLAVs in (D), and the numbers of ruptured DLAVs in (E) from data observed in equivalent fields of view (within eight somites). The severity of blood vessel defects in cep41-deficient zebrafish: B (narrowed ISVs) < C (shorten, fused, and missing ISVs) < D and E (ruptured DLAVs). Data are shown as mean ± SD of three independent experiments with ≥ 20 embryos per condition.


CV protrusions ( arrowheads ) and vascular loops ( asterisks ) were observed in zebrafish at 35 hpf . The images within the dotted rectangles are magnified in the right panels . A , anterior ; P , posterior ; CA , caudal artery ; CV , caudal vein . Scale bars , 40 μm . Quantification of the length of the CV protrusions in ( G ) presented graphically by measuring them in equivalent fields of view ( within five somites ) .

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

CV protrusions (arrowheads) and vascular loops (asterisks) were observed in zebrafish at 35 hpf. The images within the dotted rectangles are magnified in the right panels. A, anterior; P, posterior; CA, caudal artery; CV, caudal vein. Scale bars, 40 μm. Quantification of the length of the CV protrusions in (G) presented graphically by measuring them in equivalent fields of view (within five somites).


Control or CEP41 siRNA - transfected HUVECs were immunostained with Ac - Tub - or GT335 - specific antibodies in ( A ) . The rectangles indicate the representative cells from each immunostaining experiment presented as magnified images in the right panels . Scale bars , 20 μm . Quantification of the ciliated cell numbers in the images in ( A ) from the results of three independent experiments with ≥ 200 cells per condition ( mean ± SD ) in ( B ) .

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

Control or CEP41 siRNA-transfected HUVECs were immunostained with Ac-Tub- or GT335-specific antibodies in (A). The rectangles indicate the representative cells from each immunostaining experiment presented as magnified images in the right panels. Scale bars, 20 μm. Quantification of the ciliated cell numbers in the images in (A) from the results of three independent experiments with ≥ 200 cells per condition (mean ± SD) in (B).


Wild - type and cep41 - deficient Tg ( kdrl : eGFP ) zebrafish embryos were fixed for immunostaining with Ac - Tub - or GT335 - specific antibodies at 28 hpf in ( C ) . The rectangles indicate the arterial cilia ( AC ) and venous cilia ( VC ) in zebrafish endothelial cells ( EC ) . Magnified representative images are displayed in the right panels . Scale bars , 40 μm . Quantification of the labeled cilia observed in equivalent fields of view are presented graphically in ( D ) . Data are shown as median of three independent experiments ( n ≥ 20 embryos per condition ) .

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

Wild-type and cep41-deficient Tg(kdrl:eGFP) zebrafish embryos were fixed for immunostaining with Ac-Tub- or GT335-specific antibodies at 28 hpf in (C). The rectangles indicate the arterial cilia (AC) and venous cilia (VC) in zebrafish endothelial cells (EC). Magnified representative images are displayed in the right panels. Scale bars, 40 μm. Quantification of the labeled cilia observed in equivalent fields of view are presented graphically in (D). Data are shown as median of three independent experiments (n ≥ 20 embryos per condition).


HUVECs transfected with CCP5 and / or CEP41 siRNAs were scratched and incubated to evaluate wound closure . The extent of wound closure in co - transfected cells with CCP5 and CEP41 siRNAs was compared with that in control or CEP41 siRNA - transfected cells at the indicated time points ( A ) . Scale bars , 600 μm . Quantification of the extent of wound closure in ( A ) is presented graphically by measuring the distance between the dotted lines at each time point in ( B ) . Data are shown as mean ± SD of three independent experiments ( n ≥ 3 scratches per experimental condition ) .

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

HUVECs transfected with CCP5 and/or CEP41 siRNAs were scratched and incubated to evaluate wound closure. The extent of wound closure in co-transfected cells with CCP5 and CEP41 siRNAs was compared with that in control or CEP41 siRNA-transfected cells at the indicated time points (A). Scale bars, 600 μm. Quantification of the extent of wound closure in (A) is presented graphically by measuring the distance between the dotted lines at each time point in (B). Data are shown as mean ± SD of three independent experiments (n ≥ 3 scratches per experimental condition).


The indicated siRNA - transfected HUVECs were subjected to in vitro angiogenesis assays . Tubulogenesis of HUVECs depleted for CEP41 and CCP5 was compared to that of control , CEP41 single - knockdown , or CCP5 single - knockdown cells ( C ) . Scale bars , 600 μm . Quantification of tube node number in ( D ) and tube length in ( E ) from data examined within equivalent fields of view at the indicated time points using the ImageJ angiogenesis analyzer . Data are shown as mean ± SD of five independent experiments with ≥ 5 tubulogenesis regions per condition .

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

The indicated siRNA-transfected HUVECs were subjected to in vitro angiogenesis assays. Tubulogenesis of HUVECs depleted for CEP41 and CCP5 was compared to that of control, CEP41 single-knockdown, or CCP5 single-knockdown cells (C). Scale bars, 600 μm. Quantification of tube node number in (D) and tube length in (E) from data examined within equivalent fields of view at the indicated time points using the ImageJ angiogenesis analyzer. Data are shown as mean ± SD of five independent experiments with ≥ 5 tubulogenesis regions per condition.


The blood vessels in the trunks of cep41 - mutant / morphant zebrafish were compared to those of zebrafish co - injected with ccp5 MOs ( 2 ng ) at 40 hpf in ( F ) . Asterisks and arrowheads indicate impaired ISVs and DLAVs , respectively . A , anterior ; P , posterior . Scale bars , 100 μm . Quantification of the number of defective ISVs and DLAVs from data observed in equivalent fields of view ( within eight somites ) . Data are median of three independent experiments with ≥ 20 embryos per condition .

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

The blood vessels in the trunks of cep41-mutant/morphant zebrafish were compared to those of zebrafish co-injected with ccp5 MOs (2 ng) at 40 hpf in (F). Asterisks and arrowheads indicate impaired ISVs and DLAVs, respectively. A, anterior; P, posterior. Scale bars, 100 μm. Quantification of the number of defective ISVs and DLAVs from data observed in equivalent fields of view (within eight somites). Data are median of three independent experiments with ≥ 20 embryos per condition.


HUVECs co - transfected with CEP41 and CCP5 siRNAs were immunostained with ARL13b - and GT335 - specific antibodies . The staining results were compared to those of control , single CEP41 , or CCP5 siRNA - transfected cells . Representative images indicated with rectangles appear in magnified images in the lower panels . Scale bars , 20 μm . Quantification of relative intensity of GT335 / ARL13b signals in the cilia ciliary length ( J ) in images ( H ) are the result of three independent experiments with ≥ 200 cells per condition ( median ) . * *

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

HUVECs co-transfected with CEP41 and CCP5 siRNAs were immunostained with ARL13b- and GT335-specific antibodies. The staining results were compared to those of control, single CEP41, or CCP5 siRNA-transfected cells. Representative images indicated with rectangles appear in magnified images in the lower panels. Scale bars, 20 μm. Quantification of relative intensity of GT335/ARL13b signals in the cilia ciliary length (J) in images (H) are the result of three independent experiments with ≥ 200 cells per condition (median). **


Control or CEP41 siRNA - transfected HUVECs cultivated under either static or shear stress states and examined in a wound healing assay in ( A ) . Scale bars , 600 μm . Quantification of the extent of wound closure in ( A ) displayed graphically by measuring the distance between the lines at the indicated time points in ( B ) . Data are presented as mean ± SD of three independent experiments ( n ≥ 3 scratches per experimental condition ) . Statistical significance was determined using the two - way ANOVA followed by Tukey ' s post hoc test ( * * * P < 0 . 01 , ns : non - significant ) .

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

Control or CEP41 siRNA-transfected HUVECs cultivated under either static or shear stress states and examined in a wound healing assay in (A). Scale bars, 600 μm. Quantification of the extent of wound closure in (A) displayed graphically by measuring the distance between the lines at the indicated time points in (B). Data are presented as mean ± SD of three independent experiments (n ≥ 3 scratches per experimental condition). Statistical significance was determined using the two-way ANOVA followed by Tukey's post hoc test (***P < 0.01, ns: non-significant).


in vitro angiogenesis assay was performed in static or shear stress - exposed HUVECs transfected with the indicated siRNAs for 18 h in ( C ) .

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

in vitro angiogenesis assay was performed in static or shear stress-exposed HUVECs transfected with the indicated siRNAs for 18 h in (C).


in vitro angiogenesis assay was performed in static or shear stress - exposed HUVECs transfected with the indicated siRNAs for 18 h in ( C ) . Scale bars , 600 μm . Quantification of tube node number in ( D ) and tube length in ( E ) from data examined within equivalent fields of view at each time point using the ImageJ angiogenesis analyzer . Data are shown as mean ± SD of five independent experiments with ≥ 5 tubulogenesis regions per condition .

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

in vitro angiogenesis assay was performed in static or shear stress-exposed HUVECs transfected with the indicated siRNAs for 18 h in (C). Scale bars, 600 μm. Quantification of tube node number in (D) and tube length in (E) from data examined within equivalent fields of view at each time point using the ImageJ angiogenesis analyzer. Data are shown as mean ± SD of five independent experiments with ≥ 5 tubulogenesis regions per condition.


Control or CEP41 - deficient HUVECs cultivated under laminar shear stress and immunostained with ARL13b - specific antibodies to measure deciliation . Representative images are indicated by rectangles with numbers and shown in magnified images in the right panels . The resulting data were compared to those of cells under static conditions in ( F ) . Scale bars , 20 μm . Quantification of ARL13b - positive ciliary cells ( G ) and of relative intensity of GT335 / ARL13b signals in the cilia ( H ) in images ( F ) is the results of three independent experiments with ≥ 200 cells per condition ( mean ± SD ( G ) , median ( H ) ) . *

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

Control or CEP41-deficient HUVECs cultivated under laminar shear stress and immunostained with ARL13b-specific antibodies to measure deciliation. Representative images are indicated by rectangles with numbers and shown in magnified images in the right panels. The resulting data were compared to those of cells under static conditions in (F). Scale bars, 20 μm. Quantification of ARL13b-positive ciliary cells (G) and of relative intensity of GT335/ARL13b signals in the cilia (H) in images (F) is the results of three independent experiments with ≥ 200 cells per condition (mean ± SD (G), median (H)). *


Control , CEP41 - , or CCP5 - depleted hTERT - RPE1 cells cultured under serum starvation for 48 h and fixed at 18 h after serum retrieval for immunostaining with both ARL13b - and GT335 - specific antibodies in ( I ) . Images of the results 18 h after serum addition . Representative cells are indicated by rectangles . Scale bars , 20 μm . Quantification of ARL13b - positive ciliated cells ( J ) and relative intensity of GT335 / ARL13b signals in the cilia ( K ) under either serum starvation or serum retrieval ( 18 h ) conditions are the results of three independent experiments with ≥ 200 cells per condition ( mean ± SD ( J ) , median ( K ) ) .

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

Control, CEP41-, or CCP5-depleted hTERT-RPE1 cells cultured under serum starvation for 48 h and fixed at 18 h after serum retrieval for immunostaining with both ARL13b- and GT335-specific antibodies in (I). Images of the results 18 h after serum addition. Representative cells are indicated by rectangles. Scale bars, 20 μm. Quantification of ARL13b-positive ciliated cells (J) and relative intensity of GT335/ARL13b signals in the cilia (K) under either serum starvation or serum retrieval (18 h) conditions are the results of three independent experiments with ≥ 200 cells per condition (mean ± SD (J), median (K)).


VEGFA ( A ) and VEGFR2 ( B ) mRNA levels quantified by qRT - PCR in control or CEP41 - knockdown HUVECs under static or shear stress conditions . The expression of GAPDH was quantified for the normalization of the qRT - PCR results . Data are shown as mean ± SD of three independent experiments . Statistical significance was determined with the Brown - Forsythe ANOVA followed by Dunnett ' s T3 post hoc test ( * * P < 0 . 01 , * * * P < 0 . 001 , ns : non - significant ) .

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

VEGFA (A) and VEGFR2 (B) mRNA levels quantified by qRT-PCR in control or CEP41-knockdown HUVECs under static or shear stress conditions. The expression of GAPDH was quantified for the normalization of the qRT-PCR results. Data are shown as mean ± SD of three independent experiments. Statistical significance was determined with the Brown-Forsythe ANOVA followed by Dunnett's T3 post hoc test (**P < 0.01, ***P < 0.001, ns: non-significant).


Immunoblot assays for phospho - AURKA and AURKA were performed in HUVECs transfected with the indicated siRNAs . Protein levels were normalized against β - ACTIN in the same blots .

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

Immunoblot assays for phospho-AURKA and AURKA were performed in HUVECs transfected with the indicated siRNAs. Protein levels were normalized against β-ACTIN in the same blots.


Immunoblot assays for phospho - AURKA and AURKA were performed in hTERT - RPE1 cells ( D ) transfected with the indicated siRNAs . Protein levels were normalized against β - ACTIN in the same blots .

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

Immunoblot assays for phospho-AURKA and AURKA were performed in hTERT-RPE1 cells (D) transfected with the indicated siRNAs. Protein levels were normalized against β-ACTIN in the same blots.


The mRNA levels of zebrafish vegfa ( E ) and vegfr2 ( F ) were quantified by qRT - PCR in eGFP - positive ECs of control - or cep41 - MO - injected Tg ( kdrl : eGFP ) zebrafish at 18 hpf ( low shear stress ) and 26 hpf ( high shear stress ) . The expression of vegfa and vegfr2 was quantified in neuronal cells from control MO - injected zebrafish for comparisons with that of ECs . The expression of zebrafish β - actin was quantified for the normalization of those qRT - PCR results . Data are shown as mean ± SD of three independent experiments . Statistical significance was determined using the one - way ANOVA followed by Tukey ' s post hoc test ( * * * P < 0 . 001 , ns : non - significant ) .

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

The mRNA levels of zebrafish vegfa (E) and vegfr2 (F) were quantified by qRT-PCR in eGFP-positive ECs of control- or cep41-MO-injected Tg(kdrl:eGFP) zebrafish at 18 hpf (low shear stress) and 26 hpf (high shear stress). The expression of vegfa and vegfr2 was quantified in neuronal cells from control MO-injected zebrafish for comparisons with that of ECs. The expression of zebrafish β-actin was quantified for the normalization of those qRT-PCR results. Data are shown as mean ± SD of three independent experiments. Statistical significance was determined using the one-way ANOVA followed by Tukey's post hoc test (***P < 0.001, ns: non-significant).