EMBO/sd-character-level-ner huggingface dataset:
Bim Suppresses Autophagy by Mediating the LC8-Beclin 1 Interaction.Figure 7(A) HeLa cells were transfected with vector (vec) /GFP-LC3, BimEL-EE (EL)/GFP-LC3, BimL-EE (L)/GFP-LC3, BimS-EE (S)/GFP-LC3, or BimEL-EE-AAA (AAA)/GFP-LC3 (3:1). The percentages of cells with GFP-LC3 vesicles were assessed. Data are shown as mean ± SD. ∗∗∗p < 0.001; ∗∗p < 0.01; NS, not significant. Note that BimEL-EE-S109A, S113A, T114A is designated as BimEL-EE-AAA. (B) Bim bridges the Beclin 1-LC8 interaction. Dynein light chain1 (Myc-LC8)/empty vectors (IP negative control), Myc-LC8/Beclin 1-Flag/empty vector, Myc-LC8/Beclin 1-Flag/BimEL-EE (EL), Myc-LC8/Beclin 1-Flag/BimL-EE (L), Myc-LC8/Beclin 1-Flag/BimS-EE (S), and Myc-LC8/Beclin 1-Flag/BimEL-EE-S109A, S113A, T114A (AAA) were transfected into HeLa cells. Anti-Flag (M2) was used for immunoprecipitation. (C) Starvation reduces the ability of Bim to bridge the Beclin 1-LC8 interaction. Myc-LC8/empty vectors (IP negative control), Myc-LC8/Beclin 1-Myc/empty vector, or Myc-LC8/Beclin 1-Flag/HA-BimEL-EE (HA-Bim) (two replicates) were transfected into HeLa cells. After 20 hr, one set of cells with Myc-LC8/Beclin 1-Flag/HA-HA-Bim was starved in HBSS for 2 hr. Anti-Flag (M2) antibody was used for immunoprecipitation. (D) HeLa cells were cultured with DMEM with 10% serum (Control) or starved (Starve) in HBSS for 2 hr. The fixed cells were stained with Beclin 1 and tubulin antibodies and analyzed by confocal microscopy. White boxes show the areas where Beclin 1 is enriched. Yellow boxes show enlarged areas. (E) HeLa cells were treated with control siRNA or LC8 siRNA. After 24 hr, cells were split. Vector/GFP-LC3 or Bim(EL) EE/GFP-LC3 (3:1) were transfected into the control siRNA-transfected or LC8 siRNA-transfected cells. The percentages of cells with GFP-LC3 vesicles were assessed. Data are shown as mean ± SD. ∗∗p < 0.01; NS, not significant. (F) HeLa cells were treated with control siRNA, Bim siRNA, or LC8 siRNA for 48 hr. Cells were then fixed in 37°C, 4% PFA for 10 min. Cells were stained with Beclin 1 and tubulin antibodies and analyzed by confocal microscopy. White boxes/triangle show areas where Beclin 1 is enriched. Yellow boxes show enlarged areas. Colocalizations were quantified from images in 12-15 cells with Volocity program (Colocalization coefficient Mx). Data are shown as mean ± SD. ∗∗∗p < 0.001. (G) Bim inhibits autophagy. In normal conditions, LC8 recruits Beclin 1 to the microtubule-based dynein motor complex via Bim, thereby inhibiting autophagosome formation (Bim possesses autophagy-inhibitory activity). Under stress conditions (e.g., nutrient starvation), Bim phosphorylation at T116 by JNK leads to its dissociation from LC8, and this dissociation triggers the dissociation of Bim-Beclin 1. The Beclin 1-Vps34 can then localize to isolation membranes (IMs) to enable autophagosome synthesis. Free Bim can induce apoptosis.
Figure 3(a) Stacked bar plots of all 11 and 12-member (full) multi-species communities. The text above each subplot denotes the absent organism in the community. Time and relative abundance are represented on the x and y-axis of each subplot, respectively. Colors represent different organisms in the community. (b) Difference in community assembly for single-species dropout communities. Difference in community assembly was computed as $\sum_{i = 1,\ }^{11}{\frac{1}{7}\sum_{j = 1}^{7}{({\widehat{v}}_{ji,FULL} - v_{ji,X})}}^{2}$ where i and j represent species and time points, respectively. X represents a single-species dropout community lacking the organism X. v̂ denotes the renormalized relative abundance of the shared set of species in the full (12-member) and single-species dropout community. Here ${\widehat{v}}_{i} = \ \frac{v_{i}}{\sum_{i\ \in C}^{}v_{i}}$ where C denotes the set of 11 shared organisms in the full and single-species dropout community. Data points represent biological replicates (n = 6, circles) and the mean of biological replicates (blue squares), respectively. (c) Box plot of the Pearson correlation coefficient for three model training sets (left) including T1: monospecies (M); T2: M and pairwise 1 (PW1); and T3: M, PW1 and pairwise 2 (PW2). On each box, the red line represents the median, the edges of the box are the 25th and 75th percentiles, the whiskers extend to the most extreme data points and the outliers are plotted as red crosses. (d) Box plot of the sum of mean squared errors for the model predictions of the time-resolved measurements of multi-species communities trained on T1, T2 or T3 training sets. On each box, the red line represents the median, the edges of the box are the 25th and 75th percentiles, the whiskers extend to the most extreme data points and the outliers are plotted as red crosses. (e) Comparison between model prediction based on T1-T4 and experimental data for each organism in the full community as a function of time. On each subplot, the x and y-axis represent time and relative abundance of each species, respectively. Data points (red) and lines denote experimental data and model predictions based on T1-T4. Species names are displayed in the upper left corner of each subplot. Error bars represent 1 s.d. from the mean of six biological replicates.
Model of BRM complex action.Figure 6In the absence of Notch signaling, the BRM complex maintains the accessibility of Notch-responsive enhancers to allow Su(H) recruitment by promoting nucleosome turnover. When Notch signaling is activated, the nucleosome turnover at Notch-responsive enhancers increases, increasing the accessibility of the chromatin and allowing more Su(H) to bind. The BRM complex is essential for the process to occur. Target genes are activated via co-activators.
Pacs1 deletion warps ER Ca2+ handling.Figure 6(A) Immunoblot of Pacs1, Wdr37, IP3Rs, and ER stress markers in the parental NIH-3T3 cell line and three separate Pacs1-/- clones. (B) Real-time quantitative PCR of IP3R isoform expression WT and Pacs1-/- 3T3 cells. Expression in the Pacs1-/- cells was measured in three independent clones. Data are presented as mean ± SD. (C) Pacs1+/+ and Pacs1-/- NIH-3T3 cells were transfected with cytosolic aequorin and Ca2+ flux was measured after treatment with 1 µM bradykinin. Representative kinetic tracing is shown. (D) Peak cytosolic Ca2+ concentration based on aequorin measurements as performed in (C). Symbols represent individual wells containing ~5 x 105 transfected 3T3 or Pacs1-/- cells from one experiment. Horizontal bars indicate mean. Two-tailed unpaired t test, **P < 0.01. (E) Pacs1+/+ and Pacs1-/- NIH-3T3 cells (C1 and C2 from (A)) were transfected with ER-GCamP6-210. ER Ca2+ was measured before and after treatment with 10 µM ATP. Kinetic traces show the mean 488/405 nm excitation ratio of each cell line with error bars indicating SEM. N=30 cells (3T3), 16 cells (C1), 25 cells (C2). Data are representative of 2 independent experiments. (F) ER Ca2+ release from the NIH-3T3 cell lines imaged in (E). Symbols represent individual cells. Horizontal bars indicate mean value. One-way ANOVA with Tukey's test for multiple comparisons, **P < 0.01, ***P < 0.001.
Figure 7A Schematic of AAVretro-Cre and AAV-DIO-hM4Di-mCherry or AAV-DIO-mCherry injections. B mCherry-labeled axons in the vHIP. Scale bars, 200 μm (left panel), and 50 μm (right panel). C Representative animal tracks in three-chamber test. D Discrimination indices of the social memory session of three-chamber test. mCherry mock, n=8 mice; mCherry ZIKV, n=10 mice; hM4Di ZIKV n=10 mice, mCherry mock vs. mCherry ZIKV p=0.0041; mCherry ZIKV vs. hM4Di ZIKV p=0.0115; and mCherry mock vs. hM4Di ZIKV p=0.7076. Data information: All data are presented as mean ± SEM. *P < 0.05, **P < 0.01, ****P < 0.0001, one-way ANOVA with Tukey post hoc test. E Representative confocal images of mCherry (red) and c-Fos (green) double-labeled cells in the vHIP CA1 regions. Scale bars, 50 μm. F. Quantification of the percentage of c-fos;mCherry double positive cells out of total mCherry cells. mCherry mock, n=6 mice; mCherry ZIKV, n=4 mice; hM4Di ZIKV n=6 mice. Data information: All data are presented as mean ± SEM. *P < 0.05, **P < 0.01, ****P < 0.0001, one-way ANOVA with Tukey post hoc test.
Hdac1 deletion increases H3K79 methylation in thymocytes in vivo, and simultaneous heterozygous Dot1L deletion prolongs tumor-free survival.Figure 3Mass spectrometry analysis of H3K79 methylation in thymuses from 3-week-old mice, either wild-type (Cre-) or with deleted Hdac1 or Dot1L alleles, as indicated. Mean and individual data points of biological replicates; H3K79me0 is the predominant state, the y axis is truncated at 70% for readability. The remaining H3K79 methylation after homozygous Dot1L deletion is probably due to the presence of some cells in which Cre is not expressed (yet). p<0.01 (**) and p<0.001 (***) by two-way ANOVA, comparison to wild type. Representative H&E and immunohistochemical stainings on sequential sections of thymic lymphomas of the indicated genotypes. A picture with lower magnification of independent samples is included in Fig EV3B. Kaplan-Meier curves of mice harboring thymocytes with indicated genotypes. An event was defined as death or sacrifice of a mouse caused by a thymic lymphoma. Mice that died due to other causes or were still alive and event-free at the end of the experiment were censored. Mice for which the cause of death could not be determined were removed from the data. Wild-type mice were the Cre- littermates of the mice that were used for the other curves. Summary of the data presented in panel C. A median latency could only be calculated when the tumor incidence was >50%. The p value was determined by comparing to the Lck-Cre;Hdac1f/f curve with a Peto test, but a logrank test yielded the same conclusions.
Specific and efficient siRNA delivery to NK cells using NKp46 antibody-coated NPs.Figure 3(A) NK92-NKp46low, NK92-NKp46high, YTS KIR2DL1, K562 and 721.221 HLA-Cw4 cells were stained with anti-NKp46 monoclonal antibody followed by staining with Alexa568-Fluor goat anti-mouse IgG secondary antibody. Cells were then analyzed using flow cytometry. (B) NK92-NKp46low, NK92-NKp46+, YTS KIR2DL1, K562, and 721.221 HLA-Cw4 cells were incubated with 50 µg rhodamine-labeled NPs and analyzed using flow cytometry to confirm the internalization of the coated NPs in a NKp46-specific manner. (C-F) (C) NK92-NKp46low, (D) NK92-NKp46high, (E) YTS KIR2DL1 and (F) K562 cells were incubated with 50 µg rhodamine-labeled NPs and analyzed using confocal microscopy. Upper panel shows wide field images. Lower panel shows single-cell images. The data shown are representative of three independent experiments. (G) YTS KIR2DL1 were incubated with NKp46 antibody coated-NPs loaded with FITC labeled siRNA. After 24 hours, the cells were stained with anti-NKp46 monoclonal antibody followed by staining with Alexa568-Fluor goat anti-mouse IgG secondary antibody. Then, the cells were analyzed by flow cytometry to determine the incorporation of the rhodamine-labeled NPs loaded with FITC-siRNA and NKp46 antibody staining. Cells that were positive for both rhodamine-labeled NPs and NKp46 antibody staining, were analyzed for the presence of fluorescent FITC-siRNA.
Figure 5A) Sep+/+ or Sep-/- oocytes were matured in vitro until metaphase II. Sep-/- oocytes were injected with wild-type separase or cleavage mutant separase encoding mRNAs in metaphase II. Oocytes were either fixed in metaphase II or activated and fixed in anaphase II. Spreads were stained with Rec8 antibody (red), CREST serum (green), and DAPI (blue) to label DNA. n indicates the number of oocytes with the shown phenotype (e.g. in anaphase II, after successful activation) and the total number of oocytes analysed B) Representative image of kinetochore individualization in Sep-/- oocytes injected with wild-type separase in metaphase II and fixed after activation, from A). Shown is staining with CREST serum (green), and DAPI (blue) to label DNA. Arrows indicate separated sister kinetochores. The scheme on the left illustrates the individualization of sister kinetochores (green arrows) at anaphase II. n indicates the number of oocytes with the shown phenotype (e.g. in anaphase II, after successful activation) and the total number of oocytes analysed Scale bars: 10 µm, except bottom panel in B), where the scale bar is 2 μm.
a–f) Representative EM of WT (a), ATG5−/− (b), Ulk1gt/gt (c,d) and DKO (e,f) reticulocytes from embryonic liver (E14.f4(a-f) Representative EM of WT (a), ATG5−/− (b), Ulk1gt/gt (c,d) and DKO (e,f) reticulocytes from embryonic liver (E14.5). (a,b) Mitophagy was detected in WT and ATG5−/− cells (scale bar, 1 μm). Insets indicate mitophagy (scale bar, 0.2 μm). (c,e) In Ulk1gt/gt and DKO cells, numerous mitochondria were in contact with membrane structures (scale bar, 1 μm). Enlarged images are shown on the right side (scale bar, 0.2 μm). (d,f) Some Ulk1gt/gt and DKO cells showed plasma membrane blebs containing mitochondria (scale bar, 1 μm). (g,h) Quantitative analysis of mitophagy calculated from EM photos in the embryonic liver at E18.5. Population of reticulocytes with mitophagy (g), and number of mitochondria per reticulocytes (h) were calculated (n>35 cells per mouse). The data are shown as mean±s.d. (n=3). *P0.05 versus value of WT (analysis of variance (ANOVA)); #P0.05 versus value of Atg5−/− (ANOVA); 'NS' indicates not significant versus value of WT (ANOVA).
Figure 1A. Immunofluorescence of SATB2 (green) and the GSC marker SOX2 or OLIG2 (red) on frozen tissue sections of human GBM surgical specimens. SATB2 is preferentially expressed by GSCs in human GBMs. Scale bar, 25 μm. B. Quantification of the fraction of SATB2+ cells in SOX2+ or OLIG2+ cells in human GBMs. More than 90% SOX2+ or OLIG2+ cells showed SATB2 staining. n=3 GBMs. C. qPCR analysis of SATB2 mRNA expression in GSCs and matched non-stem tumor cells (NSTCs) (n=5). D. Immunoblot analysis of SATB2, SOX2 and OLIG2 expression in cell lysates of GSCs and matched NSTCs. E. Immunofluorescenc of SATB2 (green) and SOX2 (red) in T3359 GSCs and matched NSTCs. Scale bar, 50 μm. F. qPCR analysis of SATB2 mRNA expression in GSCs and neural progenitor cells (NPCs) (n=3). G. Immunoblot analysis of SATB2 and SOX2 expression in cell lysates of GSCs and NPCs. H. Immunoblot analysis of SATB2, GSC marker SOX2 and differentiation marker GFAP expression during serum-induced GSC differentiation.
DHR3 regulates dS6K activity.Figure 3(A) The DHR3 locus is schematized at the top with the 2 EP insertions indicated (arrows). The RA and RB transcripts encode a DBD sequence (indicated by the grey box). RS is a putative novel transcript whose first AUG is located beyond the DBD-coding sequences. (B-G) Adult wings in which the ap-Gal4 induces expression of the following UAS constructs: (B) ap-Gal4 control; (C) UAS-dS6K; (D) UAS-DHR3-RS; (E) both UAS-dS6K and UAS-DHR3-RS; (F) UAS-DHR3-RS in a dS6Kl-1 mutant escaper; (G) UAS-dS6K, UAS-DHR3-RS, and UAS-DHR3-RNAi together. (H) Upper panel, H2B substrate phosphorylation by dS6K from larval protein extracts. Ubiquitous induction of UAS-DHR3 RNAi, using either a daughterless-Gal4 (da>Ri) or an actin-Gal4 (ac>Ri) driver, provoked a drop in dS6K activity, as compared with control drivers alone (da, ac). Middle and lower panels are western blots detecting dS6K and α-tubulin, respectively.
Figure 1Primary sequence of 5-mer peptide (C5) derived from the dopamine transporter C-terminus and the TAT conjugated variant, Tat-C5. Fluorescence polarization (FP) competition derived affinity assessment of consecutive truncations of the DAT C13 C-terminus as indicated by the sequences. Bars indicate the mean fold change relative to C13 (Ki = 1.69 µM). Data is shown as mean with error-bars as SEM with n = 6. Representative binding curves are shown in EV1A. FP competition binding of C5 and Tat-C5 to PICK1 with 5FAM-Tat-C5 (20 nM) as tracer. Saturation binding curve is shown in EV1B. Backbone amide chemical shift changes (ΔδHN) of PICK1-PDZ-C10 after addition of Tat-C5 and addition of protease to cleave C10 from the PDZ domain and allow exchange. Numbers refer to residue number in PICK1 (Uniprot: Q9EP80). Red bars indicate a chemical shift larger than mean+SD. 1H-15N-HSQC 2D spectra of PICK1-PDZ-C10 (orange) following addition of Tat-C5 (green) and subsequent addition of protease (red) to allow for exchange between C10 and Tat-C5. Docking model, for visual purposes, of PICK1-PDZ (PDB: 2LUI) in complex with Tat-C5 (blue), with perturbed residues indicated from D. in red. Selectivity screen for Tat-C5 against a selection of 42 purified PDZ domains, performed with a fixed concentration of Tat-C5 (10 µM) in competition with PDZ domains and their respective fluorescent ligands. Data are normalized to binding in absence of peptide (full line). Dashed line represents the level of competition obtained for PICK1. Screen for C5 can be seen in EV1C. Affinity gain of bivalent C5 peptides (Pn-(C5)2) N-terminally conjugated with different length PEG linkers as indicated. Bars indicate the mean fold change relative to C5 (Ki = 1.69 µM). Data is shown as mean with error-bars as SEM with n = 6. Representative binding curves are shown in EV1C. FP competition binding of C5 (also used in C) and Tat-P4-(C5)2 to PICK1 with 5FAM-Tat-C5 (20 nM) as tracer. Structure and highest obtained affinity of C5, TAT11-C5 and TAT11-P4-(C5)2 towards PICK1.
Overexpression of radixin mutant mitigates glucagon-elicited hyperglycemia.Figure 6The molecular interactions between PROM1 and radixin mutants were determined by co-immunoprecipitation with an anti-FLAG antibody. HEK293 cells were co-transfected with plasmids expressing Myc-radixin and various deletion mutants of PROM1-FLAG (A) for 24 hours. The molecular interactions between PROM1 and radixin mutants were determined by co-immunoprecipitation with an anti-FLAG antibody. HEK293 cells were co-transfected with plasmids expressing PROM1-FLAG and various deletion mutants of Myc-radixin (B) for 24 hours. C. The molecular interaction between PROM1-FLAG and Myc-radixin in the presence of increasing amounts of Myc-FERM was determined by co-immunoprecipitation with an anti-FLAG antibody. WT primary hepatocytes were infected with an adenovirus harboring FERM-Myc (Ad-FERM-Myc) or LacZ (Ad-LacZ) for 48 hours. The cellular localization of radixin was determined by immunofluorescence staining. Scale bar, 20 μm. WT primary hepatocytes were infected with an adenovirus harboring FERM-Myc (Ad-FERM-Myc) or LacZ (Ad-LacZ) for 48 hours. the nuclear localization of p-CREB after glucagon stimulation for 10 minutes (E) was determined by immunofluorescence staining. Scale bar, 20 μm. WT primary hepatocytes were infected with an adenovirus harboring FERM-Myc (Ad-FERM-Myc) or LacZ (Ad-LacZ) for 48 hours. (F) Ad-FERM-Myc- or Ad-LacZ-expressing WT hepatocytes were serum-starved for 16 hours and stimulated with glucagon (10 nM) for 10 minutes. Levels of p-PKA substrates, p-CREB, CREB, FERM-Myc and GAPDH were determined by immunoblotting. Twelve-week-old male wild type mice were infected with an adenovirus harboring GFP or FERM for 3 days, fasted for 4 hours and intraperitoneally injected with glucagon. (G) Blood glucose levels (mg/dL) were measured 0, 15, 30, 60 and 120 minutes after glucagon stimulation (0.2 mg/kg body weight, n = 10 mice/ group). Twelve-week-old male wild type mice were infected with an adenovirus harboring GFP or FERM for 3 days, fasted for 4 hours and intraperitoneally injected with glucagon. (H) Levels of p-CREB, CREB, p-PKA substrates, and FERM in the liver 10 minutes after glucagon stimulation (2 mg/kg body weight) were determined by immunoblotting (n = 3 mice/ group). Twelve-week-old male wild type mice were infected with an adenovirus harboring GFP or FERM for 3 days, fasted for 4 hours and intraperitoneally injected with glucagon. (I) Glucose disposal rates in GFP- or FERM-overexpressing mice were measured using glucose, pyruvate and insulin tolerance tests (n = 10 mice/group).
Figure 4 Heatmap of the tolerance to amino acid substitutions in WT transactivation domain sequences. The tolerance of a mutation is defined as the fraction of functional sequences over all the sequences when a specific substitution was performed. The columns (amino acid in a WT TAD that is substituted) are ordered according to decreasing tolerance (from left to right) and the rows (amino acid into which a residue in the WT TAD is substituted for) are ordered according to decreasing tolerance (from bottom to top). The cells are colored on a green to red gradient for high to low tolerance respectively. Empty tiles represent data points not present in the library  Schematic describing the sequence space explored by the design library (left). Table listing the top 10 most important features. The relative feature importance is given as relative percentages in the last four columns. The size of the circles is scaled per method (lasso, ridge, xgboost, stacked). The direction column denotes the direction of enrichment of the given feature for functional sequences compared to non-functional sequences (up, positive direction and down, negative direction)
Figure 2MARCH2+/+ (n = 8) or MARCH2-/- (n = 8) mice were challenged intraperitoneally with LPS (24 mg/kg). Percentage of surviving mice (A, log-rank test, **P < 0.01) MARCH2+/+ (n = 8) or MARCH2-/- (n = 8) mice were challenged intraperitoneally with LPS (24 mg/kg). body weight changes (B) in each groups. C MARCH2+/+ (n = 4) or MARCH2-/- (n = 4) mice were challenged intraperitoneally with LPS (24 mg/kg). Representing slides of H&E staining of spleen sections from each group. Scale bar, 6mm. D MARCH2+/+ (n = 6) or MARCH2-/- (n = 6) mice were challenged intraperitoneally with LPS (24 mg/kg). Levels of IL-6, TNF-α, CXCL-10, CCL-5 in serum from mice in each groups were measured at12 hpc by ELISA. E, F MARCH2+/+ (n = 5) or MARCH2-/- (n = 5) mice were challenged intraperitoneally with LPS (24 mg/kg). cDNA was prepared from total RNA extracted from spleen and liver of mice. Expression of mRNA encoding IL-6, TNF-α, CXCL10, IL-1β in spleen (E) and liver (F) from mice in each group was examined at 6 hpc by qPCR.
a) Morphology of cultured erythroid cells on day 0, 1 and 3 of differentiation.f6(a) Morphology of cultured erythroid cells on day 0, 1 and 3 of differentiation. Erythroblasts were purified from embryonic liver (E14.5) and incubated for the indicated times in erythrocyte differentiation medium. The cells were stained with a Diff-quick kit. (b) Percentage of cells without nuclei (Syto16low cells) on the indicated days of differentiation (mean±s.d., n=6). Loss of nuclei occurred normally in all mice. (c,d) Percentage of Syto16low cells without mitochondria. Differentiated cells were stained with Syto16 and Mitotracker Deep Red. Representative histograms of mitochondrial content in Syto16low cells on day 6 are shown in (c). Numbers indicate the population of mitochondrialow cells. Representative dot plots are also available in Supplementary Fig. 13. (d) Percentage of Syto16low cells lacking mitochondria determined by gating the mitochondrialow fraction as shown in (c) (mean±s.d., n=6). *P0.05 versus value of WT (analysis of variance (ANOVA)); #P0.05 versus value of Atg5−/− (ANOVA); 'NS' indicates not significant versus value of WT (ANOVA). (e-h) Impact of various drugs on mitochondrial clearance during in vitro differentiation. Differentiated cells from WT embryos were incubated with or without wortmannin (e), 3-methyladenine (3-MA) (f), rapamycin (g) or compound C (h) at the indicated concentrations and time periods. Percentage of mitochondrialow cells/Syto16low cells was measured by flow cytometry (mean±s.d., n=3). *P0.05 versus value of no drug (ANOVA). (i) Impact of various drugs on mitochondrial clearance during in vitro differentiation.with cells from Atg5−/− embryos exposed to wortmannin or 3-MA (mean±s.d., n=3). *P0.05 versus value of no drug (ANOVA).