EMBO/sd-character-level-ner huggingface dataset:
Figure 2A) Description of RNA oligonucleotide sequences composed of multiple TDP-43 binding sites, A(GU)18, CLIP34 and (AUG12)3, NS, or A(CA)18 Sequence in different colors separates regions that accommodate one TDP-43 molecule, if known. NS, non-specific control RNA. B) TDP-43 condensates in the presence of the different RNA sequences. TDP-43 and RNA oligonucleotide concentration is 4 μM and 3.9 μM, respectively. Representative images for 3 biological replicates using 2 different protein preparations. Scale bars, 10 μm. C) LLPS measured by turbidity in the same experimental conditions as panel B. Mean and SD from 3 biological replicates with 2 different protein preparations. D) Lysate from HEK 293 cells expressing a GFP-tagged copy of either wild-type (WT) or F147/149/229/231L (F4L) TDP-43 was added to a mixture of recombinant WT or F4L TDP-43 (5.3 μM, 10% Cy3-labeled protein) and A(GU)6 (22.8 μM), A(CA)18 (7.6 μM), A(GU)18 (7.6 μM), or no RNA control at 250 mM NaCl. Droplets were imaged by brightfield and fluorescence microscopy. Representative images for 3 biological replicates using 2 protein preparations. Scale bars, 10 μm. E) LLPS measured by turbidity in the same experimental conditions as panel D. Mean and SD of 4 biological replicates using 2 recombinant protein preparations. Analyzed by one-way ANOVA (F(7,24)=69.30, P<0.0001). Sidak's multiple comparisons test was used to compare selected groups. *P<0.05, ****P<0.0001.
Nucleolar partitioning of PELP1 is controlled by the SUMO system.Figure 6(A) Flag‐SUMO2 was expressed in HeLa cells by transient transfection. Flag‐SUMO2‐expressing cells were visualized by indirect immunofluorescence. The localization of endogenous PELP1 was monitored in Flag‐SUMO2‐positive cells (green arrowheads) or untransfected cells (white arrowheads) using anti‐PELP1 antibody. (B-D) HeLa cells were transfected with siRNAs as indicated. After fixation and permeabilization, the localization of PELP1, LAS1L, TEX10 or PES1 was monitored by indirect immunofluorescence using the corresponding antibodies. (E) HeLa cells were depleted of endogenous PELP1 and reconstituted with siRNA resistant constructs expressing either wild‐type HA‐tagged PELP1 or a Flag‐tagged PELP1-SUMO2 fusion protein. The localization of the respective proteins was monitored by indirect immunofluorescence using either anti‐HA‐ or anti‐Flag‐antibodies. (F) The localization of LAS1L or SENP3 was analysed in cells expressing Flag‐PELP1-SUMO2. Indirect immunofluorescence was done using anti‐LAS1L or anti‐SENP3 antibodies. Nuclei were counterstained with DAPI.
Figure 3A Schematic showing the formation of control, MECP2-KO, and mosaic MECP2-KO/control neurospheres (WT83/Q83X cell lines were used). B, C Immunofluorescent staining (B) and quantitation (C) confirmed the graded decrease in MeCP2 expression from control to 50% MECP2 mosaic to full MECP2-KO neurospheres. Scale bar = 50 µm. D 8-week MECP2-KO neurospheres (N = 44) exhibited decreased size (diameter; **P < 0.01), but 50% MECP2-mosaic neurospheres (CTR/KO; N = 44) were unchanged from controls (one-way ANOVA, F2,150 = 6.03, P = 0.003; Dunnett's multiple comparisons test; N = 65). Left: representative brightfield images of neurospheres; Right: neurosphere size quantification. Scale bar = 200 µm. E Calcium imaging analysis showing calcium transients normalized by control (one-way ANOVA, F2,72 = 15.62, P < 0.0001; Dunnett's multiple comparisons test vs control: MECP2-mosaic, ***P < 0.001; MECP2-KO, ***P < 0.001). WT83/Q83X cell lines were used; N = 44 Q83X neurospheres and 65 WT83 neurospheres. F 8-week neurosphere size (diameter) remained unchanged by treatment with either drug (one-way ANOVA, F6,344 = 0.52, P = 0.79; N = 44 Q83X neurospheres and 65 WT83 neurospheres). G Cell viability was improved or unharmed in treated 8-week neurospheres (one-way ANOVA, F9,104 = 6.81; Dunnett's multiple comparisons test vs CTR/KO untreated: Nefi + PHA 1 µM, **P < 0.01 and Z = 2.974; Nefi + PHA 10 µM, ***P < 0.001 and Z = 3.733; N = 8-24 neurospheres per condition). H, I Calcium transient frequency (number of peaks in 10 mins recording) normalized by CTR/KO untreated (H; one-way ANOVA, F9,239 = 6.18; Dunnett's multiple comparisons test vs CTR/KO untreated: Nefi 1 µM, *P < 0.05 and Z = 0.780; Nefi + PHA 10 µM, ***P < 0.001 and Z = 1.972; N = 15-61 neurospheres per condition) and calcium transient amplitude normalized by CTR/KO untreated (I; one-way ANOVA, F9,241 = 1.27, P = 0.255; N = 16-62 neurospheres per condition). J, K MEA spike frequency heatmap (J) and quantification (K) normalized by CTR/KO untreated (one-way ANOVA, F5,38 = 1.21, P = 0.321; Dunnett's multiple comparisons test vs CTR/KO untreated: PHA, P = 0.65 and Z = 1.134; Nefi, P = 0.98 and Z = 0.06; PHA + Nefi, P = 0.86 and Z = 0.94; N = 7-8 wells per condition). 'X' on activity heatmap signifies absence of a neurosphere in that position.
Figure 6(a) SR predicts resistance to PD1/PDL1 and CTLA4 blockade in patients. Cross-validation accuracy of SR-based supervised predictors in predicting resistance to PD1 (Prat et. al. & Hugo et. al.) and CTLA4 blockade (Van Allen et al.), reported in terms of the corresponding Receiver Operating (ROC) curves. Expression of the predicted rescuer genes of PD1 (CTLA4) was used to train an SVM supervised predictor of PD1 (CTLA4) blockade. For comparison, we also display the ROC curves of supervised predictors trained on the expression of all genes (shown as "All") and on the expression of genes selected randomly and controlled for the number of rescuers predicted (shown as "Random"). The transcriptomic alterations of rescuer genes post PD1/PDL1 blockade in patient tumor biopsies: Their post (vs. pre) treatment expression changes of DU/DD rescuers after anti-PD1 (b) Each panel displays the expression fold change of each predicted rescuer gene (rows) for different tumor samples (columns) and the P-value of over-all paired Wilcoxon test of the expression changes observed in paired samples. Significantly altered up/down-regulated genes are marked by (*). Genes marked in red are those whose CRISPR knockdown enhances melanoma sensitivity to anti-PD1 blockade in mice models. The transcriptomic alterations of rescuer genes post CTLA4 blockade in patient tumor biopsies: Their post (vs. pre) treatment expression changes of DU/DD rescuers after anti-CTLA4 (c) Each panel displays the expression fold change of each predicted rescuer gene (rows) for different tumor samples (columns) and the P-value of over-all paired Wilcoxon test of the expression changes observed in paired samples. Significantly altered up/down-regulated genes are marked by (*). Genes marked in red are those whose CRISPR knockdown enhances melanoma sensitivity to anti-PD1 blockade in mice models. The transcriptomic alterations of rescuer genes post PD1/PDL1 and CTLA4 blockade in patient tumor biopsies: Their post (vs. pre) treatment expression changes of DU/DD rescuers after PD1 + CTLA4 combination therapies (d). Each panel displays the expression fold change of each predicted rescuer gene (rows) for different tumor samples (columns) and the P-value of over-all paired Wilcoxon test of the expression changes observed in paired samples. Significantly altered up/down-regulated genes are marked by (*). Genes marked in red are those whose CRISPR knockdown enhances melanoma sensitivity to anti-PD1 blockade in mice models.
Figure 5A. Immunofluorescence of control, parental and patient fibroblasts labeled for LAMP-1 and mTOR. In steady state conditions (0 hours) VPS41S285P/R662* fibroblasts show less colocalization between mTOR and LAMP-1. VPS41WT/WT, VPS41WT/S285P and VPS41WT/R662* show an appropriate mTOR response upon nutrient deprivation (2 hours) or restimulation (2 hours starvation followed by 15 minutes restimulation) (Appendix Fig S5A). >10 Cells per cell line were quantified (A'). Quantifications are performed relative to colocalization under steady state conditions per cell line (n=3). Scale bars, 10µm; zoom, 1µm. B. Immunofluorescence of VPS41WT/WT, VPS41WT/S285P, VPS41WT/R662* and VPS41S285P/R662* fibroblasts labeled for TFE3. In VPS41S285P/R662* fibroblasts, TFE3 is constitutively localized in the nucleus regardless of nutrient state (quantified in B'). >25 Cells per condition were quantified (n=3). Scale bars, 10µm. C. TFE3 immunofluorescence in HeLaWT and HeLaVPS41KO cells. In HeLaVPS41KO cells TFE3 constitutively localizes in the nucleus (quantified in C') >83 Cells per condition were quantified (n=3). Scale bars, 10µm. D. Rescue experiments of HeLaVPS41KO cells. Expression of VPS41WT-APEX2-V5, VPS41S285P-APEX2-V5 or VPS41R662*-APEX2-V5. Reintroduction of VPS41WT rescues TFE3 localization, whereas expression of mutant VPS41 has no effect (quantified in D'). >83 Cells per condition were quantified (n=3). Scale bars, 10µm. E. Western blot of phosphorylated mTORC1 substrates S6K and 4EBP1 after starvation (2 hours) and restimulation (10, 30 or 60 minutes). HeLaVPS41KO cells show comparable levels of phospho-S6K and phospho-4EBP1, with no difference in recovery after restimulation (quantified in E') (n=3).
ULK1 and 2 localize to the isolation membrane (phagophore) under starvation conditions.Figure 1.(A and B) NIH3T3 cells stably expressing GFP-ULK1 (A) and -ULK2 (B) were cultured in complete or starvation medium for 30 min. They were then cultured in fresh complete medium for an additional 30 min (starvation→complete). (C and D) NIH3T3 cells stably expressing GFP-ULK1 (C) and -ULK2 (D) were cultured in starvation medium for 120 min. The cells were fixed, permeabilized, and subjected to immunofluorescence microscopy using anti-Atg16L1 antibody and Alexa Fluor 660-conjugated secondary antibody. More than 90% of GFP-ULKs dots were positive for Atg16L1. (E-H) Wild-type (E and G) and Atg5−/− (F and H) MEFs were transfected with retroviral vectors encoding GFP-ULK1 and -ULK2. MEFs stably expressing GFP-ULK1 (E and F) and -ULK2 (G and H) were cultured in complete or starvation medium for 120 min. The cells were fixed and examined by fluorescence microscopy. Bars, 20 μm.
Figure 7(A) MEFs expressing B-RafWT or B-RafAVKA were stimulated with EGF for the indicated time points (min) and analyzed by Western blotting. (B) IP of EGF stimulated MEFs expressing B-RafWT or B-RafAVKA. Sorafenib was used to increase complex build up. (C) Proliferation of two different MEF pools expressing either B-RafWT or B-RafAVKA. MEFs expressing B-RafAVKA show a strong reduction in growth rate. n = 3, mean + SEM, t-test, ** p < 0.01, *** p < 0.001. (D) Cell morphology of B-RafAVKA expressing MEFs are enlarged and present with abnormal nuclei (E) MEFs expressing B-RafAVKA show a significant increase in caspase-3 activity. n = 3, mean + SEM, t-test, ** p < 0.01. (F) Decay of TAg expression in MEFs following dox withdrawal. (G) Morphology of MEFs expressing B-Rafwt or B-RafAVKA in the presence or absence of TAg.
Figure 2Schematic representation of two-photon microscopy experimental design Two-photon representative images of wild-type and Nlrp3-/- mice (5 and 15 months old). Scale bar: 20µm. Quantification of morphological parameters for wild-type and Nlpr3-/- mice after LPS injection
Figure 4A) Lysates from 3 control clones and 7 VPS15 CRISPR clones were analysed by Western blot. LC3 and p62 accumulation as well as depletion of VPS34 CI components was observed in all 6 sgA-derived clones. B) Controls and 6 VPS15 CRISPR KO clones were starved for 1 hr, fixed and imaged for WIPI2 (green). Depletion of VPS15 was associated with a block in WIPI2 puncta formation and large vacuoles (red asterisks). Quantification of WIPI2 spots, mean +/- SEM, n=3. C) GFP-2xFYVE was overexpressed in control (A3) and VPS15 KO clones. The GFP-2xFYVE puncta were abolished in VPS15 KO clones. D) Wild type (WT), 6SA or 6SE VPS15-HA, or empty vector (EV), were expressed in VPS15 KO or HEK293A cells. Cells were starved for 1 hr and the distributions of WIPI2 (green), HA (red) and LC3 (blue) were assessed, mean +/- SEM, n=4. E) (Top) VPS34 CI incorporating VPS15 WT (black), 6SA (blue) or 6SE (pink) were purified and thermal stability was assessed. The first derivative of the ratio of tryptophan emission at 330 and 350nm (Y axis) is plotted against temperature (X axis, °C) with threshold melting temperatures annotated. (Bottom) Representative Coomassie stained gel shows relative protein levels used. F) In vitro lipid kinase activities of WT-, 6SA- or 6SE-reconstituted VPS34 complex I on GUVs (giant unilamellar vesicles) were assessed. Representative images show GUV membranes (Lissamine Rhodamine-PE, green) and PI3P (AlexaFluor647-PX, red). Bottom left: VPS34 complex activities over time measured by recruitment of AlexaFluor647-PX to GUVs are shown. Bottom right: kinase reaction rates for GUVs phosphorylated with WT, 6SA or 6SE complexes, taken from linear portion of reactions. Representative data from 1 of 4 independent repeats are shown, with mean +/-SD. In this experiment, 67 GUVs were analysed in total (15 from WT, 17 from 6SA and 35 from 6SE).
Figure 1Schematic representation of the Rosa26LSL-CARD14-E138A transgene construct (LSL = LoxP-stop-LoxP). Ratio of genotypes obtained of 1 week-old pups after crossing K5creTg/+ CARD14E138A+/+ male mice with K5cre+/+ CARD14E138ATg/+ female mice; CARD14E138A+/+ = Rosa26+/+, CARD14E138Atg/+ = Rosa26LSL-CARD14-E138A/+ Representative Western blot showing CARD14 protein levels in skin and liver lysates of E18.5 pups. Actin is shown as a loading control. Representative images of K5creTg/+ CARD14E138A+/+ and K5creTg/+ CARD14E138ATg/+ pups at E18.5. Ratio of genotypes obtained of 1 week-old pups from crossing Malt1fl/fl K5creTg/+ CARD1414E138A+/+ male mice with Malt1fl/fl K5cre+/+ CARD14E138Atg/+ female mice. Representative image of 6 month-old Malt1fl/fl K5cretg/+ CARD14E138A+/+ and Malt1fl/fl K5cretg/+ CARD14E138Atg/+ mice. Body weight and ear thickness of untreated WT and Malt1fl/fl K5creTg/+ CARD14E138ATg/+ male and female mice (between 4 and 7 months old). Each symbol represents one mouse; the line represents the mean value (n≥4 mice per group). WT = Malt1fl/fl K5cre+/+ CARD14E138A+/+, Malt1fl/fl K5cre+/+ CARD14E138ATg/+ and Malt1fl/fl K5creTg/+ CARD14E138A+/+ mice. Statistical difference between two groups was determined using a Mann-Whitney U-test (ns: p-value>0.05) Representative H&E-stained histological sections of ear and skin tissue of Malt1fl/fl K5cre+/+ CARD14E138A+/+ and Malt1fl/fl K5creTg/+ CARD14E138ATg/+ mice (scale bar represents 100 μm).
Mec1-dependent RNAPII degradation occurs during an unchallenged S phase (A-B) RNAPII occupancy on chromatin in S phase was assessed by ChIP-qPCR.Figure 8(A-B) RNAPII occupancy on chromatin in S phase was assessed by ChIP-qPCR. (A) The level of Rpb1 was measured at snR13-TRS31 in both G1 and S phase (70 min at 16°C). Data is expressed as a percentage of input. SEM (n=3 biological replicates) is indicated. (B) Quantification of the data from (A) depicting the mean retention of Rpb1 in S phase over G1. Total Rpb1 levels of exponentially growing cells that were synchronized in G1 with α-factor and released into S phase at 25°C. Actin was used as a loading control. control. (D) Quantitation of blots Rpb1 levels in S phase are expressed as a percentage of the starting level in G1 (100%, black dashed line). (E) Quantitation of Rpb1-GFP fluorescent intensity expressed as percentage over the G1 level. SEM is indicated (n=106 for WT and n=102 for mec1Δsml1Δ).
Lam6 Is Important for Cross-Talk between Contact Sites.Figure 4(A) Fluorescent microscopy shows that the ERMES contact (as measured by the number of Mdm34-GFP puncta per cell) expanded in the Δvps39 background relative to WT. However, the expansion did not occur on the background of Δvps39 Δlam6, demonstrating that Lam6 is necessary for ERMES expansion under these conditions. Scale bar represents 5 μm.(B) Quantitation of (A). Bars represent the percentage of cells containing the specific number of puncta/cell out of total cells counted for the strain (for WT, n = 234 cells; for Δvps39, n = 246; for Δvps39 Δlam6, n = 271). (C) Fluorescent microscopy demonstrates that downregulating ERMES contacts (by growing GALp-MDM34 strains in glucose) indeed caused expansion of vCLAMP (GFP-Vps39). However, this expansion was diminished in a Δlam6 background. Scale bar represents 5 μm (see also Figure S4). (D) A model summarizing our hypothesis on the way that Lam6 functions to regulate contact site communication. In normal cells, where all contact sites are intact, Lam6 is found mostly in ERMES contact sites, and to a lesser extent in vCLAMP and NVJ. However, in case a contact site is lost, the Lam6 proteins that were localized to this contact become free to associate with the other contact sites, thus signaling that they must expand. Raising Lam6 levels in any other way in a contact site therefore would cause an expansion of this contact site.
Sequestration of ubiquitin in solid PML-NBs impairs the recruitment of 53BP1 at fragile chromosomal sites (see also ).Figure 7Subcellular distribution of 53BP1 and DRiPs in HeLa cells that were left untreated or treated with MG132 (10 µM) and OP-puro (25 µM) for 4 h, followed by 5h recovery in drug-free medium (+ rec. control), with VER (40 µM; + rec. VER) or Eeyarestatin I (5 µM; + rec. EeyI). Where indicated, cells were treated with MG132, VER or EeyI alone for 5 h. Scale bars: 20 µm. Quantitation of the % of cells with 53BP1 foci. Cells were treated as described in (A) and divided in three categories, based on the number of 53BP1 foci/nucleus: 0, 1-2 and > 3. Number of cells counted/condition: 1112 - 1537 in three independent experiments; statistical significance via One-way ANOVA; p < 0.01. HeLa cells were lipofected with cDNAs encoding for HA-Ubiquitin, Flag-Ubiquitin or Flag-VCP 24 h post-transfection, cells were either left untreated or treated with MG132 (10 µM) and OP-puro (25 µM). Cells were processed for staining of HA, Flag and endogenous 53BP1. * indicates cells that do not express HA- or Flag-Ubiquitin and show no rescue of 53BP1 foci formation after stress. Scale bars: 20 µm. Quantitation of the % of cells with 53BP1 foci. Cells were treated as described in (C) and divided in three categories, based on the number of 53BP1 foci/nucleus: 0, 1-2 and > 3. Number of cells counted/condition: 710 - 938 in three independent experiments; statistical significance via One-way ANOVA; p < 0.01. Colony formation assay. The number of colonies surviving 10 days after plating is shown. Control cells or cells treated with MG132 (10 µM) and puromycin (5 µg/ml), followed by 5h recovery in drug-free medium (+ rec. control), with VER (40 µM; + rec. VER) or Eeyarestatin I (5 µM; + rec. EeyI) are shown. Where indicated, cells were treated with VER or EeyI alone for 5 h. Number of colonies counted/condition: up to 526 in three independent experiments; statistical significance via One-way ANOVA; p < 0.03, ± s.e.m.
Figure 1Confocal images of growth stage wild-type Dictyostelium AX3 cell co-expressing tPH-CynA-KikGR (green) and an F-actin biosensor LimE-RFP (red). Front and back of the cell are shown. Time lapse confocal images of growth stage AX3 cell co-expressing tPH-CynA-KikGR (green) and an activated Ras biosensor, RBD-mCherry (red). Front and back of the cell are shown. Time-lapse confocal images of tPH-CynA-KikGR expressing AX3 cells. Back region of cells was photoconverted from green fluorescence to red at t=0s. Front and back of the cell are shown, box showing the photoconverted region. Profiles of the converted red fluorescence at the back remain almost symmetric over time. The dotted line represents the axis of symmetry of the intensity profile of the photoconverted fluorophores at t = 0. Temporal profile of normalized intensity of total converted red fluorescence. Mean ± SEM is shown for n = 14 cells. Time-lapse confocal images of tPH-CynA-KikGR expressing AX3 cells during photoconversion. Side region of cells was photoconverted at time 0. Green fluorescence was converted to red. Front and back of the cell are shown. Box shows the photoconverted region. Profiles of the converted red fluorescence at side become skewer (steeper near the front) over time. The dotted line represents the axis of symmetry of the intensity profile of the photoconverted fluorophores at t = 0. Confocal fluorescent images of tPH-CynA-KikGR (top) and CAR1-mcherry (bottom) Dictyostelium AX3 cell showing the bleached area and the side (blue) and middle (red) boxes where the fluorescence recovery is monitored. Front and back of the cell are shown. The graphs depict the recovery of CAR1-mcherry (left) and tPH-CynA-KikGR (right). Mean ± SEM is shown for n = 2 cells. Time-lapse confocal images of tPH-CynA-KikGR expressing AX3 cells showing docking events at the back of a migrating Dictyostelium cell. Front and back of the cell are shown. Time-lapse confocal images of PH-CynA-HALO expressing AX3 cells showing high PH-CynA vesicles accumulation at the back of a migrating Dictyostelium cell. Front and back of the cell are shown. Time-lapse confocal images of tPH-CynA-KikGR (green) expressing AX3 cells. Back cortical region of cell was photoconverted at time 0. Green fluorescence was converted to red. Front and back of the cell is shown, box showing the photoconverted region.
Figure 4A Western blot and qRT-PCR results showing phosphorylation levels of GSK-3β and WNT1 and WNT3 expression, respectively, in the indicated cell lines. Mean ± s.d. (n = 3). P-values by two-tailed Student's t-test. a/a, CDK12-amplified/HER2-amplified cells; n/a, CDK12 non-amplified/HER2-amplified cells; S, trastuzumab-sensitive Subcellular localization of β-catenin in the indicated stable cell lines were analysed by cellular fractionation (B) Subcellular localization of β-catenin in the indicated stable cell lines were analysed by immunofluorescence staining (C). Nuclear translocation of β-catenin in control, CDK12-overexpressing, and CDK12-knockdown stable cell lines was quantified by counting the cells harboring exclusively nuclear β-catenin (N) from the immunofluorescence image. Mean ± s.d. (n = 3). P-value was calculated using a two-tailed Student's t-test. M+C, cells containing membrane and/or cytoplasmic β-catenin; N, cells dominantly expressing nuclear β-catenin. Green, β-catenin staining; Blue, DAPI staining for visualization of nucleus; Scale bar = 10 μ. a/a, CDK12-amplified/HER2-amplified cells; n/a, CDK12 non-amplified/HER2-amplified cells; S, trastuzumab-sensitive. D Luciferase-reporter assay measuring the TCF/ lymphoid enhancer-binding factor-promoter activity in the indicated cell lines. TOP, pTOP-flash (wild-type TCF-binding sites), FOP, pFOP-flash (mutant TCF-binding sites). Data represent the mean ± s.d. P-values were calculated using a two-tailed Student's t-test. E The mRNA levels of the WNT/β-catenin/TCF-target genes in CDK12-overexpressing ZR-75-30 cells and CDK12-knockdown BT474 cells were analysed by qRT-PCR. Mean ± s.d. (n = 3). P-values were calculated using a two-tailed Student's t-test. F Effect of WNT/ β-catenin/TCF pathway on the CDK12 regulation of breast CSCs. Cells were treated with DMSO (vehicle) or 10 nM FH535, a β-catenin/TCF inhibitor, for 24 h, and changes in the CD44+/CD24-/ESA+ cell population were measured using FACS analysis. Cells seen at lower right quadrant in the dot plots indicate CD44+/CD24- population (red) and CD44+/CD24-/ESA+ population (blue). Data represent the mean ± s.d. ofP-value was calculated using one-way ANOVA with a post-hoc LSD test.