EMBO/sd-character-level-ner huggingface dataset:
Figure 4(A) Overview of a reconstructed superresolution image of GFP-Bax overexpressing HCT116Bax/Bak -/- cells stained with AF647-anti-GFP-nanobodies Image was acquired on fixed cells 7 h after apoptosis induction with STS. Dotted line shows the cell shape (see Fig EV3). Scale bar, 5 µm.(B) Gallery of typical GFP-Bax WT non-random structures during apoptosis on HCT116Bax/Bak -/- cells. Scale bars, 100 nm. (C) Comparison of confocal (left) and STED (right) images of GFP-Bax overexpressing HeLa cells 2h after STS treatment. STED reveals a notable increase in resolution. Scale bars, 500 nm.(D) Representative GFP-Bax non-random structures found with STED (lines, arcs, rings, double lines). Scale bars, 100 nm.
AtNTR1 is present at the DOG1 geneAtNTR1 antibodies were used to analyse AtNTR1 protein presence at DOG1 locus using ChIP.Figure 3AtNTR1 antibodies were used to analyse AtNTR1 protein presence at DOG1 locus using ChIP. Data shown represent enrichment above background level measured in atntr1‐1 mutant. Gene structure is shown with black boxes representing constitutive exons; grey box, alternative region; white box, promoter region; black lines, introns. Red lines show amplified regions. 0.5 kb scale is shown. Error bars represent ± SD of three independent experiments. As an additional negative control, primers amplifying an unlinked intergenic region (IGR) were used.
Methylation activity of BREX A Consensus sequence around m6A modified bases in the BREX‐containing B.Figure 4A Consensus sequence around m6A modified bases in the BREX‐containing B. subtilis genome. The modified base is marked by an arrow. Modifications were directly detected by DNA sequencing using the PacBio platform. B Statistics of modified motifs in the BREX‐containing B. subtilis genome. C, D Culture dynamics of non‐infected (C) or phage‐infected (D) cultures. Curves depict culture dynamics of strains lacking BREX (black) and BREX‐containing (red) strains of B. subtilis BEST7003, as well as a BREX‐containing strain where the pglX methylase was deleted (green). Axes and error bars are as in Fig . E Southern blot analysis of phage Φ3T genome during infection. Numbers indicate time (in min) following infection; U, uninfected. Probe was designed to match positions 94,645-95,416 in the phage genome. Each lane contains 200 ng total DNA.
Neurodegeneration by mutations in ft.Figure 4(A) Tangential eye sections through ftfd clones aged 1, 7 and 14 days, of ftfd,dDGC13 double mutant clones and of ftfd clones in a ykiB5 heterozygous background aged 14 days. Clones are marked by the absence of yellow pigment. Arrows point at degenerating photoreceptors, arrowheads point at intact wt photoreceptors in mosaic ommatidia. The circled ommatidium is genetically wt but non‐autonomously flipped in its polarity and has not degenerated. Lower panels are masks that show clonal borders and quantification of PR number per ommatidia. After 14 days, almost all ftfd cells have degenerated, whereas almost all ftfd,dDGC13 and some ftfd,ykiB5/+ neuronalphotoreceptors have not. (B) Histograms showing the quantification of degeneration in ftfd, ftfddDGC13 and ftfdykiB5/+ clones. Loss of ft leads to statistically significant loss of neurons and this is suppressed at 14 days by ykiB5and much more dramatically by dDGC13. ***P0.001, **P0.01; *P0.05 in two‐tailed t‐test. (C) Degeneration in mosaic ommatidia in ftfd clones. At this stage of degeneration approximately half of the non‐pigmented (genotypically mutant) PR cells display a degenerative phenotype, whereas virtually all pigmented (genotypically wt) PR cells are normal. χ2‐test: 82.38; P0.001. Total PR N=310 (pigmented) and 327 (not pigmented). (D) Tangential eye sections through MARCM clone mutants for ftfd that express either no transgene or an RNAi construct against yki (UAS-ykiIR) with Tub-Gal4 and aged 14 days at 29°C. Because the transgene UAS-ykiIR is on chromosomal arm 2 L, the same as ft, clones generated with this system carry two copies of UAS-ykiIR and therefore downregulate yki very effectively. No other cell outside the clones expresses the construct and is therefore wt for yki. Mask panel is presented on the right of each section. A quantification of photoreceptor numbers (far right) displays a very significant increase in the number of wt ommatidia (N=210 and 219 from four eyes). ***P0.001 in two‐tailed t‐test. If all classes of ommatidia are considered, χ2‐test=232.30; P0.001 for 3 degrees of freedom.
The membrane-binding helix is critical for autophagy GFP-Atg8 processing is monitored for either the TRAPP core binding mutations or the amphipathic helix (membrane binding) substitutions.Figure 7GFP-Atg8 processing is monitored for either the TRAPP core binding mutations or the amphipathic helix (membrane binding) substitutions. With functional Trs85, there is an accumulation of free GFP. Samples are from pre-rapamycin treatment (-) and 1hr after addition of rapamycin (+). Representative of n=3 independent experiments. (WT = wild-type, EV = empty vector)   Quantification of A. The average of at least three replicates is plotted with error bars representing the 95% confidence interval. The percent of free GFP for wild-type Trs85 is compared to each mutant using an unpaired, two-tailed Student's t-test with Welch's correction: vs. EV ** p =   0.0022, vs. R618E, R619E, R620E, K621E *** p = 0.0008, vs. R618A, R619A, R620A, K621A *** p = 0.0006, vs. 501-698 *** p = 0.0003, vs. 4A p = 0.3295, vs. 7A *** p = 0.0008, vs. 4E *** p = 0.0005, ns = not significant. (WT = wild-type, EV = empty vector) Model for TRAPPIII-mediated Rab1 activation in cells. Inactive Rab1-GDP/GDI exists in an equilibrium where Rab1-GDP samples various organellar membranes. TRAPPIII is recruited to specific membranes containing negative charges (red circles) and utilizes an amphipathic helix to bind stably to the membrane surface. Rab1 is locally activated by TRAPPIII in order to recruit downstream effectors to facilitate trafficking or autophagic events.
Figure 5(A Wip1 and Smad4 form an endogenous protein complex. Cell lysates of HEK293T cells, which were stimulated or not with Activin (10 ng/ml), TGF-β1 (40 ng/ml) or BMP4 (20 ng/ml) for 1 h, were immunoprecipitated with anti-Smad4 antibody followed by western blotting with anti-Wip1 antibody (A) (-), no treatment with protein ligand. IgG, control IgG. An arrow in (A) indicates non-specific bands. B) Wip1 and Smad4 form an endogenous protein complex. Cell lysates of HEK293T cells, which were stimulated or not with Activin (10 ng/ml), TGF-β1 (40 ng/ml) or BMP4 (20 ng/ml) for 1 h, were immunoprecipitated with anti-Smad4 antibody followed by western blotting with anti-Wip1 antibody (A) and vice versa (B). (-), no treatment with protein ligand. IgG, control IgG. (C) Upon ligand stimulation, Wip1 forms a complex with Smad4 in the nucleus. HeLa cells were treated or not with TGF-β1 (40 ng/ml) for 2 h, fixed and then subjected to proximity ligation assays with primary antibodies against Wip1 and Smad4. Blue: DAPI; Red: PLA signal. Scale bar, 20 μm. (D) Quantitative analysis of Wip1/Smad4 interactions shown in (C). Data are represented as the mean ± SEM (n=3 biological replicates). Twenty cells were analyzed per sample. ***P<0.001 by Student's t-test. (E) Wip1 interferes with the ability of Smad4 to bind to Smad2. HEK293T cells co-transfected with the indicated constructs were treated or not with TGF-β1 for 1 h and then harvested. An arrow denotes Myc-tagged Wip1 protein. (F) Wip1 has a negative effect on nuclear accumulation of Smad4. HEK293T cells were transfected with Co siRNA or Wip1 siRNA, treated with TGF-β1 (40 ng/ml, 1 h) 48 h after siRNA transfection and subjected to nuclear/cytoplasmic fractionation and western blotting analysis. β-tubulin and lamin A/C serve as cytoplasmic and nuclear markers, respectively. C, cytoplasm; N, nucleus. The experiment was repeated two times with similar results. (G) Quantification of the levels of nuclear Smad4 normalized to those of cytoplasmic Smad4 shown in (F).
Red1 co‐localizes and interacts with Mmi1 in vegetative cells.Figure 3(A) Red1 foci coincide with Mmi1 dots. Deconvolved images of a strain expressing both Red1‐tdTomato and CFP‐Mmi1 are shown. Bars, 2 μm. (B) Red1 co‐immunoprecipitates with Mmi1. Total cell lysates from strains expressing CFP‐Mmi1, Red1‐myc, and Red1‐myc/CFP‐Mmi1 were subjected to immunoprecipitation, followed by western blotting. (C) Red1 and Mmi1 have common target genes. RT-PCR of four DSR‐containing genes (mei4+, ssm4+, rec8+, and spo5+) indicates that either a red1 deficiency or a mmi1 mutation (mmi1‐619) resulted in increased levels of these meiotic mRNAs. (D) Meiotic mRNAs accumulate in conditional mmi1Δ cells. Total RNA samples from mei4‐P527, red1Δmei4‐P527, and mmi1Δmei4‐P527 cells were subjected to RT-PCR analyses. RT-PCR of mei4+, mcp5+, and SPBPB2B2.03c indicates that red1Δ or mmi1Δ in a mei4‐P527 background results in increased levels of these meiotic transcripts. Note that mmi1+ was deleted in the mei4‐P527 mutant strain because mei4 deficiency suppresses the severe growth defect caused by mmi1Δ.
Figure 8Immunofluorescence analysis of TDP-43 in sagittal brain sections. Representative images of indicated brain regions (CTX = Cortex; MB = Midbrain). White arrow heads indicate TDP-43 aggregation. Scale bar indicates 100 µm. Zoom in on TDP-43 aggregation in double knockout mouse brain sections of the midbrain, scale bar zoom in indicates 50 μm. (Animals 4.5 months of age). Immunofluorescence analysis of total TDP-43 and phosphorylated TDP-43 (p-TDP-43) in sagittal brain sections by confocal microscopy. Representative images of one confocal layer in the midbrain. Zoom in on TDP-43 aggregation in double knockout mouse brain sections of the midbrain. Scale bar indicates 25 µm. (Animals 4.5 months of age). Western blot analysis of TDP-43 and phosphorylated TDP-43 (p-TDP-43) in sequential high salt (HS)/RIPA/Urea fractionations of total brain lysates from 4.5-month-old mice with the indicated genotype. Analysis of C-terminal fragments of TDP-43 (CTF) in RIPA fraction. p-TDP-43 analyzed in Urea fraction. Quantification of (C) Protein expression was normalized to levels in wild type animals. Data represent the mean ± SD.
Figure 4(A) Control, CITK or ASPM-depleted HeLa cells immunostained for α-tubulin (red) and DNA (blue). Maximum intensity projections of confocal z-stacks are shown.(B) Quantification of astral MT number and maximal length in HeLa cells transfected with CTRL, CITK or ASPM siRNA (n > 130 cells in 3 independent experiments). (C) Quantification of the rescue of astral MT number after CITK overexpression in ASPM-depleted HeLa cells (n > 50 cells in 3 independent experiments).(D) Quantification of spindle orientation rescue after overexpression of CITK, CITN (lacking the kinase domain) or CKD (Citron kinase dead) mutants in ASPM-depleted HeLa cells (n > 50 cells in 3 independent experiments). (E) Synchronized HeLa transfected with control, ASPM- or CITK-specific siRNAs treated with DMSO or with 250 pM Paclitaxel (taxol) 30 min before immunostaining for α-Tubulin. Spindle angles were measured as above (n > 100 cells in 6 independent experiments).
Figure 3(A) Serum insulin of WT and Pdia4tg/tg (TG) mice on BKS or Leprdb/db background at the indicated ages, was measured using an ELISA kit. Data information: Data from 3 experiments more One-way ANOVA test was used for statistical analysis of differences between groups, and P (*) < 0.05; P (**) < 0.01 and P (***) < 0.001 are considered statistically significant. The number of mice (n) is indicated in parentheses. (B) Pancreata of the same batch of mice as in (A), at the age of 14 weeks, were stained with anti-insulin (αIns) antibody and DHE (left). Islet area (μm2) and relative fluorescence intensity (RFI) were quantified (right). Scale bar = 100 μm. The dash circles indicate islet regions. Data information: Data from 3 experiments more are presented as the mean ± SD. One-way ANOVA test was used for statistical analysis of differences between groups, and P (*) < 0.05; P (**) < 0.01 and P (***) < 0.001 are considered statistically significant. (C) Serum ROS of the mice from (A) were determined. Data information: Data from 3 experiments more are presented as the mean ± SD. One-way ANOVA test was used for statistical analysis of differences between groups, and P (*) < 0.05; P (**) < 0.01 and P (***) < 0.001 are considered statistically significant. (D) The islets of the mice from (C) were tested for GSIS. High glucose (HG, 16.7 mM) or low glucose (LG, 3.3 mM) were used in GSIS assays. Data information: Data from 3 experiments more are presented as the mean ± SD. One-way ANOVA test was used for statistical analysis of differences between groups, and P (*) < 0.05; P (**) < 0.01 and P (***) < 0.001 are considered statistically significant. (E) Pancreatic islets of WT, Pdia4-/- (KO) and Pdia4tg/tg (TG) BKS mice, which received an intraperitoneal dose of glucose (1 g/kg body weight) for 0.5 h, were isolated and divided into 2 aliquots. One aliquot of the islets was stained with anti-insulin antibody and measured for their basal insulin content using flow cytometry analysis (0 h). The other aliquot of the islets were grown in a complete medium for 24 h and their insulin content was measured (24 h). Data information: Data from 3 experiments are presented as the mean ± SD. One-way ANOVA test was used for statistical analysis of differences between groups, and P (*) < 0.05; P (**) < 0.01 and P (***) < 0.001 are considered statistically significant.
Arp2/3 dependent actin accumulation at the centrosome.Figure 3Maximum projection (2-z slices) view of Hela cells pre-treated with DMSO and 0.2mM CK666 for 15 mins during their mitotic exit showing that treatment with CK666 leads to reduced accumulation of actin around the centrosomes during anaphase. Scale bar-10µm. Quantification of actin around centrosomes for cells treated with DMSO or 0.2mM CK666, showing the reduction in actin accumulation around the centrosomes following CK666 treatment. DMSO-metaphase-1±0.4325, n=54, CK666-metaphase-0.936±0.4604, n=43, DMSO-anaphase-1.8±0.9736, n=76, CK666-anaphase-1.087±0.4597, n=73; one-way ANOVA, p<0.0001. Date pooled from 3 independent experiments. Z-projection of HeLa cells expressing GFP-centrin1 pre-treated with DMSO or 0.2mM CK666 during prometaphase arrest and forced mitotic exit and stained with phalloidin for F-actin. Quantification of the level of actin around the centrosome from (C), which shows the reduction in actin accumulation around centrosomes following CK666 pre-treatment. DMSO-STLC= 1 ± 0.02768, n=99, CK666 STLC= 0.776 ± 0.02186, n=87, DMSO-RO-3306= 1.339 ± 0.03048, n=127, CK666-RO-3306= 0.7699 ± 0.02246, n=70, one-way ANOVA, p<0.0001. Quantification of tubulin around centrosomes for cells treated with DMSO or 0.2mM CK666, showing the failure to reduce tubulin density around centrosomes following CK666 treatment during bipolar exit. DMSO-metaphase-1±0.1968, n=54, DMSO-anaphase-0.867±0.2345, n=78, CK666-metaphase-0.7995±0.1275, n=43, CK666-anaphase-0.7407±0.1859, n=74; one-way ANOVA, p<0.0001. Data pooled from 3 independent experiments. Error bars indicated standard deviation. Quantification of tubulin around centrosomes for cells treated with DMSO or 0.2mM CK666, during monopolar exit, showing the failure to reduce tubulin density around centrosomes following CK666 treatment. Data from three independent experiments. Prometaphase arrest (p.a.) 1±0.34 , n=27; forced mitotic exit (f.m.e) 0.78±0.30, n=26; p.a. plus CK666 0.49±0.34, n=30 ; f.m.e plus CK666 0.92±0.37, n=30. One-way ANOVA with Sidak's multiple comparisons test.
Myosin VI depletion leads to increased OFD1 and Cep164 recruitment at the centrioles.Figure 2(A) Total lysates from hTERT-RPE1 cells were IP with anti-myosin VI antibody and an anti-rabbit IgG antibody (as control). IB was performed with anti-OFD1 and anti-myosin VI antibodies. (B) A scheme of the IF analysis performed to calculate the total intensity of OFD1 staining at the mother and daughter centrioles. (C, IF analysis of centriole-associated OFD1 signal. hTERT-RPE1 cells were transfected with siRNA against myosin VI. Four days after transfection, cells were treated with nocodazole (1 hour, 6 μg/ml) and immunostained with anti-OFD1, anti-centrin1 and anti-Cep164 antibodies. Mother centrioles were identified by the coincident staining of centrin1 and Cep164, while daughter centrioles were centrin1-only stained. A scheme of the position of the markers used is depicted above. (C) Representative images, scale bar, 1 μm. D) Quantification of OFD1 intensity at the mother or daughter centrioles. Results are expressed as fold change with respect to mock average intensity. Bars represent mean ± SD. Mother centrioles: Mock, n=148 cells; MyoVI KD, n=199 cells, from four independent experiments. Daughter centrioles: Mock, n=101 cells; MyoVI KD, n=148 cells, from three independent experiments. **** P<0,0001 by Mann-Whitney test. (E) IB analysis of hTERT-RPE cells treated with the indicated siRNAs with anti-myosin VI and anti-OFD1 antibodies. Anti-GAPDH was used as loading control. (F-G) IF analysis of Cep164 signal. hTERT-RPE1 cells were transfected with siRNA against myosin VI and/or OFD1. Four days after transfection, cells were treated with nocodazole (1 hour, 6 μg/ml) and immunostained with anti-OFD1, anti-centrin1 and anti-Cep164 antibodies. (F) Representative images. Scale bar, 1 μm. (G) Quantification of Cep164 intensity at the mother centrioles. Results are expressed as fold change with respect to mock average intensity. Bars represent mean ±SD. Mock, n=147 cells; OFD1 KD, n=146 cells; MyoVI KD, n=150 cells; MyoVI + OFD1 KD, n=151 cells, from four independent experiments. ** P<0,005; **** P<0,0001 by Kruskal-Wallis test.
Figure 4(A) Immunoblot analysis of endogenous Tdp-43, C9orf72 and control Gapdh of E18 mouse cortical neurons transduced with lentivirus expressing either control shRNA or shRNA targeting C9orf72 mRNA. (B) Left panel, representative images of immunofluorescence labeling of endogenous phosphorylated Ser409/410-Tdp-43 on primary cultures of E18 mouse cortical neurons transduced with lentiviral particles expressing either control shRNA or shRNA targeting C9orf72. Right panel, quantification of cytoplasmic Tdp-43 aggregates. (C) Left panel, representative images of immunofluorescence labeling of D169G mutant GFP-tagged TDP-43 on primary cultures of E18 mouse cortical neurons transfected with either HA-tagged control or HA-C9ORF72 plasmid. Right panel, quantification of cells with cytoplasmic aggregates of TDP-43. Scale bars, 10 µm. Nuclei were counterstained with DAPI. Error bars indicate s.e.m. Student T-test, *** indicates p<0.001. n=3.
Figure 5A. Schematic representation of the experimental strategy. Four-month-old lethally irradiated mdx mice were transplanted with either GFP-CriptoMy-LOF (mdx-CriptoMy-LOF) or GFP-Control (mdx-Control) bone marrow and sacrificed at 16 weeks (wks) after transplantation. B. Representative pictures of immunostaining of mdx-Control and mdx-CriptoMy-LOF diaphragm sections with GFP (green) and CD206 (white; left panel), pSMAD3 (red; middle panel) and GFP and pSMAD3 (right panel). C-E. Quantification of GFP (C), GFP/CD206 (D) and GFP/pSMAD3 (E) positive cells per area (mm2) in mdx-Control and mdx-CriptoMy-LOF diaphragms. Data are expressed as box plots displaying minimum, first quartile, median, third quartile and maximum (n≥5 biological replicates; **P<0.01, Student's t-test). Data information: Nuclei were counterstained with DAPI (blue). Scale bar: 100 μm. Magnification of the boxes is 3.5 x. F-G. Representative pictures of Picrosirius red staining of mdx-Control and mdx-CriptoMy-LOF diaphragm sections (F) and quantification of picrosirius red staining (G). Data are expressed as percentage of stained area and are box plots displaying minimum, first quartile, median, third quartile and maximum (n=5 biological replicates; *P<0.05, Student's t-test). H. Hydroxyproline (HOP) concentration in mdx-Control and mdx-CriptoMy-LOF diaphragm muscle sections. Data are expressed as HOP levels (μg) per muscle section volume (mm3) and as box plots displaying minimum, first quartile, median, third quartile and maximum (n=5 biological replicates; **P<0.01, Student's t-test). Representative images of Laminin (red) immunostaining of mdx-Control and mdx-CriptoMy-LOF diaphragm sections of centrally nucleated myofibers. Data information: Nuclei were counterstained with DAPI (blue). Scale bar: 100 μm. Magnification of the boxes is 3.5 x. Representative mdx-Control and mdx-CriptoMy-LOF diaphragm sections minimal Feret's diameter distribution of centrally nucleated myofibers. Data are expressed as mean±SEM (n=5 biological replicates; *P<0.05; ***P<0.001; Student's t-test) Representative mdx-Control and mdx-CriptoMy-LOF diaphragm sections minimal Feret's diameter distribution Variance coefficient of centrally nucleated myofibers. box plots displaying minimum, first quartile, median, third quartile and maximum L-M. Representative images of eMHC (red) immunostaining of mdx-Control and mdx-CriptoMy-LOF diaphragm sections (L) and quantification of eMHC+ myofibers per area (mm2, M). Data are expressed as box plots displaying minimum, first quartile, median, third quartile and maximum (n=5 biological replicates; *P<0.05; Student's t-test). Data information: Nuclei were counterstained with DAPI (blue). Scale bar: 100 μm. Magnification of the boxes is 3.5 x.
Figure 1HMRs express RNAs. Heat maps show RNA expression levels in 16.5 dpc prospermatogonia. Only uniquely mapped RNA-seq reads were used for the analyses. metaplots (B) show RNA expression levels in 16.5 dpc prospermatogonia. Only uniquely mapped RNA-seq reads were used for the analyses. Positions (1)-(4) in (A and B) (green arrows) indicate the same positions (±2 kbp from the centers). The lower chart in (B) represents mappability defined as the percentage of unique sequences in all possible 50-nt sequences (see Appendix Figure S2B). TSS isoforms are included in 55798 TSSs. Low mappability at the centers of HMR_-L1s is due to high occurrence of repeat (retrotransposon) sequences. RPM, reads per million mapped reads.
binds specific mRNAs and its loss affects splicing.Figure 5Volcano plot (left) showing mass-spec data for proteins co-immunoprecipitating with HMGB1. Statistically-enriched HMGB1 interactors (orange dots) associate with the GO terms/pathways illustrated in the network analysis (right; node size reflects the number of proteins it includes; proteins are listed in Dataset EV4). Venn diagram showing 1/3 of HMGB1 interactors classifying as RNA-binding proteins (according to Castello et al, 2016). Genome browser views showing HMGB1 sCLIP data (black) along the ASH1L, CCNL2 and TET2 loci; input tracks (grey) provide background levels. *: significantly-enriched peaks. The consensus motif for HMGB1 binding on RNA is also shown (bottom right). Bar graphs showing genomic distribution of HMGB1 RNA-bound peaks (log2 enrichment). Bar plots showing relative occurrence of differential-splicing events in IMR90 undergoing senescence (left), in HMGB1-knockdown IMR90 (middle) or in HMGB1-bound mRNAs (right). The number of bound mRNAs (N) analyzed is indicated below each bar.   Venn diagram showing differential-splicing events shared between conditions from panel F.   Heatmaps showing GO terms/pathways associated with differentially-spliced mRNAs shared between senescence entry and HMGB1-knockdown from panel F. As in panel G, but for the 75 mRNAs also bound by HMGB1 in sCLIP data. Venn diagram (left) showing 15 HMGB1 interacting proteins from panel A are also downregulated upon both senescence entry and HMGB1-knockdown in IMR90. Of these, 12 are RBPs, 6 have been implicated in senescence (asterisks), and 4 are bound by HMGB1 in sCLIP data (arrows). Bar graphs showing mean fold-change of selected mRNAs (over ±S.D. from two biological replicates) from ILF3- (purple), RBMX- (white) or PNN-knockdown experiments (blue) in proliferating IMR90. *: significantly different to siRNA controls; P<0.01, unpaired two-tailed Student's t-test. Dashed line indicates no change in expression.