EMBO/SourceData huggingface dataset:
Figure 2(A) Time-lapse analysis of NF-κB-driven EGFP reporter activity in A549kB cells where cytokinesis failure was induced by treatment with ZM (2μM ) or DHCB (4μM). Single cells were imaged every 20 min using IncuCyte life cell imaging technology. Left, representative images of A549kB cells 48h after starting the treatment. Scale bars: 10 μm. Right, EGFP integrated fluorescence intensity of individually tracked cells was measured over time. Time zero was determined as the moment of cytokinesis failure (CF) of each individual cell analyzed. Dashed lines indicate the integrated EGFP intensity of the cells at time zero that was used to calculate the fold-change over time. Solid lines indicate the mean EGFP integrated fluorescence intensity of the cells 24 h after failing cytokinesis. An n ≥ 10 cells per condition were followed individually in a single experiment, data points represent means ± SEM. Data information: The statistical significance was determined using two-way ANOVA; p ≤ 0. 1 (*), p ≤ 0.001 (***), p ≤ 0.0001 (****).(B) Top, experimental design. Left, densitometric analysis of IKBα protein levels in synchronized A549 cells detected over time after release. Bars represent ZM values normalized with respect to DMSO. n=3 independent experiments, data represent mean values ± SEM. Right, time course immunoblot analysis of synchronized A549 cells, released in DMSO or ZM (2μM). Data information: The statistical significance was determined using two-way ANOVA; p ≤ 0. 1 (*), p ≤ 0.001 (***), p ≤ 0.0001 (****).(D, E) (D) Western blot analysis confirming lack of PIDD1 expression in A549 cells and (E) MDM2 processing after induction of cytokinesis failure using ZM (2µM) for 48h. Data information: The statistical significance was determined using two-way ANOVA; p ≤ 0. 1 (*), p ≤ 0.001 (***), p ≤ 0.0001 (****).(F) Live-cell imaging analysis of NF-κB EGFP-reporter activity in CRISPR-edited polyclonal A549κB cells. NF-κB activity of ZM-treated cells is represented as fold-change with respect their untreated counterparts (DMSO-treated cells, grey lines). n=5 independent experiments. Error bars represent ± SEM. Data information: The statistical significance was determined using two-way ANOVA; p ≤ 0. 1 (*), p ≤ 0.001 (***), p ≤ 0.0001 (****).(H) Live-cell imaging analysis of NF-κB EGFP-reporter activity in CRISPR-edited polyclonal RPE1kB cells analyzed as in as in F. Data information: The statistical significance was determined using two-way ANOVA; p ≤ 0. 1 (*), p ≤ 0.001 (***), p ≤ 0.0001 (****).
Figure 3(A) Representative immunofluorescence images of γ-tubulin staining to mark and enumerate centrosomes in A549κB reporter cells. Cells were pre-treated for 24 h with centrinone (125nM) or solvent control (DMSO) and cultured further in medium either containing DMSO or ZM to promote cytokinesis failure. Size bar: 10µM. Data information: The statistical significance was determined using two-way ANOVA; p ≤ 0.001 (***), p ≤ 0.0001 (****).(B) Cells treated as indicated in A were analyzed for NF-κB-driven EGFP-reporter activation by IncuCyte live-cell imaging. Left, representative pictures of live-cell imaging NF-κB activation after treatment with DMSO, ZM (2μM, 48 h) or TNF (20 ng/mL, 24h). Size bar: 20µm. Right, EGFP MFI quantification of ZM-treated cells was plotted as fold-change expression relative to DMSO-treated controls. Data points represent means of n=4 biological replicates ± SEM. Data information: The statistical significance was determined using two-way ANOVA; p ≤ 0.001 (***), p ≤ 0.0001 (****).(C) Left, representative immunofluorescence images of supernumerary centrosomes originated from Dox-inducible PLK4 overexpression in CRISPR-edited polyclonal U2OSPLK4 cells. Size scale bar: 10µm. Right, representative cell cycle profiles of U2OSPLK4 control and PIDD1-deficient cells. Data information: The statistical significance was determined using two-way ANOVA; p ≤ 0.001 (***), p ≤ 0.0001 (****).(E) Live-cell imaging analysis of NF-κB EGFP-reporter activity in CRISPR-edited polyclonal PIDD1-competent or -deficient U2OSκB-PLK4 and A549κB-PLK4 cells. NF-κB activity of ZM, Dox or TNF-treated cells is represented as fold-change with respect to their untreated counterparts. Bars represent means of n=5 independent experiments ± SEM. Data information: The statistical significance was determined using two-way ANOVA; p ≤ 0.001 (***), p ≤ 0.0001 (****).(F) Representative immunofluorescence images of A549 cells co-stained with the indicated antibodies and documenting PIDD1 localization at centrosomes. Scale bars: 10 μm. (G) Luciferase reporter analysis of NF-κB activity in CRISPR-edited SCLT1- competent or -deficient A549 cells. Luciferase activity was normalized respect renilla control. n=3 independent experiments; represented mean values ± SEM. Data information: The statistical significance was determined using two-way ANOVA; p ≤ 0.001 (***), p ≤ 0.0001 (****).
Figure 4(B) Left, immunoblot of CRISPR-edited polyclonal RIPK1- or NEMO-deficient A549κB derivatives. Right, luciferase assay of A549 cells indicated genotypes, treated for 48h with either ZM (2 μM) or DHCB (4 μM). n=4 independent experiments. Data are represented as the mean values ± SEM. The statistical significance was determined using two-way ANOVA; p< 0.01 (**);p< 0.001 (***); p< 0.0001 (****).(C) Representative confocal fluorescence images documenting centrosome accumulation, as identified by CEP63 staining in 293T-Trex-Flip-in cells. Time course analysis was performed in cells synchronized in G1/S after a 24h single thymidine block and release in either DMSO or ZM (2 μM). Cells were fixed at the indicated times for IF analyses. Scale bars: 10 μm.(D) Different time point immunoprecipitations (IP) after release in ZM (2 μM) of endogenous RAIDD, RIPK1 and NEMO proteins in PIDD1-deficient 293T-Trex-Flip-in cells re-expressing a FLAG-tagged PIDD1-FL protein. One representative experiment out of three is shown.(E) Analysis of NEMO-PIDDosome formation in PIDD1-deficient 293Trex-Flip-In cells re-expressing different PIDD1 mutants depicted in G. Cells were treated as in C and processed for FLAG-IP 11h after release into DMSO or ZM (2 μM). One out of two independent experiments is shown.(F) 293T cells were transiently transfected with constructs allowing expression of GFP, a full length (FL) single (PIDD1-FL_FLAG) or double-tagged (HA_PIDD1-FL_FLAG) version of PIDD1, or an HA-tagged N-terminal fragment of PIDD1 (HA_PIDD1-N). Co-immunoprecipitation was performed to probe for interaction with endogenous RIPK1 after either anti-FLAG or anti-HA pull-down. One out of two independent experiments is shown.
Figure 5Primary MEF from Pidd1-/- mice were treated for 72h with ZM or DMSO and subjected to mRNA-sequencing analysis. (B) qRT-PCR analysis confirm expression of different pro-inflammatory genes in Pidd1+/+ but not Pidd1-/- MEF. Hprt1, β-actin and Gapdh were used as housekeeping gene expression controls. n=3 independent samples; data represent mean values ± SEM. The statistical significance was determined using two-way ANOVA test; p ≤ 0.01 (**), p ≤ 0.001 (***).Primary MEF from Pidd1-/- mice were treated for 72h with ZM or DMSO and subjected to mRNA-sequencing analysis. (D) qRT-qPCR analysis of macrophage activation markers indicative of a pro-inflammatory state. Relative expression values are plotted as the fold-change expression relative to BMDM stimulated with conditioned medium from untreated MEFPLK4. β-actin was used as housekeeping gene for normalization. Bars represent mean ± SEM (n=9) biological replicates. The statistical significance was determined using two-way ANOVA; p ≤ 0. 1 (*), p ≤ 0.01 (**), p ≤ 0.001 (***), p ≤ 0.0001 (****).
Figure 6A549EGFP cells were treated with DMSO or ZM (2μM) for 72h, followed by co-culture with NK-92 cells and live-cell imaging analysis. (A) Representative images of target A549EGFP cells pre-treated for 72 h with ZM (2µM) and co-cultured subsequently with NK-92 cells in a 1:10 target to effector ratio. Interactions were recorded by live-cell imaging for 18 h at a 1 min interval. White arrow indicate polyploid cell undergoing cell death. EGFP release coincides with loss of cell membrane integrity. All images were acquired at the same exposure time and light intensity. Scale bar: 10 µm. Data information: The statistical significance was determined using two-tailed t-test; P < 0.001 (**); P < 0.001 (***).A549EGFP cells were treated with DMSO or ZM (2μM) for 72h, followed by co-culture with NK-92 cells and live-cell imaging analysis. (D) Confluence of ZM pre-treated A549EGFP cells expressing or lacking PIDD1 was measured over time in presence of NK cells and normalized with respect to their DMSO treated counterparts. Middle, NK cell-mediated killing interpolated from cell confluence graphs at 50 h. n≥4 independent experiments performed in duplicates, mean ± SEM. (E) Confluence measurement over time of A549EGFP derivatives impaired in either NEMO-PIDDosome (RIPK1 KO) or Caspase-2-PIDDosome formation (RAIDD KO). Middle, NK cell-mediated killing interpolated from cell confluence graphs at 50 h. n≥3 independent experiments; mean ± SEM; unpaired t-test. (F) Confluence analysis of Dox pre-treated A549EGFP cells overexpressing PLK4 was performed over time in the presence of NK cells and normalized with respect to their DMSO treated controls. n=5 independent experiments; mean ± SEM. (G) Confluence analysis of centriole-depleted and ZM pre-treated A549EGFP cells cultured in presence of NK cells. Values are normalized with respect to their DMSO ± Centrinone (125nM) treated controls. n=4 independent experiments; mean ± SEM. Data information: The statistical significance was determined using two-tailed t-test; P < 0.001 (**); P < 0.001 (***).
Figure 1Cell number in the bone marrow of wild-type and Hdac6 knockout mice (n = 12-13 mice per group). Data information: All values are presented as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns, not significant (Student's t test).Flow cytometry profiles for the LK and LSK subsets in wild-type and Hdac6 knockout mice.Frequency of the LK subset in wild-type and Hdac6 knockout mice (n = 12-13 mice per group). Data information: All values are presented as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns, not significant (Student's t test).L. number of the LSK subset in wild-type and Hdac6 knockout mice (n = 12-13 mice per group). Data information: All values are presented as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns, not significant (Student's t test).Flow cytometry profiles for the proportion of cycling HSPCs examined with EdU incorporation experiments.Representative images of colonies formed by bone marrow cells from wild-type and Hdac6 knockout mice. Scale bar, 200 µm. Number of colonies formed by bone marrow cells from wild-type and Hdac6 knockout mice (n = 10 mice per group). Data information: All values are presented as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns, not significant (Student's t test).
Figure 2Immunoprecipitation and immunoblotting showing the interaction of HA-HDAC6 with GFP-IDH1 in HEK293T cells.Immunoprecipitation and immunoblotting showing the interaction of HA-HDAC6 with GFP-IDH1 in HEK293T cells. D. Immunoprecipitation and immunoblotting showing the interaction of GFP-IDH1 with endogenous HDAC6 in HEK293T cells.J. Immunoprecipitation and immunoblotting with various truncated mutants of HA-HDAC6 to identify the domains that mediate its interaction with GFP-IDH1. K. Immunoprecipitation and immunoblotting with various truncated mutants of GFP-IDH1 to identify the domains that mediate its interaction with HA-HDAC6.
Figure 3Analysis of IDH1 acetylation via immunoprecipitation of the bone marrow cell lysate with the acetylated lysine (Ace-lysine) antibody, followed by immunoblotting with the IDH1 antibody. Analysis of IDH1 acetylation via immunoprecipitation of the bone marrow cell lysate with the IDH1 antibody, followed by immunoblotting with the Ace-lysine antibody.E. Analysis of GFP-IDH1 acetylation by immunoprecipitation with the GFP antibody followed by immunoblotting with the Ace-lysine antibody. HEK293T cells were treated with tubacin, tubastatin A (TubA), trichostatin A (TSA), or sodium butyrate (NaB) (E).Immunoprecipitation and immunoblotting showing the level of IDH1 acetylation in HEK293T cells transfected with wild-type GFP-IDH1 or various mutants, These cells were treated with the HDAC6 inhibitor, tubacinImmunoprecipitation and immunoblotting showing the level of IDH1 acetylation in HEK293T cells transfected with wild-type GFP-IDH1 or various mutants, These cells were transfected with the HDAC6 siRNAO-P. In vitro deacetylation analysis using GST-IDH1 and GST-IDH1 K233R proteins as substrates. The black arrowhead indicates HA-HDAC6, while the white arrowhead marks GST-IDH1 and GST-IDH1 K233R. P is the corresponding statistical result (n = 3 technical replicates). Data information: All values are presented as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns, not significant (Student's t test).
Figure 4C. Analysis of IDH1 activity in HEK293T cells treated with the indicated concentration of tubacin (n = 6-9 tests from three independent experiments). D. Analysis of the α-KG level in HEK293T cells treated with the indicated concentration of tubacin (n = 4 independent experiments). Data information: All values are presented as mean ± SEM. **P < 0.01, ***P < 0.001, ****P < 0.0001 ANOVA testImmunoblotting (E) in HEK293T cells transfected with plasmids expressing GFP, GFP-IDH1, GFP-IDH1 K233R, or GFP-IDH1 K233Q. In panel E, the black arrowhead indicates exogenous IDH1, while the white arrowhead marks endogenous IDH1.G-H. Immunoblotting (G) and IDH1 activity analysis (H, n = 6-9 tests from three independent experiments) in HEK293T cells transfected with control or HDAC6 siRNAs and plasmids expressing GFP, GFP-IDH1, GFP-IDH1 K233R, or GFP-IDH1 K233Q. In panel G, the black arrowhead indicates exogenous IDH1, while the white arrowhead marks endogenous IDH1. Data information: All values are presented as mean ± SEM. **P < 0.01, ***P < 0.001, ****P < 0.0001 ANOVA testCoomassie blue staining (I) of the purified proteins.K. Representative images of the colony formation assay. Scale bar, 200 µm. L. Number of colonies formed by wild-type and Hdac6 knockout mice bone marrow cells treated or untreated with 10 µM GSK321 (n = 6 mice per group). Data information: All values are presented as mean ± SEM. **P < 0.01, ***P < 0.001, ****P < 0.0001 ANOVA test
Figure 5E. IGV screenshots showing elevated hydroxymethylation of multiple candidate genes associated with negative regulation of the cell cycle.F. IGV screenshots of TET2-independent genes showing no significant changes in hydroxymethylation.
Figure 6Heatmaps based on the GO enrichment analysis showing genes that negatively regulate the cell cycle.qPCR showing that knockdown of HDAC6 significantly upregulates the expression of TET2 target genes with hyper-hydroxymethylation. Data information: All values are presented as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns, not significant (ANOVA test).qPCR showing that knockdown of HDAC6 significantly upregulates the expression of TET2 target genes with hyper-hydroxymethylation. qPCR showing that treatment with 10 µM GSK321 inhibits the upregulation of the indicated genes in HDAC6-knockdown HEK293T cells. Data information: All values are presented as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns, not significant (ANOVA test).qPCR showing that treatment with 10 µM GSK321 inhibits the upregulation of the indicated genes in HDAC6-knockdown HEK293T cells. Data information: All values are presented as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns, not significant (ANOVA test).
Figure 1C Western blot analysis of isolated mouse heart mitochondria from 12-week-old animals.D Survival of indicated mice presented as percent of total number of mice analyzed. n=60 (per genotype), median survival of TAZG197V mice is 24.5 weeks, Mantel-Cox test: p < 0.0001 * * * *.E Quantification of neutrophils, percent of white blood cells (left), white blood cells (middle), and red blood cells (right) of 12-week-old mice. Mean ± SEM, n=6, unpaired t test: p < 0.0001 * * * *, ns (non-significant).I Picrosirius red staining of histology sections of hearts from WT and TAZ mutant mice.J qPCR analysis of ANP (left) and BNP (right) from total mRNA isolated from heart lysates of 20-week-old mice. Mean ± SEM, n=5 for ANP, n=4 for BNP, unpaired t test: p < 0.05 *, p < 0.001 * * *
Figure 2A Mass spectrometric analysis of Cardiolipin and Monolysocardiolipin in WT and TAZG197V mouse heart. Mean ± SEM, n=3, unpaired t test: p < 0.05 *, p < 0.01 * *.B Western blot analysis of BN-PAGE separted protein complexes from digitonin solubilized heart mitochondria (12-week-old mice).D Steady state protein levels of isolated mitochondria from 12-week-old mouse hearts.E and F Oxygen consumption rate of isolated heart mitochondria from 12-week-old mice using succinate E and pyruvate F as substrates. Mean ± SEM, n=3, unpaired t test: p < 0.001 * * *, p < 0.0001 * * * G Flow cytometric analysis of isolated mitochondria using MitoSOX Red staining. Mean ± SEM, n=3, unpaired t test: p < 0.05 *.
Figure 3B Volcano plot of differentially expressed genes between 14-week-old WT and TAZG197V mice. X-axis denotes fold change in expression (log2 scale), y-axis denotes adjusted p-value (negative log10 scale) for the analyzed genes in the data (each dot represents one single gene). Blue and red dots represent genes significantly downregulated (DR) and upregulated (UR), respectively, in the TAZG197V mice compared to WT mice. The cut-offs used were: adjusted p-value < 0.05 and fold change > 1.15 or < -1.15. Non-significant (NS) genes are depicted in black. Selected genes involved in pathways relevant for the study are labelled in the plot.
Figure 4F Western Blot analysis of BN-PAGE separated complexes from digitonin solubilized heart mitochondria (12-week-old mice).G Steady state protein levels of isolated heart mitochondria (12-week-old mice). CPT1B and CPT2 protein levels were normalized to VDAC. Mean ± SEM, n=5, unpaired t test: p < 0.01* *, p < 0.001* *
Figure 5C Western blot steady state protein levels of CPT1B and CPT2. Values were normalized to β-tubulin in control and iTAZ iPSC-cardiomyocyte. Mean of WT values was set to 1. Mean ± SEM, n=5, unpaired t test: p < 0.05 *, p < 0.0001* * * *.D Real time respirometry. Left, Oxygen consumption measured as basal, and upon addition of oligomycin, CCCP, and antimycin/rotenone using palmitate-BSA as substrate, ETO, etomoxir. Right, quantification of basal and maximal respiration. Mean ± SEM, n=4, unpaired t test: p < 0.01* *, p < 0.0001* * * *E Extracellular acidification rate of control and iTAZ iPSC-cardiomyocytes. Basal state and upon sequential addition of glucose, oligomycin and 2 deoxyglucose (2-DG). Right, quantification of glycolytic capacity. Mean ± SEM, n=6, unpaired t test: p < 0.001* * * *
Figure 6A Western blot analysis of steady state protein levels of untreated (US) and cells starved (S) for indicated times.C Glycolytic and mitochondrial ATP production measured using a real time ATP rate assay. Mean ± SEM, n=3, unpaired t test: p < 0.001* * *.D Western blot analysis of steady state AMPK and pAMPK protein levels of starved and/or A769662 (AMPK activator) treated cells as indicated.
Figure 7A Ejection fraction from short axis expressed in %, B Fractional shortening in %, Mean ± SEM, n=15 (WT control); 4 (TAZG197V control); 9 (WT treated); 7 (TAZG197V treated), 2Way ANOVA: p < 0.05 *, p < 0.01 * *F Gene expression addressed by qPCR of ANP (left) and BNP (right) in 24-week-old WT and TAZG197V mice heart upon 6-week treatment with A769662. Mean ± SEM, n=4, unpaired t test: ns (non-significant)G Quantification of steady state levels of AMPK normalized to beta tubulin in tissue lysates from 24-week-old WT and TAZG197V mice heart from untreated control or following a 6-week treatment with A769662. The mean of the WT values was set to 1. Mean ± SEM, n=7 (control); 5 (treated), unpaired t test: p < 0.01 * *I Real time respirometry, oxygen consumption rate, of isolated heart mitochondria from 24-week-old TAZG197V mice, from untreated control or following a 6-week treatment with A769662, driven by palmitoyl/carnitine/malate/ADP. The OCR of the untreated WT samples was taken as 100%. Mean ± SEM, n=3, unpaired t test: p < 0.01 * *
Figure 1Line plots of the mean relative abundances and SD of each bacterial strain from ages 10 to 30 days. For non-AM strains, relative abundances of the 14SM groups were recalculated excluding AM in order to compare abundances with the 13SM group. Data information: Strain abundances were normally distributed within each group according to the Kolmogorov-Smirnov test. Statistical significance compared to the 14SM FR control at each time point was calculated by two-way ANOVA with false-discovery adjustment using the Benjamini and Hochberg method. Adjusted p values are indicated for 14SM FF vs 14SM FR (red) and 13SM FR vs 14SM FR (orange). Data are from two independent experiments and represent biological replicates (n = 2-11/group).
Figure 2Volcano plot showing gene abbreviations for colonic transcripts that were significantly enriched in 14SM FF or 14SM FR after correction for multiple comparisons using DESeq2mRNA expression of Ifng, Tnfa, Il17f, and Il22. Colonic transcription levels were normalized by Hprt, which was the most stable among the three housekeeping genes tested (Hprt, Hsp90, Gapdh). Longitudinal data of mean ± SEM is shown for 14SM FF, 14SM FR, and 13SM FR pups at age 10-20 days (circles) with statistical significance calculated using two-way ANOVA factored on group and time point compared to the 14SM FR control. To the right of each longitudinal plot, we also report colonic transcript levels for pups born to germ-free (GF) FF or FR dams at age 15 days (squares) with statistical significance based on a one-way ANOVA with false-discovery adjustment using the Benjamini and Hochberg method. Outliers removed using ROUT method with Q = 10%. Data are from two independent experiments and represent biological replicates (n = 3-20/group).
Figure 3For mice at age 15 days, the proportion of cytotoxic natural killer (cNK), innate lymphoid cell (ILC) type 1, ILC type 2, ILC type 3, natural cytotoxicity receptor (NCR) positive ILC3, and NCR negative ILC3 populations among CD45+ cells. Data information: Statistical significance is based on a one-way ANOVA with false-discovery adjustment using the Benjamini and Hochberg method. Boxplots show IQR with error bars showing min. and max. values. Data are from two independent experiments and represent biological replicates (n = 8-20/group).For mice at age 15 days, the proportion of CD8+ T cells, CD4+ T cells, Tregs (total FoxP3+), Th1 cells (T-bet+FoxP3-), Th2 (GATA3+FoxP3-), and Th17 (RORgt+FoxP3-) populations among CD45+ cells. Data information: Statistical significance is based on a one-way ANOVA with false-discovery adjustment using the Benjamini and Hochberg method. Boxplots show IQR with error bars showing min. and max. values. Data are from two independent experiments and represent biological replicates (n = 8-20/group).
Figure 1A Coronal section of E15.5 wild-type (WT) and DKO developing cortices were co-stained with either anti-Sox2 (green) and anti-Pax6 (red) or with anti-Sox2 (green) and anti-Tbr2 (red) and both counter-stained with DAPI (blue). Scale bars are 50 µm. Quantitation of total DAPI+ cells and Sox2+, Pax6+ or Tbr2+ progenitor cells in the SVZ/VZ in the entire image, corresponding to an area of 100 μM x 100 μM are plotted.B, D Coronal sections of WT and DKO developing cortices stained with DAPI (blue) and co-stained with anti-Ctip2 (red) and Satb2 (green) at P2 and P7. The location of the cortical plate (CP), intermediate zone (IZ), subventricular/ventricular zone (SVZ/VZ) in P2 mice and Layers I-VI in P7 WT mice are indicated. Scale bars are 50 µm.C, E Quantitation of Ctip2+ and Satb2+ progenitor cells as a percent of total DAPI+ cells in each of 8 bins (marked on left) within the cortex (dashed lines) from a total area corresponding to 100 μM x 600 μM are plotted.