EMBO/SourceData huggingface dataset:
Figure 8Lysosomes were isolated from livers of starved wild-type (WT) and Ctns−/− mice as described in Supplementary Materials and Methods and incubated at 37°C for 30 min with the CMA substrate GAPDH, in the presence or absence of ATP (necessary for CMA) and protease inhibitors (Prot. Inhib.). A fraction of the CMA reactions was then mixed with sample buffer and boiled at 95°C for 5 min, followed by SDS-PAGE and GAPDH and LAMP1 immunoblotting.Quantitative densitometry analysis of CMA activity performed in independent experiments using lysosomes isolated from a total of 11 WT and 10 Ctns−/− mice. Results are mean ± SEM. ***P = 0.0005; **P = 0.0041; N.S., not significant (unpaired t-test).The ability of lysosomal proteases to mediate GAPDH degradation was assessed by incubating Triton X-100-treated WT and Ctns−/− lysosomes with GAPDH for 30 min in acidic reaction buffer and compared with input (reaction with no lysosomes). Quantitative densitometry analysis of independent reactions performed with lysosomes from a total of 9 WT and 9 Ctns−/− mice shows no significant difference in protease activity. Results are mean ± SEM.
Figure 9WT and Ctns−/− mouse fibroblasts were treated with either 50 μM H2O2 or 1 mM paraquat (PQ) for 4 h and stained with (A) FITC-annexin V or (B) propidium iodide (PI) and analyzed by FACS. Results from 3 different experiments are shown as mean ± SEM. In (A), *P = 0.05; ***P = 0.0002. In (B), *P = 0.046; **P = 0.0065. Unpaired t-test.WT and Ctns−/− cells were treated as described above and samples analyzed by Western blot using antibodies recognizing cleaved (active) caspase 3. Data are representative of 2 independent experiments with similar results.
Figure 10Cystine content in WT,Ctns−/−, cysteamine-treated Ctns−/− fibroblasts or Ctns−/− fibroblasts expressing GFP-CTNS was assessed by mass spectrometry.WT, Ctns−/−, Ctns−/− fibroblasts treated with 1 mM cysteamine (48 h), or Ctns−/− fibroblasts expressing GFP-CTNS were fixed and stained with anti-LAMP1 and LAMP2A antibodies as indicated. For better visualization of LAMP1/LAMP2A colocalization, GFP-CTNS was pseudocolored as magenta and LAMP1 was pseudocolored as green (lower panels). Some lysosomes are indicated with arrows. Arrowheads indicate LAMP2A distribution to structures different from lysosomes (only observed in Ctns−/− cells and in cysteamine-treated Ctns−/− cells). Rescue of the LAMP2A localization to lysosomes was observed in Ctns−/− cells expressing GFP-CTNS. Scale bar: 2 μm.Quantification of the colocalization analysis described in (B). Calculation of the Pearson's colocalization coefficient was done by analyzing 113 WT, 251 Ctns−/−, 88 cysteamine-treated and 164 GFP-CTNS-expressing Ctns−/− cells, by using the ZEN 2010 software. Results are mean ± SEM. ***P < 0.001; NS, not significant (one-way ANOVA, Bonferroni's multiple comparisons test).
Figure 11Cystine content in WT,Ctns−/− and cysteamine-treated Ctns−/− mouse livers was assessed by mass spectrometry. Results are mean ± SEM (n = 4). ***P < 0.001; **P = 0.0026 (one-way ANOVA, Bonferroni's multiple comparisons test).Western blot analysis of LAMP2A expression in lysosomes isolated from WT,Ctns−/− and cysteamine-treated Ctns−/− mouse livers.Lysosomes, isolated as in (B), were incubated with the CMA substrate GAPDH, and CMA activity was evaluated as in Fig8 and as described in Materials and Methods.Quantitative analysis of substrate degradation is expressed as percent of the residual amount of GAPDH in the full reaction relative to its amount in the presence of protease inhibitors. Results are mean ± SEM (n = 4). *P = 0.0126 for WT versus Ctns−/− and *P = 0.0436 for WT versus Ctns−/− + cysteamine; NS, not significant; unpaired t-test.
Figure 1B Relative (left) and absolute (right) amount of 13C-Lys-labeled and unlabeled Aβ in APPtg mouse brains after labeling from 75-125 d (n = 1), 75-175 d (n = 4), 50-100 d (n = 5), 50-125 d (n = 2), 50-150 (n = 4) or 50-175 d (n = 9) of age. Error bars show mean ± SD.C Representative chromatogram and mass spectra of doubly charged ions of unlabeled Aβ17-28 (663.3 m/z) and 13C-Lys Aβ17-28 (666.3 m/z) in an APPtg brain after labeling from 75-175 d. %- ion intensity, m/z- mass-to-charge. Data were acquired in data-independent analysis mode (MSE). D Representative chromatograms of unlabeled Aβ17-28 (left) and 13C-Lys Aβ17-28 (right) in an APPtg brain after labeling from 50-175 d. The intensities of 4 transitions (y10, y9, y8, and y7) were assessed. Data were acquired by MRM in nanoflow mode.F Representative chromatograms of unlabeled Aβ17-28 (left) and 13C-Lys Aβ17-28 (right) in a non-APPtg brain after labeling from 90-100 d. Data were acquired by MRM in microflow mode.
Figure 2A MALDI-TOF mass spectrum of trypsin-digested, synthetic Aβ1-42 (10 pmol). The Aβ17-28 [MH]+ fragment was detected with the highest intensity. Measurements in reflectron positive mode. The sequence of Aβ1-42 with the tryptic cleavage sites is indicated at the topB MS/MS spectrum of unlabeled Aβ17-28 and 13C15N-Lys Aβ17-28 synthetic peptides in the 50 fmol range. m/z of precursors ions (m/z 663.34 for Aβ17-28 [MH]2+, m/z 667.36 for 13C15N-Lys Aβ17-28 [MH]2+) and their derived fragments are indicated as a series of y ions (y4-y10). The peptides were analyzed in data independent acquisition MSE modeC Decreasing quantities of synthetic 13C15N-Lys Aβ1-42 were trypsin-digested and analyzed by MRM. The intensities of 4 transitions (y10, y9, y8, and y7) were assessed for 13C15N-Lys Aβ17-28, showing a detection limit level of 25 amol. Measurements in technical triplicates, normalization to total ion current. Error bars show mean ± SDD Decreasing quantities of synthetic 13C15N-Lys Aβ1-42 were spiked into the insoluble fraction of APPtg brains, immunoprecipitated, trypsin-digested, and analyzed by MRM. Assessment of the intensities of y10, y9, y8 and y7 showed a detection limit level of 50 amol for 13C15N-Lys Aβ17-28 and detected an excess of brain-derived unlabeled Aβ17-28. Measurements in technical triplicates, normalization to total ion current. Error bars show mean ± SD
Figure 3B Temporal MRM analysis of 13C-Lys Aβ17-28 and unlabeled Aβ17-28 in the liver, spleen, mesenteric lymph nodes, and peritoneal cells after 13C-Lys-APPtg brain extract i.p. injection (n = 3 mice per time point and tissue at 1-10 d, except for n = 2 mice for peritoneal cells at 3 d; n = 4 mice per time point and tissue at 25-100 d, except for n = 3 mice for spleen at 100 d). Measurements in technical triplicates, normalization to 13C15N-Lys Aβ17-28 intensity as a spiked internal standard and additionally to organ weight (mg) for lymph nodes. Error bars show median ± interquartile range.C Temporal 4G8 immunoassay quantification of Aβ42 in the liver, spleen, mesenteric lymph nodes, and peritoneal cells after unlabeled APPtg (n = 8 mice at 5 h - 7 d, except for n = 5 mice for peritoneal lavage, n = 5 mice at 10-14 d, n = 4 mice at 21 d) or non-tg brain extract injection (n = 2 mice at 5 h and 3 d, n = 3 mice at 1 d, n = 1 mouse at 5 and 7 d, except for peritoneal cells where n = 1 mouse at 5 h and 3 d, and n = 2 mice at 1 d). Error bars show median ± interquartile range. D Normalization of APPtg extract group data presented in (C) to organ weight (mg) and injected Aβ amount (range 1,027-1,426 ng). Error bars show median ± interquartile range.
Figure 2A) Evaluation of RB mRNA levels, normalised by GAPDH, in an MDM2 dose-dependent manner. The H1299 cells were treated with doxorubicin during 16 h to induce genotoxic stress. Endogenous levels (upper panel); exogenous levels (middle panel); endogenous levels of RB but using the MDM2(S395D) under normal conditions (lower panel).B) Kinetic of the endogenous RB expression in H1299 cell line from 0 to 48h. The cells were treated with cichlohexamide (CHX) was added to stop translation, under normal conditions (upper panel) and treated with doxorubicin (lower panel). The cells were transfected with MDM2.C) Kinetic of the endogenous RB expression in H1299 under genotoxic stress conditions in presence (upper panel) or in absence (lower panel) of CHX.D) Evaluation of the levels of expression of RB treated with doxorubicin to generate stress in absence of CHX.
Figure 3Different constructs of RB were transfected in H1299-RBKO treated with doxorubicin 16 h to induce genotoxic stress; the RB constructs levels of expression were evaluated using HA antibody. The bar diagrams show the quantification of the western blots data. A) Evaluation of the small pocket expression in an MDM2 dose-dependent manner. B) Cells were transfected with the RB large pocket construct. Different constructs of RB were transfected in H1299-RBKO treated with doxorubicin 16 h to induce genotoxic stress; the RB constructs levels of expression were evaluated using HA antibody. The bar diagrams show the quantification of the western blots data. C) Cells were transfected with RB full length without the 5´URT (named RB-HA). D) Cells were transfected with the RB full-length plus 260 nt of its own 5´ UTR (named RB+5´UTR). E) The binding of MDM2 to the RB mRNA in H1299 cells as estimated using RNA CoIP. Cells were treated with doxorubicin (1 µM for 16 h) or DMSO.F) RT-qPCR of RB and GAPDH of the MDM2-immunoprecipitation from the polysomal fractionation (see EV2D). Data are representative of three similar independent experiments.G) PLA assay using anti-RB and anti-MDM2 antibodies, H1299 cell were treated or not with etoposide to induce stress, the endogenous proteins were visualised. Cell nuclei were visualised with DAPI blue. The red dots show the interaction between the two proteins. The scale bar corresponds to 20 μm.H) RNA-PLA assay using rabbit anti-MDM2 and a mouse biotin-antibody. H1299- RBKO was stable transfected with RB-HA or RB+5´UTR. Cell nuclei were visualised with DAPI blue. The red dots show the interaction between the protein and the mRNA. The scale bar corresponds to 20 μm.
Figure 4A) Cell cycle analysis (FACS) of C33 cell line after treatment with etoposide. The DNA contents of the cells were evaluated by propidium iodide staining, (PE-A; Phycoerythrin). The inset shows the % of the cells in each cell cycle phase.B) The cell cycle analysis of synchronised C33 cell line during the time.C) Evaluation of increasing concentrations of MDM2 on the cell cycle of C33 cell line after genotoxic stress induced with etoposide, (EV; empty vector). D) The same as in (C) but under normal cellular conditions.E) RB levels of the cells after 14 h of the release of nocodazole treatment. Etoposide was used to induce genotoxic stress.F) Evaluation of the G1 cell cycle phase after 14 h of the release of nocodazole treatment. Data information: Data are means ± s.d. of three independent experiments, student t test was used to calculate statistical significance (*p <0.05, **p <0.01).
Figure 5A) Ubiquitination of endogenous RB in C33 cells synchronised in the G1 or G2/M cell cycle phases under stress conditions.B) In vitro RB ubiquitination by MDM2 wild type and the phosphomimetic mutant MDM2(S395D).C) In vitro RB ubiquitination by MDM2(S395D) in presence or absence of RB mRNA.D) A representative clonogenic assay. Increasing concentrations of MDM2 were transfected in H1299-RBKO stable transfected with RB-HA or RB+5´UTR. The cells were treated with doxorubicin to induce genotoxic stress conditions. Data are means ± s.d. of three independent experiments, student t test was used to calculate statistical significance (*p <0.05).E) Evaluation of cyclin E mRNA levels in an MDM2 dose-dependent manner. The H1299 cells were treated with doxorubicin during 16 h to induce genotoxic stress. Data are means ± s.d. of three independent experiments, student t test was used to calculate statistical significance (*p <0.05, ***p< 0.001).
figf1b-d, MEFs were cultured in nutrient-rich medium (fed) or Hanks' buffer (starved) for 1 h. Total cell lysates were prepared, and blotted with the indicated antibodies (b).α, β, indicate Atg16L1 isoform α, β; ∆CCD indicates Atg16L1∆CCD; asterisk indicates a non-specific band. Atg16L1∆/∆ indicates mice expressing ∆Atg16L1. The number of endogenous LC3 or GFP-Atg5 dots was counted (c, d). The results shown are mean + s.d. (n > 20).e, Electron micrograph of starved MEFs.f, Degradation of long-lived proteins in MEFs. Wortmannin is an autophagy inhibitor. The results shown are mean1s.d. Statistical significance (P value) was determined by the Student's t-test.
figf2a, Cytokine production from macrophages stimulated with LPS (100 ng ml-1) for 24 h. Statistical significance was determined by the Student's t-test. *P  0.01.b, IL-1b production from macrophages stimulated with indicated ligands.c, IL-1b production from LPS-primed macrophages infected with S. typhimurium (multiplicity of infection, m.o.i., 1), or stimulated with ATP or MSU for 1 h.d, Expression levels of caspase-1 and IL-1b in macrophages. Lys., cell lysates; Sup., culture supernatants.e, LPS-induced production of IL-1b from macrophages with the indicated phenotype.f, Expression levels of caspase-1 and IL-1b in macrophages treated as in e.g, Effect of the ROS scavenger FeTPPS (25 mM), N-acetyl-L-cysteine (NAC; 25mM) or P2X7 receptor antagonist oxidized ATP (250 mM) on IL-1b production.h, ROS in LPS-stimulated macrophages were detected by CM-H2DCFDA staining.
figf3a, Macrophages were stimulated with LPS (100 ng ml-1) for the indicated time period. The number of endogenous LC3 dots within each cell was counted. The results shown are mean + s.d. (n > 100).b, Expression levels of p62 and LC3 in macrophages.c, IL-1b and IL-6 production by wild-type or Atg7-deficient macrophages stimulated with LPS. Statistical significance was determined by the Student's t-test. *P,0.01.d, Macrophages were pre-treated with or without 10 mM 3MA and then stimulated with the indicated ligands.
figf4a, b, Fetal liver chimaeric mice were given 5% DSS in drinking water for 7 days. The survival (a) and weight loss (b) of each mouse genotype were plotted. The results shown are mean ± s.d. Statistical significance was determined by the Students t-test.c, Typical distal colon appearance 6  days after the initiation of DSS administration. The results shown are mean ± s.d. (n = 3, each group).d, Expression levels of IL-1b and IL-18 in serum (n55, each group).e, f, Atg16L1-deficient chimaeric mice given 5% DSS in drinking water were intraperitoneally injected with both anti-IL-1b and anti- IL-18 neutralizing antibodies (squares; n55) or isotype control IgG (circles;n55) at days 1, 3, 5 and 7. The survival (e) and weight loss (f) of each mouse genotype were plotted.
Figure 1B Social interaction ratio in control, susceptible and resilient mice following the CSDS protocol (n = 44, 55, 44 mice).C Sucrose preference in control, susceptible and resilient mice (n = 20, 20, 12 mice. Treatment F2, 49 = 16.05, P < 0.0001, one-way ANOVA with Fisher's LSD test).D The immobility time in TST in susceptible mice, compared to control mice (n = 16, 32 mice. Susceptible vs control, P = 0.002, Student's t test).E The immobility time in FST in susceptible mice, compared to control mice (n = 17, 35 mice. Susceptible vs control, P = 0.0003, Student's t test).F, G The time in the open arms (F) and closed arms (G) in the EPM in susceptible mice, compared to control mice (n = 19, 33 mice. Susceptible vs control, for open arms, P < 0.0001; For closed time, P < 0.0001, Student's t test).H The central distance in the OFT in susceptible mice, compared to control mice (n = 13, 28 mice. Susceptible vs control, P = 0.0089, Student's t test).I The mRNA level of CD39, ENTPD2 and ENTPD3 in the hippocampus of susceptible mice after CSDS (n = 7, 8 mice. CSDS vs control, CD39 P = 0.0002, ENTPD2 P = 0.0025, Student's t test).J The CD39 protein expression in the hippocampus of susceptible mice induced by CSDS (n = 6, 7 mice. CSDS vs control, P = 0.0003, Student's t test).K The ATPase activity in the hippocampus of stressed animals (n = 6 mice/group. CSDS vs control, P =0.0041, Student's t test).
Figure 2B The effects of apyrase (40, 80 and 160 U/ml) infusion into cerebral ventricle on social interaction time (n = 8, 10, 10, 10 mice. Interaction F3, 34 = 3.917, P = 0.0167; Target F1, 34 = 0.0004, P = 0.9834; Drug F3, 34 = 2.295, P = 0.0954; For target, Apyrase 80 U/ml vs control, P = 0.0008, Two-way ANOVA with Tukey's post-test).C The effects of apyrase (40, 80 and 160 U/ml) infusion into cerebral ventricle on sucrose preference (n = 8, 10, 9, 11 mice. Treatment F3, 34 = 2.87, P = 0.0505; Apyrase 80 U/ml vs control, P = 0.007, one-way ANOVA with Fisher's LSD test).E The effect of apyrase (40 U/ml) infusion into hippocampal on the social interaction time (n = 14, 20 mice. Interaction F1, 32 = 7.0754, P = 0.0121; Target F1, 32 = 0.68447, P = 0.4142; Drug F1, 32 = 18.211, P = 0.0002; Apyrase vs control, P < 0.0001, Student's t test).F The effect of apyrase (40 U/ml) infusion into hippocampal on the sucrose preference (n = 10, 16 mice. Apyrase vs control, P = 0.0003, Student's t test).G The social avoidance behavior in mice with ATP intra-hippocampal infusion after apyrase exposure (n = 14, 10, 10 mice. Interaction F2, 31 = 2.051, P = 0.1457; Target F1, 31 = 2.666, P = 0.1127; Drug F2, 31 = 3.711, P = 0.0359; For Target, Apyrase-ACSF vs Vehicle, P = 0.0387; Apyrase-ATP vs Apyrase-ACSF, P = 0.0099, Two-way ANOVA with Tukey's post-test).H The sucrose preference in mice with ATP intra-hippocampal infusion after apyrase exposure (n = 10, 8, 8 mice. Treatment F2, 23 = 3.32, P = 0.0540; Apyrase-ACSF vs Vehicle, P =0.0466, one-way ANOVA with Fisher's LSD test).
Figure 3B The ATPase activity of mice exposure to ARL67156 (n = 7, 7, 5, 5 mice. Treatment F3, 20 = 16.81, P < 0.0001; CSDS - ACSF vs Ctrl - ACSF, P < 0.0001; CSDS - ARL67156 vs CSDS - ACSF, P = 0.0405. One-way ANOVA with Fisher's LSD test).C The social avoidance behavior of susceptible mice with intracerebroventricular infusion of ARL67156 (n = 12, 10, 9, 9 mice. Interaction F3, 36 = 5.119, P = 0.0047; Target F1, 36 = 0.005, P = 0.9437; Drug F3, 36 = 3.626, P = 0.022. For Target, CSDS - ACSF vs Ctrl -ACSF, P = 0.0001; CSDS - ARL67156 vs CSDS - ACSF, P = 0.0054. Two-way ANOVA with Tukey's post-test).D The sucrose preference of susceptible mice with intracerebroventricular infusion of ARL67156 (n = 12, 10, 10, 10 mice. Interaction F1, 38 = 5.352, P = 0.0262; Group F1, 38 = 5.711, P = 0.0219; Drug F1, 38 = 9.703, P = 0.0035; CSDS - ACSF vs Ctrl - ACSF, P = 0.0083; Ctrl - ARL67156 vs CSDS - ACSF, P = 0.0027; CSDS - ARL67156 vs CSDS - ACSF, P = 0.0031. Two-way ANOVA with Tukey's post-test).The social avoidance behavior (E) in mice with intra-hippocampal infusion of ARL67156 after CSDS (n = 9, 9, 9, 8 mice. For social behavior, Interaction F3, 31 = 5.579, P = 0.0035; Target F1, 31 = 0.5121, P = 0.4796; Drug F3, 31 = 8.367, P = 0.0003; CSDS - ACSF vs Ctrl - ACSF, P < 0.0001; CSDS - ARL67156 vs CSDS - ACSF, P = 0.0131.sucrose preference (F) in mice with intra-hippocampal infusion of ARL67156 after CSDS (n = 9, 9, 9, 8 mice. Sucrose, Interaction F1, 30 = 0.3342, P = 0.5675; Group F1, 30 = 17.22, P = 0.0003; Drug F1, 30 = 0.05903, P = 0.8097; CSDS- ACSF vs Ctrl - ACSF, P = 0.0761. Two-way ANOVA with Tukey's post-test).G The immobility time in the TST of mice with intra-hippocampal infusion of ARL67156 after CSDS (n = 16, 12, 21, 19 mice. Treatment F 3, 64= 13.61, P < 0.0001; CSDS - ACSF vs Ctrl - ACSF, P = 0.0059; Ctrl - ARL67156 vs Ctrl - ACSF, P = 0.0011; CSDS - ARL67156 vs CSDS - ACSF, P = 0.0002. One-way ANOVA with Fisher's LSD test).H The immobility time in the FST with intra-hippocampal infusion of ARL67156 after CSDS (n = 21, 12, 23, 19 mice. Treatment F 3, 71 = 12.94, P < 0.0001; CSDS - ACSF vs Ctrl - ACSF, P = 0.0036; Ctrl - ARL67156 vs Ctrl - ACSF, P = 0.0428; CSDS - ARL67156 vs CSDS - ACSF, P < 0.0001. One-way ANOVA with Fisher's LSD test).Data information: data are expressed as the means ± SEM. NS P > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001. Ctrl, control.
Figure 4B Imaging of GFP expression in the hippocampus 3 weeks after virus vector injection. Scale bar: 250 μm.C Expression of CD39 in the hippocampus of mice injected with LV-siCD39 (n = 6 mice/group. LV-siCD39 vs Control, P = 0.0006, Student's t test).D The social avoidance behavior for the stressed mice after knocking down CD39 (n = 15, 15, 14 mice. Interaction F2, 41 = 17.96, P < 0.0001; Target F1, 41 = 3.75e-005, P = 0.9951; Drug F2, 41 = 6.099, P = 0.0048; For Target CSDS - LV-GFP vs Ctrl - LV-GFP, P < 0.0001; CSDS - LV-siCD39 vs CSDS - LV-GFP, P < 0.0001. Two-way ANOVA with Tukey's post-test).E The immobility time in the TST of mice injected with LV- siCD39 (n = 11, 19, 15 mice. Treatment F 2, 42 = 5.807, P = 0.0059; CSDS - LV-GFP vs Ctrl - LV-GFP, P = 0.0069; CSDS - LV-siCD39 vs CSDS - LV-GFP, P = 0.0065. One-way ANOVA with Fisher's LSD test).F The immobility time spent in the FST of mice treatment with LV-siCD39 (n = 12, 19, 15 mice. Treatment F2, 43 = 5.237, P = 0.0092; CSDS - LV-GFP vs Ctrl - LV-GFP, P = 0.0095; CSDS - LV-siCD39 vs CSDS - LV-GFP, P = 0.0093. One-way ANOVA with Fisher's LSD test).G The ATPase hyperactivity of mice after knocking down CD39 in the hippocampal (n = 5, 6, 5 mice. Treatment F2, 13 = 8.45, P = 0.0044; CSDS - LV-GFP vs Ctrl - LV-GFP, P = 0.0016; CSDS - LV-CD39 vs CSDS - LV-GFP, P = 0.0166. One-way ANOVA with Fisher's LSD test).H The ATP level of mice after knocking down CD39 in the hippocampal (n = 5 mice/group. Treatment F2, 12 = 6.04, P = 0.0153; CSDS - LV-GFP vs Ctrl - LV-GFP, P = 0.0444; CSDS - LV-CD39 vs CSDS - LV-GFP, P = 0.0051. One-way ANOVA with Fisher's LSD test).
Figure 5B Immunostaining for BrdU (blue) and DCX (green) in the DG region of control, susceptible and ARL67156 treatment mice. Arrows indicate the BrdU+ DCX+ cells. Scale bar: 50 μm.C, D Quantification of BrdU+ cells (C) and BrdU+ DCX+ cells (D) in the DG of susceptible mice with ARL67156 compared with vehicle treatment (n = 4 mice/group. For BrdU+ Treatment F2, 9 = 10.07, P =0.0051; CSDS - ACSF vs Ctrl - ACSF, P = 0.0147; CSDS - ARL67156 vs CSDS - ACSF, P = 0.0018; BrdU+ DCX+ Treatment F2, 9 = 9.005, P = 0.0071; CSDS - ACSF vs Ctrl - ACSF, P = 0.0211; CSDS - ARL67156 vs CSDS - ACSF, P = 0.0024, one-way ANOVA with Fisher's LSD test).F Confocal images of BrdU (blue) and DCX (red) in mice treatment with LV-siCD39 in the DG. Arrows indicate the BrdU+ DCX+ cells. Scale bar: 50 μm.G, H Quantitative of BrdU+ (G) and BrdU+ DCX+ (H) cells in the DG of susceptible mice with LV-siCD39 compared with LV-GFP treatment (n = 3, 6, 6 mice. For BrdU+ Treatment F2, 12 = 15.79, P =0.0004; CSDS - LV-GFP vs Ctrl - LV-GFP, P = 0.0012; CSDS - LV-siCD39 vs CSDS - LV-GFP, P = 0.0003; BrdU+ DCX+ Treatment F2, 12 = 12.79, P = 0.0011; CSDS - LV-GFP vs Ctrl - LV-GFP, P = 0.05; CSDS - LV-siCD39 vs CSDS - LV-GFP, P = 0.0003, one-way ANOVA with Fisher's LSD test).