EMBO/sd-character-level-ner huggingface dataset:
(G) Western blot analysis at 2-cell stage showed the levels of p-IκBα were downregulated in the exogenous Tab1 mRNA injected groups, ddH2O was injected as control. Dotted line indicates a cropped lane.
(h) Plasma membrane calcium influx was measured by analysing the Fura-2 fluorescence quench rate upon addition of extracellular Mn2+.
A.) Construct used to express the E. coli DPCK in the apicoplast. Appended are the N-terminal signal sequence and transit peptide from ACP (api) and a C-terminal mCherry tag.
A. A close-up view of the sucrose-binding pocket. Residues hydrogen-bonding with sucrose are shown as yellow sticks, and the sucrose molecule is shown as light pink stick.
(F) Quantification of TIF assay for % cells with > 4 TIFs per cell for p53+/+ (black) or p53-/- (red) cell.
E. LDH release at DIV 25 is strongly reduced due to long term treatment with ceftriaxone in hTauAT slice cultures (n=8-10 culture inserts containing 6 slices each / group and condition). One Way ANOVA F(3,32)=7.685; p=0.0005).
(J) The levels of UBTD1 mRNA in lung adenocarcinoma patients (522 patients) are correlated with OS.
(F) GST EBNA2 fragment 246-487 wt and mutant S457A/T465V were treated with recombinant active PLK1 (+) in the presence of [γ-32P] ATP in vitro or left untreated (-). CRS, an artificial PLK1 test substrate was used as a positive control.
f, Confocal microscopy images of humanlung sections from e immunostained for M. tuberculosis and parkin; DNA was visualized using DAPI (4′,6-diamidino-2-phenylindole). Scale bars, 25 µm.
C-D Confocal images or (D) quantification of the macrophages in germband in Stage 12 embryos from the control, atosPBG, and atosPBG expressing Atos itself or variants lacking particular domains. Transgene expression directly from macrophage specific promoter (mac-). For control (n=32 embryos) vs atosPBG mutant (n=56) p<0.0001; vs rescue with mac-atos (n=18) p>0.99; vs rescue with mac-atosDUF- (n=17) p=0.0003; vs rescue with mac-atosChrSeg-(n=21) p=0.0003; vs rescue with mac-atosDUF-/ChrSeg-(n=19) p=0,00014; vs rescue with mac-atosTAD1-/ TAD2- (n=25) p=0.0009, atosPBG mutant vs. rescue with mac-atos p=0.0031. Data information: Germband edge: dotted white line. Mac- indicates direct expression from the srpHemo promoter. (C, macrophage nuclei (red), actin by Phalloidin staining (green). (D One-way ANOVA with Tukey. Mean±SEM, ns=p>0.05, **p<0.01, ***p<0.001, ****p<0.0001. Scale bars: 50µm (C
A. DNA-RNA hybrids in IGVL and IGCL in wild type and h3.3. The DRIP signal is normalised to total input and to wild type IgVλ (*** = p<0.001, ns = not significant, unpaired T-test).
(A) Survival model #1 with low dose treatment after 4 hr of CPA injection. Mice were infected with 5 x 106 CFU of S. aureus via intravenous injection on day 3. Moribund mice were euthanized and recorded as deceased on the day of euthanasia. Control mice without CPA.
(C) Photocurrent action spectrum of a ReaChR-expressing RGC (1.2 × 1016 photons cm-2 s-1). (D) Normalized action spectrums of ReaChR-RGCs. Solid line represents patch-clamp data (n = 8 cells; 1.2 × 1016 photons cm-2 s-1). The mean photocurrent amplitude at 550 nm was −324 ± 97 pA. Dashed line represents multi-electrode array data (n = 168 cells; ~1016 photons cm-2 s-1).
Glucose tolerance test (GTT) and area under the curve (AUC) data for GTT in WT and DKO mice after HFD feeding (n=10-11).
(D) Preservation of ACE2-Fc peptidase activity. The peptidase activity of ACE2-Fc was measured by cleavage of the fluorescent peptide substrates.
Predicted binding sites of compounds 9, 10 and 13. The top set of images gives a global view of the binding sites. The middle set of images gives a 3D view of residue intermolecular interactions with the compounds of interest. The bottom set of images gives a diagrammatic 2D representation of the same interactions. Hydrogen bonds are represented by arrows (arrowhead pointing to acceptor) and salt bridges by a red to blue gradient line (color change red:negative, and blue:positive).
(A) qPCR analysis of DLL4 levels in TREx cells 0 and 24 hours post induction. GAPDH was used a housekeeper (n=3). Data information: data are presented as mean ± SD. *P<0.05, **P<0.01, ***P<0.001 (Unpaired Student's t-test). All repeats are biological.
i, Effect of bafilomycin and etomoxir on spherogenic potential of +/−Kras cells (n = 6). Data are mean ± s.d.
In vivo validation of S100a6 expressing DIE astrocytes in Aldh1l1-eGFP mouse (3 mo) with immunolabelling for S100a6 (B: in the SEZ, LV: lateral ventricle, CC: corpus callosum. Scale bars: 200 µm
Representative double immunofluorescence confocal images of coronal sections of the dorsal striatum showing p62 immunolabelling (red) in choline acetyltransferase (ChAT)-positive (cyano) ChIs of Tor1a+/+ and Tor1a+/- mice. A1) Two representative ChIs identified by the white box in the low magnification images (bar = 20 µm) are shown at higher magnification in (A2) (bar = 5 µm). A3) The graph reporting mean ± SEM values of p62/ChAT signal overlap in ChIs, normalized to Tor1a+/+ values of the same experiment, shows a significant increase of p62 in Tor1a+/- neurons (Tor1a+/+ n=20 cells, N=6 mice; Tor1a+/- n=20 cells, N=6 mice, t test ***P=0.00008)
The nuclear and cytoplasmic fractionation was valid by immunoblotting using cytoplasmic and nuclear protein markers (GAPDH and Lamin B1) to show the purity of fractions.
(B) Ratio of HW/TL (heart weight/tibia length) in WT and miR-574-/- mice (n=8/7/8/6 for 4 groups of WT, Veh.; WT, ISO; KO, Veh.; KO, ISO).
E, F. Lineweaver-Burk plot with varying concentration of H2O2 (E) and GSH (F) in presence of Vs nanozyme, respectively. The concentration of NADPH was constant (0.2 mM) in all the assay conditions.
C Representative phenotypes of tailbud stage Xenopus leavis embryos injected with ctrl or znrf3ΔRING mRNAs as indicated. mRNAs were injected in pre-mature oocytes and embryos were obtained through Host transfer technology. Scale bars: 1 mm.
B Correlation between response in GB-PDOC (left side of the graph) and its respective patient (right side of the graph). Response in GB-PDOCs was obtained by quantification of number of proliferative cancer cells found in Rx + TMZ treated normalized to DMSO treated (100%) PDOCs from the same patient. Representative GB-PDOC responding (R) or not (NR) to the standard of care that was provided ex vivo (Radiation (Rx): 2x 10 Gy + temozolomide (TMZ) 250 µM) are shown. Scale bar: 50 µm. Values are shown as mean ± s.e.m. (n=3-6 PDOCs per experimental condition per patient, each patient is represented in an individual bar. Fourteen patients included; each patient is an independent experiment). Time to progression of the patients after neurosurgery is represented in months. Response to Rx + TMZ was evaluated by volumetric measurement of the lesion (dashed line) based on MRI before and after the treatment. Representative patients responding (R) or not (NR) to the standard of care are shown. A white circle indicates progressive disease. Patients with ongoing response to Rx + TMZ (stable disease) are indicated with green bars. MGMT promoter methylation status is shown for each patient (met: methylated; unmet: unmethylated). N/A: not available.
D. ssDNA-bound Streptavidin beads incubated with cell extracts as in (C) were washed with buffer containing indicated salt concentrations prior to immunoblotting.
(B-C) CAMDI blocked HDAC6-mediated α-tubulin deacetylation. (C) CAMDI N-terminal domain alone increased Ac-tubulin level. n=3 independent experiments. **, p<0.01, One-way ANOVA with Bonferroni's post hoc test. Data are presented as mean ± SEM.
A Kaplan-Meier curves for overall survival of patients at all stages of cancers (breast, gastric, ovarian, and lung cancer) depending on TRF2 expression level. Data information: For all panels, the optimal cut-off is determined on KMplot. The p value (log Rank test), the Hazard Ratio and number of patients are indicated.
representative KRAS CNA segments (F) from Affymetrix CytoScanHD Arrays of 33 primary MPM cell lines (called MESO #) from Nantes, France (GEO dataset GSE134349). Red lines denote the KRAS locus on chromosome 12p12.1.
(F) Immunodetection of complex II structural subunits on Western blots of total cell lysates separated by SDS-PAGE, from three independent replicates of WT and ∆4-CYB cells. The graph shows the densitometric quantification of the signals corresponding to each subunit normalized to that of the β-Actin. The mean of the three control (WT) samples was set to 1.0 and all the measurements were referenced to that value. The values plotted in the graphs are the mean ± SD (n=3). 2-way ANOVA with Sidak's multiple comparisons test ****p<0.0001.
D Images revealing the collagen (blue) by Masson's trichrome staining in diaphragm of WT, mdx, and mdx/CD38-/- mice. Quantification of collagen staining area (% total area) in the diaphragm of WT, mdx and mdx/CD38-/- mice (n=4 per group). Scale bars: 200 µm. Data information: Each dot of the graphs represents a mouse. D in duplicate; After normality and variance comparison tests, significance was assessed using: : ANOVA followed by Fisher's LSD test Values are expressed as means ± SEM. Significance: *p<0.05, **p<0.01, ***p<0.001.
C-J. Confocal images and cell density quantifications as in A using molecular markers for different cell types:E, F. E14 brains stained with anti-Tbr1 antibody to label layer IVneurons p-value = 0.01392 (Het vs. KO) (n = 3 and 4 brains per genotype group from 2 independent litters). Scale bars: 50 μm.
Western blot detection of p-Beclin 1 Ser90, p-Beclin 1 Ser93 and Beclin 1 in MEFs indicated genotype in normal medium or after 1 hr HBSS starvation (D)
Triple transgenic showing endothelial membrane (red), neurons (green) and erythrocytes (red). Surrounding neurons were excluded from the volume of the kugel (unfilled arrowhead) and erythrocytes were not observed inside kugeln (arrowhead).
(B) PAS staining for total glycogen in APBD87 fibroblasts glucose-starved for 48 h (left), or glucose starved and then replenished for the last 24 h to induce glycogen burden (right). Image acquisition performed by Nikon Eclipse Ti2 microscope using a 40x PlanFluor objective and CY3 filter. Scale bar, 100 µm.
(K) Serum insulin in Abca12tm1d +/- HCD mice after i.p glucose challenge (mean±SEM, n=3 mice/genotype, **p=<0.01, Students t-test).
(D) Boxplots summarizing the data shown in panel (C) using the area under the curve (AUC) as a measure for drug sensitivity. High AUC values indicate drug resistance. Again, it is evident that cell lines predicted to be more resistant to a certain drug were in fact significantly (p≤0.05, one-sided Mann-Whitney test) more resistant than cell lines predicted to be more sensitive.
(A) Examples of simulated hemifusion-to-fusion transitions in the presence of modified vacuolarSNARE complexes. Upper panel: Short spacer. Lower panel: Long spacer. These stirred molecular dynamics simulations illustrate that progression of fusion is sterically achievable for both of the fluorescent protein-tagged SNARE complexes, even when the geometry of the fusion zone is extremely confined, i.e. the fusion between two highly curved membrane dimples (1/8 nm-1). This scenario suggests that the fusion-inhibiting effect of a fluorescent protein added via a short peptide spacer is unlikely to result from a direct "steric clash" with the membrane. Vam3 in red; Nyv1 in blue; Vti1, Vam7 in green.
C. Analysis of Gli1 mRNA levels in MEFs Ptch1+/+ and Ptch1-/- upon Fbxl17 depletion using two different siRNAs. (mean ± SEM from 3 independent experiments, ***p<0.0005).
A) Plots showing deuterium-exchanged peptides, after deuterium exchange of 10 sec of Okp1 in Ctf19D-RWD-Mcm21D-RWD-Okp1229-336, in full-length COMA, or in Ame11-260-Okp1123-336. Plots for full time course are in Appendix Fig S6C.
Chromatin IP analysis of binding of Glis2 to Stat3 promoter (see Appendix Fig S9D) in the absence or presence of SHH at different concentration as indicated on triplicated samples, using specific anti-Glis2 antibodies and rabbit IgG as control.
D. Surface representation of the Sec62 domain colored according to the electrostatic potential. The viewing angle is the same as in the right panel in (B).
(A) Representative images from ARPE-19 cells treated with DMSO and NSC668394. All cells were fixed and stained with anti-LC3 (green) and anti-LAMP1 (red) antibodies and nuclei were counterstained with DAPI (blue). Scale bar 5 μm. (B) The plot shows the quantification of the numbers of LC3/LAMP1-positive vesicles per cell (n ≥ 100). Values represent means ± s.e.m from 3 independent experiments. Student's t-test (NSC668394 vs DMSO) ***p ≤ 0.005.
CLSM images of UBF (immunofluorescence, green) or rDNA (DNA FISH, green) and DNA (DAPI, blue) after knockdown of NCL and NPM by siRNA in HeLa cells.
MDA-MB-231 cell were treated with different concentration of PARG inhibitor PDD0017273 for 48 h, cell lysates were analysed by Western blot (n=3).
A, B Sirt7 mRNA (A) and protein (B) abundance in microdissected renal tubules normalized to Rpl26 or Ponceau, respectively. Enrichment for each segment was previously validated by immunoblotting against segment-specific markers as indicated in material and methods (Spirli et al, 2019).
(H,I) I-Ab presentation by cDC isolated from naïve mice. (I) I-Ab-IR17 presentation by DC incubated with OVA-expressing E. coli at a ratio of 20:1, assessed with the OTIIZ hybridomas. H,I: representative of 2 independent experiments with 20 naïve mice pooled per experiment. Dotted line in D,E,F,H,I shows the basal response of the hybridomas in the absence of antigen.
D Average frequency distribution for sequence read mid-point data in the size range of 122-137 bp for pl-iPSC and NPC cells centred on the 2,522 CTCF binding sites identified from NPC specific ChIP-seq data. A distinctive peak corresponding to the CTCF protein complex is present in the NPC state, but reduced to background levels in pl-iPSC. E Average frequency distribution for sequence read mid-point data centred on the CTCF binding sites in the size range of 162-188 bp, corresponding to larger nucleosome footprints, for pl-iPSC and NPC centred on the 2,522 CTCF binding sites identified from NPC specific ChIP-seq data. Nucleosome arrays are present in both cell states, independent of the CTCF protein complex. Nucleosomes in the CTCF sites in pl-iPSC tend to position closer to the CTCF site than in NPC. The data derived from the unique NPC CTCF sites has greater noise due to the smaller number of sites analysed.
A Gene expression analysis of myokines (table EV3) during a five day time course of myogenic differentiation going from cycling myoblasts in growth media (Ham's F10 supplemented with 20% Fetal Bovine Serum, 1% penicillin/streptomycin, 2.5 ng/ml basic Fibroblast Growth Factor) to terminally differentiated myocytes (2 days in differentiation media, DMEM supplemented with 5% horse serum) to post mitotic multinucleated myotubes (5 days in differentiation media). Gene expression was assayed in biological triplicate by microarray (Soleimani et al., 2018; Soleimani et al., 2012a). Expression of each gene was averaged across three replicates and normalized to mean zero and standard deviation of one. Expression values were then truncated at +/- 3 for color display. Blue indicates lower than average expression, while red indicates higher than average expression. B Distribution of Myf6 peaks within 100kb of the TSS of the myokine genes (table EV3), similar to the analysis shown in (A).
(B) Experimental design: fluorescently labeled recombinant hGBP1 variants and nucleotides were mixed with broth-cultured live or fixed bacteria; bacterial binding was monitored by confocal microscopy.
A. Reduced weight (in grams) of adult Rb∆K7 (n=11) vs control littermate (n=17) males. Results are presented as mean ±SD; P values calculated by two-tailed unpaired student's t-test.
B and C. Changes in Ucp1 (B) and Pgc1α(C) mRNA expression in the differentiated brown adipocytes from WT and TRPV2KO mice with and without 10 µmol/L isoproterenol (ISO) for 4 h. Data are presented as mean ± SEM, n = 5; ** P < 0.01 vs. DMSO group; # P < 0.05; ## P < 0.01 vs. WT group. One-way ANOVA followed by 2-tailed t-test with Bonferroni correction.
D BMDMs and PMs derived from wild-type and M-Shp2-/- mice were stimulated by IMQ (10 µg/ml) for indicated times. Whole cell lysates were subjected to Western blotting.
D. MC3T3E1 cells were treated with indicated concentrations of KY-02327 for 14 days. The mRNA level of collagen 1a (Col1a) was measured by quantitative real-time qRT-PCR. [n=3]
(C) qPCR of DLL4 levels in scr and circHIPK3 KD stable TREx cells at 0 and 24 hours post induction with GAPDH as a housekeeper (n=3). Data information: data are presented as mean ± SD. *P<0.05, **P<0.01, ***P<0.001 (Unpaired Student's t-test). All repeats are biological.
(A) Nuclear and cytosolic extracts of 22Rv1-M cells transfected with either shScramble or shCTNNB1 were prepared and the levels of indicated proteins were detected by immunoblotting.
Large (>1Mb) CNA (amplifications- shades of violet; deletions - shades of red) identified by low coverage whole genome sequencing in each of the patients and corresponding PDXs
A. Astrocyte-specific Daam2 cKO mice show delays in buried food finding. N=13, 15 mice per genotype. Data is presented as mean ± SEM. Student's t-test was used, *p<0.05.
Rab8a (T22N) protein accumulated in BAG6-knockdown cells. HeLa cells were transfected with siRNA duplexes for BAG6 or control siRNA. At 48 h after siRNA transfection, Flag tagged-Rab8a (T22N) was expressed in the cells. At 24 h after Rab8a (T22N) transfection, the cells were chased with 50 μg/mL CHX and harvested at the indicated time after CHX addition. Actin was used as a loading control.
(D) Primary hippocampal neurons (DIV6+3) were transfected shTDP or shCtrl and GFP as transfection marker. After 3 days neurons were subjected to Alexa-Tf for 20 min and intracellular Alexa-Tf movement was imaged as in Figure 1 afterwards. Quantitative analysis of Alexa-Tf movement (E) and vesicle number (F) from kymographs.
(C) PI4KB is potently inhibited by c10orf76 in a dose-dependent manner in vitro. Kinase assays of PI4KB (20 nM) in the presence of varying concentrations of c10orf76 (1.6 nM-1 μM) were carried out on pure PI lipid vesicles (0.5 mg/L) in the presence of 100 μM ATP. The data was normalized to the kinase activity of PI4KB alone. IC50 values were determined by one binding site, nonlinear regression (curve fit) using Graphpad. Error bars represent standard deviation of independent technical replicates (n=3).
H, I In-gel assay of OST1 activity in 10-d-old wild-type and EGR2-overexpressing plants under cold stress. Data information the ratio of band intensity of OST1 to RuBisCO in the wild type with cold treatment for 2 h was set to 1.00. In H), representative pictures are shown: Top, autoradiograph; bottom, CBB staining. In I), relative kinase activity is shown. Each bar represents the mean ± SEM of three biological repeats. **P < 0.01 (two-tailed t-test).
F Binding of Usp27xto BimEL does not require β-TrCP293FT-TetR-3xFlag-Usp27x cells were transfected with control siRNA (siCo3) or siRNA specific for β-TrCP. 48h later cells were stimulated with PMA plus dox plus QVD for additional 24h. After cell lysis, Flag-Usp27x was immuno-precipitated using anti-Flag-matrix, and BimEL and β-TrCP bound to Usp27x were identified by Western blotting. Blots are representative of n=2 independent experiments.
(F) Stills of representative Hela cells expressing Lifeact-GFP and H2B-mCherry captured at early anaphase under different conditions i.e. Control siRNA, knock-down of RACGAP1, in the presence and absence of Aurora B kinase inhibitor (2µM ZM). The masks below were generated for each cell and the shortest distance between the centroid of DNA (+) and the masked cortical actomyosin network (*) was quantified in (G) with Matlab. Scale bar - 10 µm
K - Relative mRNA expression of Adrb1, Adrb2 and Adrb3 in BMDMs (n = 3). mRNA expression was measured by qPCR and normalized to levels of Tbp using the 2-ΔΔCt method.
A) We identified previously unknown telomere regulators in fission yeast using the haploid S. pombe whole-genome gene deletion library including ssu72+ (SPAC3G9.04). B) Telomere length of wt, ssu72Δ and ssu72-C13S (point mutation at the phosphatase active site) strains were measured by Southern blot in ApaI digested genomic DNA using a telomeric probe.
B) Guide:off-target pairs examined listed with increasing values of N (the length of the matched region between miRNA and target RNA). N = 7 is the fully seed paired target.
i) Scatter plot correlating the expression changes induced by miR-29a antagonization in adult mice at the transcript- (log2FC, x-axis) and protein-level (log2FC, y-axis). Spearman coefficient correlation, r=0.11; p=0.006, Fisher test showing significant enrichment in the I quadrant p=0.03. The number of genes for each quadrant is reported in figure.
Cellgrowth of siRNA-transfected Capan-1 or Panc8680 cells; n = 3 cell culture wells per group; significantly different (P < 0.05) from control (no siRNA) *, siNC #1 #, siNC #2 $; P-values: Capan-1 cells 48 h time-point: 0.0408 (*) for siTBL1 #1 / 0.0464 for siTBL1 #2, Capan-1 cells 72 h time-point: 8.60 × 10−5 (*), 1.82 × 10−3 (#), 3.24 × 10−5 ($) for siTBL1 #1 / 2.39 × 10−10 (*), 3.34 × 10−9 (#), 1.08 × 10−10 ($) for siTBL1 #2; Panc8680 72 h time-point: 1.55 × 10−4 (*), 2.73 × 10−5 ($); two-way ANOVA with Bonferroni post-test.
Western blot analysis and quantitation of Crebh in Crebh-transfected WT (B) and HRD1-/- (C) HEK293T cells pre-treated with the proteasomal inhibitor bortezomib (BTZ) or lysosomal inhibitor chloroquine (CHQ) for 2 hours and then with CHX for additional 1 hour (n=2 per group, 2 independent repeats). Hsp90, loading control for Western blot analysis.
F Proportion of mitochondria with 0 to 5 MAMs are shown for each. Data information: n=214-246 mitochondria per cell line.
Average enrichment of density plots are plotted for different subsets of Mta ChIP-seq peaks.
Aβ profiles (% contribution of individual Aβ37, Aβ38, Aβ40, and Aβ42 peptides to the total Aβ levels (Aβ37+38+40+42)) in the conditioned medium collected from WT or mutant (NCT) MEF cells lines co-expressing WT or mutant APPC99 substrates were assayed by ELISA. Schematic models of the tested GSEC-APP complexes are shown.
(C) Patient (F7/7F-healthy carrier, F7/2M-RIRCD, F7/5F-healthy carrier, F7/1M-RIRCD without a clear second mutation) and control fibroblasts were grown for 12 days in media with no added glutamine or glutamic acid and 5% dialyzed FBS. Cells were lysed and the above mentioned mitochondrial proteins were checked via Immunoblotting. VCL was used as a loading control while the densitometry analysis (D) was done based on the mitochondrial proteinTOM20. Control bar represents an average of the two control fibroblast lines employed (F008 and F154). Although we could observe significant changes upon cysteine depletion in all conditions, the phenotype was exacerbated in the digenic fibroblasts compared to the controls.
(A) The protein expression of AR and FNTA (circFNTA host gene) were determined using Western blot and showed that shAR can decrease FNTA protein expression in UMUC3 cells and oeAR can increase FNTA protein expression in J82 cells.
(b) Expression of hlh-30 was measured by quantitative PCR (qPCR) in day 1 adult WT (N2), eat-2(ad1116) (dietary restriction, DR), daf-2(e1370) (insulin/IGF-1 signaling), clk-1(e2519) (mitochondrial respiration) and rsks-1(sv31) (mRNA translation) animals (mean±s.d. of three biological replicates, *P0.05, Student's t-test).
I, J Knockdown of RNF123 enhances SeV- and EMCV- induced IFN-β expression in HeLa cells. HeLa cells were infected with SeV or EMCV for 12 h, total RNA was isolated, and Q-PCR (J) was performed using the indicated primers.
C Quantitative RT-PCR analysis of BMP2-induce Sp7 transcription. Prx1-Cre;Usp34f/f MSCs were treated with Smurf1 or control siRNAs, and depleted from serum overnight before BMP2 stimulation. Results are shown as mean ± SEM; n=3; *: p<0.05 and **: p<0.01 by ANOVA with Tukey's post hoc test