EMBO/sd-character-level-ner huggingface dataset:
mediated evasion of ferroptosisA) MCF10A cells were treated for 60 minutes with either DMSO, 25uM 4HNE, 5uM RSL3, 4HNE and 10uM KRIBB11 or RSL3 and KRIBB11.Figure 4MCF10A cells were treated for 60 minutes with either DMSO, 25uM 4HNE, 5uM RSL3, 4HNE and 10uM KRIBB11 or RSL3 and KRIBB11. mRNA was isolated and PROM2 expression was quantified by qPCR. Shown are three independent experiments with standard deviation (n=3 experiments per group). P‑values were obtained by unpaired Student's t‑test with *P<0.05, **p<0.01, ***p<0.005. Exact p‑values are reported in the Appendix Table S1. MCF10A cells were treated for 60 minutes with either DMSO, 25uM 4HNE, 5uM RSL3, 4HNE and 10uM KRIBB11 or RSL3 and KRIBB11. mRNA was isolated and HSF1 expression was quantified by qPCR. Shown are three independent experiments with standard deviation (n=3 experiments per group). P‑values were obtained by unpaired Student's t‑test with *P<0.05, **p<0.01, ***p<0.005. Exact p‑values are reported in the Appendix Table S1. MCF10A cells were plated on slides pre-coated with laminin. Cells were treated for 60 minutes with either EtOH, DMSO, 25uM 4HNE, 5uM RSL3, 4HNE and 10uM KRIBB1 or RSL3 and KRIBB11. Cells were stained for total HSF1 and counterstained with DAPI. Images were taken at 20x magnification. Scale bar is 50 microns. Shown is one replicate of three independent experiments (n=3). MCF10A cells were treated for 60 minutes with either EtOH, DMSO, 25uM 4HNE, 5uM RSL3, 4HNE and 10uM KRIBB11 or RSL3 and KRIBB11. Isolated protein was assessed by immunoblotting for prominin2, phospho‑HSF1 (Ser326), total HSF1, phospho-p38 (Thr180/Tyr182), total p38, and β-actin expression. Shown is one replicate of three independent experiments (n=3). MCF10A cells were treated with either RSL3, FIN56 or IKE at the range of concentrations shown with either DMSO or 10uM KRIBB11. Cells were assessed for viability after 24 hours. Absorbance was normalized to DMSO control. Shown are three independent replicates with standard deviation (n=3 experiments per group). P‑values were obtained by unpaired Student's t‑test with *P<0.05, **p<0.01, ***p<0.005. Exact p‑values are reported in the Appendix Table S1. HMLE cells were treated with IKE at the range of concentrations shown with either DMSO or 10uM KRIBB11. Cells were assessed for viability after 24 hours. Absorbance was normalized to DMSO control. Shown are three independent replicates with standard deviation (n=3 experiments per group). P‑values were obtained by unpaired Student's t‑test with *P<0.05, **p<0.01, ***p<0.005. Exact p‑values are reported in the Appendix Table S1. S1 cells were treated with IKE at the range of concentrations shown with either DMSO or 10uM KRIBB11. Cells were assessed for viability after 24 hours. Absorbance was normalized to DMSO control. Shown are three independent replicates with standard deviation (n=3 experiments per group). P‑values were obtained by unpaired Student's t‑test with *P<0.05, **p<0.01, ***p<0.005. Exact p‑values are reported in the Appendix Table S1.
Figure 2A Doubling time in pRH6 versus 2a2iL-RH6. **P<0.01 (t-test). Data presented as mean ± SD (n=3). B Cloning efficiency in the presence (+) and absence (−) of ROCK inhibitor (ROCKi) in pRH6 versus 2a2iL-RH6. ROCKi (−)*P<0.05, ROCKi (+) *P<0.01 (t-test), data presented as mean ± SD (n=3). C Firefly luciferase expression of constructs that contain distal and proximal enhancers of OCT4 after transfection of pRh6 and 2a2iL-RH6. The ratio of firefly luciferase expression to Renilla luciferase expression (expressed from co-transfected plasmid) measured 48 h after transfection showed proximal enhancer activity in pRH6 and distal enhancer activation in 2a2iL-RH6. OCT4 PE-Luc ***P<0.001, OCT4 DE-Luc *P<0.05 (t-test). Data presented as mean ± SD (n=3). D qRT-PCR analysis of pluripotency-related genes in pRH6 and 2a2iL-RH6. *P<0.05, **P<0.01, ***P<0.001 (t-test). Data presented as mean ± SD (n=3). E Immunofluorescence staining for several naïve specific markers in 2a2iL-RH6. Scale bar: 100 µm. F Western blot analysis of naïve-related pluripotency markers in pRH6 and 2a2iL-RH6. **P<0.01, ***P<0.001 (t-test). Data presented as mean ± SD (n=3). G Immunofluorescence staining against pRH5 and 2a2iL-RH5 (46, XX) for H3K27me3. Nuclei were stained with 4′, 6-diamidino-2-phenylindole (DAPI). Reactivation of inactive X-chromosome in female 2a2iL-RH5 was confirmed by the lack of H3K27me3 nuclear foci. Scale bar: 50 µm.
Figure 3(A) Motif-wise combinatorial mutations within the core promoter: motif strength and motif position are changed individually. From top to bottom: knockout of motifs (individual or pairwise knockout of motifs, and knockout of all motifs); replacing the original motif with its computationally (XXmotif) derived sequences with different PWM scores (consensus with the highest score), or insertion of the consensus into the motif-less promoter sequences; point mutation of motifs; substitution with functionally or positionally equivalent motifs from other architectures; shift of motif positions; sequence context exchange between different core promoters. The Mec2 motif composition is shown here as an example. (B-C) Comparison of normalized expression levels between wild-type configuration and motif knockouts for two types of core promoters (developmental: CG8157 (B); constitutive: RpL5 (C)). Upper panels: schematic depiction of the wild-type motif compositions (TTGTT motif in RpL5 is ignored due to its strong overlap with R-INR). Two-sample t-test: ns, not significant, *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001; ****p ≤ 0.0001. The middle hinge represents the median. The Interquartile range the difference between the 75th and 25th percentiles. 3-4 biological replicate measurements. (D) Mean expression fold changes compared to wild-type expressions for individual knockout of motifs in different core promoters. Constitutive and developmental promoters are highlighted in red and green, respectively. (E-F) Effect of pairwise motif knockout (log2 scale) in core promoters CG7712 (E) and pain (F), respectively. The heatmaps display the mean expression fold changes compared to wild-type expressions for pairwise knockout of motifs compared to individual knockouts (diagonals). Additivity was calculated as the difference between the pairwise effect and the sum of two individual effects, Subadditive (in blue): > 0; superadditive (in yellow): < 0; Additivity values for effects > 3×SDnoise shown in the right lower corner of each pairwise effect.
Figure 4(A) Trypan blue counting was used to analyze growth curves for P493-6 cells that expressed NTC or SPI1 shRNA or PHF12 shRNA. ** P<0.01 as compared between indicated groups. (mean ± SD, n = 4 biological replicates, Student's t‐test) (B, C) Trypan blue counting was used to analyze growth curves for P493-6 cells overexpressing ALKBH5 and further infected with viruses expressing SPI1 (B) or PHF12 (C). ** P<0.01 as compared between indicated groups (mean ± SD, n = 4 biological replicates, Student's t‐test). (D, E) Raji cells stably expressing EV or MYC were infected with viruses expressing shALKBH5. Cells were injected subcutaneously into nude mice (n=5 for each group). Tumor growth curves were measured starting from 12 days post injection (D). Photo of tumors collected at the end of the experiment (day 27) (E). Data are presented as mean (±SEM). ** P<0.01 or *** P<0.001 as compared between indicated groups (Student's t‐test). (F P493-6 cells stably expressing NTC or shSPI1 or shPHF12 were injected subcutaneously into nude mice (n=5 for each group). Tumor growth curves were measured starting from 12 days post injection (F). Data are presented as mean (±SEM). ** P<0.01 as compared between indicated groups (Student's t‐test). G) P493-6 cells stably expressing NTC or shSPI1 or shPHF12 were injected subcutaneously into nude mice (n=5 for each group). Photo (G) of tumors collected at the end of the experiment (day 25). (H, I) P493-6 cells stably expressing EV or ALKBH5 were infected with viruses expressing SPI1 or PHF12. Cells were injected subcutaneously into nude mice (n=5 for each group). Tumor growth curves were measured starting from 17 days post injection (H). Photo (I) of tumors collected at the end of the experiment (day 32). Data are presented as mean (±SEM). * P<0.05 as compared between indicated groups (Student's t‐test). (J, K) Representative IHC images (J), and analysis (K) of MYC, ALKBH5, SPI1 and PHF12 expressions in normal lymphocyte tissue (normal) and lymphoma specimens. Scale bar: 50 um. Data are presented as mean (±SEM), clinical samples n = 30 for normal group, n = 28 for low MYC expression group, n = 27 for high MYC expression group, P value was presented between indicated groups (Student's t‐test).
Figure 3The promoter design is based on the CYC1 core promoter. The relevant structural elements of the CYC1 core promoter elements, which are required for transcription, were conserved in the synthetic promoter design. These elements comprised two TATA boxes at position -221 and -117 (relative to the start of the CYC1 ORF), and the 5'UTR of the CYC1 core promoter (including transcriptional start site, TSS). In the promoter design, three CggR operator sites were inserted adjacent to the two TATA boxes. All functional elements were conserved (blue colored region) during the optimization of the promoter sequence. Nucleotide sequences between the functional elements (grey colored region) were allowed to be optimized by the algorithm. Nucleotides that got optimized are indicated with a black line. A total of 75 sequence versions were generated, where each sequence differed in one mutation from the progenitor sequence. The sequences were optimized for low nucleosome affinity. After optimization, all sequences were checked for synthesis feasibility. The synthesis of the sequences was feasible (green) until the 46th round. After this round, the sequences (not feasible in red) reached a GC content insufficient for proper synthesis. The promoter sequence, which was generated in round 38 (black), showed the best compromise between minimal nucleosome affinity and the possibility to synthesize the sequence.
Dynamic composition of oskar mRNPs ex vivo.Figure 3(A, B) Kymographs of oskMS2-GFP mRNPs (green) associated with Khc-mKate2 (A, red) and mCherry-Tm1-I/C (B, red) ex vivo. Arrows indicate motile RNPs in stable complex with Khc (A) or Tm1-I/C (B), the arrowheads point to non-motile oskMS2-RNPs showing no obvious accumulation of the tagged protein. Note that mCherry-Tm1-I was exposed twice as long as Khc-mKate to obtain comparable red fluorescence signals. Scale bars represent 1 µm and 1 second, respectively.(C-E) Relative Khc-mKate2 (C), mCherry-Tm1-I/C (D) coverage of motile (white) and non-motile (grey) oskMS2-GFP trajectories. Numbers within the boxes indicate the number of trajectories and the number of ooplasms (in brackets) analysed. Percentages above the plots show the fraction of RNPs that were found stably and reliably associating with the indicated protein (for at least half of the duration of the trajectory, p<0.01, binominal distribution, see also Fig EV2B). P values of pairwise Mann-Whitney U tests are indicated above the boxplots. (C-E) Relative GFP-Staufen (E) coverage of motile (white) and non-motile (grey) oskMS2-GFP trajectories. Numbers within the boxes indicate the number of trajectories and the number of ooplasms (in brackets) analysed. Percentages above the plots show the fraction of RNPs that were found stably and reliably associating with the indicated protein (for at least half of the duration of the trajectory, p<0.01, binominal distribution, see also Fig EV2B). P values of pairwise Mann-Whitney U tests are indicated above the boxplots. (F) Example kymograph of Khc molecules (red) associating with an oskMS2-GFP mRNP (green) before and during its run. White arrowheads indicate two association events, yellow arrow indicates the onset of motility. Scale bars represent 5 µm and 1 second, respectively.(G) Frequency of Khc-mKate2 appearance on motile (white), before (motility-primed, checked) and after (dotted) the onset of motility, and non-motile (grey) oskMS2-GFP trajectories. Fractions within the bars indicate number of association events that lasted longer than a single frame over the total number of frames analysed. Indicated P values show results of pairwise Fisher's exact test. The Khc association frequency observed on RNPs before (checked) and during (dotted) their motility is not significantly different from wild-type motile RNP controls (p>0.01).
Figure 6(A-B') Lateral (A) and ventrolateral (B, B') images of the cranial vessels in kdrl:nlsKaede;kdrl:EGFP embryos, with unconverted tissue shown in green (A-B) while photoconverted tissue is shown in red (B'). The early kdrl-positive VA-L was photoconverted in 34 hpf embryos (A), and followed through to 54 hpf (B, B'). B' is a higher magnification image of the box in B and is accompanied by a red channel only (traced photoconverted cells) image with the VA-L (dotted line) demarcated from the adjacent VA. The VA-L photoconverted cells are indicated by arrowheads. (C) Still images from Movie EV5 of VA-L development in a kdrl:EGFP;kdrl:nlsmCherry compound transgenic from 31-35.5 hpf. This movie shows that the VA-L begins expressing kdrl just after 32 hpf, in a manner that is isolated and independent of any neighbouring kdrl-positive tissue or vessel. Putative kdrl-positive primitive myeloid cells (light blue asterisks) can also be seen migrating along the surface of the yolk in the immediate vicinity of the VA and VA-L. (D) Still images from Movie EV6 depicting a 3D reconstruction of the 35.5 hpf timepoint from Movie EV5. A dorsal view that scrolls through the Y-stack of the VA-L is shown, which illustrates that the VA-L is closely associated with, but not connected to the VA. (E-E') Still images from Movie EV7 of VA-L development in an etv2:EGFP (E), kdrl:nlsmCherry (E') compound transgenic from 28-35.2 hpf. This movie shows that the VA-L begins expressing etv2 (E) at 29 hpf and is independent of any neighbouring vessel. kdrl expression (red arrowheads) occurs 4 hours later in the VA-L (demarcated by the green dotted line) (E'). (F-G') Lateral images of the cranial vessels in etv2:Kaede embryos, with unconverted tissue shown in green (F-G) while photoconverted tissue is shown in red (G'). The earliest visible form of the VA-L was photoconverted at 29 hpf (F), then followed through to 54 (G). G' is a higher magnification image of the box in G and is accompanied by a red channel only (traced photoconverted cells) image with the VA-L (dotted line) demarcated from the adjacent VA, and VA-L photoconverted cells indicated (arrowheads).
Figure 3Effects of Ttyh1 expression on the generation of NICD at 2 days posttransduction of E14.5 neural progenitor cells were analyzed by Western blotting using an anti-NICD antibody. Quantification of band intensities Effects of Ttyh1 knockdown (C) on the generation of NICD at 2 days posttransduction of E14.5 neural progenitor cells were analyzed by Western blotting using an anti-NICD antibody. (B, D) Quantification of band intensities Schematic representation of the γ-secretase reporter assay. Following cleavage at the γ-secretase site in Notch∆E-Gal4-VP16 (Notch∆E-GVP), the intracellular domain containing the GVP domain translocates into the nucleus and triggers luciferase expression driven by the UAS promoter Ttyh1-transduced E14.5 neural progenitors were incubated with DAPT for 2 days, then cells were harvested and lysates were used for luciferase assay (n = 3). Effects of Ttyh1-knockdown on γ-secretase activity was also assessed using E14.5 neural progenitor cells Ttyh1-transduced E14.5 neural progenitors were incubated for 2 days in the presence or absence of DAPT, and samples were prepared for Western blotting using anti-NICD antibody Ttyh1-transduced E14.5 neural progenitors were incubated for 2 days in the presence or absence of DAPT, and samples were prepared for qPCR for Notch target genes (I) The indicated plasmids were electroporated into E13.5 brains, and brain samples were harvested at E15.5 for anti-GFP immunolabeling. GFP and DAPI signals are shown in green and blue, respectively. Quantification of GFP+ cell localization in (J) dnPS1, dominant negative presenilin 1; VZ, ventricular zone; SVZ, subventricular zone; IZ, intermediate zone; CP, cortical plate. Scale bar: 100 μm.
Fig.Figure 2Phosphatidic Acid (PA) levels in HSA-Cre and control GC muscles. Phospholipidome in HSA-Cre and control GC muscles. Proportions of indicated phospholipid classes in total phospholipids are represented. PC; Phosphatidylcholine, PE; Phosphatidylethanolamine, PI; Phosphatidylinositol, PS; Phoshpatidylserine, PA; Phosphatidic Acid, PG; Phosphatidylglycerol. Amounts of indicated phospholipid classes normalized by protein content in HSA-Cre and control GC muscles. (D) Total phospholipid (PL) amount normalized by protein content in HSA-Cre and control GC muscles. Distribution of the total carbon number in acyl chains of PC in HSA-Cre and control GC muscles. Distribution of the number of double bonds in acyl chains of PC in HSA-Cre and control GC muscles. Distribution of the total carbon number in acyl chains of PE in HSA-Cre and control GC muscles. Distribution of the number of double bonds in acyl chains of PE in HSA-Cre and control GC muscles.
Figure 3(A) Expression of PARP1 and KLF4 in mammary gland epithelial cell and various types of breast cancer cell lines. (B). Accumulation of PARP1 and KLF4 protein were detected in human breast cancer tissues with 183 breast invasive ductal carcinoma and 10 pairs of primary cancer and adjacent normal tissue specimens by IHC in comparison with adjacent normal breast tissues. Scale bars, 100 μm. Statistic results of KLF4 IHC staining. Normal (n=10), tumor (n=183); p = 4.36x10-7 (KLF4) one-way ANOVA was used for the statistical analysis. Statistic results of PARP1 IHC staining. Normal (n=10), tumor (n=183) p = 0.0473 (PARP1). one-way ANOVA was used for the statistical analysis. (E) Statistical analysis of PARP1 staining among normal (n=10), ER/PR positive (ER/PR) (n=68), HER2 positive (n=62), and triple negative breast cancer (TNBC) (n=48). The p values were labeled in figure, one-way ANOVA was used for the statistical analysis. (F) Statistical analysis of KLF4 protein staining among normal (n=10), ER/PR positive (n=68), HER2 positive (n=62) and TNBC (n=47). The p values were labeled in figure, one-way ANOVA was used for the statistical analysis. Elevated expression of PARP1 and KLF4 are significantly correlated in 183 breast invasive ductal carcinoma human breast cancer tissue specimens (same batch of staining as (B)). Represents of paired IHC staining of PARP1 and KLF4 (case 1-3) are shown (G). Scale bars, 100 μm. (H) Statistic analysis of IHC staining indicates that PARP1 expression is positively correlated with KLF4 expression in breast cancer. n=183, r=0.434, p=8.68x10-10, Pearson correlation coefficients. (I) Survival analysis of KLF4 protein expression in 117 breast cancer patients. Compared to patients with low KLF4 protein expression, patients with high KLF4 levels had an inferior cumulative survival rate (Kaplan-Meier assay, LogRank P=0.012); Low, staining weak and moderate; High, staining strong. (J) Survival analysis of both KLF4 and PARP1 protein expression in 66 breast cancer patients. Compared to patients with both low KLF4 and PARP1 protein expression levels, patients with both high KLF4 and PARP1 levels had lower cumulative survival rate (Kaplan-Meier assay, LogRank P=0.022).
Genetic evidence for cyclin D1b as a credentialed oncogene.Figure 3Murine adult fibroblasts (MAFs) were harvested from the peritoneum of Ccnd1+/+ and Ccnd1KI/KI mice, and stable cell lines were generated utilizing a 3T3 protocol. Indicated passage-matched MAF lines were grown to 70% confluency and then harvested for RNA and protein extraction. Top: qPCR analysis of cyclin D1 transcript using primer pairs common to both transcript a and transcript b. Bottom: Immunoblot of cyclin D1 levels in Ccnd1+/+ and Ccnd1KI/KMAF lines using an antibody common to both isoforms. Percentage of tumor-free mice post-injection of 1 million cells mixed with Matrigel (1:1 ratio) into the flanks of nude mice over a period of 10 months (n = 10 per genotype). Indicated tumors were harvested 3 weeks after detectable tumor formation, fixed in formalin, and stained with H&amp;amp;E. Tumors were analyzed for features of malignancy by a board-certified veterinary pathologist. Arrows indicate the specific tumor-associated features noted. Top panels were taken at 40× magnification, and lower panels were taken at 400× magnification. Ccnd1+/+ or Ccnd1KI/KI MAF lines were stably transfected with either cyclin D1a (+/+), cyclin D1b (KI/KI) or vector control constructs. After selection with puromycin, individual lines were assessed for the induction of cyclin D1a in the +/+ line and cyclin D1b in KI/KI lines via immunoblot. One million cells from the indicated stable cell lines were injected subcutaneously into the flanks of nude mice and monitored for tumor formation over a period of 365 days. Indicated time points represent time of palpable tumor detection and are plotted as % of tumor-free mice over time. Sections from KI/KI vec and KI/KI-D1b tumors were stained for the proliferative marker Ki67. Three random fields from each tumor were quantified for Ki67 positivity, and are plotted as the mean ratio of Ki67-positive cells/total cell number for each individual tumor.