EMBO/sd-character-level-ner huggingface dataset:
Figure 1(A) Representative western blots of Nedd4-2 and GAPDH from Nedd4-2 WT and Nedd4-2 cKO forebrains at postnatal day 14. (B) Patch-clamp recording from Nedd4-2 WT or Nedd4-2 cKO cultured hippocampal neurons at DIV 14-17. Representative mEPSC traces (left) and quantification of mEPSC amplitude and frequency are shown (right) (n = 17 and 18 for Nedd4-2 WT and Nedd4-2 cKO neurons, respectively). (C) Immunocytochemistry showing post-synaptic marker PSD-95 (red), pre-synaptic marker Synapsin-I (green), dendritic marker Map2 (blue) and co-localization of PSD-95 and Synapsin-I from dissociated Nedd4-2 WT or Nedd4-2 cKO hippocampal neurons at DIV 16. Representative secondary dendrites (left) and quantification of colocalized synaptic puncta number and area (right) (n = 36 for Nedd4-2 WT and Nedd4-2 cKO neurons) are shown. Scale bar: 5 µm. (D) Synaptic input-output relationship was obtained by initial slope of fEPSPs against the stimulus intensity at Schaffer collateral synapses of hippocampal slices from Nedd4-2 WT (n = 14 slices from 11 mice) and Nedd4-2 cKO (n = 17 slices from 12 mice) mice at 6-8 weeks of age. (E) Paired-pulse ratio of fEPSPs at Schaffer collateral synapses over an interpulse interval range of 25-400 ms from Nedd4-2 WT (n = 13 slices from 9 mice) and Nedd4-2 cKO (n = 12 slices from 8 mice) slices. (F) A train of high frequency stimulation (HFS; 100 Hz for 1s) induced early-phase LTP (E-LTP) at Schaffer collateral synapses in Nedd4-2 WT (n = 7 slices from 6 mice) and Nedd4-2 cKO (n = 6 slices from 5 mice) mice. Representative fEPSP traces were recorded before and 60 min after LTP induction (Scale bars: 0.5 mV, 5 ms). Summary bar graphs showing the fEPSP slopes measured 50-60 min after a HFS at Schaffer collateral synapses was on the right. (G) Late-phase LTP (L-LTP) induced by four trains of HFS in Nedd4-2 WT slices (n = 7 slices from 7 mice) and Nedd4-2 cKO slices (n = 9 slices from 8 mice). Representative fEPSP traces were recorded before and 180 min after LTP induction (Scale bars: 0.5 mV, 5 ms). Summary bar graphs showing the fEPSP slopes measured 160-180 min after L-LTP induction at Schaffer collateral synapses was on the right.
a,b) Quantitative analysis of TMRM fluorescence changes over mitochondrial regions in MEFs of the indicated genotype.figf5(a,b) Quantitative analysis of TMRM fluorescence changes over mitochondrial regions in MEFs of the indicated genotype. Where indicated, cells were starved for 5 h before TMRM loading. Where indicated (arrows), 2.5 μg ml−1 oligomycin and 2 μM carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP) were added. Data represent mean ± s.e.m. of seven independent experiments. (c) Total cellular ATP levels were measured in cells of the indicated genotype starved for the indicated times. Data represent mean ± s.e.m. of five independent experiments. (d) Mitochondrial ATP measured in situ by mitochondrially targeted luciferase in cells of the indicated genotype starved for the indicated times. Data represent mean ± s.e.m. of five independent experiments and are normalized to the initial value. (e,f) Cells of the indicated genotype were starved for the indicated times. Viability was determined by flow cytometry as the percentage of annexin-V- and propidium iodide (PI)-negative events. Data represent mean ± s.e.m. of five independent experiments. (g) MEFs of the indicated genotype were starved for 2.5 h. Where indicated, cells were treated with 20 μM H89. Viability was determined as in e,f. Data represent mean ± s.e.m. of five independent experiments. (h) Cells of the indicated genotype were starved for the indicated times. Viability was determined as in e,f. Data represent mean ± s.e.m. of five independent experiments. (i) Drp1−/− MEFs were transfected with the indicated plasmids and after 24 h starved for 5 h where indicated. Cell death was determined by flow cytometry as the percentage of YFP- and propidium-iodide-positive events. Data represent mean ± s.e.m. of four independent experiments. (j,k) MEFs of the indicated genotype were starved for 5 h in the presence of 2.5 μg ml−1 oligomycin where indicated. Viability was determined as in e,f. Data represent mean ± s.e.m. of five independent experiments.
Figure 1A) Scheme depicting the topology of the CDP-DAG -and Kennedy (red) pathways producing PE and PC in wild-type (WT) yeast cells (left panel), and an example of a rewired condition in which PE synthesis is directed to the mitochondrial inner membrane (MIM) and PC synthesis to peroxisomes (pex) (right panel). Black arrows indicate PS (dashed) and PE (dotted) lipid transport that must occur to enable the sequential enzymatic reactions. B) Localization of the GFP-tagged PE synthesizing enzyme (Psd) targeted to the indicated organelles in choppΔ cells, grown in SD medium supplemented with 10 mM choline. Images shown are either a single Z-slice (ER construct) or a maximum intensity projection of several Z-sections (other constructs); LD, lipid droplets; MM, mitochondrial matrix. A scheme depicting each chimeric Psd construct is shown under the corresponding microscopy image. C) Localization of the GFP-tagged PC synthesizing enzyme (Pmt) targeted to the indicated organelles in choppΔ cells, grown in SD medium supplemented with 10 mM ethanolamine. Images shown are either a single Z-slice (ER construct) or a maximum intensity projection of several Z-sections (other constructs). A schematic depicting each chimeric Pmt construct is shown under the corresponding microscopy image.
Autophagic clearance of α-synuclein inclusions.Figure 4(A) Autophagy flux was analyzed by LC3 protein monitoring. α-Synuclein fibrils-introduced HEK293 cells were treated with (+)/without (−) bafilomycin A1 (BafA1) for 2 h. Cell lysates were subjected to immunoblotting analysis using anti-LC3 (upper panel) or anti-actin (lower panel) antibodies. Quantification of the relative levels of LC3-II/Actin or autophagic flux (Baf+/Baf-) is shown with the ratio. Relative level of LC3-II/Actin was represented by mean ± s.d. as a graph. Statistical analysis was performed with one-way ANOVA (post-hoc Tukey's test). *p<0.05. This experiment was repeated three times. (B) α-Synuclein fibrils were introduced into HEK293 cells, followed by addition of DMSO, 100 nM bafilomycin (BafA1), or 200 nM rapamycin (Rapa). Cells were stained by anti-phosphorylated α-synuclein antibody (green) and DAPI (blue) 1 h (left panels) or 24 h (right panels) after introduction. (C) The number of phosphorylated α-synuclein-positive cells per unit area (0.6 mm2) was assessed in randomly chosen fields (n = 6). Data represent mean ± s.e.m. Statistical analysis was performed with one-way ANOVA (post-hoc Tukey's test). *p<0.05, **p<0.001. (D) Colocalization of α-synuclein inclusions and lysosomes. α-Synuclein inclusions (P-αSyn) and lysosomes (LAMP1) were immunostained with anti-phospho-α-synuclein polyclonal antibody (green) and anti-LAMP1 antibody (red) 1 h (left panel) or 4 h (middle panel) after fibril introduction. Fibril-unintroduced cells were used as mock control (right panel). Arrowheads and arrows indicate localized or unlocalized α-synuclein inclusions to lysosomes, respectively. Blue, DAPI. Scale bars, 10 µm. (E) Colocalization of α-synuclein inclusions (n = 41-73) to lysosomes were measured in randomly chosen fields. The experiment was repeated three times. Data represent mean ± s.e.m. Statistical analysis was performed with one-way ANOVA (post-hoc Tukey's test). *p<0.01.
– Autophagy flux is unaffected by sensors.Figure 4A-C. Analysis of autophagy flux in stable inducible HeLa cell lines expressing mCherry-fusion proteins of indicated ATG8 sensor upon doxycycline (DOX) induction: Autophagy flux is monitored in basal conditions (A) indirectly via lipidation of LC3B and turnover of the autophagy receptor p62. A-C. Analysis of autophagy flux in stable inducible HeLa cell lines expressing mCherry-fusion proteins of indicated ATG8 sensor upon doxycycline (DOX) induction: Autophagy flux is monitored upon starvation (EBSS) (B) indirectly via lipidation of LC3B and turnover of the autophagy receptor p62. A-C. Analysis of autophagy flux in stable inducible HeLa cell lines expressing mCherry-fusion proteins of indicated ATG8 sensor upon doxycycline (DOX) induction: Autophagy flux is monitored after autophagy induction by Torin I (C) indirectly via lipidation of LC3B and turnover of the autophagy receptor p62. D. FACS based mitophagy flux analysis performed in HeLa cell line stably expressing HA-Parkin, mt-mKeima, and a GFP fusion of the indicated sensor. Cells were treated with OAQ (oligomycin, antimycin, Q-VD) for indicated times and analyzed by FACS for lysosomal-positive mt-mKeima (561nm). The average of three independent experiments is presented. Representative FACS data from experiments (GFP control and AS3_67) are shown in the lower panels. Error bars indicate standard deviation. Significance calculation: 2-way anova of one-way anova. n.s.= not significant. *** indicates p-value of 0.0001.
Figure 2Time lapse images of T6SS activity in tagged strain (VipA-mCherry2 TssAVC-mNeonGreen). White arrow indicates path of TssAVC localization during one cycle of T6SS dynamics. Scale bar: 1 µm. Kymographs of dynamics observed for TssAVC. Frequency of specific dynamics is indicated in the upper right corner of the merge image. (B) Sheath and TssAVC dynamics from A. TssAVC localizes to T6SS initiation site and to distal end of growing sheath (white arrow). (C) Once sheath reaches cell periphery, TssAVC dissociates from distal end (white arrow) and sheath stays extended a prolonged period of time. (D) TssAVC dissociates from growing sheath structure and re-associates at a later time point (white arrows) to continue catalysis of polymerization. (E) TssAVC stays attached to sheath distal end after contraction (white arrow). Fluorescence microscopy of T6SS dynamics in a double tagged strain (∆retS TssB2-mCherry2 TssA2PA-mNeonGreen). White arrow indicates full cycle of extending and contracting T6SS sheath. Scale bar: 1 µm. Kymographs of TssB2 and TssA2PA dynamics. TssA2PA localizes at sheath initiation site and on distal end during polymerization (G), dissociates before contraction (H), dissociates and re-associated during sheath extension (I) or stays attached to distal end after contraction (J).
a) We achieved specific siRNA knockdown of overexpressed, Flag-tagged ATG16L1 in HEK293 cells within 48 h of transfection with oligonucleotide duplexes.figf3(a) We achieved specific siRNA knockdown of overexpressed, Flag-tagged ATG16L1 in HEK293 cells within 48 h of transfection with oligonucleotide duplexes. Flag-ATG16L1 was undetectable by protein blotting after treatment with specific siRNA constructs, but expression was maintained with control duplexes. (b) Endogenous ATG16L1 mRNA knockdown (mean relative abundance ± s.d.) by siRNA 2 in HeLa cells within 48 h of transfection, as assessed by real-time quantitative RT-PCR normalized to GAPDH. Compared with control duplex, siRNA 2 yielded an 89% reduction in transcripts. RT-PCR was performed in triplicate; results represent two independent experiments. c) Knockdown of ATG16L1 prevented effective autophagy of S. typhimurium in HeLa cells. Forty-eight hours after cotransfection with control siRNA or duplex 2 and LC3-GFP plasmid, HeLa cells were infected for 1 h with Salmonella typhimurium SL1344, fixed and examined microscopically. Mean percentages (± s.e.m.) of bacteria per cell encapsulated by LC3+ membranes (autophagosomes) are shown. Bacterial counts were pooled from two separate experiments; each counted a minimum of 100 infected cells. Significance was assessed by two-tailed Students t-test, assuming unequal variances. (d) Representative images of intracellular S. typhimurium show normal autophagic encapsulation by LC3+ membranes in control siRNA-treated cells but loss of autophagic targeting after ATG16L1 knockdown. Channels are separated to show both the LC3-GFP fusion protein (autophagosomal marker) and S. typhimurium SL1344 dsRed2 (SL1344), with a merged image in the final column. Rows 2 and 4 show magnified views of boxed areas in rows 1 and 3, respectively. Images were obtained using confocal microscopy and are a flat projection of z stacks taken throughout the plane of the infected cell. Scale bars = 5 μm.
Fig.Figure 3The ABA-dependent regulation of the PP2Cs ABI1 (A,D,G), ABI2 (B,E,H), and HAB1 (C,F,I) by RCAR12 and RCAR13 were analyzed in yeast (A-C), Arabidopsis protoplasts (D-F), and in vitro (G-I).
Figure 3Representative plots of γ-H2AX intensity vs DAPI signal of CCNF K/O cells transfected with empty GFP and GFP-CCNF untreated (top panels) and after treatment with 1μM Chk1i (LY2603618) for 20h (bottom panels). Normalised γ-H2AX intensity in CCNF K/O cells transfected with either GFP empty vector or GFP-Cyc F and treated with 1μM Chk1i for 20 hours. GFP positive cells were gated. Data from 3 independent experiments were plotted with Mean % ± SD. Representative images of neutral and alkaline comet assay from HeLa and CCNF K/O cells treated with Chk1i. Cells were treated with 1μM Chk1i for 24h before being harvested for neutral and alkaline comet assays as indicated. Quantification of comets in alkaline gels. At least 100 cells, across two slides, were analysed in each condition in two biological replicates. Data are shown as medians, with 25/75% percentile range (box) and 10-90% percentile range (whiskers). Fold changes of median values are shown in bold. p-values were calculated using the Mann-Whitney test (two-tailed). Olive tail moment = (Tail.mean - Head.mean)*Tail%DNA/100. Quantification of comets in neutral gels. At least 100 cells, across two slides, were analysed in each condition in two biological replicates. Data are shown as medians, with 25/75% percentile range (box) and 10-90% percentile range (whiskers). Fold changes of median values are shown in bold. p-values were calculated using the Mann-Whitney test (two-tailed). Olive tail moment = (Tail.mean - Head.mean)*Tail%DNA/100.
Figure 4A pcDNA-FLAG-TRADD and pcDNA-FLAG-TRAF3IP3 were transfected into HEK293T cells at increasing amount respectively. ISG54 and IL6 induction were measured thirty-six hours post-transfection by qPCR. Expression levels were shown in Fig EV4B. All data are presented as the mean values based on three independent experiments, and error bars indicate s.d. P values were determined by unpaired two-tailed Student's t-test. *P<0.05, **P<0.01 and ***P<0.001 NS indicates no statistically significant difference. B HEK293T cells were transfected with pcDNA3-HA-TRAF3IP3, in combination with pcDNA3-FLAG- RIG-I, MAVS, TBK1, IRF3, TRAF2, TRAF3, TRAF5, or TRAF6 as indicated. Thirty-six hours after transfection, cells were collected and anti-HA immunoprecipitations were performed. IP products were subjected to immunoblotting. C shRNA targeting TRAF3IP3 (sh-TRAF3IP3) was transduced into Mavs-/- HEK293T cells. Twenty-four hours after transduction, cells were treated with puromycin (2 μg/mL) for forty-eight hours and then transfected with pcDNA3-FLAG-MAVS-(Region III only). Twenty-four hours after transfection, cells were infected with or without VSV for twelve hours. Cells were then collected and subjected to immunoprecipitation assay and immunoblotting. D Wild type and Traf3ip3-/- HEK293T cells were transfected with pcDNA3-FLAG-MAVS-(Region III only). Twenty-four hours post-transfection, cells were infected with or without VSV. Twelve hours post-infection, cells were collected and subjected to immunoprecipitation assay and immunoblotting.
Figure 7Kaplan-Meier curves for the overall survival of breast cancer patients based on BRCC3 (A), XIST (B), and BRCC3/XIST(C) expression levels. HR: hazard ratio. Schematic model for how X dosage reduces HR efficiency. Numerous genes encoded on autosomes and the X chromosome and involved in HR may create a complex and balanced network for proper HR. In undifferentiated female ESCs or in some breast cancers, two active X chromosomes lead to excess doses of X-linked gene expression, including for BRCC3, which causes an unbalanced network and reduces the HR efficiency. XCI, induced by Xist RNA, restores the network and proper HR efficiency in a reversible way.
is important for GSC growth, neuro-like sphere forming frequency, and tumorigenicity qRT-PCR analysis of LINC00115 knockdown using two different shRNAs, shL115-1 and shL115-2 in 1123 and 528 GSCs.Figure 2qRT-PCR analysis of LINC00115 knockdown using two different shRNAs, shL115-1 and shL115-2 in 1123 and 528 GSCs. Data information: data are representative of three independent experiments. Error bars, ± SD. *, p < 0.05, **, p < 0.01, by One-way ANOVA Effects of LINC00115 knockdown on GSC proliferation (B) data are representative of three independent experiments. Error bars, ± SD. *, by two-tailed t-test in B. C. Effects of LINC00115 knockdown on neuro-like sphere formation (C). Data information: data are representative of three independent experiments. Error bars, ± SD. *, p < 0.05, **, p < 0.01, by One-way ANOVA in D. Representative bioluminescence images of brains with indicated 528 GSC tumor xenografts expressing shC, shL115-1, or shL115-2 at 45 days after implantation. Images represent results of 5 mice per group of 2 independent experiments. E. Quantification of the bioluminescence activity in D. F. Representative hematoxylin and eosin (H&E)-stained images of mouse brain sections with GSC1123 xenografts expressing shC, shL115-1, or shL115-2. Images represent results of five mice per group of two independent experiments. Scale bars: 1 mm. G. Quantification of tumor volume in F. H. Immunofluorescence (IF) analysis of Nestin expression in 1123 GSC control and LINC00115 knockdown xenograft tumors from F. Images are representative of two independent experiments. Scale bars: 50 μm. I. Kaplan-Meier analysis of animal implantation with 1123/shC, 1123/shL115-1 or 1123/shL115-2 GSCs. n = 10. Data information: **, p < 0.01, by log-rank test.
Figure 2(A) Cells in early G1 stably expressing nAC-mCherry and doxycycline-inducible ACTN4-SNAP were analyzed by time-lapse microscopy. ACTN4-SNAP was labeled by SNAP-Cell 647SiR dye (green). Different arrows mark dynamic ACTN4 clusters. The white square 1 is shown as a timeseries on the right. The white square 2 is shown as a magnification below. The white square 3 shows an actin filament decorated with ACTN4 that was analyzed by linescan in Fig 2B. Scale bar overview (nAC) represents 10 µm. Scale bar time series and magnification 5 µm. Cells in early G1 stably expressing nAC-mCherry and doxycycline-inducible ACTN4-SNAP were analyzed by time-lapse microscopy. ACTN4-SNAP was labeled by SNAP-Cell 647SiR dye (green). The white square 3 shows an actin filament decorated with ACTN4 that was analyzed by linescan in Fig 2B. B) Linescan of an actin filament with associated ACTN4 from square 3 in A. (C) Automated tracking (autoregressive motion) of nuclear actin and ACTN4 in nuclei by Imaris software. Cells were treated like in A. Tracks are visualized by red (actin) or green (ACTN4) lines in a representative image (see corresponding Movie EV2). Scale bar is 2 µm. The track length, track displacement (distance between starting- and end point), average speed and maximum speed were quantified from 3 independent experiments. The black line shows the median and grey dashed lines show the quartiles. Quantifications and images show similar motion characteristics for actin and ACTN4.
Figure 4Bone marrow-derived myeloid cells were treated with BSA-control (Con) or oleate in the presence or absence of 10 nM rapamycin (Rapa). The polarization of myeloid cells was detected via flow cytometry as indicated by CD11b and CD206 expression. Purified Gr1-CD11b+ myeloid cells were co-cultured with CD4+ T cells for proliferation assay. The supernatant was collected for nitric oxide (NO) detection. The oxygen consumption rate (OCR) of 1×105 differentiated myeloid cells were monitored after the addition of oligomycin (OA) (1 µM), the uncoupler carbonyl cyanide-4-(trifluoromethoxy) phenylhydrazone (FCCP, 1 µM), and the electron transport inhibitor rotenone and antimycin A (R/AA, 5 µM) at indicated time points. The basal OCR, basal extracellular acidification rate (ECAR), spare respiratory capacity, ATP production and maximal respiration based on the OCR value were quantified. Mean fluorescence intensity (MFI) of intracellular phosphor-mTOR (pS2448) in polarized myeloid cells as determined by flow cytometry Mean fluorescence intensity (MFI) of intracellular phosphor-mTOR (pS2448) in polarized myeloid cells lysed for western blot.
Figure 6Percentages of axons that initiated regeneration 24 h after laser surgery are shown. The numbers (n) of animals examined are shown. Error bars indicate 95% CI. Data information statistical significance was determined by Fisher's exact test; *P < 0.05; **P < 0.01; ***P < 0.001; NS; not significant. Percentages of axons that initiated regeneration 24 h after laser surgery are shown. The numbers (n) of animals examined are shown. Error bars indicate 95% CI. Data information: statistical significance was determined by Fisher's exact test; *P < 0.05; **P < 0.01; ***P < 0.001; NS; not significant. Percentages of axons that initiated regeneration 24 h after laser surgery are shown. The numbers (n) of animals examined are shown. Error bars indicate 95% CI. Data information: statistical significance was determined by Fisher's exact test; *P < 0.05; **P < 0.01; ***P < 0.001; NS; not significant.
Figure 6A-H Live confocal images of ddaC neurons at WP stage and 16 h APF. Treatment with colchicine significantly suppressed the pruning defects of efa6 RNAi neurons (A, B), while taxol treatment enhanced the pruning defects (C, D). Removal of one copy of γTub23C (F) or expression of γTub23C RNAi (H) almost fully rescued the dendrite pruning defects in efa6 mutant (E) or RNAi neurons (G), respectively. Red arrowheads point to the ddaC somas. I-K Quantification of unpruned dendrite lengths at 16 h APF.
S rRNA.Figure 4(A) The secondary structure of the HCVIRES domain III (hIII1, hIII2, IIIe and IIId) and helix 26 (h26) from the 18S rRNA. Colors of HCVIRES Domains are the same as in Fig 3. Interaction between the HCVIRES and 18S rRNA are indicated as red lines and a tertiary IRES interaction is indicated as black line.(B) The interaction core between IIId, IIIe, hIII1 of HCVIRES (colored as in (A)) and Expansion segment 7 (ES7) of h26 (dark gray).(C) The rearrangement of h26 (red) upon binding of HCVIRES. The 80S*HCVIRES complex (yellow) is compared to the elongating POST state (gray; PDB 5Aj0) (Behrmann et al, 2015). The loop closing the helix is highlighted in red and dark gray. (D) Primary sequence comparison of helix 26. ES7 is boxed in green. The three apical cytosine bases in the loop region of human and rabbit h26 are boxed in red. (E) Close up on the interactions between GGG266-268 of IIId (cyan) and CCC1116-1118 of ES7 (gray).(F) Close up on the interactions between G295, A296 and U297 of IIIe (green), A136 and A288 of hIII1 (yellow) and U1114, U1115 of ES7 (gray).