EMBO/sd-character-level-ner huggingface dataset:
Figure 3A. Western blot confirming knockdown of Rab11-FIP1 by shRNAs. Relative intensity by densitometry of Rab11-FIP1 results are shown at the bottom. 3D organoids were generated from WT and mutant p53 cells expressing non-targeting control (NT) or Rab11-FIP1 shRNAs (sh1 and sh2). B. Diameter of organoids (n= 80-130 organoids). Bar graph represents mean diameter ± SEM. ****p<0.0001 WT or mutant p53 non-targeting control vs Rab11-FIP1 shRNA1, ***p=0.0006 WT non-targeting control vs Rab11-FIP1 shRNA2, ****p<0.0001 mutant p53 non-targeting control vs Rab11-FIP1 shRNA2 (One-way Anova). ****p<0.0001 WT vs mutant p53 non-targeting control (Unpaired t-test). 3D organoids were generated from WT and mutant p53 cells expressing non-targeting control (NT) or Rab11-FIP1 shRNAs (sh1 and sh2). C. Percent of WT p53 (top panel) and mutant (bottom panel) organoids exhibiting a solid, intermediate, or stratified morphology (n>50 organoids generated from three different cell passages quantified). Bar graph represents mean diameter ± SEM. n.s. WT p53 non-targeting control vs Rab11-FIP1 shRNAs, *p=0.046 mutant p53 non-targeting control vs Rab11-FIP1 shRNA1, ***p=0.0002 mutant p53 non-targeting control vs Rab11-FIP1 shRNA2 (One-way Anova). 3D organoids were generated from WT and mutant p53 cells expressing non-targeting control (NT) or Rab11-FIP1 shRNAs (sh1 and sh2). D. Percent of WT p53 and mutant organoids exhibiting a basaloid-like morphology (n>50 organoids generated from three different cell passages quantified). Bar graph represents mean diameter ± SEM. n.s. WT p53 non-targeting control vs Rab11-FIP1 shRNAs, *p=0.046 mutant p53 non-targeting control vs Rab11-FIP1 shRNA1, ***p=0.0002 mutant p53 non-targeting control vs Rab11-FIP1 shRNA2 (One-way Anova). 3D organoids were generated from WT and mutant p53 cells expressing non-targeting control (NT) or Rab11-FIP1 shRNAs (sh1 and sh2). E. WT p53 organoids stained for H&E (left), Rab11-FIP1 (middle), and the basal cell marker p63 (right) by IHC. 40X. Scale bar = 100 μm. F. Mutant p53 organoids stained for H&E (left), Rab11-FIP1 (middle), and the basal cell marker p63 (right) by IHC. 40X. Scale bar = 100 μm.
Aurora B is required for polarization of actomyosin network in the absence of a midzone(A) Maximum projection of 2 z slices of Hela cells immunostained for Aurora B (top) and actin-membrane cross-linker pERM (bottom), showing the change in localization upon Cdk1 inhibition with RO3306 (exit onset).Figure 2(A) Maximum projection of 2 z slices of Hela cells immunostained for Aurora B (top) and actin-membrane cross-linker pERM (bottom), showing the change in localization upon Cdk1 inhibition with RO3306 (exit onset). As DNA moves to one side, Aurora B re-localizes from DNA to cortical region of the side opposite the DNA. In addition to Aurora B, the membrane-actin cross-linker pERM also polarizes in the direction opposite to DNA localization during the course of mitotic exit. Scale bar-10 µm. (B) Schematic representation of cell, showing the quantification of actin and pERM along the cortex during mitotic exit. Regions 5 and 6 are cortex close to the DNA, while regions 1 and 10 are the furthest away. (C) Maximum projection of 2 z slices of Hela cells immunostained for pERM following 10 mins of forced mitotic exit in presence of DMSO and 2µM ZM447439. Cells fail to polarize their pERM along the perimeter with Aurora B inhibition, quantified in (D). Scale bar-10 µm. (D) Quantification of pERM intensity across the perimeter of the cells as shown in (B) where 5 and 6 represent regions closest to DNA, and 1 and 10 furthest. DMSO (n=28 cells) show increase in pERM levels away from the DNA, cells treated with ZM447439 (n=26 cells) during exit, fail to polarize the pERM proteins. Data presented as mean ± SD. (E) Stills of representative Hela cells expressing Lifeact-GFP and H2B-mCherry at 10 mins following forced mitotic exit with siControl, siRACGAP1, DMSO and 2µM ZM447439 treatment. Control siRNA, RACGAP1 siRNA and DMSO treated cells show clearance of actin from the cortex close to the DNA, while ZM447439 treated cells fail to clear actin, quantified in (F). Note, failure in actin accumulation in RACGAP1 depleted cell, asterisk. Scale bar - 10 µm (F) Quantification of actin intensity across the perimeter of the cells based on distance from DNA, with 5 and 6 being closest to DNA and 1 and 10 furthest away, as in cartoon (B). Graph shows decrease in actin intensity closer to the DNA in siControl (n=26), siRACGAP1 (n=37) and Control DMSO treated cells (n=38 cells), seen by levels lower than 1 (dotted red line), while this decrease is absent in ZM447439 treated cells (n=42 cells). Data presented as mean ± SD.
Photoreceptors form nanotube-like processes to connect with neighbouring photoreceptors in culture a-d, Representative 3D deconvolved MIP images from live imaging of Nrl.Figure 3a-d, Representative 3D deconvolved MIP images from live imaging of Nrl.Gfp+/+ (green) P8 photoreceptors at DIV1-3, labelled with SiR-actin (red) (N = 7 independent cultures). Photoreceptors exhibit a variety of processes, including (a) neurites (blue arrow), (b) nascent inner segment-like protrusions (yellow arrow), and (c) short and (d) long (magenta arrows) actin+ve nanotube-like (herein termed PhNTs) connections; Scale bar = 5 µm; e-g, Representative 3D deconvolved MIP images from live imaging of Nrl.Gfp+/+ (green) P8 photoreceptors at DIV1-3 labelled with SiR-tubulin (red) (N = 7 independent cultures). Photoreceptors exhibit a variety of processes, including nascent inner segment-like protrusions (yellow arrows), neurites (blue arrows) and rare tubulin+ve PhNTs (magenta arrows); Scale bar = 5 µm; h, Representative SEM images with digitally enhanced microphotographs of cultured P8 Nrl.Gfp+/+ photoreceptors. Red arrows indicate PhNT connections between neighbouring photoreceptors, while blue arrows indicate broken PhNT connections (N = 3 cultures). Scale bar = 5 µm; i & j, Representative images of depth colour coding (ImageJ) of x,y,z live imaging of Nrl.Gfp+/+ photoreceptor cultures showing (i) neurites (blue arrows) growing along the substrate (asterisks denote secondary branching of long GFP+ neurites), and in (j), a free-floating PhNT (magenta arrow). Scale bar = 10 µm; k & l, Quantification of the localization of Nrl.Gfp+/+ processes in the z-axis (depth) (N = 4 independent cultures, n = 1096 cells, nneurites = 440, nnanotubes = 31 where (k) neurites. P values = **** p < 0.0001 (bottom vs top); **p = 0.002 (bottom vs middle); **p = 0.003 (middle vs top) and (l) PhNTs; P-values = **** p < 0.0001 (bottom vs middle & middle vs top) . One way ANOVA, non-parametric, Kruskal-Wallis with Dunns' multiple comparisons test shows means vary significantly. m & n, Diameter and length quantification of SiR-actin+ve PhNTs (N = 7 cultures; ncells = 646 cells; nnanotubes = 35, red dots) compared to SiR-tubulin+ve PhNTs (N = 7 cultures; ncells = 885 cells, nnanotubes = 15; blue dots). One way ANOVA, non-parametric, Kolmogorov-Smirnov test shows non-significant (ns) difference in length but significant difference in diameter between actin+ve versus tubulin+ve PhNTs. P values; ** p = 0.001, and p = 0.245, in (m) and (n), respectively.
Figure 4A Stretch-induced conformational states of A170-M1 corresponding to main mechanical events. In brief, after 20 ns the frontal α-helix in the NL has been lost, at 30 ns the frontal fraction of the NL is completely unfolded, at 31.6 ns the C-terminal β-strand in the CRD starts to unravel, and by 36 ns the NL has unwrapped and become completely disassociated from the TK domain. The main force peak corresponds to the unfolding of the 'knot' formed by the NYDEE motif in the NL segment, which occurs just before the final complete detachment of the NL. At this point, the kinase N-lobe started to unfold and the simulations were terminated. B Force versus pulling time curves obtained by SMDS of wild-type A170-M1 (black) and A170-M1D24728V (orange). C, D The change in secondary structure (SS) composition of the NL segment during the simulation is shown for wild-type and D24728V proteins. Here, the secondary structure content of NL is shown vertically for each time point. Secondary structure is as defined in VMD (Humphrey et al, 1996) where: pink, α-helix; teal, turn; blue, 310 helix; yellow, β-strand. Histograms showing the number of hydrogen bonds established between regulatory tails (NL, yellow; CRD, red) and the TK kinase domain over the course of the simulation are also shown. Hydrogen bonds were measured using VMD (Humphrey et al, 1996) applying distance and angle cutoffs of 3Å and 20o, respectively. Values averaged over 1 ns are shown as a black line in the histogram profile. The graphs show that the α-helical composition of the NL tail is lost early in the simulation of both wild-type and D24728V models and that, under the simulation conditions, the NL detaches completely from TK but the CRD does not. The differences in the histograms of wild-type and D24728V models are due to the formation of the resistant 'knot' structure in the NL of wild-type A170-M1, which causes the CRD to react to stretch by the detachment of its β-R1 strand. In contrast, the lax NL of the D24728V model prevents stretch from impacting the CRD sequence, which remains largely unaltered throughout the simulation (number of hydrogen bonds remain approximately constant).
Specificity of the NeutrobodyPlex in determining individual neutralizing immune responses.Figure 6A For the NeutrobodyPlex on RBD, two concentrations of NM1267 (1 µM and 1 nM) were applied (n = 1). Light-colored squares (high MFI (%control)) are indicative for IgGs outcompeting NM1267 from the RBD:ACE2 interface, dark-colored squares (low MFI (%control) show a continuous displacement of IgGs from serum samples in the presence of NM1267. B For the virus neutralization assay, serial dilutions of the serum samples (1:40 - 1:5120) were applied (n = 1). Dark colored squares are indicative for a low infection level (low % Infection), light colored squares show a high infection rate (high % Infection). C The mean percent of infection (% Infection) derived from all individual serum dilutions obtained by the virus neutralization assay was calculated and plotted against the respective MFI (% control) obtained from the NeutrobodyPlex on RBD in the presence of NM1267 (1 nM). The Pearson correlation determined a negative correlation with r = -0.77 and R2 = 0.59 (p <0.05).
Appearance of tracer in AV.Figure 3Appearance of tracer in AV. Cells were allowed to endocytose tracer particles continuously for the designated periods of time. At each time, random sections were searched for AV+, and AV+/500 cell profiles were plotted. The number of cell profiles examined (denominator) and the respective number of AV+ scored (numerator) are listed above the graph.
Overview of small molecule compound library composition and hit triage.Fig. 1.(A) The small molecule compound library used in this study was mainly composed of bioactives and FDA-approved drugs, with a small proportion of natural products and kinase inhibitors. (B) High-content screening (HCS) of 5,406 compounds in two batches in duplicate and calculation of Z'-factors between high (MERS-CoV infection, black) and low (mock, green) values. (C) Correlation of duplicate screening sets. Compounds with MERS-CoV inhibition and cell viability >70% were regarded as primary hits. Scatter plot shows MERS-CoV inhibition ratios overlaid with cell viability ratios. (D) Flowchart of HCS hit selection, and confirmation of final hit selection. Compounds (cpds).
ESCRT machinery is required for completion of microautophagy of ER whorls during ER stress.Figure 6A Electron micrographs of wild-type and ∆snf7 cells treated with DTT for 3 h. Cy, cytosol; V, vacuole. Scale bars: 300 nm. B Quantification of vacuolar whorls for which the uptake process was complete or incomplete. The observed distributions in the two strains were analyzed with a Fisher's exact test. C, D Electron micrographs of serial thin sections of a ∆snf7 cell (C) or a wild-type cell (D) treated with DTT for 3 h. Note that the whorl appears separated from the cytosol in some sections (e.g. section 300 nm) but is still in contact with the cytosol (see sections 600 nm and 700 nm). In the wild-type cell (D), the vacuolar whorl is completely separated from the cytosol. Scale bar: 300 nm.
a) Endogenous TR3 is localized in the MIM in response to THPN stimulation.figf5(a) Endogenous TR3 is localized in the MIM in response to THPN stimulation. Left, isolated mitochondria from cells treated with THPN (20 μM, 6 h) were incubated with proteinase K (20 μM and 40 μM) for 30 min. TR3 expression was determined. Tom20 in MOM and Tim23 in MIM were used as controls. Right, the mitochondria were further subjected to submitochondrial fractionation. VDAC1, Tim23 and Hsp60 were used to represent MOM, MIM and mitochondrial matrix protein, respectively. '-' represents vehicle treatment only. (b) Knockdown of Tom40 and Tom70 blocks TR3 transit to MIM. Endogenous Toms were separately knocked down by their corresponding shRNAs, and the TR3 expression of mitochondrial fractions was detected as described above. Ctrl, control. (c) Tom40 and Tom70 are required for the THPN-associated dissipation of ΔΨm (left), conversion of LC3-I to LC3-II (right and top) and cell death (right and bottom). Endogenous Toms were separately knocked down by the corresponding shRNAs. ΔΨm, LC3-II and cell death were detected as described above. Images of full blots are shown in Supplementary Figure 7. (d) The mitochondria were markedly eliminated after treatment with THPN for 24 h. Right, endogenous Tom40 and Tom70 were knocked down, and the amount of mitochondria was measured by the fluorescence intensity of Tom20 (green). Left, the volume of mitochondria from five randomly picked regions was quantified by ImageJ software. All of the data are presented as the mean ± s.e.m. of three independent experiments. *P 0.05, **P 0.01, ***P 0.001; NS, not significant.
mAtg9 binds to p38IP in vitro and in vivo.Figure 1(A) The AH109 strain was co‐transformed with pGBKT7 containing the C‐terminal (CT) domain of mAtg9 (bait) and pGADT7, or pACT2, containing the CT domain of p38IP (prey). The interaction was then visualized by growth of co‐transformed yeast on SD‐Trp/Leu/His plates for 3 days at 30°C. (B) Domain organization of p38IP, the N‐ (1-487 aa) and C‐ (487-733 aa) terminal domains. p38IP has a predicted nuclear localization signal (NLS), a PEST domain, and two serine‐rich domains. The region of interaction with p38α and mAtg9 is indicated. (C) HEK293A cells were transfected with RFP-mAtg9 and HA-p38IP. HA-p38IP was immunoprecipitated with an anti‐HA antibody and the co‐immunoprecipitated RFP-mAtg9 was detected with an anti‐mAtg9 antibody. HA-p38IP was detected with an anti‐HA antibody. Asterisk indicates a non‐specific band. Indirect immunofluorescence analysis of mAtg9 with (D)HA-full‐length p38IP (HA-FL), (E)HA-C‐terminal p38IP (HA-CT), and (F) HA-N‐terminal p38IP (HA-NT). Bars=5 μm. The co‐localization seen in (D) and (E) was not because of overlap of randomly distributed vesicles. Cross‐correlation function (CCF) analysis ( van Steensel et al, 1996) showed a peak and maximum Rp value around ΔX=0, indicating that co‐localization between mAtg9 and p38IP is positively correlated and non random ( Supplementary Figure S1).
a zinc finger-2 (ZF2) plays an important role in CSR and epigenomic regulation required for DNA repair.Figure 8(A) A representative FACS profile of CSR complementation assay using WT and mutant Phf5 as indicated and describe in Fig 7B. (B) Western blot showing expression of endogenous and exogenously introduced Phf5a. (C) H2A.Z ChIP assay was performed using CH12F3-2A cells transfected with indicated siRNA and the Phf5a expression constructs. The data are compiled from 2 experiments and presented relative to siControl treated sample, which was set as 100. The error bars represent the mean ± sd. (D) The schematic illustration of Phf5 zinc fingers (ZF1-3) was adapted from van Roon et al., 2008. The Cys75 and Cys72 are indicated by thick and thin arrows, respectively. (E) Illustration depicting AID-induced DNA breaks and repair in the S region, which leads to CSR and IgH/c-Myc chromosomal translocation. The scheme also highlights the involvement of the Phf5a-p400 pathway, which promotes H2A.Z and H2AX deposition in the recombining S regions and facilitates NHEJ-mediated DNA repair.
a) Fluorescence microscopy images showing HACE1 (green) co-aggregating with.f5(a) Fluorescence microscopy images showing HACE1 (green) co-aggregating with p62 (red) in the perinuclear space on puromycin treatment (8 h) in Hace1−/− NCM transiently expressing HACE1-GFP. p62 was visualized by immunofluorescent staining. Arrows indicate co-localized puncta. (b) Representative confocal images showing HACE1 (red) localizes to LC3+ puncta (green) induced by MG132 treatment (8 h) in Hace1−/−GFP-Lc3Tg NCM cells transiently expressing HACE1-RFP. Arrows indicate co-localized puncta. (c) Primary NCMs from Hace1+/+Lc3Tg and Hace1−/−Lc3Tg newborn pups were treated with or without MG132 for 8 h and the cell lysates were subjected to western blot with anti-GFP antibodies. Gapdh was used as a loading control. (d) Fluorescence microscopy images showing co-localization of HACE1 (green) with Lamp1 (red) on MG132 treatment (8 h) in Hace1−/−NCM cells transiently expressing HACE1-GFP. Lamp1 was visualized by immunofluorescent staining. Scale bar, 10 μm in all images.
Figure 4(A) Phosphoproteome-wide correlation between the effects observed upon UPS7i and OA treatments (4 and 2 hrs, respectively) in RPE cells. The p-value was obtained by a t-test to evaluate the Pearson correlation between both samples. (B) Venn-diagrams representing the overlap between the peptides that show increased levels of phosphorylation upon USP7i and OA treatments. The p-value was obtained by a hypergeometric test. (B) Venn-diagrams representing the overlap between the peptides that show increased levels of phosphorylation upon USP7i and OA treatments. The p-value was obtained by a hypergeometric test. (C, D) Reactome analysis illustrating the biological pathways related to the factors that show increased phosphorylation upon treatment with USP7i, or that are commonly upregulated in response to USP7i and OA. (E) Representation of the phosphorylation levels at epitopes that follow the CDK consensus motif p(S/T) Px(K/R) upon USP7i or OA treatments from data obtained at phosphoproteomic analyses. (F) Western blot showing the levels of MPM-2, phosphor-CDK substrate motif [(K/H) pSP] and β-Actin in whole cell extracts of RPE cells treated with DMSO (Control), 0.5 μM Okadaic acid (OA) for 2 hrs, 10 nM Calyculin A for 2 hrs, 25 μM P22077 for 4 hrs or 25 μM BAY 11-7082 for 2 hrs. Calyculin A was added to this experiment to illustrate the gain in CDK activity induced by an additional PP2A inhibitor. Ponceau staining is shown as loading control. (G, H) Representation of the phosphorylation levels at epitopes from nucleoporins (NUPs; (G)) or Retinoblastoma (RB1; (H)) upon USP7i or OA treatments from data obtained at phosphoproteomic analyses.
Fig.Figure 5A. TEM images of longitudinal sections of lateral ventricle CP villi of P18-P21 Mpdz+/+ and Mpdz-/- mice. Scale bar, 2 μm. B. A large number of voids of varying sizes was evident in the CPECs of Mpdz-/- mice. Higher magnification images of the areas in the rectangles are shown underneath the panels. Adherens junctions are denoted by horizontal lines and arrows; voids are indicated by arrows. Scale bar, 1 μm; insets, 200 nm. C. Triplicate images of tight junctions proximal to the apical faces of CPECs in Mpdz+/+ and Mpdz-/- mice. Scale bar, 100 nm D. Mitochondria were smaller and frequently lacked large portions of their cristae. Autophagosomes are present in some of them (arrows). Scale bar, 200 nm. The images are representative of 2 Mpdz+/+ mice and 2 Mpdz-/- mice. E. Time course and standard deviations of the impedance of confluent hpCPEC monolayers that were transduced by either MPDZ or non-targeting (Ctrl) shRNA. Each record represents 4 wells. MPDZ immunoblot of each cell group is shown in the inset.
Figure 5(A-D) Confocal z-projections of brains expressing DIP-δGFSTF (DIP-δ-GFP) together with the indicated Gal4s and transgenes. Expressing diphtheria toxin (DTi) and the membrane bound RFP (mCD8-RFP; CD8) driven by the γ4>γ1γ2 MBON driver MB294B-Gal4 (MBONγ4>γ1γ2-Gal4) did not affect DIP-δ-GFP expression (A, n=16/16; B, n=14/14). In contrast, similar expression of DTi and membrane bound Tomato (CD4-tdT; CD4) in PAM-DANs (using the GMR58E02-Gal4; PAM-DAN-Gal4) abolished the normal DIP-δ-GFP expression in the γ4/5 zone (compare D, n=18/18, to C, n=16/16) and within the PAM-DAN cell bodies (compare D' to C'). Magenta is CD8-RFP (A-B), CD4-mtdT (C-D). Green is GFP, grayscale depict individual channels as labeled. Scale bar is 20 µm. Yellow dashed line demarcates the γ-lobe based on FasII staining (not shown). (E-G) Confocal z-projections of MARCM clones labeled by DIP-δT2A-Gal4 (DIP-δ Gal4) driving the expression of membrane bound GFP (mCD8-GFP; CD8) and heat shocked at 24hr after egg laying. Clones innervate the γ4/5 zones at 24hr APF (E; n=8), 48hr APF (F; n=8) and adult (G; n=16). Clones become tyrosine hydroxylase (TH) positive only at 48hr APF onwards (F,G). Magenta is FasII, green is mCD8-GFP, cyan is TH antibody staining, grayscale single channels are shown as indicated. White dashed line demarcates the γ-lobe. Scale bar is 20 µm.
TSC1 is crucial for survival of Burkitt's lymphoma (BL) cells.Figure 3Left graph shows the multiplication rate of P493-6 (-Tet) cells expressing either a scrambled control sh-RNA or a TSC1-specific sh-RNA determined by viable cell counting 3 days after seeding of equal number of viable cells (determined by Trypan blue exclusion; mean ± st.dev., n=3 biological replicates). Right graph shows percentage of apoptotic P493-6 (-Tet) cells expressing scrambled control shRNA or a TSC1-specific sh-RNA determined by FACS analysis of AnnexinV/7AAD stained cells (mean ± st.dev., n=3 biological replicates). Rapamycin treatment recovers survival of TSC1 knockdown in P493-6 cells. Relative viable cell number counts of P493-6 (-Tet) cells expressing scrambled control shRNA or TSC1-specific sh-RNA 14 days after seeding equal number of viable cells (Trypan blue exclusion), in the presence of 30pM rapamycin where indicated (mean ± st.dev., n=3 biological replicates). TSC1 knockdown is synthetic lethal with MYC deregulation. U2OS-MYC-ER cells expressing either scrambled control sh-RNA or TSC1-specific sh-RNA were treated with hydroxytamoxifen to induce MYC and rapamycin (100 nM) where indicated. Percentage of apoptotic cells was determined with Annexin/7AAD staining 4 days after MYC induction (mean ± st.dev., n=3 biological replicates). Survival rate of different BL cell lines upon TSC1 knockdown. Graphs show numbers of viable cells expressing either a scrambled control sh-RNA or a TSC1-specific sh-RNA 3 days after seeding of equal number of viable cells (determined by trypan blue exclusion; mean ± st.dev., n=3 biological replicates). Immunoblots of control- or TSC1-shRNA expressing BL2 or DG75 cells treated with different concentrations of Rapamycin to either completely inhibit mTORC1 activity (10nM) or titrate the activity to control levels (30 pM), and survival rate of these cells over 7 days (mean ± st.dev., n=3 biological replicates); (Bl2 cells were selected for stable knockdown with Puromycin, DG75 cells without selection). Ramos cells expressing either a TSC1-specific or a control shRNA were inoculated into NOD/SCID mice and tumor volume was measured regularly. The immunoblot shows the level of knockdown of TSC1 (sh-TSC1b) compared to control (sh-contr), and levels of TSC2 and phosphorylated mTORC1 target proteins before inoculation. Tumor growth curves show mean ± SEM (n=8/group). Pictures of xenotransplanted human Ramos lymphoma tumors 35 days after inoculation. The top panels show tumors derived from Ramos cells stably expressing control sh-RNA (sh-contr); the lower panels show tumors derived from Ramos cells stably expressing TSC1-specific sh-RNA (sh-TSC1b).