EMBO/sd-character-level-ner huggingface dataset:
Deletion of INO80 prevents linc-MYH dependent changes in MuSC proliferation and gene expression A-D Isolated linc-MYH KO MuSCs reach confluence much faster than WT MuSCs indicating higher proliferation rates.Figure 4A-D Isolated linc-MYH KO MuSCs reach confluence much faster than WT MuSCs indicating higher proliferation rates. Deletion of INO80 eliminates the difference between Pax7-Crepos / INO80-/- (Ino80 KO) and linc-MYH-/- / linc-MYH-/-/ Pax7-Crepos / INO80-/- (dKO). Images are representative images for cultures after 13 days in culture. E Differences in proliferation rates are abolished by additional deletion of INO80 in MuSCs. Cell confluence was monitored by a live cell imaging analysis system for 13 days (n = 7 WT / 6 linc-MYH KO / 3 Ino80 KO / 3 dKO independent preparations of MuSCs). F Statistical analysis of cell confluence after 13 days of muscle stem cell proliferation (n = 7 WT / 6 linc-MYH KO / 3 Ino80 KO / 3 dKO independent preparations of MuSCs, Student's t test, two-tailed; *p < 0.05, ns = not significant). Data are mean ± S.E.M. G-I Gene set enrichment analysis (GSEA, broadinstitute.org) of microarray data from proliferating MuSCs. Comparison of linc-MYH-/- vs. WT proliferating MuSC transcriptomes shows enrichment of genes related to cell proliferation (G), enrichment of genes containing cis regulatory elements for YY1 (H) and p53 (I). Enrichments are highly significant using the most conservative familywise-error rate method (FWER). Enrichments were lost in linc-MYH-/- MuSCs after additional deletion of INO80 (dKOs) compared to Pax7-Crepos / INO80-/- MuSCs. Heat maps of the 10 most upregulated genes of the respective gene sets depicted in G-I in linc-MYH-/- vs. WT MuSCs and in linc-MYH-/-/ Pax7-Crepos / INO80-/- dKO vs. Pax7-Crepos / INO80-/- MuSCs. Expression of up-regulated genes in linc-MYH-/- was normalized after additional deletion of INO80 (dKOs), indicating that effects of linc-MYH completely depend on INO80 (WT n = 4, linc-MYH-/- n = 4, Pax7-Crepos / INO80-/- n = 4, Pax7-Cre dKO n = 3 animals used for independent isolations of MuSCs). Heat maps of the 10 most upregulated genes of the respective gene sets depicted in G-I in linc-MYH-/- vs. WT MuSCs and in linc-MYH-/-/ Pax7-Crepos / INO80-/- dKO vs. Pax7-Crepos / INO80-/- MuSCs. Expression of up-regulated genes in linc-MYH-/- was normalized after additional deletion of INO80 (dKOs), indicating that effects of linc-MYH completely depend on INO80 (WT n = 4, linc-MYH-/- n = 4, Pax7-Crepos / INO80-/- n = 4, Pax7-Cre dKO n = 3 animals used for independent isolations of MuSCs). Heat maps of the 10 most upregulated genes of the respective gene sets depicted in G-I in linc-MYH-/- vs. WT MuSCs and in linc-MYH-/-/ Pax7-Crepos / INO80-/- dKO vs. Pax7-Crepos / INO80-/- MuSCs. Expression of up-regulated genes in linc-MYH-/- was normalized after additional deletion of INO80 (dKOs), indicating that effects of linc-MYH completely depend on INO80 (WT n = 4, linc-MYH-/- n = 4, Pax7-Crepos / INO80-/- n = 4, Pax7-Cre dKO n = 3 animals used for independent isolations of MuSCs).
Glucose pulses incorporate directly into the de novo biomass in non-dividing cells.Figure 5Percentage of labeled threonine and deoxyribose from protein and DNA hydrolysate shows de novo protein and DNA synthesis in non-dividing cells. After 6 hours of pulsing uniformly labeled 13C-glucose, cultures were lysed, and their macromolecules were washed free of latent metabolites and hydrolyzed to monomers. Labeling data are presented as the mean ± standard error of independent biological replicates (n = 3, all pairwise P < 0.02 as determined by one sided Student"s t-
Figure 4(A) Increased SCAP polyubiquitination in TMEM33 transfected HEK293T cells. 293T cells were transiently transfected with Halo-tagged TMEM33 for 18 hours followed by treatment with MG-132 for 3 hours. (B) Western blot analysis of PKM2, PKM1, NRF1, TMEM33, RNF5 and SCAP levels in MEF (PKM2fl/fl, Cre-ERT2) cells treated with 4-OHT in a time course experiment. (C) Co-immunoprecipitation of endogenous SCAP and RNF5 in Flag-tagged TMEM33 stably expressed MCF7 cells. SEC61β is an ER protein, which is found to interact with TMEM33. (D) Reciprocal co-immunoprecipitation of TMEM33 and RNF5 in MDA-MB-231 cells. TMEM33, RNF5 and SCAP were detected by Western blotting. (E) Proximity ligation assays of TMEM33 and RNF5 in MDA-MB-231 cells using confocal imaging. NC means negative control. Scale bar, 10μm. (F) Interaction between in vitro translated TMEM33 and RNF5. FLAG-tagged vector or TMEM33 and HA-tagged RNF5 were expressed using an in vitro transcriptional and translational kit, followed by FLAG antibody pull down. (G) Increased polyubiquitination of SCAP in RNF5-transfected HEK293 cells. 293T cells were transiently transfected HA-tagged RNF5 for 18 hours followed by MG-132 treatment for 3 hours. Arrow indicated HA-tagged RNF5. (H) RNF5 is involved in TMEM33 mediated SCAP polyubiquitination in 293T cells. 293T cells were stably transfected with shCtrl or shRNF5 constructs, then the indicated plasmids were co-transfected into shCtrl or shRNF5 expressing 293T cells for 18 hours followed by MG-132 treatment for 8 hours. FLAG M2 beads were used for pull-down. (I) The relative binding affinity of FLAG-tagged full-length and truncated SCAP proteins with endogenous TMEM33 and RNF5. The band intensity of TMEM33 and RNF5 in Appendix Fig S2D were quantified and normalized with the FLAG control. SCAP protein encompasses eight transmembrane (TM) helices in N-terminus (TM 2-6: sterol-sensing domain) and a WD40 domain in C-terminus with total of 1279 aa. (J) The relative binding affinity of HA-tagged full- length and truncated TMEM33 with endogenous SCAP and RNF5. The band intensity of SCAP and RNF5 in Appendix Fig S2E were quantified and normalized to the HA control. TMEM33 protein encompasses three transmembrane (TM) helices in N-terminus and a C-terminal cytosolic domain with a total of 247 aa.
Figure 5A) The chest computed tomography (CT) was normal five days after symptom onset. Disease aggravation was evidenced by progressed chest CT images ten days after symptom onset displaying bilateral multiple patchy ground glass opacities. Disease alleviation was evidenced by improved chest CT images 25 days after symptom onset. B) Procalcitonin (PCT) levels stayed within normal range throughout the course of disease. C) Lymphocyte count was normal five days after symptom onset, while firstly decreasing then returning to normal range during the course of disease. Both abnormal C-reactive protein (CRP) (D) were detected five days after symptom onset when the chest CT and lymphocyte count were still normal. In association with changes in chest CT scans, CRP further increased ten days after symptom onset and returned to normal range 25 days after symptom onset. IL-6 (E) were detected five days after symptom onset when the chest CT and lymphocyte count were still normal. In association with changes in chest CT scans, IL-6 further increased ten days after symptom onset and returned to normal range 25 days after symptom onset. F) The body temperature was elevated when CRP and IL-6 levels were rising, but returned to normal when CRP and IL-6. Levels were declining.
Figure 5(a) Total cellular lysates from HEK293T cells transfected with myc-VPS39 or VPS41 were incubated with GST-empty vector (EV), RAB2A-S20N (SN), or RAB2A-Q65L (QL) proteins bound to glutathione sepharose beads. Immunoblotting was performed with anti-myc antibody. Input lysates are also shown. GST proteins loaded in gels were stained with CBB. (b) FLAG-EV or RAB2A were transiently co-transfected with EGFP-VPS39 or VPS41 into HEK293T cells. Immunoprecipitated (IP) fraction from cell lysates with anti-FLAG antibody and total lysates were immunoblotted with the antibodies indicated on the right. (c, d) MDA-MB-231 cells were transfected with EGFP-fused empty EV, VPS39, or VPS41. Cells were immunostained with RAB7 (c, red) or anti-MT1-MMP (d) antibodies. Merged images with DAPI (blue) are shown on the right. Enlarged endosomes in the boxed region are highly magnified in the insets. Diameters of RAB7 positive vesicles are also shown. n, number of cells scored for each cell population. Bars, 10 μm, (inset 5 μm). (e-i) Scrambled siRNA (siNC) or siRNA against RAB2A (siRAB2A), VPS39 (siVPS39), or VPS41 (siVPS41) were transfected into MCF10.DCIS.com cells. Serum-starved cells were then plated onto fluorescent-conjugated gelatin (red), stimulated with HGF (100 ng/ml) overnight, and stained with phalloidin (green) and DAPI (blue). Quantification of gelatin degradation (f) and efficacy of targeted gene silencing verified by qPCR (g-i). Data are the mean ± SEM (error bars; n > 100 cells/experiment in at least 4 independent ones). Bars, 50 µm. (j-l) Inverse invasion assay of MDA-MB-231 cells transfected with siRNA oligonucleotides targeting interest genes. Invasive activity is the numbers of cells/field that migrate through Matrigel further than 50 µm (k). Efficacy of targeted gene silencing was verified by qPCR (l). Data are mean ± SD from 3 different fields. Bar, 250 µm. (m-q) Doxycycline-inducible murine RAB2A-expression MCF10.DCIS.com cells were transfected with siNC, siRAB2A, or siVPS39 in the absence (CTR) or presence (RAB2A) of doxycycline. Their ECM degradation activities were quantified (n). Data are the mean ± SEM (error bars; n > 100 cells/experiment in 3 independent ones). Total cell lysates were immunoblotted by anti-RAB2A and anti-tubulin antibodies (o). Efficacy of targeted gene silencing was verified by qPCR (p-q). Bar, 50 μm. **, P < 0.01, ***, P < 0.001.
Figure 6Schematic representation of how Kap114p regulates gene expression. Under normal growth conditions, RanGTP liberates yTBP from Kap114p, allowing gene expression. As nuclear Ran levels are reduced under salt stress, binding of Kap114 to yTBP is enhanced, thereby trans-repressing yTBP activity and down-regulating gene expression.
Figure 2(a) Reaction performed as in Fig. 1a, except Cdc45 was omitted. (b) Reaction performed as in Fig. 1g, with the indicated proteins omitted. (c) Schematic depicting MCM-binding by Sld3 fragments. Two essential CDK phosphorylation sites are shown by vertical bars. The Sld3-Treslin Domain (S.T.D.) is shown as a black box. Also see Fig. EV1 (d) Schematic showing position of MCM-interacting region in Sld3. Alignments were generated as in Fig. 1e. Residues were substituted for Glu. as indicated. (e) Reaction performed as in (a), except Sld3/7 was incubated with Rad53 before addition to reactions. ATP was omitted from this pre-incubation as indicated. (f) Sld3 recruitment reaction performed as in (a). Sld7 was omitted from this experiment.
Figure 4(A) Effects of different adenine nucleotides or their washout on the ClC-5 current amplitudes (n=4-7). The data were normalized to the initial current amplitude determined shortly after establishing whole-cell configuration. Some of the data points are omitted for clarity. For comparison, the ADP data set from Figure 1 is also depicted. Red lines indicate monoexponential fits to the averaged data. (B) Averaged residual currents determined after 600-s cell dialysis as shown in (A), and normalized to the current at the start of the measurements (n=4-7). Asterisks indicate statistically significant differences from a two-sample t-test analysis with p < 0.001; n.d. - not defined. Averaged rundown time constants obtained from fits with monoexponential decay functions as shown in (A).
Figure 5DC3.2-Rab39a-H2-Ld cells (Rab39a KO with dox-inducible Rab39a) were incubated with or without 1 µg/ml dox overnight to induce Rab39a expression. 1x106 cells were fed at 4 beads/cell of biotinylated-magnetic-beads for 4 hours. Magnetic bead phagosomes were isolated using the listed protocol. Phagosomes were washed, fixed, permeabilized and stained for Open (64-3-7) and Closed (30-5-7s) forms of H2-Ld. Representative histograms shown in (B). The background fluorescence of unstained beads was subtracted from the resulting phagosome gMFI and the fold change of open or closed H2-Ld (dox/no dox) was calculated. Three independent experiments were combined for analysis and error bars show the SD between experiments. Open H2-Ld, but not Closed H2-Ld was statistically significant (p<0.05) between KO and rescued cells using two-tailed ratio-paired Student's t-test. DC3.2-Rab39a-H2-Ld cells (Rab39a KO with dox-inducible Rab39a) were incubated with or without 1 µg/ml dox overnight to induce Rab39a expression. 1x106 cells were fed at 4 beads/cell of biotinylated-magnetic-beads for 4 hours. Magnetic bead phagosomes were isolated using the listed protocol. Phagosomes were washed, fixed, permeabilized and stained for Open (64-3-7) and Closed (30-5-7s) forms of H2-Ld. H2-Ld cell surface levels of KO and induced cells as in (A). Phagosomes from KO and induced cells were isolated as in (A). Permeabilized, unfixed phagosomes were incubated with 10 µM of the indicated peptides for 3 hours at 25oC in Perm buffer. Phagosomes were then washed and stained for H2-Ld. Shown are histograms of indicated stains. Phagosomes from KO and induced cells were isolated as in (A). Permeabilized, unfixed phagosomes were incubated with 10 µM of the indicated peptides for 3 hours at 25oC in Perm buffer. Phagosomes were then washed and stained for H2-Ld. Shown are histograms of indicated stains. Flow cytometry plots of (D). Analysis of phagosomes shown in (D) and (E). The background fluorescence of unstained beads was subtracted from the resulting phagosome gMFI and the fold change of pulsed / unpulsed phagosomes was calculated. DC3.2 Rab39a-H2-Ld cells were incubated with or without 1 µg/ml dox overnight to induce Rab39a expression. 1x106 cells were fed at 4 beads/cell of biotinylated-Ova-magnetic-beads for 4 hours in the presence or absence of BFA. Isolated phagosomes were stained for H2-Ld. Representative of two independent experiments.
Figure 4A Tandem affinity purification of MAGE-A3 from HEK293 cells identified DDB1, DCAF5, and DCAF12 as binding partners. Mascot score, number of peptides identified, and percent coverage is shown. B MAGE-A3 interacts with DCAF12, but not DCAF5. HeLa cells were transfected with the indicated constructs (Myc-MAGE-A3 and HA-DCAF5 or HA-DCAF12). Two days later cells were treated with complete media or EBSS for 4 hrs. Cells were treated in the presence of 1 µM MLN4924 to stabilize MAGE-A3. Cell extracts were subjected to anti-Myc IP. C Nutrient deprivation increases endogenous DCAF12 binding to MAGE-A3. HeLa cells were transfect with Myc-vector or Myc-MAGE-A3 for 48 hrs. Cells were then treated with 10 µM MG132 in complete media, EBSS, or EBSS containing 20 µg/mL insulin for 6 hrs. Cell extracts were subjected to anti-Myc IP. Knockdown of DCAF12, but not DCAF5, altered MAGE-A3/6 protein levels and partially rescued its instability upon nutrient deprivation. HeLa cells were treated with control, DCAF5 siRNAs for 72 hrs before incubation in complete media or EBSS for the indicated times with or without MLN4924 (1 µM). E Knockdown of DCAF12, but not DCAF5, altered MAGE-A3/6 protein levels and partially rescued its instability upon nutrient deprivation. HeLa cells were treated with control or DCAF12 siRNAs for 72 hrs before incubation in complete media or EBSS for the indicated times with or without MLN4924 (1 µM). F Knockdown of DCAF12 with individual siRNAs blocks MAGE-A3/6 degradation upon nutrient deprivation. HeLa cells were treated with the indicated siRNAs for 72 hrs before incubation in EBSS for the indicated times. Note, time 0 samples between siRNA groups were normalized to more readily discern differences in MAGE-A3/6 degradation upon nutrient deprivation. G Ubiquitination of MAGE-A3/6 upon nutrient deprivation is dependent on DCAF12. A375 parental or DCAF12 KO cells were treated with MG132 (10 µM) in complete media or EBSS for 6 hrs. Cell extracts were incubated with control agarose or agarose-TUBE2 to isolate ubiquitinated proteins. Input and pulldown samples were probed for the indicated proteins.
Figure 1A Co‐localization of endogenous TBC1D5 and VPS35 with mCherry‐ATG9A, in steady state and autophagy induced by mTOR inhibitor KU0063794 (6 h). B, C Quantification of co‐localization experiments presented in (A). D Co‐immunoprecipitation of HA‐Flag‐TBC1D5 with myc‐VPS29, transiently overexpressed in 293T cells, DMSO or KU0063794 (6 h) treated. E, F Co‐immunoprecipitation of endogenous TBC1D5 and ATG9A with myc‐VPS29(E) transiently overexpressed in 293T cells or with HA‐Flag‐VPS35 (F) transiently overexpressed in 293T cells, treated with DMSO or KU0063794 (6 h). G Co‐immunoprecipitation of endogenous VPS29 and ATG9A with HA‐Flag‐TBC1D5stably expressed in 293T cells, treated with DMSO or KU0063794 (6 h). H Co‐immunoprecipitation of endogenous ULK1 and pATG13 (Ser318) with HA‐Flag‐TBC1D5, stably expressed in T‐REx 293T cells, induced by doxycycline (50 ng/ml) for 20 h prior to DMSO or KU0063794 (6 h) treatment. I Magnified still images extracted from Supplementary Movie S2. mCherry‐TBC1D5 co‐localizes with GFP‐ATG9 upon autophagy induction by mTOR inhibitor, KU0063794. H Co-immunoprecipitation of endogenous ULK1 and pATG13 (Ser318) with HA-Flag-TBC1D5, stably expressed in T-REx 293T cells, induced by doxycycline (50 ng/ml) for 20 h prior to DMSO or KU0063794 (6 h) treatment.
Figure 5(A) Lysine 48 linked polyubiquitinated SCAP levels in parental and PKM2 KO MDA-MB-231 cells. (B) SCAP-GFP stability in parental and PKM2 KO MDA-MB-231 cells (n=5). Cells stably expressing SCAP-GFP were treated with cycloheximide (CHX). GFP signal was measured at the indicated time. Data information: Data are displayed as means ± SD. Statistical significance was assessed using two-tailed Student's t-test, ∗p < 0.05, ∗∗p < 0.01 and ∗∗∗p < 0.001. (C) Western blot analyses of RNF5 and SCAP in shCtrl and shRNF5 PKM2 KO MDA-MB-231 cells. (D) qRT-PCR analyses of selected SREBPs downstream genes in shCtrl and shRNF5 PKM2 KO MDA-MB-231 cells (n=3). Data information: Data are displayed as means ± SD. Statistical significance was assessed using two-tailed Student's t-test, ∗p < 0.05, ∗∗p < 0.01 and ∗∗∗p < 0.001. (E) Western blot analyses of RNF5, SCAP and IDI1 in Ctrl and HA-RNF5 OE MCF7 cells. (F) qRT-PCR analyses of SREBPs downstream genes in Ctrl and HA-RNF5 OE MCF7 cells (n=3). Data information: Data are displayed as means ± SD. Statistical significance was assessed using two-tailed Student's t-test, ∗p < 0.05, ∗∗p < 0.01 and ∗∗∗p < 0.001. (G) Cell proliferation of Ctrl and HA-RNF5 OE MCF7 cells measured by MTT assays (n=8). Data information: Data are displayed as means ± SD. Statistical significance was assessed using two-tailed Student's t-test, ∗p < 0.05, ∗∗p < 0.01 and ∗∗∗p < 0.001.
ATRA-dependent anti-tumor activity in HCC-1599-derived xenografts in vivo.Figure 2SCID mice were xenografted subcutaneously with 10 × 106HCC-1599 cells on both sides. One week after transplantation 10 animals/experimental group were treated intraperitoneally with vehicle (DMSO) or two doses of ATRA (7.5 and 15.0 mg/kg) once/day, 5 days a week for a total of 24 days. At the end of this period, treatment was discontinued until sacrifice.The size of the tumors was determined with a caliper and the volume plotted. Each point is the mean ± SE of 20 tumors. *Significantly lower than the corresponding vehicle-treated group (P-value < 0.05, Student's t-test). **Significantly lower than the corresponding vehicle-treated group (P-value < 0.01, Student's t-test). Magnetic resonance imaging (MRI) analysis was performed on five animals/experimental group on day 24. The picture shows representative 2D images of tumor sections from one animal treated with vehicle and one animal treated with 15.0 mg/kg ATRA. The bar graph shows the volume of the tumors calculated after 3D reconstruction of the MRI images. Each point is the mean ± SE of five tumors. The P-value of the comparisons of ATRA versus vehicle is shown.
ALKAL2-driven NB is sensitive to treatment with the ALK TKI lorlatinib in vivo.Figure 7A. Cells derived from tumours arising in Rosa26_Alkal2;Th-MYCN (#4953) and Alk-F1178S;Th-MYCN (#4938) mice are sensitive to both lorlatinib and brigatinib. The effect of increasing concentrations of each ALK TKI (as indicated) on cell confluence was analyzed by IncuCyte Live Cell Analysis. Data is presented as mean ± SEM from three independent experiments. *p<0.05, **p<0.005; two-tailed paired student t-test. B. Tumour volume changes over time for Rosa26_Alkal2;Th-MYCN mice treated with lorlatinib (10 mg/kg; twice daily) or vehicle control. Tumour volume was measured by ultrasound on Day 0 and 7, and by direct measurement at Day 14. Day 0 (lorlatinib n=7, Ctrl n=6), Day 7 (lorlatinib n=2, Ctrl n=5) and Day 14 (lorlatinib n=7, Ctrl n=6). Data shown represents mean ± SD. **p<0.005; two-tailed unpaired student t-test. C. Rosa26_Alkal2;Th-MYCN animals treated with lorlatinib did not display any significant loss of body weight compared with vehicle controls. Data shown represents mean ± SD. D. Representative ultrasound images of tumours observed in Rosa26_Alkal2;Th-MYCN mice with annotated measurements at Day 0 and Day 7. Tumours arise in the retroperitoneal space ventral to the aorta, Ao. E. Representative MRI imaging of Rosa26_Alkal2;Th-MYCN tumours in response to lorlatinib at 4d and 14d. F. Rosa26_Alkal2;Th-MYCN tumours from lorlatinib or vehicle controls were analysed for pHis. A representative field of view for each tumour at 40x (175740μm2) were manually counted. Data shown represents mean ± 95% CI. p=0.0286; Mann-Whitney test.