EMBO/soda-nel huggingface dataset:
Ribosome slowdown triggers codon-mediated mRNA decay independently of ribosome quality control (2021)
Figure 5

Figure 5. The effect of ribosome stalling on mRNA stability.

(A) A scheme of the hCMV uORF2 reporter mRNAs. Wild-type hCMV uORF2 (light blue) or its mutant (dark blue) was inserted at the end of the sfGFP ORF. Asterisks indicate stop codons.

(B) Western blotting to detect the sfGFP levels at 6 hpf. Tubulin (Tub) was detected as a loading control.

(C) qRT-PCR analysis of hCMV uORF2 reporter mRNA levels in wild-type embryos at 6 hpf relative to 2 hpf.

(D) Poly(A) tail analysis of hCMV uORF2 reporter mRNAs at 2 and 6 hpf. The developmental stages are shown above as hpf. The lane labeled A0 shows the 3´ UTR fragment without a poly(A) tail.

(E) Quantification of the PAT assay at 6 hpf in (D).

(F) Scheme of human and zebrafish Znf598 proteins. The sgRNA target site is indicated by an arrowhead. Genome sequences of wild-type and znf598 mutant zebrafish are shown below. The predicted amino acid sequence of mutated Znf598 is shown in red. The asterisk indicates the premature stop codon.

(G) qRT-PCR analysis of hCMV uORF2 reporter mRNA levels in MZznf598 embryos at 6 hpf relative to 2 hpf.

(H) qRT-PCR analysis of hCMV uORF2 reporter mRNA levels in wild-type embryos, MZznf598 embryos and MZznf598 embryos injected with a mRNA encoding Myc-tagged full-length (full) or RING domain deleted (∆RING) Znf598. mRNA levels at 6 hpf relative to 2 hpf are shown.

(I) Western blotting to detect the Myc-tagged Znf598 proteins at 6 hpf. Tubulin (Tub) was detected as a loading control.

(J) Results of PACE in MZznf598 zebrafish embryos. Black circles show the relative stability of the reporter mRNAs (averages of two biological replicates) in MZznf598 embryos. Gray circles show the relative stability of reporter mRNAs in wild-type embryos shown in Figure 2B. The stability of a codon-tag reporter with a UGA stop codon was set to one. Error bars represent maximum and minimum data points. The relative effect of each codon on mRNA stability in wild-type embryos measured in Figure 2B is shown as a color gradient from red (destabilizing) to blue (stabilizing).

Data information: In C, G, and H, bar charts show the average of three independent biological replicates. Error bars show SD. Individual data points are shown as dots. P-values were calculated using two-sided Student's t-test.
Function of the SNARE Ykt6 on autophagosomes requires the Dsl1 complex and the Atg1 kinase complex (2020)
Figure 1

Figure 1. Ykt6 is located to different organelles during starvation.

(A) Localization of Ape1 relative to Ykt6 and Sec63 during nitrogen starvation. Cells expressing GFP-tagged Ykt6 under the control of the GAL1 promoter and tdTomato-tagged Sec63 were transformed with a centromeric plasmid expressing BFP-Ape1. Cells were grown in SGC-LEU, shifted to SG-N for the indicated time, and analyzed by fluorescence microscopy. Images are shown as single planes. Scale bar, 5 µm. Arrows indicate Ykt6 dots colocalizing with Ape1.

(B) Percentage of Ykt6 puncta simultaneously co-localizing with both Ape1 and Sec63. The data were quantified from (A). Error bars represent standard deviation of three independent experiments. Cells (n≥250) and Ape1 dots (n≥50) were quantified.

(C) Localization of Ykt6 relative to different organelle marker proteins during nitrogen starvation. Cells expressing GFP-tagged Ykt6 under the control of the GAL1 promoter and tdTomato-tagged Snx41 (endosomes), Sec63 (ER), Vac8 (vacuole), or Mnn9 (Golgi) were were grown in SGC-LEU, then shifted to SG-N for 2 h, and analyzed by fluorescence microscopy. Single imaging planes are shown. Scale bar, 5 µm. Arrows indicate Ykt6 dots that colocalize with marker proteins.
Function of the SNARE Ykt6 on autophagosomes requires the Dsl1 complex and the Atg1 kinase complex (2020)
Figure 2

Figure 2. Ykt6 is recruited to the PAS at an early stage.

(A-B) Localization of Ykt6 in multiple atg mutants. (A) Wild-type or the respective mutant cells expressing mCherry-tagged Ape1 and GFP-tagged Ykt6 were grown in SGC to allow for Ykt6 expression from the GAL1-promoter and analyzed as in Figure 1A. Scale bar, 5 µm. (B) Quantification of colocalization of Ykt6 relative to Ape1 as a PAS marker. The data were quantified from (A). Error bars represent standard deviation of three independent experiments. Cells (n≥250) and Ape1 dots (n≥70) were quantified.

(C) Localization of Atg9 relative to Ykt6. Cells expressing GFP-tagged Ykt6 and mScarlet-tagged Atg9 were grown and analyzed as in Figure 1. Scale bar, 5 µm. The left is the quantification of colocalization of Ykt6 relative to Atg9. Error bars represent standard deviation of three independent experiments. Cells (n≥200) and Atg9 dots (n≥80) were quantified.

(D-F) Ykt6 localizes to the isolation membrane during starvation. (D) Cells expressing mCherry-tagged Atg8 and GFP-Ykt6 carrying the Ape1 encoding plasmid pRS315-CUP1pr-BFP-APE1 were grown in SGC medium containing CuSO4 and starved for 1 h, before to be analyzed by microscopy. Scale bar, 5 µm. (E) Volume rendering of LLSM image from a single time point after 3D deconvolution from 3D view. Cells expressing mCherry-tagged Atg8 and GFP-Ykt6 carrying plasmid pRS315-CUP1pr-BFP-APE1, cells were grown as Figure 2D, images were analyzed by Imaris. Scale bar, 5 µm. (F) The only partial time points (90 s to 600 s) of images from (E) were assembled, and analyzed by Imaris and image J. Scale bar, 5 µm. 500-1000 cells were analyzed in each independent experiment. The 3D stacks were cropped by Imaris and different channels were split by Image J.

Data information: Arrows in the images indicate dots of Ykt6 colocalizing with Ape1 (A), Atg9 (C), and Atg8 (D,E).
Function of the SNARE Ykt6 on autophagosomes requires the Dsl1 complex and the Atg1 kinase complex (2020)
Figure 3

Figure 3. The Dsl1 complex is required for autophagy.

(A) Effect of ykt6 and dsl1 complex ts mutants on autophagosome-vacuole fusion. Cells carrying a CEN plasmid expressing GFP-Atg8 were grown at 24°C in SGC and then shifted to SG-N for 2 h at 24°C or 37°C. Vacuoles were stained by FM4-64 and analyzed by fluorescence microscopy. Scale bar, 5 µm. Scale bar of inset is 0.6 µm.

(B) Analysis of autophagy over time. Cells were grown at 24°C and shifted to starvation medium at 24°C or 37°C for the indicated time periods. The cells were harvested and proteins were extracted from cells by TCA precipitation. The extracted proteins were analyzed by SDS-PAGE and Western blotting against GFP.

(C) Protease protection assay of ykt6-11 and dsl3 mutants. Lysates of atg1∆, ypt7∆, ykt6ts and dsl3ts cells carrying a CEN plasmid expressing GFP-Atg8 were grown at 37°C and subjected to the protease (PK)-protection (see Materials and methods for details).

(D) Localization of Atg8 relative to Atg1 during nitrogen starvation condition in sec12-1 ts mutant. Cells carrying a CEN plasmid expressing GFP-Atg8 and encoding 3xmCherry-tagged Atg1 were grown at 24°C in SDC and then shifted to SD-N for 2 h at 24°C or 37°C, and analyzed by fluorescence microscopy. Individual slices are shown. Scale bar, 5 µm. Scale bar of inset is 0.6 µm.

(E) Localization of Ykt6 relative to Dsl1 during growth and nitrogen starvation conditions. Cells expressing GFP-tagged Ykt6 under the control of the PHO5 promoter and genomically 3xmCherry-tagged Dsl1 were grown in SDC or SD-N for 2 h, and analyzed by fluorescence microscopy. Individual slices are shown. Scale bar, 5 µm.

(F) Percentage of Ykt6 puncta co-localizing with Dsl1. The data was quantified from (E). Error bars represent standard deviation of three independent experiments. Cells (n≥250) and Dsl1 dots (n≥50) were quantified.

(G) Localization of Dsl3 relative to Atg8 during growth and nitrogen starvation. Cells expressing GFP-tagged Dsl3 and mCherry-tagged Atg8 were grown in SDC or SD-N for 2 h, and analyzed by fluorescence microscopy. Scale bar, 5 µm.

Data information: Arrows in images indicate dots colocalization of Atg1 with Atg8 (D) and Ykt6 with Dsl1 (E).
Function of the SNARE Ykt6 on autophagosomes requires the Dsl1 complex and the Atg1 kinase complex (2020)
Figure 4

Figure 4. Ykt6 targeting to the PAS requires the Dsl1 complex.

(A) Effect of Dsl and COPII ts mutants on Ykt6 localization to autophagosomes. Wild-type and selected ts strains (sec12-1, tip20-5 and dsl3-2) expressing GFP-tagged Ykt6 and mCherry-tagged Atg8 were grown at 24°C in SGC for 8 h and then shifted to SG-N for 2 h at 37°C. Vacuoles were stained by CMAC and analyzed by fluorescence microscopy. Scale bar, 5 µm. Scale bar in inset, 0.7 µm.

(B) Percentage of Ykt6 puncta co-localizing with Atg8. The data was quantified from (A). Error bars represent standard deviation of three independent experiments. Cells (n≥150) and Atg8 dots (n≥100) were quantified.

(C) Localization of Ykt6 relative to Sec63 and Sec13 during growth and nitrogen starvation conditions. tip20-5 cells expressing GFP-tagged Ykt6 and 3xmCherry-tagged Sec63 or Sec13 were grown at 24°C in SGC for 8 h and then shifted to SG-N for 2 h at 24°C or 37°C, and analyzed by fluorescence microscopy. Scale bar, 5 µm. Scale bar of inset, 0.7 µm.

(D) Effect of dsl mutation on autophagosome-vacuole fusion in vitro. The vam3∆ or dsl ts strain expressing Atg9-3xFLAG and GFP-Atg8 were starved for 3 h at 30°C before purifying autophagosomes, which was sufficient to induce the ts phenotype. Vacuoles were isolated from pep4∆ cells expressing Vac8-3xmCherry and then incubated with autophagosomes at 26°C for 1 h with or without ATP (Gao et al., 2018b). Scale bar, 5 µm.

(E) Quantification of (D). Error bars represent standard deviation of 3 independent experiments. Fluorescence intensity of GFP-Atg8 in the vacuolar lumen was quantified by image J using the ROI (region of interest) manager tool. Vacuoles (n≥40) for each experiment were quantified.

Data information: Arrows indicate dot colocalization of Ykt6 and Atg8 (A) and Ykt6 with Sec63 (C).
Function of the SNARE Ykt6 on autophagosomes requires the Dsl1 complex and the Atg1 kinase complex (2020)
Figure 5

Figure 5. Atg9 and the Dsl1 complex independently contribute to the autophagosome biogenesis.

(A-B) Quantification of size and number of autophagosomes in the indicated ts mutant strains. Error bars represent standard deviation of 3 independent experiments. GFP-tagged Atg8 in the different mutants were grown at 23°C (ts mutant) or 30°C (atg1∆) in SDC and then shifted to SD-N for 2 h at 30°C (atg1∆) or 37°C (ts mutant), and analyzed by fluorescence microscopy. Scale bar, 1 µm.

(C) Localization of Atg8 relative to Atg9 during nitrogen starvation. dsl3 cells expressing mCherry-tagged Atg9 and carrying a CEN plasmid expressing GFP-Atg8 were grown at 23°C in SDC and then shifted to SD-N for 2 h at 24°C or 37°C. Cells were analyzed by fluorescence microscopy. Scale bar, 5 µm. Scale bar for inset is 0.6 µm.

(D) Atg9 localization in dsl3 cells during starvation. dsl3 ts cells expressing mCherry-tagged Atg9, and carrying a CEN plasmid expressing GFP-Atg8 and plasmid pRS315-CUP1pr-BFP-APE1 were grown in SDC medium containing CuSO4 to induce formation of the giant Ape1 structure, and starved for 1 h. Cells were analyzed by fluorescence microscopy. Scale bar, 5 µm. Scale bar for inset is 0.6 µm.
Function of the SNARE Ykt6 on autophagosomes requires the Dsl1 complex and the Atg1 kinase complex (2020)
Figure 6

Figure 6. Ykt6 is a direct target of the Atg1 kinase complex during autophagy.

(A) Alignments of Ykt6 core domain with identified phosphorylation sites in SNARE domain. Sc, Saccharomyces cerevisiae; Cg, Candida glabrata; Ca, Candida albicans; Ce, Caenorhabditis elegans; Dm, Drosophila melanogaster; Hs, Homo sapiens; Mm, Mus musculus; Rn, Rattus norvegicus. Red symbols refer to consensus sequence of Atg1 kinase (Papinski et al., 2014). The conserved L is shown in green. The CaaX box is framed, the conserved geranylgeranylated C next to the farnesylation site is marked in orange.

(B) Localization of Ykt6 relative to Atg1 during growth and nitrogen starvation conditions. Cells expressing GFP-tagged Ykt6 under the control of the PHO5 promoter and genomically 3xmCherry-tagged Atg1 were grown in SDC or SD-N for 2 h, and analyzed by fluorescence microscopy. Individual slices are shown. Scale bar, 5 µm. Scale bar of inset is 0.6 µm. Right, percentage of Ykt6 puncta co-localizing with Atg1. Error bars represent standard deviation of three independent experiments. Cells (n≥200) and Atg1 dots (n≥100) were quantified.

(C) In vitro phosphorylation of purified Ykt6 by the Atg1 kinase complex. Purified Ykt6 was incubated with the Atg1-Atg13 complex in the presence or absence of ATP, and Ykt6 band was cut out and analyzed by mass spectrometry.

(D) Localization of Ykt6 relative to Atg8 under growth and nitrogen starvation conditions. Wild-type and atg1D211A cells expressing GFP-tagged Ykt6 under the control of the PHO5 promoter and genomically Cherry-tagged Atg8 were grown in SDC or SD-N for 2 h, and analyzed by fluorescence microscopy. Individual focal planes are shown. Scale bar, 5 µm.

(E) Percentage of Ykt6 puncta co-localizing with Atg8. Error bars represent standard deviation of three independent experiments. Numbers in the column indicates the percentage. Cells (n≥300) and Atg8 dots (n≥60) were quantified.

Data information: Arrows indicate Ykt6 positive dots colocalizing with Atg1 (B) or Atg8 (D).
Function of the SNARE Ykt6 on autophagosomes requires the Dsl1 complex and the Atg1 kinase complex (2020)
Figure 7

Figure 7. The Atg1 kinase complex controls the fusogenic activity of Ykt6 during autophagy.

(A) Growth test of Ykt6 wild-type and mutants. ykt6∆ cells carrying a URA3 plasmid coding for wild-type Ykt6 and pRS413-GAL1pr-YKT6-GFP (wild-type or mutant) were spotted as serial dilutions on SGC-HIS to select for the mutant plasmid or 5-Fluoroorotic acid (5-FOA) to force the loss of the plasmid coding for wild-type Ykt6.

(B) Effect of Ykt6 phospho-mutants on autophagy. ykt6∆ cells expressing wild-type Ykt6 plus a pRS413 plasmid encoding GAL1pr-YKT6 (wild-type or mutants with S to A mutations [SA; S182, S183A], or S to D mutations [SD; S182D, S183D]), and a plasmid expressing GFP-Atg8 were grown in SGC and then shifted to SG-N for 2 h. Cells were analyzed by fluorescence microscopy. Individual slices are shown. Scale bar, 5 µm.

(C) Quantification of Atg8 dots per cell from images in (B). Error bars represent standard deviation of three independent experiments. Cells (n≥100) and Atg8 dots (n≥50) were quantified.

(D) Effect of Atg1-Atg13 subcomplex on autophagosome-vacuole fusion in vitro. The vam3∆ strain carrying pRS413 plasmid encoding GAL1pr-YKT6 (wild-type or mutant), and expressing Atg9-3xFLAG and GFP-Atg8, were starved for 3 h at 30°C before purifying autophagosomes. Vacuoles were isolated from pep4∆ cells expressing Vac8-3xmCherry and then incubated with autophagosomes in the presence or absence of 50 nM Atg1-Atg13 subcomplex at 26°C for 1 h with or without ATP (Gao et al., 2018b). Scale bar, 5 µm.

(E) Quantification of (D). Error bars represent standard deviation of 3 independent experiments. Fluorescence intensity of GFP-Atg8 in the vacuolar lumen was quantified by image J using the ROI (region of interest) manager tool. Vacuoles (n≥40) for each experiment were quantified.

(F) Working model for the recruitment and regulation of Ykt6 during autophagy. Ykt6 arrives either after prenylation of via COPI-coated vesicles at the ER, where it binds the Dsl1 complex. Ykt6 is packaged into COPII-coated vesicles, which deliver Ykt6 independently from Atg9 vesicles to the PAS. Atg1-Atg13 kinase controls Ykt6 activity until autophagosomes are completely closed.

Expanded View Figures
Predicting Infectivity:Comparing Four PCR Assays to Detect Culturable SARS-CoV-2 in Clinical Samples (2021)
Figure 2

Figure 2. Kinetics of SARS-CoV-2 growth in Vero-E6 and Vero E6-TMPRSS2 cells.

(A-F) Vero E6-TMPRSS2 (A, C, E) or Vero-E6 cells (B, D, F) were infected with SARS-CoV-2 WA1 at an MOI of 0.001. Supernatants were collected twice daily for two days, daily for an additional two days, and cells and supernatants were collected for a final time point at day 7 (for Vero E6-TMPRSS2 cells) or day 8 (for Vero E6 cells). A, B) Viral titer was determined by immune-focus forming assay (focus-forming units [FFU]/mL), day 0 values are calculated based on known titer of inoculum. RNA levels were determined by RT-PCR from cells (C, D) or viral supernatants (E, F) for each time point using the indicated primer-probe sets. Note that CT is plotted inversely on the Y axes, so that lower RNA concentrations are shown lower on the axes. All data points represent four technical replicates from two independent experiments and are plotted as mean with error bars plotted as +/- SEM. Limit of detection for infectious titer is plotted as the minimum y value.
Predicting Infectivity:Comparing Four PCR Assays to Detect Culturable SARS-CoV-2 in Clinical Samples (2021)
Figure 4

Figure 4. Viral isolation from SARS-CoV-2 RT-PCR-positive clinical samples in Vero-E6 and Vero-E6 TMPRSS2 cell lines.

(A_B) Vero E6-TMPRSS2 (A) or Vero E6 (B) cells were inoculated with a total of 126 clinical NP swab samples representing a range of viral RNA levels, both total E RNA (Clinical Sample E CT detected by WHO-E primer/probe set) and subgenomic E RNA (CT detected by the Mills-SgE primer set developed in this study). Culture supernatants were harvested when cells displayed CPE or at day 7 if no CPE was observed earlier, clarified, and presence (blue square) or absence (yellow circle) of SARS-CoV-2 determined by immuno-focus assay.
Predicting Infectivity:Comparing Four PCR Assays to Detect Culturable SARS-CoV-2 in Clinical Samples (2021)
Figure 5

Figure 5. Kinetics of SARS-CoV-2 isolation.

(A-B) Supernatants from the panel of infected (A) Vero E6-TMPRSS2 and (B) Vero E6 cells shown in Fig. 4 were harvested when CPE was observed and the viral titer (FFU/mL measured by focus forming assay) was determined for the final time point of each sample that successfully isolated. Samples are colored according to the day CPE was observed and the sample was harvested. Total E RNA CT of the original clinical sample used for viral isolation is plotted on the X axis. Data shown is from the subset of samples that successfully isolated in each cell line. Limit of detection for infectious titer is 400 FFU/mL.
Predicting Infectivity:Comparing Four PCR Assays to Detect Culturable SARS-CoV-2 in Clinical Samples (2021)
Figure 6

Figure 6. Detection of sgE by Wölfel-sgE primer set, or negative-strand RNA in clinical samples.

(A) The amount of subgenomic E RNA in the panel of clinical samples used to infect Vero E6-TMPRSS2 cells in Fig. 4 was measured using a previously-published primer-probe set (Wölfel-sgE) (Wolfel et al., 2020).

(B) For a subset of 85 NP swab samples for which sufficient material remained, SARS-CoV-2 negative-strand E RNA levels were determined by strand-specific RT-PCR with tagged primers. Data information: Culture supernatants were harvested when cells displayed CPE or at day 7 if no CPE was observed earlier, clarified, and presence (blue square) or absence (yellow circle) of infectious SARS-CoV-2 determined by immuno-focus assay.
Predicting Infectivity:Comparing Four PCR Assays to Detect Culturable SARS-CoV-2 in Clinical Samples (2021)
Figure 8

Figure 8. Relative abundance of sgE, negative-strand E, GAPDH mRNA, and 18S rRNA in cells, supernatants and purified virus.

Levels of the cellular housekeeping genes GAPDH and 18S rRNA, negative-strand E (negE), and sgE relative to total levels of E RNA were measured in infected cells, clarified supernatants (4 dpi Vero-E6 samples from Fig. 2) and virus concentrated through a sucrose cushion by ultracentrifugation. Data points represent four technical replicates from two independent experiments (Cell and Supernatant) or three replicates (Concentrated Virus) and are normalized to the total levels of E for each sample, plotted as mean with error bars plotted as +/- SEM.
The onset of PI3K-related vascular malformations occurs during angiogenesis (2022)
Figure 1

Figure 1. PI3K pathway activation in ECs leads to enhanced cell cycle progression.

(A) Immunoblot showing the activation of PI3K/AKT pathway (by assessing the levels of p-AKT Ser473) of ECs 24h after induction of Pik3caH1047R expression.

(B) Volcano plot of genes analysed by RNAseq in ECs upon Pik3caH1047R expression. Differentially expressed genes (DEGs) (p adj < 0.05) in Pik3caH1047R over Pik3caWT ECs at 24h (grey, unchanged; orange, downregulated; purple, upregulated). n=4 biological replicates per genotype.

(C) Pathway enrichment analysis showing most represented biological functions altered upon Pik3caH1047R expression in ECs.

(D) Immunoblot showing the activation of PI3K/AKT pathway of Pik3caWT and Pik3caH1047R ECs after 2h starvation (-) and 30 min stimulation with 20% fetal bovine serum (FBS) (+).

(E, F) (E) Cell cycle analysis by flow cytometry of Pik3caWT and Pik3caH1047R ECs after cultured either without serum (-) or 20% serum (+) for 24h. (F) Percentage of proliferative ECs (in S and G2M phases) from Fig 1E. Error bars are s.e.m. n ≥ 3 biological replicates per genotype. Statistical analysis was performed by nonparametric Mann-Whitney test. **p < 0.01 and ****p < 0.0001 were considered statistically significant.
The onset of PI3K-related vascular malformations occurs during angiogenesis (2022)
Figure 2

Figure 2. Pathogenesis of Pik3ca-driven vascular malformations depends on active angiogenesis.

(A) Experimental setup scheme showing analysed angiogenic stages in the retina models.

(B) Representative images of control and EC-Pik3caH1047R/WT mouse retinas isolated at indicated time points stained with IB4 for blood vessels. Scale bars: 150 μm.

(C) Pie charts showing the incidence of vascular lesions at different angiogenic stages. Lesions were categorised according to their expanse (generalised or focal). Quantification was performed per retina petal. Numbers below show the presence of any type of lesions per retina petal.

(D) Quantification of IB4-positive areas per retina. Error bars are s.e.m. n ≥ 3 retinas per genotype. (E) Representative images of EC-mTmG mouse retinas isolated at indicated time points stained with IB4 and GFP. Scale bars: 150 μm.

(F) Quantification of GFP/IB4 ratio per retina. Error bars are s.e.m. n ≥ 3 retinas per genotype. Data information: Statistical analysis was performed by nonparametric Mann-Whitney test. *p < 0.05 was considered statistically significant.
The onset of PI3K-related vascular malformations occurs during angiogenesis (2022)
Figure 3

Figure 3. Modelling PI3K-vascular malformations in murine retinas by mosaic expression of Pik3caH1047R in ECs.

(A, B) Representative images of EC-Pik3caH1047R/WT (A) and EC-mTmG (B) P6 retinas from mice treated with decreasing doses of 4-OHT on P1. Retinas were stained for blood vessels (IB4) and GFP as indicated. Scale bars: 150 μm.

(C) Quantification of GFP/IB4 ratio of EC-mTmG retinas. Error bars are s.e.m. n ≥ 3 retinas per genotype.

(D) Quantification of IB4-positive area per retina in Pik3caH1047R/WT retinas. Data presented as a percentage of the control for each 4-OHT dose. Error bars are s.e.m. n = 4 retinas per genotype. Data information: Statistical analysis was performed by nonparametric Mann-Whitney test. *p < 0.05 was considered statistically significant.