EMBO/sd-character-level-ner huggingface dataset:
PLCβ1 and PTPRN2 regulate membrane PI(4,5)P2 levels.Figure 3(A) Mean fluorescence intensity of membrane PI(4,5) P2 was analyzed in MDA-MB-231 and LM2 cells immunostained with PI(4,5) P2 antibody using fluorescence microscopy. N = 50 cells/group. Scale bar, 10 μm. (B) Bioluminescence imaging quantification of lung colonization by 40,000 LM2 cells treated with carrier incubated with PI(4,5) P2 or carrier alone for one hour and then immediately injected. Right, H&amp;amp;E staining of representative lung sections. N=6 mice/group. (C,D) MDA-MB-231 cells overexpressing PTPRN2, PLCβ1, or a control vector (C) or LM2 cells transfected with siRNA targeting PTPRN2, PLCβ1, or a control siRNA (D) were immunostained for PI(4,5) P2 levels and analyzed by fluorescence microscopy. Mean fluorescence intensity of plasma membrane levels of the lipid were quantified. N = 50 cells/group. Left, representative immunofluorescence images of cells stained with anti-PI(4,5) P2 antibody (red) and 4',6-diamidino-2-phenylindole (DAPI, blue). Scale bar, 10 μm.
Figure 4(A) A table summarizing the centrosomal localization of cilium-related proteins in ENKD1-depleted RPE1 cells. Immunofluorescence images (B) for RPE1 cells transfected with control or ENKD1 siRNAs, cultured in serum-free medium, and stained with antibodies against CP110 and acetylated α-tubulin and DAPI. quantification of the percentage of cells with one CP110 dot (C, n = 3 independent experiments) for RPE1 cells transfected with control or ENKD1 siRNAs To quantify the percentage of cells with one CP110 dot (C), >120 cells were analyzed for each experiment. (D, E) Immunofluorescence images (D) and quantification of the percentage of cells with one CEP97 dot (E, n = 3 independent experiments) for RPE1 cells transfected with control or ENKD1 siRNAs, cultured in serum-free medium, and stained with antibodies against CEP97 and acetylated α-tubulin and DAPI. To quantify the percentage of cells with one CEP97 dot (E), >150 cells were analyzed for each experiment. Scale bar, 1 µm. (F-H) Immunoblot analysis (F), immunofluorescence images (G), and quantification of the percentage of cells with one CP110 dot (H, n = 3 independent experiments) for RPE1 cells transfected with control or ENKD1 siRNAs, cultured in serum-free medium for 120 h, and stained with antibodies against CP110 and acetylated α-tubulin and DAPI. To quantify the percentage of cells with one CP110 dot (C), >80 cells were analyzed for each experiment. Scale bar, 1 µm.
Whole-genome CRISPR-Cas9 genetic screen reveals mechanisms of resistance to IMT1.Figure 1A Dose-response viability curves in presence of increasing concentrations of IMT1 for one-week, viable cells were counted and normalized to DMSO-treated controls. Data are expressed as mean ± SD of n=3 independent experiments with four technical replicates. IC50 values were calculated with non-linear least square fit, RKO: 521.8 nM, MiaPaCa-2: 291.4 nM and HeLa: 29.9 nM. B Mitochondrial transcript levels measured by qRT-PCR in RKO cells after 96 hours of 1 μM IMT1 treatment. Data are relative to DMSO-treated controls and are expressed as mean ± SD of n=4 independent experiments. Ordinary one-way ANOVA was used for comparisons to DMSO-treated controls (RNR1, RNR2, MT-COX2, MT-ATP6, MT-ND1: ***p<0,0001). C Representative western blot images of OXPHOS protein levels of RKO cells at increasing concentrations of IMT1. ACTINB is showed as loading control. D Volcano plot showing proteomic changes of RKO cells treated with 1 μM IMT1 for 96 hours. Data are plotted as average log2-fold change versus log10 of adjusted p-value of DMSO-treated controls; mitochondrial protein are highlighted in red. E Schematic description of the experimental CRISPR-Cas9 approach: Cas-9 expressing-RKO cells were transduced with a genome-wide pooled knockout CRISPR library and passaged in the presence of either IMT1 or DMSO for guide selection. After cell collection and DNA isolation, the guides were sequenced and quantified in each population. F CRISPR guides enriched (positive hits) in IMT1-treated RKO cells plotted as log2 fold change (LFC) versus log10 of adjusted p-value. mTORC1 and VHL pathway-related genes are reported in green and blue, respectively.
Figure 5 Figure 5. The effect of cell-shape change during growth on the stability of Min protein patterns. A. Cytosolic fluorescence during growth of a cell from a small elliptical form into a large rectangular shape. Numbers in red indicate time in hours. Illustrations show the positions and orientations of the cell in the first and last time frames. Green and blue lines indicate the maximum and minimum Feret diameters, respectively. B. sfGFP-MinD patterns during the growth of the cell shown in A. Illustrations indicate the cell boundaries and oscillation angles observed in the first and last frames (not to scale). C. Quantitative data obtained from the cell shown in A and B. The maximum and minimum Feret diameters (green and blue), and the measured MinD oscillations (red) were expressed in terms of length (top) and angle (bottom) and plotted against time. The number of cells that fit this category was 41/97. Arrows indicate the time when cell width reached the chamber width of 5 μm. D-F. Data are presented as in A-C for another cell that showed persistent oscillations along the horizontal axis throughout growth. The number of cells that fit this category was 28/97. G. Time-lapse images of sfGFP-MinD that reveal stochastic switching of patterns in a cell with an asymmetric shape and a low aspect ratio. White arrows indicate the oscillation axes. H. The angles of the maximum and minimum Feret diameters (green and blue), and the measured MinD clusters (red) for the cell shown in panel G plotted against time. The number of cells that fit this category was 10/97. All scale bars correspond to 5 μm.
Figure 3A) Schematic representation of the structure of the CENP-A nucleosome binding region in CENPC-CT. The canonical CENPC motif segment is shown in pink. The CDK1 phosphorylation site (T651) is also indicated. The CM upstream and downstream regions are shown in magenta. Dot circles indicate the recognition sites for H2A/B acidic patch, CENP-A C-terminal tail, and the RG loop in the CENP-A nucleosome. B) Schematic diagrams of deletion mutants of chicken CENPC-CT. The fragments containing the canonical CENPC motif and the CM downstream region. CENPC643-864, CENPC643-740, CENPC643-740, and CENPC643-683 lack the CM upstream region. CENPC-CT and CENP-C619-689 contain the CM upstream region. C) EMSA results of the binding affinities of CENP-C fragments indicated in (B) to the CENP-A nucleosome. All fragments were phosphorylated by CDK1. D) Stable expression of GFP-fused CENPC-CT and CENPC643-864 (shown in (B)) in CENP-C knockout chicken DT40 cells. α-Tubulin (Tub) was probed as a loading control. E) Localization analysis of GFP-fused CENPC-CT and CENPC643-864 lacking the CM upstream region (green) on mitotic chromosomes in CENP-C knockout DT40 cells. CENP-T (red) was used as a centromere marker. DNA was stained using DAPI (blue). Scale bar indicates 10 μm.
Figure 6Left: Eleven primary AML specimens with different cytogenetic and molecular genetic aberrations were treated with THZ1 or YKL-5-124 alone or in combination with venetoclax. IC50 values in presence of increasing doses of venetoclax are visualized for THZ1. The gray-scale bar indicates the fraction of viable cells in presence of a high dose of venetoclax (500nM) compared to DMSO to provide an approximation whether a sample was rather sensitive or resistant against venetoclax alone. Note that the addition of venetoclax reduces the IC50 in all samples regardless of their baseline resistance against venetoclax alone. The last four columns provide the average and maximum BLISS scores reached by a combination of THZ1 and venetoclax (n=11) Middle: representative dose response curves of THZ1 in presence of increasing doses of venetoclax for one primary AML specimen with intermediate venetoclax sensitivity. Right: representative 3D-synergy plot showing the BLISS synergy scores at indicated concentrations of THZ1 and venetoclax for AML E204098. Dose response curve for the CDK7i CT7001 in presence of increasing doses of venetoclax for primary AML E2113590. The indicated doses on the right represent the IC50 dose. Schematic visualizing the setup for the combinatory in vivo treatment. AML-661 cells were injected in NSG mice. Bones indicate the timepoints of bone marrow aspirations to monitor engraftment of human leukemic cells in mice before treatment start and during treatment. Percentage of human CD45+ leukemic cells three weeks post injection, i.e. before treatment start (left), and 4 and 6 weeks after treatment start (right). Dots represent individual mice, horizontal lines represent means. Unpaired t-test, *** p<0.0005, ** p<0.005. Percentage of human CD34+GPR56+ cells in the mouse bone marrow at 4 and 6 weeks post treatment start with the indicated compounds. Dots represent individual mice, horizontal lines represent means. Unpaired t-test, *** p<0.0005, ** p<0.005, * p<0.05. Left: Representative FACS plots showing CD34 and GPR56 expression on AML cells after 4-week treatment with the indicated compounds. Right: Statistical analysis of the geometric mean intensity of CD34 APC (left) and GPR56 PE (right) in the four treatment groups. Horizontal lines represent means. Unpaired t-test, *** p<0.0005, ** p<0.005, * p<0.05. Cartoon visualizing how CDK7i and venetoclax synergize to suppress both GPR56+ compartments in AML.
Figure 3(A) Interaction between endogenous PAQR3 and the components of VPS34 complex in HEK293T cells. The cell lysates were used for immunoprecipitation (IP) and immunoblotting (IB) with the antibodies as indicated. (B) Cytosol and Golgi fractionations were isolated from the livers of PAQR3-deleted mice and their littermate controls. Equal protein amounts of cytosol and Golgi fractions were subjected to IB. (C) HEK293T cells were transiently transfected with Myc-tagged PAQR3. At 24 h after transfection, the cells were treated with normal medium (NM)，amino acid starvation (AS) or glucose starvation (GS), HBSS solution, or rapamycin (Rapa, 50 nM) for 4 h respectively. The cell lysates were then used in IP and IB. (D) Four VPS34 complexes of MEFs were immunopurified using the indicated antibodies under NM, AS or GS conditions. The relative abundance of VPS34-binding partners was determined by IB. Each IP was normalized to the amount of VPS34. (E) HEK293T cells were transfected with the plasmids as indicated and the cell lysates were used in IP and IB. (F) The lysates of WT or PAQR3 knockout MEFs were used in IP and IB. (G) MEFs were infected with control or PAQR3-expressing lentivirus. Then a quantitative immunodepletion assay was performed with increasing amounts of the indicated antibodies. Supernatants of MEF lysates after immunodepletion (Post-IP) were examined to determine the level of VPS34 complex proteins.
Figure 4A) Left panels: representative images of ORO staining on ACM and HC C-MSC in AM supplemented or not with 150µg/ml oxLDL. Middle panel: image quantification (n=11 biological replicates; Two-Way Anova). Right panels: representative images of Western Blot of CD36, PPARγ and GAPDH protein expression of ACM and HC C-MSC protein extracts in AM supplemented or not with 150µg/ml oxLDL (n=5 biological replicates) and d.a. normalized on the house-keeping GAPDH (Two-Way Anova). Data information: mean ± SEM. * p<0.05, ** p<0.01, *** p<0.001. B) Left panels: representative images of ORO staining on ACM and HC C-MSC in AM supplemented or not with 20μg/ml 13HODE, 5mmol/L NAC or both. Right panel: image quantification (n=13 biological replicates; Two-Way Anova). Data information: mean ± SEM. * p<0.05, ** p<0.01, *** p<0.001. C) Left panel: representative images of Western Blot of CD36, PPARγ and GAPDH protein expression of ACM and HC C-MSC protein extracts in AM supplemented or not with 20μg/ml 13HODE, 5mmol/L NAC or both (n=8 biological replicates). Right panels: d.a. normalized on the house-keeping GAPDH (Two-Way Anova). Data information: mean ± SEM. * p<0.05, ** p<0.01, *** p<0.001.
Effects of Beclin-1 haploinsufficiency on AR113Q muscle.Figure 5A. Muscle fiber size (100 fibers/mouse) was quantified from proximal hind limbmuscle of AR113Q (red, n = 6) or AR113Q, Beclin-1 +/− (blue, n = 6) mice at 16 wks. Left panel shows fiber size distribution, and right panel shows relative fiber cross sectional area (mean +/− SEM). Left panel, p<0.0001 by Mann-Whitney test. Right panel, p<0.0001 by Student's t test. B. AR expression in skeletal muscle of 16 wk mice was assessed by western blot. GAPDH controls for loading. Right panel shows quantification of signal relative to GAPDH (mean +/− SEM). Differences not significant (n. s.).
Figure 6Diploid (2n) and tetraploid (4n) strains expressing a Gam-GFP reporter were cultured on YPD or PRE-SPO medium at 30°C or 37°C, respectively, in the presence (Gam-GFP ON) or absence (Gam-GFP OFF) of doxycycline (DOX) for 24 h. Cell images indicate calcofluor white staining (cell wall; blue), GFP, and a merged image of GFP/DAPI/DIC channels. Scale bar = 10 μm. Flow cytometric analysis of the tetraploid Gam-GFP reporter strain grown on YPD or PRE-SPO medium at 30°C or 37°C, respectively, in the presence or absence of doxycycline for 24 h. Flow cytometric analysis of the tetraploid Gam-GFP reporter strain grown on PRE-SPO medium with or without 20 mM ascorbic acid, 1 mM DTT, or 1 mM glutathione at 37°C for 24 h.
Bile acid induced YAP activation is dependent on the acto-myosin system A Fluorescence stainings for YAP (green), F-actin (magenta) and with the nuclear marker DAPI (blue) of primary hepatocyte cultures treated with DMSO (control), Y27, deoxycholic acid (DCA) or DCA+Y27 for ~18 h.Figure 5Fluorescence stainings for YAP (green), F-actin (magenta) and with the nuclear marker DAPI (blue) of primary hepatocyte cultures treated with DMSO (control), Y27, deoxycholic acid (DCA) or DCA+Y27 for ~18 h. Indicated areas (dashed rectangle) are shown as magnifications in insets. Quantification of the mean nuclear YAP intensity from fluorescence images of primary hepatocytes treated with DMSO (control), Y27, DCA or DCA+Y27 for ~18 h as representatively shown in (A). Data is normalized to untreated cells (not shown). Mean ± s.e.m., n = 7; DCA vs. DMSO p=8.98*10-5; DCA+Y27 vs. DMSO, p=0.05; Y27 vs. DMSO, p=0.55 (n.s.); DCA+Y27 vs. DCA p = 0.05 Western blot of pMLC and GAPDH (loading control) of primary hepatocyte culture lysates. Cells were untreated or incubated for 18 h with the indicated compounds Fluorescence stainings of primary hepatocytes for YAP (green), F-actin (magenta) and with the nuclear marker DAPI (blue). Cells were treated with DMSO (control), Y27, SMIFH2 or Cytochalasin D (CytoD) for 6 h. Indicated areas (dashed rectangle) are shown as magnifications in insets. Note the dilation of canaliculi (asterisks) and fragmentation of F-actin (arrows) upon CytoD treatment. Quantification of the mean nuclear YAP intensity from images of primary hepatocytes treated with DMSO, Saline, Y27, Fasudil, SMIFH2, CK666, CytoD or Latrunculin A (LatA) for 6 h. Saline serves as control for Fasudil, DMSO serves as control for all other conditions. Inhibitors affecting similar actin processes are displayed in the same grey level. Saline, Fasudil, CK666 and LatA conditions are not shown in (d). Data is normalized to untreated cells (not shown). Mean ± s.e.m., n=3-5; DMSO vs. Y27, p=0.002; DMSO vs. CytoD, p=5.11*10-7; DMSO vs. LatA, p=0.01; DMSO vs. CK666, p=0.15 (n.s.); DMSO vs. SMIFH2, p= 0.11 (n.s.); Saline vs. Fasudil, p= 0.92. Western blot of pMLC and GAPDH (loading control) in untreated or actin inhibitor-treated primary hepatocyte culture lysates. Inhibitor treatments are the same as in (E). Scale bars, 20 µm (A, D).
Figure 3(A) Representative immunofluorescence images of p21 levels in p53-WT or KO RPE1 cells, 48 hours after release from 1, 4 or 7 days palbociclib (1.25μM) treatment. Zoom inserts are 3x magnification of the indicated regions. Scale bars = 250 μM. (B) Quantification of p21 intensities in cells treated as in panel A. At least 100 cells were analysed per experiment and graph shows data from 3 experimental repeats. Violin plots display the variation in intensities between individual cells. Horizontal lines display the median, and error bars show 95% confidence intervals. ( (C) Immunofluorescence images of DAPI and γH2AX staining in p53-WT or KO RPE1 cells either before or 48 hours after release from a 7-day treatment with palbociclib (1.25μM). Scale bar = 250 μM, zoom inserts = 3x magnification of highlighted regions. (D-E) Quantification of nuclear morphologies (D) and γH2AX-positive DNA damage foci (E) following palbociclib (1.25μM) treatment in p53 WT and KO RPE1 cells. Cells were treated for 1, 4 or 7 days and then analysed before or after drug washout for 48 hours. 100 cells (nuclear morphology) or 50 cells (γH2AX foci) were scored per condition per experiment, and bar graphs represent mean data +SEM from 6 experiments. (F) Immunofluorescence images showing symmetrical 53BP1 staining following mitotic exit in p53-KO cells after release from 7 days of palbociclib arrest. 3 separate examples are displayed. Scale bar = 25 μM (G-I) Quantification of the first mitosis in GFP-53BP1/H2B-RFP RPE1 cells after release of from a 1-day or 7-day palbociclib (1.25μM) arrest. Quantified from the same movies are nuclear morphology after mitosis (G), chromosome segregation defects during mitosis (H) and appearance of 53BP1 foci after mitosis (I). 54 cells (1-day) or 80 cells (7-day) were analysed in total from 2 experiments. Errors bars display SD. (J-K) Representative immunofluorescence images (J) and quantifications (K) of mitotic DNA replication assays (MiDAS) in p53-KO RPE1 cells released from 7 days of palbociclib (1.25μM) treatment or following 0.4uM aphidicolin treatment for 40Hrs. EdU foci were quantified in nocodazole-arrested cells. Scale bar= 5 μM, zoom inserts = 3x magnification of highlighted areas. 10 cells were analysed per experiment and the bar chart shows the mean + SEM from 3 experimental repeats.
Figure 5A. Schematic of TAM treatment and collection of brain tissue. Representative confocal images of the SGZ 6.5 months after the treatment with TAM. C. Representative confocal images of the SGZ 6.5 months after the treatment with TAM. Quantification of cell numbers 6.5 months after the induction of KO (n = 4 for Cont-8M, n = 3 for cKO-8M, n = 4 for Cont-15M,). (D) The density of RGL-ANSCs tends to be reduced in cKO. F (2, 8) = 3.98, P = 0.063, One-way ANOVA. (E) The density of ANPCs was reduced in cKO-8M and Cont-15M mice. F (2, 8) = 11.49, P = 0.0044, One-way ANOVA followed by Tukey's HSD test (** P < 0.01). Quantification of cell numbers 6.5 months after the induction of KO (n = 4 for Cont-8M, n = 3 for cKO-8M, n = 4 for Cont-15M,). (F) The density of DCX-positive cells was reduced in WT-old and cKO mice. p = 0.0091, Kruskal-Wallis test followed by Dunn's test (* P < 0.05). (G) The density of Ki67+RGL-ANSCs was reduced in cKO-8M mice (* P = 0.029, Kruskal-Wallis test followed by Dunn's test). (H) The density of Ki67+ANPC was not significantly different among three groups (P = 0.49, Kruskal-Wallis test). (I) The density of NeuN+EYFP+ neurons was reduced in cKO-8M mice. F (2, 8) = 34.85, P = 0.00011, One-way ANOVA followed by Tukey's HSD test (** P < 0.001). (n = 4 for Cont-8M, n = 3 for cKO-8M, n = 4 for Cont-15M).
Secretagogin regulates CRH release in vitro and in vivo A, A1 siRNA‐mediated secretagogin (scgn) knockdown in cultured hypothalamic neurons, as indicated by reduced secretagogin immunoreactivity (A) and decreased CRH content in the culture medium (A1).Figure 6A, A1 siRNA‐mediated secretagogin (scgn) knockdown in cultured hypothalamic neurons, as indicated by reduced secretagogin immunoreactivity (A) and decreased CRH content in the culture medium (A1). B-B2 Transient overexpression of secretagogin in immortalized CRH‐expressing mHypoE‐N44 hypothalamic cells significantly reduced CRHimmunofluorescence microscopy intensity in secretagogin+cell bodies, indirectly supporting enhanced CRH release. All experiments were performed in triplicate. Scale bar: 50 nm. C, C1 siRNA‐mediated in vivo silencing of secretagogin mRNA expression in the PVN provoked somatic CRH accumulation (arrowheads). Scale bar: 150 μm.C2 Quantitative analysis demonstrating significantly increased somatic CRH contents. Note that somatic secretagogin levels remained unchanged, which we interpret as data on a neuronal contingent not affected by siRNA silencing. The lack of secretagogin/CRH co‐localization suggests that secretagogin expression fell below detection threshold in many CRH+ neurons. D, D1 Individual data points show maximal CRH fluorescence intensity (gray scale arbitrary unit (a.u.) expression) in PVN neurons that have low or no secretagogin expression after siRNA infusion.
Figure 2(A) Tumor volume of MDA-MB-468 mammary fat-pad xenografts treated with vehicle, AZD8186 (50 mg/kg, og twice/day), erlotinib (50 mg/kg IP once/day) alone or in combination (5-6 mice per group, mean±SEM). Statistical significance of two-way ANOVA statistical test **P=0.0028 and ***P<0.0001. (B) Tumor volume of HCC-70 mammary fat-pad xenografts treated with vehicle, AZD8186 (150 mg/kg, og once/day), erlotinib (50 mg/kg IP once/day) alone or in combination (6-7 mice per group, mean±SEM). Growth curves were compared using two-way ANOVA statistical test. Statistical significance of two-way ANOVA statistical test ***P=0.0006 and ****P<0.0001. (C) Waterfall representation of changes in the volume of individual HCC-70 tumors during the treatment. Statistical comparison between different treatment groups by Mann-Whitney test **P=0.0023 and ***P=0.0006. (D) Change in the body weight of mice harbouring HCC70 xenograft tumors (6-7 mice per group, mean±SEM) during 21 days of treatment. (E) A cell line derived from a mammary tumor spontaneously developed in a Wap-cre:Ptenfl/fl:Tp53fl/fl mouse was cloned and injected in the mammary fat pad of syngeneic C57BL6/J recipient mice. Tumours grew in 12 out of 35 transplanted mice and only these tumours were selected for the treatments described in the figure. When tumours reached an average volume of 100mm3, they were treated with vehicle or a combination of AZD8186 (150mg/kg, og once/day) and erlotinib (50mg/kg IP once/day). Tumours were then measured during the treatment (6 mice per group, mean±SEM). Statistical significance of two-way ANOVA statistical test ****P<0.0001.
Figure 5(A) Fluorescence-based degradation assay in S. cerevisiae. The substrate proteins consisted of a UbL domain, followed by a yellow fluorescent protein (YFP) domain and a disordered tail (51 amino acids derived from subunit 9 of the Fo component of the Neurospora crassa ATPase, Su9). Control substrates lacked the disordered tail. Substrates and a red fluorescent protein were expressed from consecutive constitutive promoters on the same CEN plasmid (YCplac33). (B) Fluorescence profiles of cells expressing different substrates monitored by flow cytometry. UbLRad23 targeted YFP-Su9 protein for degradation. Removing the Su9 tail or replacing UbLRad23 with a DHFR domain stabilizes YFP protein in cells. (C) UbL domains targeted YFP substrates to degradation in the presence of an initiation region. The Y-axis plots abundance of the UbL-YFP-tail proteins normalized by a translation control, which is the same UbL-YFP protein without the tail. The ratio is equal to one for domains that are not recognized by the proteasome, and smaller than one if the UbL is recognized by the proteasome and the UbL-YFP-tail protein is degraded. YFP fluorescence is also corrected for plasmid copy number using RFP fluorescence. The median of YFP/RFP ratio for each construct was calculated from 10,000 cells collected in one flow cytometry run and reflected the abundance of YFP substrates in yeast cells as described in Methods. Data points represent mean values determined from at least three repeat experiments; error bars indicate s.e.m.