EMBO/sd-character-level-ner huggingface dataset:
Figure 3(A) Effect of ykt6 and dsl1 complex ts mutants on autophagosome-vacuole fusion. Cells carrying a CEN plasmid expressing GFP-Atg8 were grown at 24°C in SGC and then shifted to SG-N for 2 h at 24°C or 37°C. Vacuoles were stained by FM4-64 and analyzed by fluorescence microscopy. Scale bar, 5 µm. Scale bar of inset is 0.6 µm. (B) Analysis of autophagy over time. Cells were grown at 24°C and shifted to starvation medium at 24°C or 37°C for the indicated time periods. The cells were harvested and proteins were extracted from cells by TCA precipitation. The extracted proteins were analyzed by SDS-PAGE and Western blotting against GFP. (C) Protease protection assay of ykt6-11 and dsl3 mutants. Lysates of atg1∆, ypt7∆, ykt6ts and dsl3ts cells carrying a CEN plasmid expressing GFP-Atg8 were grown at 37°C and subjected to the protease (PK)-protection (D) Localization of Atg8 relative to Atg1 during nitrogen starvation condition in sec12-1 ts mutant. Cells carrying a CEN plasmid expressing GFP-Atg8 and encoding 3xmCherry-tagged Atg1 were grown at 24°C in SDC and then shifted to SD-N for 2 h at 24°C or 37°C, and analyzed by fluorescence microscopy. Individual slices are shown. Scale bar, 5 µm. Scale bar of inset is 0.6 µm. Arrows in images indicate dots colocalization of Atg1 with Atg8 (D) (E) Localization of Ykt6 relative to Dsl1 during growth and nitrogen starvation conditions. Cells expressing GFP-tagged Ykt6 under the control of the PHO5 promoter and genomically 3xmCherry-tagged Dsl1 were grown in SDC or SD-N for 2 h, and analyzed by fluorescence microscopy. Individual slices are shown. Scale bar, 5 µm. (F) Percentage of Ykt6 puncta co-localizing with Dsl1. The data was quantified from (E). Error bars represent standard deviation of three independent experiments. Cells (n≥250) and Dsl1 dots (n≥50) were quantified. Arrows in images indicate dots colocalization of Ykt6 with Dsl1 (E). (G) Localization of Dsl3 relative to Atg8 during growth and nitrogen starvation. Cells expressing GFP-tagged Dsl3 and mCherry-tagged Atg8 were grown in SDC or SD-N for 2 h, and analyzed by fluorescence microscopy. Scale bar, 5 µm.
The MaMTH-HTS workflow.Figure 11) Grow up cell lines containing stably integrated prey and reporter. 2) Transfect cells with Bait of Interest. 3) Induce cells with tetracycline to express Bait and Prey. Continue to grow cells for 48 hours. At this point will have a mixed population containing cells expressing combinations of Bait (along with tagBFP / blue fluorescence) and Prey (along with mCherry / red fluorescence). Cells expressing interacting Bait/Prey pairs will induce eGFP reporter and have additional green fluorescence. 4) Carry out sequential FACS sort, first selecting cells with red fluorescence (indicating proper Prey expression), then blue fluorescence (indicating proper Bait expression) and finally green fluorescence (indicating Bait/Prey interaction). 5) Expand/grow cells to increase overall cell number, isolate genomic DNA and PCR amplify using prey cassette specific primers. 6) Deep sequence amplified PCR products to identify prey ORFs enriched in population (indicating potential interactors)
HSCs induce autophagy following metabolic stress.Figure 1Fluorescent microscopy of cultured Gfp-Lc3 GMPs (a) and HSCs (b) Electron microscopy images of WT HSCs. Arrowheads indicate autophagic vesicles. d, GFP-LC3 loss in cultured Gfp-Lc3 HSCs (n = 3). Results are expressed as percentage of GFP-LC3 MFI in -BafA compared to +BafA conditions. GFP-LC3 levels in HSCs of fed and starved Gfp-Lc3 mice (n = 3). Results are expressed as percent of GFP-LC3 MFI in fed mice. Data are means ± s.d. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001.
Figure 5(A) Volcano plot of up - (log2-fold change for transcripts with counts per million, CPM >1; red dots) and downregulated (log2-fold change CPM <1; blue dots) transcripts in LV after Cand2 KO. Differential expression analysis performed with edgeR; Benjamini-Hochberg method for p-value calculation, cut off 0.05. Cand2 and Grk5 marked among depleted transcripts in Cand2 KO vs. WT mice. Enrichment of Kyoto Encyclopedia of Genes and Genomes (KEGG Pathways) terms for differentially expressed genes in LV of Cand2 KO vs. WT mice. (B) Immunoblots and quantification of Grk5 protein levels in vitro after Cand2 KD with siRNA and after Cand2 OE with AAV6. t-test, n=4-5 biological replicates per group. Data information: Error bars indicate means ± SEM. * - P ≤ 0.05, *** - P ≤ 0.001. (C) Grk5 mRNA levels in NRCMs depleted of and overexpressing Cand2 measured by RT-qPCR. t-test, n=6-9 biological replicates. Data information: Error bars indicate means ± SEM. * - P ≤ 0.05, *** - P ≤ 0.001. (D) Grk5 protein levels in vivo in LVs of WT and Cand2 KO mice 4 weeks after sham and TAC analyzed by immunoblotting. (E) Quantification of Grk5 protein levels and Grk5 mRNA levels in indicated conditions. Analyzed by One-way ANOVA, n=4-15 biological replicates . Data information: Error bars indicate means ± SEM. * - P ≤ 0.05, *** - P ≤ 0.001. (F) Representative immunoblot of Grk5 protein levels and quantification in cytoplasmic and nuclear fractions from left ventricle lysates of WT and Cand2 KO mice after TAC surgery. Lamin B - nuclear marker, Gapdh - cytoplasmic marker. Analyzed by One-way ANOVA, n=3 biological replicates. Data information: Error bars indicate means ± SEM. * - P ≤ 0.05, *** - P ≤ 0.001.
Figure 7A Fluorescence micrographs of resistant WM3248 cells plated on unpatterned slides (control) and fibronectin-coated micropattern (1600 &amp;amp;#x3BC;m2) slides. Cells were double-stained with phalloidin-AlexaFluor594 and anti-YAP/TAZ antibody. B Quantification of the experiment presented in (A). C Fluorescence micrographs showing YAP/TAZ and actin filaments in resistant WM3248 cells treated with the indicated drugs for 3 hr. Blebbistatin (Blebb) was treated at 50 &amp;amp;#x3BC;M. D Quantification of YAP/TAZ localization in the experiment presented in (C). E Cell viability assay of resistant melanoma cells treated with 2 μM of PLX4032 alone or in combination with either cytochalasin D (200 nM) or blebbistatin (50 &amp;amp;#x3BC;M). Relative cell viability was analyzed by CCK8 assay after drug treatment for 72 hr. F qRT-PCR analyses of YAP-target genes in resistant SKMEL28 and WM3248 cells after treatment of cytochalasin D (200 nM) or blebbistatin (50 μM) for 12 hr. G Cell viability assay of mock or YAP5SA retrovirus transfected resistant melanoma cells. Cells were transfected with indicated retrovirus for 24 hr, and then treated with 2 μM of PLX4032 alone or in combination with either cytochalasin D (200 nM) or blebbistatin (50 μM). Relative cell viability was analyzed by CCK8 assay after drug treatment for 72 hr.
Bim Physically Interacts with Beclin 1 Independently of the Bim BH3 Domain.Figure 1(A) Bcl-xL was cotransfected into HeLa cells with empty vector (IP negative control), Beclin 1-Flag, Beclin 1-Flag/HA-Bim(EL), Beclin 1-Flag/HA-Puma, Beclin 1-Flag/HA-Noxa, or Beclin 1-Flag/Bad. Anti-Flag (M2) was used for immunoprecipitation, and proteins were detected with anti-Bcl-xL, anti-HA, or anti-Bad, anti-Flag (Rabbit). (B) HA-Bim (wild-type) was cotransfected into Bax/Bak double-knockout (DKO) mouse embryonic fibroblasts (MEFs) with empty vector (IP negative control) or Beclin 1-Flag. Immunoprecipitations were performed as in (A). (C) Beclin 1-Flag was cotransfected into Bax/Bak DKO MEFs with empty vector (IP negative control) or HA-Bim (wild-type). Anti-HA was used for immunoprecipitation. ∗, antibody heavy chain. (D) HeLa cell lysates were subjected to GFP antibody (negative control) and anti-Bim antibody immunoprecipitation. Proteins were detected with anti-Beclin 1 and anti-Bim. ∗∗, antibody heavy chain; ∗, antibody light chain. (E) Bcl-xL was cotransfected into HeLa cells with empty vector (IP negative control), wild-type HA-Bim(EL), or HA-Bim L152E F159E [Bim(EL) EE]. Anti-Bim was used for immunoprecipitation. ∗, antibody light chain. (F) HeLa cells were transfected with vector, wild-type HA-Bim, or HA-BimEE. Lysates were probed with the indicated antibodies. HeLa cells treated with staurosporine (STS) for 5 hr were a positive control. Note that wild-type Bim causes apoptosis, which reduces its levels compared to BimEE. (G) Vector, wild-type Bim(EL), or Bim(EL) L152E F159E (BimEE) was transfected into HeLa cells. Transfection efficiency was >90%. After 20 hr, cell viability was determined by measurement pf ATP levels. Data are shown as mean ± SD. ∗∗∗p < 0.001. (H) HeLa cells were transfected with vector/GFP (3:1), wild-type HA-Bim/GFP (3:1), or HA-BimEE/GFP (3:1) (to ensure that Bim-transfected cells also contain GFP). After 16 hr, cells were stained with Annexin V-APC and analyzed by FACS. The percentage of APC and GFP double-positive cells/GFP-positive cells are shown. Data are shown as mean ± SD. ∗∗∗p < 0.001. (I) Bim(EL)/Bcl-xL were cotransfected into HeLa cells with empty vector (IP negative control) or Beclin 1-Flag; Bim(EL)-EE/Bcl-xL were also cotransfected into HeLa cells with Beclin 1-Flag. Anti-Flag (M2) was used for immunoprecipitation. Data are shown as mean ± SD. NS, not significant. (J) BimEL-EE (EL)/vector (IP negative control), BimEL-EE (EL)/Beclin 1-Flag, BimL-EE (L)/Beclin 1-Flag, and BimS-EE (S)/Beclin 1-Flag were transfected into HeLa cells. Anti-Flag was used for immunoprecipitation. Data are shown as mean ± SD. ∗∗∗p < 0.001; NS, not significant.
a) α-syn immunoblot of rat liver lysosomes incubated with WT or mutant (A53T) α-syn alone (−) or with WT LRRK2 or G/S LRRK2.figf8(a) α-syn immunoblot of rat liver lysosomes incubated with WT or mutant (A53T) α-syn alone (−) or with WT LRRK2 or G/S LRRK2. Inp, input; Mon, monomers. Right: quantification of α-syn oligomers, expressed as a multiple of amounts in samples without LRRK2 (n = 4 or 5 independent experiments). (b-d) Degradation of radiolabeled proteins (b), binding of GST-α-syn (c) and levels of endogenous α-syn (d) in intact brain lysosomes (Lysos) from non-transgenic (Ctr) and G/S LRRK2 transgenic mice. Bottom panel in c shows lower exposure to visualize monomers of α-syn. Homog, homogenates; olig, oligomers; I, input (n = 3 independent experiments). (e) Ventral midbraindopaminergic neuronal cultures from control (Ctr) or WT-LRRK2 or G/S-LRRK2 transgenic mice untreated or treated with RNAi for LAMP-2A (L2A(−)). (f) Colocalization between α-syn and total LAMP-2 from cells in e (n > 50 tyrosine hydroxylase (TH)-positive neurons). Total LAMP-2 is not reduced in L2A knockdown cells because LAMP-2B is upregulated. (g) iPSC lines from controls and from patients with G/S LRRK2-mutation Parkinson's disease, differentiated for 30 d and immunostained for LAMP-2A, α-syn and TH. Merged channels (left) and magnified green and red channels from boxed region (right). Arrows, colocalization LAMP-2A and α-syn. (h) Percentage of LAMP-2A+ puncta colocalizing with α-syn in TH+ cells (n > 50) for two control and two LRRK2 iPSC lines treated as in g. (i,j) iPSC lines as in g, differentiated for 30 or 75 d and untreated or treated with RNAi for LAMP-2A. (i) Immunofluorescence for LAMP-2A, α-syn and GFP in LAMP-2A-RNAi iPSC lines differentiated for 30 d. Bottom: higher magnifications of boxed areas. (j) Percentage of TH+ cells showing cytoplasmic accumulation of α-syn. n ≥ 2 experiments using two control and two LRRK2 iPSC lines (n > 70 and n > 150 TH+ cells in Ctr and LRRK2, respectively). Scale bars, 10 μm. All values are mean + s.e.m. Differences from WT or control (*) or between control or knockdown cells (§) are significant at P 0.05; unpaired t-test in a,h and ANOVA and Bonferroni test in f,j. Full-length blots and gels in Supplementary Figure 12.
Fig.Figure 8A The indicated overexpression and RNAi constructs were co-expressed with markers for the plasma membrane (Src-GFP, green) and nuclei (DsRed2-Nuc, magenta) in the larval epidermis. The effects of upregulating autophagy (top row) are suppressed when autophagy is blocked by simultaneously downregulating Atg1 or Atg5. n=20-40 larvae each genotype. B-D Atg1-induced syncytium formation is abolished when Atg5 function is downregulated (compare to rows 2 and 3 in Fig. 5A, B); same representation as shown in Fig. 5A-D.
Figure.Figure 7Model of the regulation of ferritin fate by NCOA4 in cellular iron homeostasis. Under iron depletion, ferritin is degraded by the NCOA4 dependent macroautophagy pathway. In the early phase of iron treatment, NCOA4 forms condensates that sequester ferritin for ferritin accumulation. During prolonged iron treatment, NCOA4 condensates bind ferritin, forming a complex that is trafficked to the lysosome in a TAX1BP1-dependent manner to avoid iron deficiency.
Hippocampal FMN2 is essential for extinction learning and cognitive flexibility.Figure 3Figure 3: Hippocampal FMN2 is essential for extinction learning and cognitive flexibility. A. Experimental design. Anti-Fmn2 or scrambled siRNA oligomers were injected via microcannulae into the hippocampus of 3-month old wild type mice every 12h for 3 days. Figure 3: Hippocampal FMN2 is essential for extinction learning and cognitive flexibility.B. Fmn2 mRNA levels were determined in the hippocampus of mice injected with either scrambled or Fmn2 siRNA via qPCR (n= 4/group). Fmn2 levels are reduced in mice injected with Fmn2 siRNAi when compared to the scrambled control group (*P < 0.05). Error bars indicate SEM. Figure 3: Hippocampal FMN2 is essential for extinction learning and cognitive flexibility. C. Similarly, hippocampal FMN2 protein levels were decreased in mice injected with Fmn2 siRNA when compared to the scramble control group (*P < 0.05).Error bars indicate SEM. Figure 3: Hippocampal FMN2 is essential for extinction learning and cognitive flexibility.D. Experimental Design. Microcannulae were implanted into the hippocampus of wild type mice. All mice were subsequently subjected to contextual fear conditioning training. Fmn2 siRNA or scrambled control RNA was injected into the dentate gyrus 24h later and then every 12h for 3 days. On subsequent days mice were subjected to extinction trial (E) 1, E2, E3 and E4 (Fig 3B). The injection protocol was continued until E3. E. Fear Extinction is significantly impaired (P < 0.001, F = 15.55; * P < 0.05, ***P < 0.001 for post hoc analysis) in 3-month old mice that received intra-hippocampal injections of Fmn2 siRNA when compared to the scrambled control group (n=9/group). When siRNA injection was terminated, all mice exhibited normal extinction behavior. Error bars indicate SEM. Figure 3: Hippocampal FMN2 is essential for extinction learning and cognitive flexibility.F. Left panel: Experimental design. Right panel: Injection of Fmn2 siRNA before fear conditioning training does not affect the acquisition of fear memories. Error bars indicate SEM.
Figure 2(A) Cell growthn (lower subpanel left patient 1 and right patient 2) and morphological aspect (upper subpanel) with MCC950 determined in healthy and representative HGPS fibroblasts. **P < 0.01, *P < 0.05 patient cells no treatment vs treatment. Results are presented as the mean ± SD of three independent experiments. (B) Western blot analysis with representative blots including lamin A/C, NLRP3, IL-1β and actin levels in skin fibroblats from control and HGPS patient after 48 hr of vehicle and MCC950 treatment. (C-E) Representative fluorescence images of HGPS and control fibroblasts to evaluate the effect of the MCC950 in the nuclear morphology (D) and NLRP3 expression (E). Scale bar: 30µm. Results are presented as the mean ± SD of three independent experiments. ***P < 0.01 patient vs control cells, aaaP < 0.01 patient cells no treatment vs treatment. One-way ANOVA test was used for statistical analysis. (F) Kaplan-Meier graph showing a significant increase in the maximum lifespan in WT mice compared with Zmpste24-/- mice. N=7 per group. (G) Body weights evolution of the groups over time. Data are showed means ± SD, n = 6 mice per group. (H) Analysis of serum concentrations of IL-1β measured by ELISA. N=6 per group. Data are shown as means ± SD. ***P < 0.001, wild-type vs Zmpste24-/- mice; aaaP < 0.001; vehicle vs MCC950.
Increase in BP proliferation in 11B mouse neocortex at E14.Figure 1A Representative immunofluorescence for PH3 (green), combined with DAPI staining (white), of E14.5 wildtype (WT) and 11B mouse dorsolateral neocortex rostrally. B Representative immunofluorescence for pVim (yellow), combined with DAPI staining (white), of E14.5 WT and 11B mouse dorsolateral neocortex rostrally. Boxes (50 µm x 65 µm) in (B) indicate areas that are shown at higher magnification in (B'). Arrow, pVim+ BP with basal process (mitotic bRG); notched arrowheads, basal process; arrowhead, pVim+ BP without basal process (mitotic bIP). C-E Quantification of PH3+ APs (C), PH3+ BPs (D) and pVim+ bRG (E left) and bIPs (E right) in a 200 µm-wide field of E14.5 WT (white) and 11B (black) mouse dorsolateral neocortex rostrally. Note the difference in ordinate scales in (E). F Data information: Images are single optical sections Scale bars, 50 µm 10 µm Data are the mean of 6-19 (WT) and 6-26 (11B) littermate embryos, which were derived from 3-9 separate litters. Error bars indicate SD, two-tailed unpaired Student's t-test, * P < 0.05; ** P < 0.01 P = 0.0269 (D); P = 0.0302 (E, left); P = 0.0139 (E, right) F Representative immunofluorescence for Pax6 (cyan) and Tbr2 (magenta), combined with DAPI staining (white), of E14.5 WT and 11B mouse dorsolateral neocortex rostrally. Boxes (25 µm x 25 µm) in (F) indicate areas that are shown at higher magnification in (F'). Outlined by dashed white lines, Pax6+ & Tbr2+ BP; outlined by solid white lines, Pax6+ & Tbr2- BP. G-K Quantification of Pax6+ & Tbr2- cells in VZ (G), Pax6+ & Tbr2+ cells in VZ (H), Pax6+ & Tbr2- cells in SVZ and IZ (I), Pax6+ & Tbr2+ cells in SVZ and IZ (J) and Pax6- & Tbr2+ cells in SVZ and IZ (K) in a 200 µm-wide field of E14.5 WT (white) and 11B (black) mouse dorsolateral neocortex rostrally. L Data information: Images are Z projections of 4 optical sections Scale bars, 50 µm 10 µm Data are the mean of 6-19 (WT) and 6-26 (11B) littermate embryos, which were derived from 3-9 separate litters. Error bars indicate SD, two-tailed unpaired Student's t-test, * P < 0.05; ** P < 0.01 P = 0.0281 (I); P = 0.0088 (J); P = 0.0014 (K) L-N, Sequential BrdU-EdU labelling. BrdU was injected into pregnant mice at E13.5, i.e. 24 hr before sacrifice at E14.5, and EdU was injected into the same pregnant mice 0.5 hr before sacrifice at E14.5. (L) Representative immunofluorescence for BrdU (magenta), combined with EdU staining (cyan) and DAPI staining (white), of E14.5 WT and 11B mouse dorsolateral neocortex rostrally. (M, N) Quantification of BrdU+ & EdU+ cells (M) and the percentage of BrdU+ cells that are BrdU+ & EdU+ (N) in VZ and SVZ in a 200 µm-wide field of E14.5 WT (white) and 11B (black) mouse dorsolateral neocortex rostrally. Data information: Data are the mean of 6-19 (WT) and 6-26 (11B) littermate embryos, which were derived from 3-9 separate litters. Error bars indicate SD, two-tailed unpaired Student's t-test, * P < 0.05; ** P < 0.01 P = 0.0049 (M, SVZ); P = 0.0323 (N, SVZ).
Figure 3(A) Percent of original wound area over 7 days after wounding (n=3 pairs; *p<0.05; **p<0.005) (B) Length of the neo-epidermis (outer contour of the migrating front up to the wound margin) at PWD 3 (n=3 pairs). (C) Morphology of the leading edges in control (Ovol2f/+;K14-Cre;ROSA26R) and Ovol2 SSKO (Ovol2f/-;K14-Cre;ROSA26R) wounds. Yellow arrow points to disseminating cells (D, E) Proliferation analysis by Ki67 immunostaining. Percent Ki67+ cells (E) was calculated as the number of Ki67+ cells within the neo-epidermis over the total number of DAPI-stained nuclei (n=3 pairs) (F) Representative wound explants stained with crystal violet. (G) Quantification of the outgrowth distance of multiple explants from (F) (n=3 pairs) (H) Movement tracks of individual cells in control and Ovol2 SSKO explants over 18 hours of live imaging (I-L) Quantitative analysis of total distance traveled (I), directionality (J), full step length, and straightness distribution of multiple cells in control and SSKO explants (n=3 pairs)
stabilizes telomeric R-loops to counteract accelerated senescence.Figure 4A) Serial dilutions of indicated strains were assayed on YPD media. Cells were plated after 30-50 generations propagated on YPD agar media. Two consecutive passages from one representative biological replicate are shown. Plates were imaged after 72h growth at 30°C. B) Serial dilutions of indicated strains were assayed on YPD media. Cells were plated after 30-50 generations propagated on YPD agar media. Two consecutive passages from one representative biological replicate are shown. Plates were imaged after 48h growth at 30°C. C) Serial dilutions of indicated strains were assayed on SC-His media. Cells were plated after 30-50 generations propagated on SC-His agar media. Two consecutive passages from one representative biological replicate are shown. Plates were imaged after 72h growth at 30°C. D) Proposed model for Npl3 function at telomeres. At normal length telomeres, Npl3 associates transiently to telomeres, likely through interaction with the TERRA nascent transcript. As TERRA and R-loops are removed in late S phase, Npl3 is also lost. When telomeres shorten, increased levels of telomeric R-loops and TERRA are a result of decreased Rat1 and RNase H2 localization to telomeres. This has the effect of allowing Npl3 to remain associated with telomeres. The continued presence of Npl3 further promotes R-loop stability, potentially by preventing access of RNA-DNA hybrid removal factors. The continued presence of stable R-loops at telomeres promotes HDR at the short telomeres, likely upon interaction with the oncoming replisome, which in turn prevents the early onset of replicative senescence.
Figure 3A. I.29µ+ B lymphoma cells were treated with LPS to induce differentiation towards IgM secretion. At the indicated times, cells were lysed in TritonX114 to separate soluble and integral membrane proteins (aqueous and detergent phases, respectively). Aliquots from the two phases were resolved electrophoretically and the blot was decorated by anti-µ antibodies. The blue arrow points at a band likely corresponding to µs2L2C575OX, the intensity of which increases upon LPS stimulation. B. After sucrose gradient fractionation, µs2L2 extracted from the aqueous phases of I.29µ+ cells were sequentially alkylated with NEM and AMS as described in the scheme. C. µ-chains with oxidized C575 bind AMS after reduction and migrate more slowly than those which were reduced at the time of lysis and hence alkylated by NEM. D. The bar graphs show the levels of C575 and C414 oxidation, expressed as the ratio between IAA and NEM modified peptides.