EMBO/soda-nel huggingface dataset:
The RNA polymerase II subunit Rpb9 activates ATG1 transcription and autophagy (2022)
Figure 2

Figure 2. Rpb9 is essential for autophagy process.

A Flow chart of the genome-wide screening for novel essential factors of autophagy in yeast cells. For the screen performance, the knockout library (4857 non-essential genes) strains were grown on YPD plates followed by starvation on nitrogen lacking plates for 7 days. After starvation, the yeast cells were re-grown on YPD plates to detect their viability. The potential candidates from this screen were selected based on the starvation-resistance observation (deletion of an autophagy gene causes cell death after starvation).

B N-terminally GFP-tagged 50Q was checked for its autophagic degradation in indicated yeast cells by GFP processing assays after 1, 4 and 16 h starvation. Yeast cells with ATG1 deletion were used as positive controls.

C WT and Rpb9-deleted yeast cells were starved for 3 days in SD-N medium and then detected for cell viability by serial dilution spotting and growth on YPD plates.

D-E Deficiency of Rpb9 caused blockage of autophagosome formation in yeast cells. Autophagosome marker Cherry-Atg8 expressed in indicated cells was observed by fluorescence microscope before and after nitrogen starvation. In WT yeast cells, Cherry-Atg8 was transferred to vacuoles by autophagy leading to the detection of Cherry signal in vacuoles. The Cherry-Atg8 dots in vam3∆ cells showed blockage of autophagosome fusion and diffused cytosolic Cherry-Atg8 in rpb9∆ or in atg1∆ cells showed blockage of autophagosome formation. Experiments were conducted for at least three times and representative images were shown. Scale bars: 5 μm.

F Autophagic transport of substrate Ape1 was blocked in Rpb9-deficient cells. Autophagy substrate Ape1 was labeled with RFP and expressed in WT and rpb9Δ cells. RFP-Ape1 translocation to vacuoles by the autophagy process leading to the detection of RFP signal in vacuoles and blockage of autophagy in rpb9∆ cells caused cytosolic retention of Ape1. Experiments were conducted for at least three times and representative images were shown. Scale bars: 5 μm.

G Detection of Rpb9-dependent transport of autophagic substrate Pho8Δ60 by ALP assays. Yeast cells were starved for 4 h. Activated Pho8Δ60 activity in vacuoles was measured with indicated cells. Bars represent mean, error bars represent standard deviation, significance was determined by one-way ANOVA (unpaired) followed by Tukey's multiple comparison test, ** indicates P < 0.01, *** indicates P < 0.001 (n = 5 biological replicates).

H Vacuole localization and degradation of endocytosis substrate GFP-Sna3 in WT, atg1∆ and rpb9∆ cells. Scale bars: 5 μm.

I Carboxypeptidases Y (Cpy1), a vacuolar-resident hydrolases, was analyzed for its transporting to vacuoles by the secretory pathway in WT, atg1∆ and rpb9∆ cells. Scale bars: 5 μm.
The RNA polymerase II subunit Rpb9 activates ATG1 transcription and autophagy (2022)
Figure 3

Figure 3. Rpb9 functions in autophagy through regulation on ATG1 transcription.

A WT and Rpb9-deleted yeast cells were grown in YPD to log phase and then cultured in SD-N media for 3 h. Yeast cells were collected and subject to genome mRNA sequencing and quantification.

B Clustered analysis of differential gene sets from RNA-seq data was shown with a heat map.

C Genome-wide analysis of differential gene expression in rpb9∆ yeast cells compared to WT cells (p value <0.05, blue: downregulated, red: upregulated). Significance was determined by one-way ANOVA (unpaired) followed by Tukey's multiple comparison test (n = 3 biological replicates).

D WT and rpb9∆ yeast cells were gown to log phase in YPD (+N) and then shifted to nitrogen starvation (-N) for 3 h. ATG1 mRNA levels were quantified by qRT-PCR. Bars represent mean, error bars represent standard deviation, significance was determined by one-way ANOVA (unpaired) followed by Tukey's multiple comparison test, *** indicates P < 0.001 (n = 5 biological replicates).

E-I Overexpression of ATG1 could partially restore autophagy in rpb9∆ cells. ATG1, ATG9, ATG13, ATG17 and ATG5 were overexpressed in rpb9∆ yeast cells, GFP-50Q was checked for its autophagic degradation by GFP processing assays after 1, 4 and 16 h starvation.
The RNA polymerase II subunit Rpb9 activates ATG1 transcription and autophagy (2022)
Figure 4

Figure 4. The N-terminal zinc figure domain and linker region of Rpb9 are important for its regulation on ATG1 transcription and autophagy.

A Schematic representation of yeast Rpb9 and indicated truncates checked for function in autophagy and regulation of ATG1 transcription.

B Indicated Rpb9 truncates and mutants were expressed in rpb9∆ cells and autophagic degradation of GFP-50Q was checked by GFP-processing assays.

C The truncates and mutants of the Rpb9 were expressed in rpb9∆ yeast cells and cell viability after 3-day starvation in SD-N medium was detected by serial dilution spotting and growth on YPD plates.

D Indicated Rpb9 truncates and mutants were expressed in rpb9∆ cells and ATG1 mRNA levels were analyzed by qRT-PCR. Bars represent mean, error bars represent standard deviation, significance was determined by one-way ANOVA (unpaired) followed by Tukey's multiple comparison test, * indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001 (n = 5 biological replicates).
The RNA polymerase II subunit Rpb9 activates ATG1 transcription and autophagy (2022)
Figure 5

Figure 5. Rpb9 binds the promoter of ATG1 through Gcn4.

A Chromatin immunoprecipitation (ChIP) assay was performed in Rpb9-HA yeast cells and precipitated DNA was extracted and detected by qRT-PCR analysis with primers spanning the indicated regions upstream and downstream of ATG1 starting code. TFC1 was used as negative control. Dotted line indicates no enrichment compared to control. Bars represent mean, error bars represent standard deviation, significance was determined by one-way ANOVA (unpaired) followed by Tukey's multiple comparison test, * indicates P < 0.05, ** indicates P < 0.01, (n = 5 biological replicates).

B His-tagged Rpb9 and GST-tagged Gcn4 was purified from E. coli cells and subject to an electrophoretic mobility shift assay (EMSA) together with DNA probes spanning the indicated promoter regions of ATG1.

C WT, rpb9∆ and gcn4∆ yeast cells were gown to log phase in YPD (+N) and shifted to nitrogen starvation (-N) for 3 h. ATG1 mRNA levels were quantified by qRT-PCR. Bars represent mean, error bars represent standard deviation, significance was determined by one-way ANOVA (unpaired) followed by Tukey's multiple comparison test, * indicates P < 0.05, *** indicates P < 0.001 (n = 5 biological replicates).

D ChIP assays were performed with WT Rpb9-HA or gcn4∆ Rpb9-HA yeast cells followed by qRT-PCR analysis. TFC1 was used as a negative control. Bars represent mean, error bars represent standard deviation, significance was determined by one-way ANOVA (unpaired) followed by Tukey's multiple comparison test, * indicates P < 0.05, ** indicates P < 0.01, (n = 5 biological replicates).

E Purified GST-Gcn4 and His-Rpb9 proteins from E. coli cells were mixed and GST pulldown assays were performed.

F-G Neither overexpression of Gcn4 in rpb9∆ yeast cells nor overexpression of Rpb9 in gcn4∆ yeast cells can restore autophagy. Gcn4 or Rpb9 were overexpressed in rpb9∆ or gcn4∆ yeast cells respectively and autophagic degradation of GFP-50Q was detected.
The RNA polymerase II subunit Rpb9 activates ATG1 transcription and autophagy (2022)
Figure 6

Figure 6. The function of Rpb9 in regulating ATG1 transcription and autophagy is conserved in eukaryotes.

A Schematic map of protein domains of Rpb9 orthologs in yeast and complex eukaryotic organisms. S. cerevisiae: Saccharomyces cerevisiae, H. sapiens: Homo sapiens, M. musculus: Mus musculus, D. melanogaster: Drosophila melanogaster, C. elegans: Caenorhabditis elegans.

B Phylogenetic tree of Rpb9 orthologs from indicated eukaryotic species.

C Rpb9 orthologs from complex eukaryotic species could restore autophagy in rpb9∆ yeast cells. Indicated Rpb9 orthologs were expressed in rpb9∆ yeast cells and autophagic degradation of GFP-50Q was detected.

D N-terminal Zinc finger domain and linker region of Rpb9 orthologs from different species could restore autophagy defects in rpb9∆ yeast cells. Indicated truncates of Rpb9 orthologs were expressed in rpb9∆ yeast cells and autophagic degradation of GFP-50Q was detected.

E Rpb9 orthologs restored starvation resistance in rpb9∆ yeast cells. Indicated Rpb9 orthologs were expressed in rpb9∆ yeast cells and cell viability after SD-N starvation was detected.

F Rpb9 orthologs restored ATG1 transcription in rpb9∆ yeast cells. Indicated Rpb9 orthologs were expressed in rpb9∆ cells and ATG1 mRNA levels were analyzed by qRT-PCR. Bars represent mean, error bars represent standard deviation, significance was determined by one-way ANOVA (unpaired) followed by Tukey's multiple comparison test, * indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001 (n = 5 biological replicates).

G POLR2I, the human ortholog of yeast Rpb9, regulates ULK1 transcription. Human HEK293T cells were transfected with siRNA targeting POLR2I and 72 h later the mRNA levels of autophagy genes were analyzed by qRT-PCR. Bars represent mean, error bars represent standard deviation, significance was determined by one-way ANOVA (unpaired) followed by Tukey's multiple comparison test, * indicates P < 0.05, (n = 5 biological replicates).

Expanded View Figure Legends
Peripheral  blood  transcriptome  profiling  enables  monitoring  disease progression in dystrophic mice and patients (2021)
Figure 2

FIGURE 2. Results of the analysis of muscle tissue (MoMus dataset).

A) Principal component analysis of the MoMus RNA-seq counts.

B) Volcano plot for the F test on differential expression between WT and mdx mice.

C) Volcano plot for the F test on differential expression between WT and mdx++ mice.

D) Volcano plot for the F test on differential expression between WT and mdx+- mice.

E, F) Boxplot comparing the distribution of the Dmd and Prune2 genes in the WT, mdx, mdx++ and mdx+- mice groups. The central band of the boxplot denotes the median; the box denotes the first and third quartile; the whiskers are computed as min(max(x), Q3 + 1.5 IQR) and max(min(x), Q1 - 1.5 * IQR), where min(x) and max(x) denote the minimum and maximum of the distribution, Q1 and Q3 the first and third quartile, and IQR the interquartile range.

G) Volcano plot for the F tests on differential expression between mdx++ and mdx+- mice.

H-J) Scatter plots comparing the estimated logFCs across mice groups. Panel H: mdx vs mdx++. Panel I: mdx vs mdx+-. Panel J: mdx++ vs mdx+-.

K) Heatmap obtained by Ingenuity Pathway Analysis showing the most affected pathways across mdx, mdx++ and mdx+- mice.
Peripheral  blood  transcriptome  profiling  enables  monitoring  disease progression in dystrophic mice and patients (2021)
Figure 3

FIGURE 3. Results of the analysis of blood tissue (MoLong dataset).

A) Histogram of the likelihood ratio p-values for the test on differences between WT and mdx at any time point.

B) Distribution of the estimated logFCs for mdx versus WT mice at weeks 6, 12, 18, 24 and 30 for the 1532 genes with significant differences between WT and mdx.

C) Venn diagram showing the overlap between the sets of genes with significant differences between WT and mdx mice at different weeks.

D) Trajectory plots comparing the trajectories of Chordc1 and Psat1 in WT and mdx mice.

E) Trajectory plots comparing the trajectories of Cd59a, Chordc1, Hpgd, Ldhb, Lincred1, Myo6, Slc39a11 and Tlr1 in WT, mdx++ and mdx+- mice.

F) Scatter plots comparing the estimated logFC of mdx++ vs WT (x axis) and of mdx+- vs WT (y axis) at weeks 6, 12, 18, 24 and 30.

G, H) Pathway analysis (G) and upstream regulator analysis (H) performed by Ingenuity Pathway Analysis of the comparison between mdx and wt mice.
Peripheral  blood  transcriptome  profiling  enables  monitoring  disease progression in dystrophic mice and patients (2021)
Figure 5

FIGURE 5. Analysis of the effect of dystrophin restoration by antisense oligonucleotides on gene expression in blood (MoTreat dataset).

A) RT-PCR (30 cycles) showing skipping of exon 23 for PMO and PS49 oligos. No skipping was visible in saline treated mice.

B) Western blot showing the percentage of restoration of dystrophin protein in mdx mice following antisense oligonucleotides administration. M is molecular ladder. WT gastrocnemius represents the standard curve using protein extracts from gastrocnemius muscle. NT is saline treated mdx mice.

C) Barplot comparing the estimated logFC for the PS49 and PMO treatments in the MoTreat dataset to the desired drug effect obtained from the MoLong dataset (logFC WT vs mdx at week 12).

D, E) Histograms comparing the distribution of selected genes across different mice groups (PMO, PS49 and saline in the MoTreat dataset, WT and mdx at week 12 in the MoLong dataset).
The tumour suppressor OPCML promotes AXL inactivation by the phosphatase PTPRG in ovarian cancer (2018)
Figure 1

Figure 1: OPCML interacts with AXL.

(A) Western blotting of AXL and OPCML protein levels in AXL-expressing, OPCML-null SKOV3 and PEA1 cell lines transduced with OPCML "O" or control Empty vector "E". (B) Mammalian 2-Hybrid assay between OPCML and AXL. (C) Western blotting of the OPCML-GST pull down. Input: 1/20 of SKOV3-Empty whole cell lysate. (D) Western blotting of the anti-AXL immunoprecipitation. Input: 1/100 of SKOV3-OPCML whole cell lysate. IP: Immunoprecipitated protein, IB: Immunoblotted protein. (E) Immunostaining of OPCML (red) and AXL (green) in SKOV3-OPCML and PEA1-OPCML cells. Scale bar = 10μm. (F) Pearson"s Correlation R for AXL-OPCML colocalisation in (E). (G) FRET efficiency for AXL-OPCML interaction in SKOV3-Empty "E" and SKOV3-OPCML "O", using AXL-AXL FRET as a positive control. Cells were labeled with Anti-Axl rabbit (conjugated to donor probe) and Anti-OPCML mouse (conjugated to acceptor probe), or Anti-Axl mouse (conjugated to acceptor probe) and Anti-Axl rabbit (conjugated to donor probe). (H) PLA of AXL-OPCML (red) in SKOV3 and PEA1 cells transduced with empty vector "E" or OPCML "O". Scale bar = 50μm. Primary ovarian tumour cells were characterised for AXL and OPCML levels by (I) Western blotting and (J) Immunostaining. PAX8 was used to identify ovarian cancer cells. (K) Quantitation of AXL-OPCML PLA in primary cells. (L) Western blotting of membrane fractionation of SKOV3-Empty cells: total cell lysate "T", liquid-disordered soluble fraction "S", detergent resistant membrane fraction "R". Caveolin-1 is used as a marker of the R fraction and Calnexin is used as a marker for the S fraction, E: SKOV3-Empty, O: SKOV3-OPCML. (M) Densitometry of AXL band intensities in (J) in S and R, normalized to AXL intensity in input.

Data in (A) to (M) are representative of at least three experiments with graphs depicting means ± SEM, (E) was performed once due to the limited numbers of primary cells; *P < 0.05, ***P < 0.01, and ****P < 0.0001 by Student"s t tests.
The tumour suppressor OPCML promotes AXL inactivation by the phosphatase PTPRG in ovarian cancer (2018)
Figure 2

Figure 2: Gas6 stimulation promotes OPCML-AXL interaction, thereby altering AXL localisation and activation.

SKOV3-OPCML cells stimulated with Gas6 over a 12 hour time course and (A) stained with DAPI (cyan), Anti-OPCML (green) and Anti-AXL (red) antibodies (scale bar = 10μm), and (B) Pearson"s Correlation R was calculated; (C,D) OPCML-AXL interaction (red) was visualised by PLA (scale bar = 50μm) and quantified, n = 3. (E) Quantitation OPCML-AXL PLA interaction in primary ovarian tumour cells, n=1. (F) Co-immunoprecipitation with anti-AXL antibody in cells stimulated with Gas6 as indicated. Input: 1/50 of whole cell lysate from SKOV3-OPCML cells. IP: Immunoprecipitated protein, IB: Immunoblotted protein. (G) Co-immunoprecipitation with anti-pAXL antibody in SKOV3-OPCML cells stimulated with Gas6. (H) OPCML-GST pull down assay in SKOV3-Empty cells stimulated with Gas6. (I) Membrane fractionation in SKOV3-Empty "E" and SKOV3-OPCML "O" cells into the detergent resistant "R" membrane fraction and the detergent soluble "S" fraction upon Gas6 stimulation. (J) Ratio of AXL band intensity in "R" to total AXL "T" (T = S + R) from panel (I). (K) FRAP recovery curves, (L) Diffusion Co-efficient, and (M) percentage of AXL localised in insoluble lipid domain or soluble plasma membrane fraction in SKOV3-Empty "E" and SKOV3-OPCML "O" cells treated with Gas6. (N) FRET Efficiency in SKOV3-OPCML "O" cells stimulated with Gas6, and labeled with Anti-Axl (donor probe) and Anti-OPCML (acceptor probe). (O) Western blotting of the "S" and "R" membrane fractions in SKOV3-Empty "E" and OCPML "O" cells stimulated with Gas6 for 3 hours, for Gas6, AXL and pAXL. Data in (A) to (O) are representative of at least three experiments with graphs depicting means ± SEM, (E) was performed once due to the limited numbers of primary cells; *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 by Student"s t tests.
The tumour suppressor OPCML promotes AXL inactivation by the phosphatase PTPRG in ovarian cancer (2018)
Figure 5

Figure 5: The integrity of lipid domains is crucial for OPCML modulation of AXL signalling.

(A) Filipin staining of SKOV3-OPCML cells treated for 30 minutes with 5mM MBCD and allowed to recover for 0, 30 minutes, and 12 hours in serum free (SF) medium. (B) Western blotting of SKOV3-OPCML cells treated with MBCD and separated into the detergent resistant "R" membrane fraction and the detergent soluble "S" fraction upon Gas6 stimulation. (C) Ratio of AXL band intensity in "R" or pAXL band intensity in "S" relative to total AXL "T" from SKOV3-OPCML "O" in (B). (D) MBCD treatment of SKOV3-EMPTY and SKOV3-OPCML cell lines was followed by the analysis of Gas6 signalling kinetics by western blot. GAPDH was used as loading control. (E,F) Signalling kinetics are presented for pAXL and pERK in SKOV3-EMPTY "E" and SKOV3-OCPML "O", treated with MBCD and no treatment control, normalised to GAPDH, from (D). (G) PLA assay of AXL-OPCML interaction (red) upon Gas6 stimulation in both MBCD treated and non-treated SKOV3-OPCML cells. Scale bar = 50μm. (H) Quantification of PLA signals from (G). Data in (A) to (H) are representative of at least three experiments with graphs depicting means ± SEM.
The tumour suppressor OPCML promotes AXL inactivation by the phosphatase PTPRG in ovarian cancer (2018)
Figure 7

Figure 7: OPCML potentiates the effects of the AXL inhibitor R428 in vitro and in vivo

(A) Mean GI50 of SKOV3-Empty "E" and SKOV3-OPCML "O" cells upon R428 treatment. (B) Western Blotting of SKOV3-Empty "E" and SKOV3-OPCML "O" cells pre incubated with either no drug (DMSO only control), 1.09 μM R428, or 2.84 μM R428 for an hour and stimulated with Gas6 for the indicated times. GAPDH was used as loading control. (C) Immunofluorescence staining of pERK (red) and OPCML (magenta) in SKOV3-Empty "E" and SKOV3-OPCML "O" cells treated with R428 and stimulated with Gas6 for the indicted time points. Scale bars = 50μm. (D) CAM assay of SKOV3-Empty "E" and SKOV3-OPCML "O" cells pre-treated with R428. Scale bars = 1mm. (E) Quantification of the fluorescence intensity of the tumours from (D). Data in (A) to (E) are representative of at least three experiments with graphs depicting means ± SEM: n.s. not significant, , **P < 0.01, Student"s t tests.
Endothelial cell death after ionizing radiation does not impair vascular structure in mouse tumor models (2022)
Figure 4

Figure 4. TEC apoptosis and proliferation after single and fractionated IR. (A-B) Representative immunofluorescent micrographs of control and treated tumor vasculature from MC38 tumors. Tumor vessels (anti-CD31) and apoptosis (anti-cleaved caspase 3) in (A) control and (B) 48 hours after 15 Gy IR. Arrows indicate apoptotic TEC. (C) quantification of apoptotic vessels after single dose 15 Gy IR from immunofluorescent images of whole MC38 tumor section (n= 6-8 biological replicates per group). (D) Quantification of apoptotic vessels after fractionated 5×3 Gy IR from immunofluorescent images of whole MC38 tumor sections (n= 6-8 biological replicates per group). (E) Quantification of apoptosis in TECs from B16F10 tumor sections (n= 4-6 biological replicates per group). (F) Quantification of apoptotic vessels according to size on thin whole MC38 tumor sections (Ctrl n=4 and treatment n=7 biological replicates). (G) Quantification of apoptotic vessels according to size in B16F10 tumor sections (Ctrl n=4 and treatment n= 10 biological replicates). (H-I) Area (H) and diameter (I) of apoptotic tumor vessels vs all tumor vessels from segmented thick 80 μm MC38 tumor sections (n=4 biological replicates). (J-K) Representative immunofluorescence image of control (J) and 15 Gy IR treated (K) MC38 and B16F10 tumors stained for proliferation (EdU), EC nuclei (anti-ERG) and tumor vessels (anti-CD31). (L-M) Quantification of proliferating cells (L) and proliferating TECs (M) in MC38 tumors (n= 8 biological replicates per group). (N-O) Quantification of proliferating cells (N) and proliferating TECs (O) in B16F10 tumors (n= 4 to 9 biological replicates per group). (P-Q) Quantification of proliferating cells (P) and TECs (Q) in MC38 tumors after fractionated IR (n=5-7 biological replicates per group). (R-S) Quantification of proliferating cells (R) and TECs (S) in B16F10 tumors after fractionated IR (n= 5-7 biological replicates per group). (T-U) Measurement of proliferating TECs in (T) MC38 tumors (n= 9 biological replicates) and (U) B16F10 tumors (n= 5 biological replicates) in small (area <250 µm2) and large (area <250 µm2) tumor blood vessels.

Data information: Error bars represent mean ± SD, except in (T, U) where box and whiskers plots with min-max are shown with median being the middle line, *P <0.05, **P <0.01, ***P <0.0001 by analysis of variance (ANOVA), except in (T, U) where *P < 0.05 by paired t-test. Scalebar: 1 mm in A-B (first column) and 100 μm in A-B (all panels except first column) and in J-K.
The retinal pigment epithelium as a gateway for monocyte trafficking into the eye (2016)
Figure 1

Figure 1. ONC elicits an immune response in the retina, including the infiltration of monocyte-derived macrophages.

(A) Representative micrographs of optic nerve sections collected from mice 8 days after ONC, showing the accumulation of IB-4+ activated myeloid cells in the injury site, which is demarcated by GFAP staining. Scale bar, 50 µm. (B) Representative micrograph of the lesion site in optic nerve sections 7 days after crush injury in [Cx3cr1gfp/+ WT] BM chimeric mice, immunostained for GFP (red), to depict monocyte-derived macrophages, and IB-4 (green) to label activated myeloid cells. Scale bar, 100 µm. Inset scale, 20 µm. (C) Representative micrograph of optic nerve section 7 days after crush injury, immunostained for CD3 (red, arrows). Scale bar, 100 µm. (D) Representative dotplots showing the general gating strategy used in flow cytometry experiments for the analysis of immune cells in the retina. Leukocytes were identified by gating on CD45+ cells, and were further gated on CD11b+ or TCRβ+ to detect monocytes/macrophages and T cells, respectively. Of the CD11b+ cells, infiltrating monocytes were regarded as CD45hi. (E) Representative contour plot from [Cx3cr1gfp/+ WT] BM chimeric mouse, verifying that infiltrating blood-derived macrophages (CX3CR1-GFP+) are indeed CD45hi, and showing that they are detectable only in retinas from eyes that underwent ONC, while absent in noninjured controls (red vs. blue line). Cells were pre-gated on CD11b. n = 3-4 per group. (F) Quantitative analysis of flow cytometric data, showing the induction of an immune response in the retina after ONC, evident by a general increase in leukocytes (CD45+), including CD11b+CD45hi cells (representing infiltrating macrophages) and T cells (TCRβ+). n = 4 per group. Data shown are representative of six independent experiments. Graphs show mean ± SE of each group. *** P < 0.001. FSC – forward scatter; noninj – noninjured control; ONC – optic nerve crush.