EMBO/sd-character-level-ner huggingface dataset:
Figure 4(A) Selected stills from live cell imaging of neuroblasts expressing UASPoloWT, UASPoloT182D, or depleted of dynein heavy chain (UASDhcRNAi) and expressing Rod-GFP under control of its endogenous promoter. CID-RFP was used as a KT reference. Insets display magnifications of the outlined regions, which highlight late congressing KTs. Asterisks indicate direction of chromosome segregation (putative spindle pole positions). (B) Graph represents the mean fluorescence intensity (MFI) for Rod-GFP determined relative to CID-RFP MFI for the KT pairs highlighted in (A). Time was measured from the first frame a KT pair moved away from the other congressing KTs, until metaphase alignment (vertical dashed lines). Data information: Statistical analysis was calculated using an unpaired t-test (Mann-Whitney). p values: **, <0.01; ****, <0.0001. Data are shown as mean ± SD. Scale bar: 5 μm. (C) Graph represents the levels of KT Rod-GFP determined relative to CID-RFP measured throughout mitotic progression for control neuroblasts (UASPoloWT, n= 24 neuroblasts, n=4 independent experiments). Data information: Statistical analysis was calculated using an unpaired t-test (Mann-Whitney). p values: **, <0.01; ****, <0.0001. Data are shown as mean ± SD. Scale bar: 5 μm. (D) Graph represents the levels of Rod-GFP determined relative to CID-RFP measured at late congressing KTs (n≥15 neuroblasts for each condition, n≥3 independent experiments). Data information: Statistical analysis was calculated using an unpaired t-test (Mann-Whitney). p values: **, <0.01; ****, <0.0001. Data are shown as mean ± SD. Scale bar: 5 μm. (E) Selected stills from live imaging analysis of Rod-GFP streaming from KTs in neuroblasts expressing either PoloWT or PoloT182D. Asterisks indicate direction of chromosome segregation (putative spindle poles positions). Arrowheads highlight streaming of Rod-GFP from unaligned KTs. Representative kymographs are shown for Rod-GFP streaming from metaphase until anaphase onset (AO). Frames were acquired every 5sec. Data information: Scale bar: 5 μm.
Figure 3Violin plots of DATP cell markers (Cldn4, Ndrg1) and essential amino acid (EAA) transporters (Slc7a5, Slc3a2, Slc6a9) in control and Lats1/2 dKO lung cells at day 5 post tamoxifen treatment. Flow cytometry sorting scheme for isolation of secretory cells (EpCAM+KDR+) (B) and experimental designs for organoid cultures (C). Secretory cells were isolated from unlabeled Lats1/2 dKO lungs, followed by 3D organoid co-cultures with stromal cells. 3D normal media was changed to experimental media 1 day prior to 4-hydroxytamoxifen (4-OHT) or ethanol (EtOH) treatment. Organoids were analyzed at day 8 post 4-OHT or EtOH treatment. Representative brightfield and H&E images of 3D organoids. Arrow indicates transitioned 3D organoids retaining DATP and AT1 cells. Scale bars, 1mm for brightfield images, 200μm for H&E images. Representative IF images of secretory, DATP, or AT1 cells in secretory organoids in indicated culture conditions. Tomato (for Scgb1a1 lineage, red), CC10 (E, green), CLDN4 (E, white), AGER (F, green), and DAPI (blue). Quantification of transitioned 3D organoids in 3D normal media (3D), amino acid-limited Basal MEM media (BME), or affluent repletion of EAA in BME media (BME+EAA) with 4-OHT or EtOH treatment. Representative IF images of YAP and TAZ expression of secretory organoids in indicated culture conditions. Tomato (for Scgb1a1 lineage, red), YAP (green), TAZ (white), and DAPI (blue). Scale bars, 100μm.
Figure 6Under low-sugar conditions, KIN10 is activated by GRIK1, and phosphorylates PIF4. The phosphorylation induces the degradation of PIF4 through 26S proteasome, suppressing PIF4-mediated thermoresponsive hypocotyl growth. An increase in sucrose results in a rise in T6P. T6P inhibits the GRIK1-mediated KIN10 activation, which allows an increase in PIF4 protein in response to high temperatures. The increased PIF4 induces hypocotyl elongation by activating auxin signaling.
dCas9 Activator Mouse (dCAM) based reprogramming of astrocytes A, Representative photomicrographs taken from the dorsal striatum 13 weeks after AAV-injection.Figure 2A, Representative photomicrographs taken from the dorsal striatum 13 weeks after AAV-injection. In mice injected with GFP control virus, virtually all GFP positive cells depict an astrocytic morphology, many GFP positive cells in ALNe-218 and ALN-treated animals show a neuron-like morphology. Data information: Scale bars indicate 20 µm. B, Immunohistochemical analysis showing GFP+/GFAP+ double positive cells 13 wpi. Arrows indicate double positive GFP+/GFAP+ cells, arrowheads indicate GFP+/GFAP- cells. Quantification of GFAP+/GFP+ cells shows a significant decrease upon ALNe-218 and ALN-treatment (GFP vs. ALNe-218 P=0.0064, GFP vs. ALN P=0.0002 and ALN vs. ALNe-218 P=0.0127). Multiple comparison ANOVA F(2,7)=32.06. C, Double immunostaining for GFP and the neuronal marker NeuN. Arrowheads indicate double positive GFP+/NeuN+ cell 13 wpi. Quantification demonstrate a significant increase in NeuN+/GFP+ cells upon ALNe-218 and ALN-induction (GFP vs. ALNe-218 P<0.0001, GFP vs. ALN P<0.0001 and ALN vs. ALNe-218 P=0.0012). Multiple comparison ANOVA F(2,6)=170.3. Data information: Scale bars indicate 20 µm. Tukey's multiple comparisons test * P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. n = 3-4 mice per condition. Error bars represent mean ± SD. D, Double immunostaining for GFP and TH as a marker for dopaminergic neurons in the dorsal striatum of GFP-control, ALNe-218 and ALN-treated animals. Arrowheads indicate GFP+/TH- cells with neuronal morphology. No GFP+/TH+ cell could be detected in any of the experimental groups. Scale bars indicate 20 µm. Data information: Scale bars indicate 20 µm.
Figure 2(A) Hybridization Chain Reaction (HCR) detection of CGG repeat RNA along with immunocytochemistry (ICC) to detect SRSF1-FLAG in U2OS cells. Expanded view of merged channels shows the CGG RNA foci (arrowhead) and an example of SRSF1-CGG RNA foci co-localization (arrow). DAPI marks the nucleus. Scale bars are 10 µm. (B) Co-localization analysis of CGG RNA (red traces) and SRSF1 (green traces) showing overlaps of CGG RNA and SRSF1. (a.u. = arbitrary unit) (C) Co-immunoprecipitation of indicated SRSF proteins with PP7-tagged control sequence (partial nLUC sequence) to correct for non-specific binding, AUG and CGG reporter RNAs. SRSF1 and 2 specifically immunoprecipitated with PP7-tagged CGG reporter.
Figure 2Representative immunofluorescence of Mecp2 null cortical neurons infected at DIV0 with lentiviruses expressing GFP or MeCP2iresGFP. Neurons were fixed at DIV7 and immunostained with anti-AC3 antibody to detect the primary cilium. GFP-positive cells represent infected cells. Scale bar 20 µm, and 10 µm in the enlarged image. Western blot comparing MeCP2 levels in Mecp2 null neurons infected with lentiviruses expressing GFP or MeCP2iresGFP with respect to WT neurons. Data are normalized to total protein content, visualized by a TGX stain-free technology. MECP2 expression in null neurons induced a significant recovery of ciliogenesis. (C) The graph represents the percentage of ciliated cells, counting n=50 WT and n=100 null cells of two different preparations (mean ± S.E., ***p<0.001, by two-way ANOVA followed by Bonferroni post-hoc test). Statistical analysis reported a significant interaction (F (1,135) = 8.264), a significant genotype effect (F (1,135) = 37.59) and a significant infection effect (F (1,135) = 26.89). (D) The graph represents the mean ± S.E. of cilium length in WT and Mecp2 null neurons infected with GFP or MeCP2iresGFP expressing lentiviruses, counting n=28 cells of two different preparations (**p<0.01, ***p<0.001, by two-way ANOVA followed by Bonferroni post-hoc test). Two-way ANOVA reported a significant interaction (F (1,103) = 6.405), a significant genotype effect (F (1,103) = 35) and a significant infection effect (F (1.103) = 4.039).
Figure 2Cartoon depicting the mutagenesis strategy, including the genomic target locus, as well as two repair templates, either introducing a silent mutation (silent oligo) or the R749H mutation (slow oligo). Multiple repair oligo templates were tested with different composition of silent restriction sites. Total number of WT, heterozygous and homozygous embryos (E3.5 and slow (R749H) RNAPII mutation or repaired with the WT sequence (silent oligo). Repair oligos and the stage that embryos/mice were analyzed are indicated.
a) HeLa cells were immunostained for endogenous ATG9A and VPS35 and subjected to confocal microscopy.f6(a) HeLa cells were immunostained for endogenous ATG9A and VPS35 and subjected to confocal microscopy. Magnified areas are shown on the right of the pictures. (b) HeLa cells were transfected with ATG9A-GFP for 24 h, and subsequently fixed, immunostained for endogenous WASH1 and subjected to confocal microscopy. (c) HeLa cells were transfected with ATG9A-GFP as in b, but immunostained instead for endogenous FAM21. (d) HeLa cells stably expressing GFP-VPS35 WT and D620N were depleted of endogenous VPS35 using 40 nM of siRNA, and subsequently immunostained for TGN46 and endogenous ATG9A and subjected to confocal microscopy. (e) Colocalization between TGN and ATG9A is expressed in terms of the Pearson's Coefficient. n=25 cells (WT) and 33 cells (D620N). Error bars represent s.e.m. and **P=0.01 by 2-tailed Student's t-test. Scale bars in (a-d), 10 μm.
Quantification of Kcnq1-positive marginal cells and the effect of treatment on the cellular organization of the marginal cells in the SV.Figure 3Quantification of Kcnq1-positive marginal cells and the effect of treatment on the cellular organization of the marginal cells in the SVThe membranes of marginal cells are labeled (red) by phalloidin conjugated with rhodamine.A Immunolabeling results (Kcnq1 labeled in green) in WT mice.B, C Immunolabeling results (Kcnq1 labeled in green) of treated Kcnq1−/−mice, middle (B) and apical (C) turns, respectively.D The percentage of marginal cells having positive Kcnq1immunolabeling signal above a visually detectable level is shown for WT (gray bars, left), untreated Kcnq1−/− (middle), and treated Kcnq1−/−mice (black bars, right). Data are given as mean ± SD (n = 6).E, F The organization of marginal cells in the SV is outlined by labeling with phalloidin conjugated with rhodamine. Results from WT (E) and untreated Kcnq1−/−mice (F) are compared.Data information: Scale bars represent approximately 50 μm.
Fig.Figure 1Nurr1 (N) and Foxa2 (F) synergistically downregulate SGK1 expression in cultured glia. (A) Up- and downregulated genes in the microarray data for the cultured glia transduced with N+F (vs. mock-transduced control). Sgk1 is marked with an arrow in the top downregulated genes. Effect of N and F on SGK1 expression in glial cells shown in the microarray (B), RNA-seq (C), Data in (B and C) represent values of fold change (FC) or read count (RC) with color intensities. n=3. One-way ANOVA. Data are represented as mean ± SEM. Significantly different at p= 0.0493*, 0.0092**, 0.0009***. Effect of N and F on SGK1 expression in glial cells shown in the RT-PCR analyses. One-way ANOVA. Data are represented as mean ± SEM. Significantly different at p= 0.0493*, 0.0092**, 0.0009***. Downregulation of SGK1 is responsible for N+F function to inhibit NFκB-mediated pro-inflammatory cytokine expression. (E) WB analysis exhibiting N+F-induced inhibition of NFκB intracellular signaling in cultured VM-glia. The N+F-mediated downregulation of NFκB signaling is attained by inhibiting IκB phosphorylation WB analysis exhibiting N+F-induced inhibition of NFκB intracellular signaling in cultured VM-glia. The N+F-mediated downregulation of NFκB signaling is attained by inhibiting IκB phosphorylation (E) and thus blocking the release of NFκB from the NFκB-IκB inhibitory complex (F). N+F-mediated downregulation of NFκB signaling (phosphorylation of p65, p-p65, G) were alleviated by forced SGK1 expression in the VM-glial cultures. H) N+F-mediated downregulation of pro-inflammatory cytokine expression (IL-1β, TNFα, H) were alleviated by forced SGK1 expression in the VM-glial cultures. (I) SGK1 inhibition blocks glial NFκB signaling. Data are represented as mean ± SEM. n=4-6. One-way ANOVA. Significantly different at p=0.002*, 0.00004**, 0.00001#, 3.01E-10##, 3.78E-06+, 1.59E-07++ in graph I. Glial pro-inflammatory cytokine expression regulated by SGK1 inhibition Glial pro-inflammatory cytokine expression regulated by SGK1 inhibition (J-M) and overexpression (N). O, Schematic summary to show how SGK1 inhibition mediates the N+F-induced anti-inflammatory action in glia.
Structure of SARS-CoV-2 hexamer in surface representation with each subunit shown in different color.Figure 4.Structure of SARS-CoV-2 hexamer in surface representation with each subunit shown in different color. The active site residues are colored red.
Figure 5A. Rabl2Q80L localized in multicilia. Cultured mEPCs were infected with adenovirus to express GFP-Rabl2 or GFP-Rabl2Q80L at 24 h prior to and fixed at day 7 post serum starvation. Rabl2Q80L selectively induced ciliary accumulation of BBS proteins. Multicilia were purified from adenovirus-infected mEPCs harvested at day 10 and subjected to label-free quantitative (LFQ) mass spectrometric analyses The abundance (B) is relative to LFQ intensities in the GFP-Rabl2 sample. Relationships among the BBS proteins, IFT-B, and IFT-A are depicted according to literature (Eguether et al, 2014; Nakayama & Katoh, 2018). Lztfl1 has been proposed to link BBSome to IFT-B through interaction with Ift27 (Eguether et al, 2014). C. Rabl2Q80L selectively induced ciliary accumulation of BBS proteins. Multicilia were purified from adenovirus-infected mEPCs harvested at day 10 and subjected to immunoblotting Lamin B1, GM130, Cep162, and Gapdh served as quality controls for the ciliary preparations (C). The values in red are relative band intensities. 1/400 of the mEPC lysates and 1/20 of the cilia lysates were loaded per lane. D, E. Ciliary accumulation of BBS proteins in Rabl2Q80L-expressing RPE1 cells. The white arrows (D) point to positions of cilia. Quantification results (E) were from three independent experiments. At least 100 GFP-positive cells were scored in each experiment and condition. F. FLIP assays revealed that ciliary Rabl2Q80L impaired BBSome export. RPE1 cells co-expressing 3NG-Bbs5 with RFP-Rabl2Q80L or RFP-Rabl2 were serum-starved to induce ciliogenesis. Photobleaching of 3NG in cells around the ciliary base was performed at t = 0 s as illustrated. Ciliary 3NG autofluorescence in the living cells was imaged at 30-s intervals after a snapshot for RFP. Images of representative cilia and half-life (t1/2) of ten cilia from one typical set of experiments are shown. G. Ciliary SMO was accumulated above the transition zone (TZ) (yellow line) in RPE1 cells expressing GFP-Rabl2Q80L. Images were acquired through 3D-SIM. CEP290 served as TZ marker. DA, distal appendage. H, I. BBSomes and IFT-B were able to reach the ciliary base through retrograde IFT in the presence of Rabl2Q80L. Ciliary 3NG-Bbs5 (H) or Ift27-GFP (I) in IMCD3 cells co-expressing RFP-Rabl2 or RFP-Rabl2Q80L was live imaged at 4 fps. Snapshots for the ciliary RFP proteins, the first frames from Movies EV2-EV5, corresponding kymographs, and quantification results from the indicated numbers of cilia are presented. For processivity analysis (I), only traceable puncta were scored.
Figure 6A, B ISH of miniature pig canine frontal sections showing expression patterns of RUNX2 at embryonic day 60 (E60) and E90. (A', B') are magnifications of boxed regions in the corresponding figure panels. Data information: Scale bars = 50 µm. C Relative ISH expression levels of RUNX2 in epithelium (Epi.) and mesenchyme (Mes.) at E60 and E90. Data information Data represent the means ± SEM. n = 3 for all experiments. Unpaired t-tests for C , *P < .05, **P < .01, ***P < .001. D, E ISH of miniature pig canine frontal sections showing expression patterns of β-catenin at E60 and E90. (D', E') are magnifications of boxed regions in the corresponding figure panels. Data information: Scale bars = 50 µm. F Relative ISH expression levels of β-catenin in epithelium (Epi.) and mesenchyme (Mes.) at E60 and E90. Data information: Data represent the means ± SEM. n = 3 for all experiments. Unpaired t-tests , *P < .05, **P < .01, ***P < .001. G, H IF of Lef1 at E60 and E90. (G, H') are magnifications of boxed regions in the corresponding figure panels. Data information: Scale bars = 50 µm. I Relative IF expression levels of Lef1 in epithelium (Epi.) and mesenchyme (Mes.) at E60 and E90. Data represent the means ± SEM. n = 3 for all experiments. Unpaired t-tests , *P < .05, **P < .01, ***P < .001. J FISH of β-catenin in E60 and E90 mandible slices and E60 mandible slices after 12 and 24 h in culture via RNAscope. Boxed regions of mesenchyme are magnified in the right panels. Epithelium is indicated with a yellow arrow. Data information: Scale bars = 50 µm. K Relative FISH expression levels of β-catenin in the nucleus and cytoplasm of epithelium and mesenchyme at E60, E90, and E60 cultured for 12 and 24 h. Data information: Data represent the means ± SEM. n = 3 for all experiments. One-way ANOVA for K, *P < .05, **P < .01, ***P < .001. L, M IF of β-catenin at E60 and E90; The yellow arrows indicate the localization of β-catenin in the nucleus. Data information: Scale bars = 50 µm. N Relative IF expression levels of β-catenin in the nucleus of epithelium and mesenchyme at E60 and E90. Data information: Data represent the means ± SEM. n = 3 for all experiments. Unpaired t-tests , *P < .05, **P < .01, ***P < .001. O, P FISH of β-catenin in mandible slices infected with RUNX2 shRNA lentivirus (knockdown) or scrambled shRNA lentivirus (scramsh) via RNAscope. Data information: Scale bars = 50 µm. Q Relative FISH expression levels of β-catenin in the nucleus and cytoplasm of epithelium and mesenchyme in knockdown and scramsh groups. Data information: Data represent the means ± SEM. n = 3 for all experiments. Unpaired t-tests , *P < .05, **P < .01, ***P < .001.
Jumpy colocalizes with and regulates Atg9 distribution to autophagic organelles.Figure 6(A-T) C2C12 cells were transfected for 24 h with GFP, GFP‐Jumpy WT or GFP‐Jumpy C330S (Jumpy CS) and Tomato‐LC3 (LC3) and HA‐Atg9, fixed and immunostained with anti‐HA. GFP (I), Jumpy WT (M), Jumpy CS (green) (A, E, Q), LC3 (red) (C, G, J, N, R) and Atg9 (white in B, F, K, O, S or blue in D, H, I, P, T). Boxed areas (A-D) are shown at higher magnification in the corresponding panel below (E-H). White arrows indicate colocalization between Jumpy CS, LC3 and Atg9, blue arrow indicates colocalization between Jumpy CS and Atg9 but not LC3 and yellow arrow indicates colocalization between Jumpy CS and LC3 but not Atg9. White boxes (I-T) show LC3 puncta location. Scale bars, 5 μm. (U) Percentage of LC3 puncta colocalizing with Atg9. n, number of puncta counted.
Figure 6Western blot analysis of p97 levels in RAW264.7 cells treated with additional NaCl (+17, 34 and 51 mM) for 12 hrs. Data information: Data are representative of at least two biological replicates. B Western blot analysis of p97 levels in HT1080 (B) cells treated with additional NaCl (+17, 34 and 51 mM) for 12 hrs. Data information: Data are representative of at least two biological replicates. C Immunoprecipitation analysis of the interaction between USP33 and p97 in RAW264.7 treated with additional NaCl (+34 mM) for 12 hrs. Data information: Data are representative of at least two biological replicates. D Immunoprecipitation analysis of acetylation of Flag-p97 in HEK293T cells transfected with Flag-p97 and then treated with additional NaCl (+17, 34 and 51 mM) for 12 hrs. Data information: Data are representative of at least two biological replicates. E, F Mass spectrometry analysis of acetylation of Flag-p97 (E) in RAW264.7 transfected with Flag-p97 and then treated with additional NaCl (+34 mM) or control ddH2O (Ctrl) for 12 hrs. The intensity of p97-K663 site was shown in (F). G Immunoprecipitation analysis of acetylation of Myc-p97 in HEK293T cells transfected with Myc-p97 (WT, K663R or K651R) and then treated with additional NaCl (+34 mM) for 12 hrs. Data information: Data are representative of at least two biological replicates. H Immunoprecipitation analysis of the interaction between USP33 and Myc-p97 in HEK293T cells transfected with Myc-p97 (WT or K663R). Data information: Data are representative of at least two biological replicates. I Western blot analysis of USP33 levels in HEK293T cells transfected with empty vectors (-) or Myc-p97 (WT or K663R). Data information: Data are representative of at least two biological replicates. J Western blot analysis of Flag-Viperin levels in HEK293T cells co-transfected with Flag-Viperin and Myc-p97 (WT or K663R). Data information: Data are representative of at least two biological replicates. K Immunoprecipitation analysis of the interaction between USP33 and Myc-p97 in HEK293T cells transfected with Myc-p97 (WT or K663R) and then treated with additional NaCl (+34 mM) for 12 hrs. Data information: Data are representative of at least two biological replicates. L Western blot analysis of FH-USP33 levels in p97-KO HEK293T cells co-transfected with Myc-p97 (WT or K663R) and Flag-USP33, and then treated with additional NaCl (+34 mM) for 12 hrs. Data information: Data are representative of at least two biological replicates. M Immunoprecipitation analysis of Viperin ubiquitination in p97-KO 2fTGH cells co-transfected with Myc-p97 (WT or K663R), Flag-Viperin and HA-Ub as indicated, and then treated with additional NaCl (+34 mM) for 12 hrs. Data information: Data are representative of at least two biological replicates.
Figure 10Under low sterol and oxygen conditions, Sre1 exits the ER and travels to the Golgi. Golgi-localized Sre1 is ubiquitinylated by E2 Ubc4 and Dsc E3 ligase. Ubiquitinylated Sre1 is recruited by Cdc48 and cleaved by Rbd2. The intermediate cleaved product is further processed by a second, unknown protease to generate Sre1N that traffics to the nucleus to activate transcription. When Rbd2 is inactive (dashed box), ubiquitinylated Sre1 is subjected to proteasomal degradation. SRE, sterol regulatory element; U, either mono-ubiquitin or poly-ubiquitin.
a–e, Representative examples of autophagic vacuoles detected by TEM in retinal sections used to generate the quantitative data shown in (f).figf2a-e, Representative examples of autophagic vacuoles detected by TEM in retinal sections used to generate the quantitative data shown in (f). a, An autophagosome (red arrow) containing cytoplasmic contents in a photoreceptor neuron from an AR52fly reared on DHT. b, Higher magnification of the autophagosome in a. c, Multiple autophagolysosomes (red arrows) containing dense, amorphous material from an AR52fly reared on DHT. d, A juxtanuclear multilamellar body (red arrow) from a DTS7fly reared at 28 °C. e, A multivesicular body (red arrow) from a DTS7fly reared at 28 °C. f, A significant increase in the frequency of neurons with autophagic figures in DTS7 flies reared at 28 °C compared to those reared at 22 °C, and in AR52 flies reared on DHT compared to those reared off DHT. Data show mean ± s.d., n = 59-82 neurons in 5 sections per condition. No accumulation of autophagic figures was found in AR12 flies. g-l, SEM images of fly eyes expressing the indicated transgenes. RNAi knockdown (KD) of atg6 and atg12 enhances degeneration in DTS7 flies reared at 28 °C (compare h, i to g) and AR52 flies reared on DHT (compare k, l to j). 200 to >1,000 fly eyes of each genotype were examined. Quantitative analyses of eye phenotypes are presented in Supplementary Fig. S2. (N, nucleus; Rh, rhabdomere.)