EMBO/sd-character-level-ner huggingface dataset:
Pyridostatin (PDS) was administered intravenously (i.v.; 7.5 mg/kg/day), NU-7441 was administered intraperitoneally (i.p.; 10 mg/kg/day), over the indicated periods of time, and paclitaxel (PACLI) was administered intravenously (i.v.; 20 mg/kg/day) on indicated single days. Vertical dashed line indicates end of treatment. Tumour volume was measured at the timepoints shown on the graph and expressed relative to tumour volume at the beginning of treatment. Each experimental group included n = 5 mice. Error bars represent SEM. P values were calculated at day 63 using an unpaired two-tailed t-test between the NU-7441+PDS+PACLI combination and each double treatment. ****, P ≤ 0.0001.
(A) Representative immunoblots of indicated proteins from whole-cell lysates of MCF7 cells transfected with indicated siRNAs for 56 h and subjected to 0-40 min of amino acid starvation [−AA]).
(A) FIP200+/− and FIP200−/− MEFs stably expressing GFP-ULK1 or -ULK2 were cultured in starvation medium for 120 min. The cells were fixed and examined by fluorescence microscopy. Bar, 20 μm.
E HeLa cells were transfected with USP19-specific siRNA or scrambled siRNA and treated with indicated concentration of rapamycin for 12 h, p62 levels were detected by immunoblot.
2B) Cortical neurons from 3 different isolations (10-20 DIV) of WT and PINK1 KO mice were either non-starved or starved for 2 h in HBSS and the LC3-II and actin content was determined by western blotting. A representative blot is shown on the right. The relative LC3-II content of WT and PINK1 KO mice, respectively, was set as 1. Primary neurons showed a strong upregulation of autophagy in reaction to starvation but cells derived from PINK1 KO mice exhibited a reduced LC3-II/actin ratio compared to the WT cells; n = 5, p<0.001.
(C, F, I) Gold particles co‐localizing with bacteria were manually counted in ten macrophages of each condition. Means ± SEM are depicted and differences between treatments are indicated, *p 0.05 using a two‐tailed paired t‐test. (B) Transmission electron microscopy (TEM) magnification ×50,000; the rest of the micrographs are TEM magnification ×80,000. Data were generated from one experiment.
(B) COS-2 cells were cotransfected with plasmids expressing either SipB428-593-YFP or SipB-CFP, and transfected cells were examined under a confocal microscope. Expression of SipB428-593-YFP prevented wild-type SipB-CFP from forming multivesicular structures (notice that vesicular structures are only apparent in the absence, but not in the presence, of SipB428-593-YFP; right).
(G) ChIP analysis of RNA Polymerase II enrichment on the transcriptional regions of Acta1 and Nppa in ventricular samples. n=3:3.
Cell-type specific AGO1-IP signals for each miR169 sequence-group with (upper) or without (lower panel) the miRoot scale normalization option.
(e,f) Proliferation (e) and IL-2 production (f) by CD4+ T cells isolated from the spleen of L2A-cKO mice and their control littermates and stimulated ex vivo for 48 h with anti-CD3 and anti-CD28 (assessed as in Fig. 2c (e) or Fig. 2b (f)). *P = 0.0062 (e) or 0.006 (f) (t-test).
(F) Flow cytometric analysis of PD-1 expression on T cells in response to different treatments (control n=4, all other groups n=3).
G, H. Clonogenic assay showing that HUWE1 knockdown in 8988T cells are sensitive to UV (G), and HU (H). As controls, both cells transfected with non-targeting siRNA (siControl), and non- transfected cells, were used. Note that the siHUWE1#2 oligonucleotide shows a stronger downregulation of HUWE1, and confers increased sensitivity compared to siHUWE1#1. The average of nine experiments is shown. Error bars represent SEM. P-values (representing the statistical difference between the samples at the highest dose treatment) are: 4.55x10-9 (G) and 1.8x10-8 (H).
(A) Atg16L1Δ/Δ MEFs were transiently transfected with either mock, FLAG-tagged Atg16L1 WT, ERER, or DRDR. After 2 hr in EBSS, cells were fixed and labeled with anti-LC3 and anti-FLAG antibodies and visualized by confocal microscopy. Scale bars, 20 μm.(B) Statistical analysis of (A) was performed using one-way ANOVA with Dunn's posttest. ∗p < 0.05. SEM for n = 3.
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) Non-recycled BACE1 (green) detected with M1 (15 min pulse, acid stripping and 20 min chase) in Rab5-positive early endosomes (magenta) in axons of siControl- and siBin1-treated neurons expressing Rab5-mCherry and BACE1-GFP, analysed by spinning-disk confocal microscopy. Scale bar, 10 µm. White squares indicate a Rab5-positive endosome magnified on the right. Scale bar, 1 µm.(D) Quantification of colocalisation between non-recycled BACE1 and Rab5 in axons (n=3, NsiControl=19, NsiBin1=24; ****P<0.0001, t-test; mean ± SEM).
(a) Diagram illustrating how lysosomal positioning coordinates mTOR signalling and autophagy. Peripheral lysosomal localization (nutrient induced) increases mTOR activity (blocking autophagosome synthesis) and reduces autophagosome-lysosome fusion. Starvation-induced lysosomal clustering reduces mTOR activity (activating autophagosome synthesis) and facilitates autophagosome-lysosome fusion.
Cortical malformations observed in six novel BBSOAS patients Patient (P) 1 shows abnormal gyrification of supramarginal and angular gyri is present in five out of six individuals (P2-P6;
Western blot analysis performed with lysates of (M) IRGM+/+ and IRGM+/- HT29 cells
(C-D) Spearman correlations between KLF10 and zDHHC7 in liver tissues from NASH and NAFL patients using GEO datasets (GSE48452 and GSE61260). In the dataset GSE61260 (C), n=83 (including 38 healthy controls, 21 NAFL patients and 24 NASH patients). In the dataset GSE48452 (D), n=73 (including 41 healthy controls, 17 NAFL patients and 15 NASH patients). Data information: * P <0.05, ** P <0.01, *** P <0.001. Results are shown as mean ± SD. Spearman's correlation (C, D).
D Densiometric quantification of COX2 protein levels from n=4 experiments. Data are expressed as mean values ± SD. Statistical significance was calculated with one-way ANOVA. IMT1 vs IMT1 + RAPA: *p=0,016; IMT1 vs IMT1 + FG4592: non-significant.
(K) The numbers of Nissl-stained interneurons in the spinal cord are similar in control and Epg5−/− mice. Means ± SEM of three mice are shown in J and K.
Schematic diagram of the experiment in which PS exposure was measured in the brains of 1-month- and 3-month-old wild-type mice by injecting AAV9-GFAP-secA5-mCherry in the neonatal stage (P0). Representative confocal z-stack colocalization images of Gephyrin (green), VGAT (blue), and secA5 (red) in the HP (hippocampus) of 1-month- and 3-month-old wild-type mice. The white circles indicate secA5-positive inhibitory synapses. Scale bar, 10 μm. Bar graphs showing the expression of secA5 (B) and IBA1 (C) in inhibitory post-synapses (Gephyrin-positive, green) and pre-synapses (VGAT-positive, blue) in the HP of 1-month- and 3-month-old wild-type mice (n = 4 for each group, ***P = 0.0009, *P = 0.0248, n.s., not significant).
C. Immunoprecipitation of PTENε with endogenous CDC42, ACTR2, VASP, and FSCN1. The plasmid expressing PTEN, PTENε, or mock plasmid carrying C-terminal S-tag and HA tag was transfected in HEK293 cells separately. Cell lysates were incubated with S protein agarose followed by western blotting with antibodies against CDC42, VASP, ACTR2, FSCN1, and HA tag.
Correlation (Pearson) of RAIDD and PIDD1 mRNA with transcript levels of Ki67 in the TCGA data set.
(e, f). Overexpression of Drp1WT, but not phosphorylation mutant Drp1S616A, suppresses mitochondrial fusion in dendrites of PINK1KO primary neurons. (e). DIV4 neurons generated from PINK1 WT control or PINK1KO mice were co-transfected mito-GFP with either pcDNA3.1 (3.1) or Drp1WT or Drp1S616A. Neurons were immuno-detected with an antibody against myc-tag (Myc) (red, to detect exogenous Drp1). Mito-GFP: green, to detect mitochondria. Representative images were shown, scale bar=25 μm. Magnified dendritic shafts gated by a dot line frame are shown (Zoom, right panels, scale bar=5 μm).(f). Quantification of Mito-index in dendrites of neurons from experiments of (e). 10 neurons were randomly selected and analyzed in each experiment. One-way ANOVA followed with Tukey's test. *p<0.05, ***p<0.001, ns: no significance. Data was presented as mean ± SEM of three independent experiments.
Transcriptional activity in U2OS/GFP-FAM111A cells treated or not with DOX pulse-labeled with EU were analyzed by QIBC (red bars, mean; n>2000 cells per condition).
Differentiated lung bud tip organoids at air-liquid interface in co-culture with donor-specific human fetal lung fibroblasts. After 14 days of differentiation at air-liquid interface, cells express basal cell (TP63 (P63), green, markers in areas containing one cell layer. Dotted lines indicate the barrier between multilayered and single layered epithelium. Data information: Nuclei are stained with Hoechst Scale bars indicate 50 μm.
(d) Confocal microscopy of differentiated THP-1 cells transfected with poly(dA:dT) and immunostained for LAMP-1 and ASC; below, z-axis projections. Scale bars, 10 μm.
F: Intracellular ATP production in iPSCs generated from Detroit 551, WS5A and CP2A fibroblasts (n=3, technical replicates per clone for control and WS5A; n=4, technical replicates per clone for CP2A).
(B) AR occupancy (blue) and levels of the histone marks H3K27ac (green) and H3K27me3 (red) at ARBS within the regulatory regions of Fkbp5, Kap (both androgen-regulated in kidney) and Tox3 (androgen-regulated in prostate but not in kidney and hence used as a negative control) were assessed by ChIP-qPCR in kidneys from 13-week-old ARLmon/Y mice and WT males.
d. Accuracy (as described in c) of calling mRNA spots to cells when using JSTA to segment cells with a lower selection of cell type marker genes (8-44 genes tested). The color of the line gets progressively darker as the number of genes used increases.
(I) qPCR levels of LOX-L2 and HIF-1A in PSCs, normalized to RPLP0 and relative to control
(C) Gene set enrichment analysis of the global expression profiles of the Black versus White HKC strains (GSE156011) using 50 HALLMARK signatures (https://www.gsea-msigdb.org/gsea/msigdb/collections.jsp#H), revealing a significant association with the OXPHOS signature. Genes are ranked by signal-to-noise ratio based on their differential expression in Black versus White HKC strains. Position of genes in each gene set is indicated by black vertical bars and the enrichment score (ES) by the green line. Normalized Enrichment Score (NES), nominal p-value and FRD q-value, based on the permutation-generated null distribution, are as shown.
E. H3K4me3 at TSS region of genes from RNA module1. Inset shows statistical comparisons among groups (One-way ANOVA, ***P<0.001, ****P<0.0001). Bars and error bars indicate mean ± SEM. Number of replicates analyzed for H3K4me3 profile at TSS (Wildtype vehicle: 3; CamkIIδc TG-vehicle n = 4; CamkIIδc TG-Vorinostat n = 4).
(D, E) Performance in the Rotarod test of ASMko and wt mice treated or not with PLX for 2 months expressed as the mean ± SEM time spent in the rotating rod on each of four trials (D) or the mean ± SEM time of the four trials (E) (n = 7 mice per group, Two-way Anova, Games-Howell (D) and Bonferroni (E) post hoc).
(E) Representative micrographs showing the expression of ICAM-1 and VCAM-1 (red) by the RPE (green) 1 day after GT intoxication. Bar graphs throughout the figure show mean ± SE of each group. *, P < 0.05; **, P < 0.01; *** P < 0.001. GT - glutamate; noninj - noninjured control.
cell death induction by cl-HK2pep; in the cytofluorimetric analyses viable cells are double negative for Annexin V-FITC and 7-AAD staining and measured 15 min after peptide treatment data are presented as mean±SD and they were obtained from 3 different experiments or more; 3 technical replicates in all analyzed experiment. Data information: Experiments throughout the Figure are carried out on HeLa cells; cl-SCRpep, negative control of cl-HK2pep (2 μM each) where indicated calpain inhibitor PD150606 (50 µM each) are pre-incubated 1 h before peptide treatment. data are presented as mean±SD of at least 3 independent experiments. p<0.0001 with a two-way ANOVA (cl-HK2pep treatment vs cl-HK2pep+PD150606 or cl-SCRpep treatments); Bonferroni post-test in graph ***p<0.001 (cl-HK2pep vs cl-SCRpep and cl-HK2pep vs PD150606+cl-HK2pep).
B. Sir3-binding at TEL6R in untagged, WT, sir3Δ cells or in cells overexpressing Sir3 (oeSir3). Binding is probed by ChIP-qPCR 0.2 (red arrows) and 1kb (blue arrows) from telomeres and at the OGG1 control locus using antibodies against Sae2-GFP. The mean of three independent biological replicates is shown and error bars correspond to the variation between replicates (SEM).
C. RT-qPCR analysis of relative mRNA expression of Pde10a in BAT, peri-ovarian visceral white adipose tissue (VAT) and inguinal subcutaneous white adipose tissue (SAT) of lean mice (n = 5). ** P = 0.0063 (SAT vs VAT), ** P = 0.0011 (SAT vs BAT) and ** P = 0.0037 (VAT vs BAT) using unpaired 2-tailed Student's t test.
B Schematic representation of the human ARL13B structure depicting the guanine nucleotide-binding (G), coiled coil (CC) and proline-rich (PR) domains as well as the point mutations affecting N-terminal palmitoylation or C-terminal ciliary targeting RVEP motif.
C: Autoradiography of Gt1 cells transfected with PIKfyve-GFP or PIKfyveΔacyl (24h), metabolically labeled with 3H-palmitate (2 hours), and immunoprecipitated with anti-GFP antibodies. Lower panel: Western blot on immunoprecipitates using anti-GFP antibodies. Error bars represent s.e.m. (n=3 individual biological replicates).
A. Human ileum-derived organoids were seeded in 2D on iBIDi chamber slides. 12 and 24 hpi cells were fixed and the amount of SARS-CoV-2 infected cells (red) and the induction of ISG15 (white) was analyzed by single molecule RNA FISH. Nuclei were visualized with DAPI (blue). White arrows indicate SARS-CoV-2 positive cells. N=3 biological samples. Representative image is shown. Scale bar=100 um.
C Heat map of interferon-related genes in U2OS cells with TERT-WT or TERT-K626A ectopic expression.
F Staining for FLAG at at P14 of the organ of Corti of a mouse injected at P1 with AAV.Syne4.
g, Immunohistochemistry of −KRas tumours for CD44 (blue) and CD133 (red) (×20-40).
G. Nppa expression in mice subjected to sham or I/R procedure. ANOVA followed by post-hoc Tukey test was performed. * P=0.002 PBS-sham vs. PBS-I/R. * P=0.02 DFO-sham vs. DFO I/R. * P=0.007 PBS-I/R vs. BPD I/R. N=6 mice for PBS-sham and DFO-sham, N=4 mice for all other groups.H. Nppb expression in mice subjected to sham or I/R procedure. ANOVA followed by post-hoc Tukey test was performed. * P= 0.0007 PBS-sham vs. PBS-I/R. * P= 0.014 DFO-sham vs. DFO I/R. * P= 0.003 PBS-I/R vs. BPD I/R. N=6 mice for PBS-sham and DFO-sham, N=4 mice for all other groups.I. Myh7 expression in mice subjected to sham or I/R procedure. ANOVA followed by post-hoc Tukey test was performed. * P=0.0001 PBS-sham vs. PBS-I/R. * P=0.038 DFO-sham vs. DFO I/R. * P=0.006 PBS-I/R vs. BPD I/R. N=6 mice for PBS-sham and DFO-sham, N=4 mice for all other groups. All data are expressed as mean ± SEM. N.S. = not signigicant.
A Inhibition of substrate crosslinking by the GSIs identifies substrate residues binding in the active site. L, L-685,458; D, DAPT.
Gene expression profiles of bulk PBMCs from P. falciparum-infected asymptomatic individuals and healthy immune controls were compared. A. Hierarchical clustering heat map of the 171 differentially expressed genes (DEGs) in P. falciparum asymptomatic and healthy immune control participants. C. Mean-difference plot displaying DEGs between asyptomatic P. falciparum malaria and healthy immune controls. Each gene is plotted as a single point determined by log-fold-change and average transcript abundance. Red genes are overrepresented, and blue genes are underrepresented in asymptomatic malaria.
B) Control VillinCreERt mouse small intestine immunostained for YAP shows a gradient of expression with mostly cytoplasmic localisation. Irradiation with 14Gy of X-rays drives strong nuclear localisation of YAP along the entire crypt-villus axis. Deletion of Src within VillinCreERt Srcflox/flox intestines prevents YAP nuclear localisation and normal regeneration after 14Gy irradiation. Arrows point to villar cells with nuclear YAP localisation. Lower panels show high-mag views of a different example than those shown in upper panels.
Primary astrocytes from OTUB1fl/fl (n = 3) and GFAP-Cre OTUB1fl/fl (n = 3) mice were stimulated with IFN-γ (10 ng/ml) for indicated times. mRNA was isolated and analyzed by quantitative real-time PCR for SOCS1. Data show the relative increase over untreated controls (mean + SEM).
(A) BMDMs were not infected (NT) or infected with L. pneumophila JR32 or the legA9 mutant at an MOI of 0.5, 1 or 5 for 24 h. Apoptosis was quantified by Photometric enzyme immunoassay analysis of cytoplasmic (apoptosis) histone−associated−DNA fragments (ELISA).
D Immunofluorescence microscopy images illustrating the association of GFP‐KSHV‐TK, but not GFP, with actinstress fibres. Scale bar, 10 μm.
14C accumulation (cpm/mg x ml-1) in H1299 R175H cells (+/- doxycycline) and H1299 -/- cells after 24h treatment with 14C-APR-246 (n = 2). Data information: TP53 status is indicated for each cell line. Data are represented as mean ± SEM. See also Figure EV5 and Appendix Figure S5.
F. RT-PCR of NCX1 mRNA expression following 200 μM D-Asp exposure, in absence or in presence of 10 μM MK-801. Graphs show quantification of ratio of NCX1 and NCX3 to L19
b, Plot showing the proportion of the four stromal subsets across all five patients.
(C) Cells were pre‐treated with a cocktail of lysosomal inhibitors (200 nM bafilomycin A1, 10 μg/ml pepstatin, and E64d; left panel) or 10 mM 3‐methyladenine (3‐MA; right panel) for 12 h and then exposed to starvation. LC3 levels monitored by western blot (D) and quantification of LC3‐II levels relative to Hsp90 were performed in the experimental conditions described in (C).
(E) Co-IP analysis of endogenous galectin-8 and TRIM16 in protein complexes from LLOMe-treated THP-1 cells.
(I) Number of GnRH neurons in E14.5 embryos injected with neutralizing antibodies to Neuropilin-1 (Nrp1Ab) compared to an isotype goat antibody, or the antiapoptotic compound ZVAD compared to its vehicle, together with fold change in GnRH neuron number as well as mutant and control embryos at the same embryonic age. Note that the data are represented as fold change, as raw values are not comparable with transgenic animals that did not undergo surgery. Unpaired t-test, n=5 to 7 embryos from at least 2 litters.
C HeLa cells were control or UBE2QL1-depleted for 60 h, fixed after 1 h of LLOMe or control treatment and stained for LAMP1 and p62 and analyzed by confocal microscopy. Arrows indicate colocalizing or non-colocalizing vesicles. Scale bars: 20 µm, 5 µm for inlays.
(B) Expression of 9 neuropsychiatric disorder-related genes in poly(I:C)-treated Tlr3-/- neurons. Relative expression levels compared with vehicle control are shown. In (B-D), dashed lines indicate the level of saline control.
(A-B) A total of 13 SNPs pre-selected according to the SOP presented in Fig 2 underwent PGAS screening as exemplified here: (A) PAUSS association pattern of an exemplary fictitious autosomal (upper panel) and a sex-chromosomal (lower panel) SNP which are eligible for the accumulation model. The genotype associated with the highest average PAUSS (in this example CC) is the 'proautistic genotype' (indicated by the black arrow) and is assigned a score of 1. The heterozygous genotype is assigned a score of 0.5 and the homozygous genotype associated with the lowest PAUSS receives a score of 0. Please note that the difference between genotypes does not have to be statistically significant. (B) PAUSS association pattern of an exemplary fictitious autosomal (upper panel) and a sex-chromosomal (lower panel) SNP which would not be selected for the accumulation model because of unclear phenotypical/biological relevance.
A EM analysis of the yNap1c2-H2AΔ14-H2BΔ28 complex. Representative rawimage of negatively-stained particles.B Representative particles (1st row), class averages coming from the projection matching procedure (2nd row) and comparison of the class averages with projections from a final model generated after four projection matching cycles (3rd row).
D Translation of HXT2 mRNA is more active late in the cell cycle. The HXT2 mRNA level bound to either di-/trisomes and polysomes (≥ 4 ribosomes) over monosomes was determined by extraction of mRNA from cells elutriated and harvested at G1/S or M-phase followed by qPCR. At G1/S similar levels of the HXT2 mRNA were found in the different ribosome fractions, whereas in M-phase more mRNA was bound to polysomes, suggesting a more active translation in M-phase.
(D) Phosphosite enrichment analysis of phosphopeptides from CK-p25, 5XFAD, and Tau mice. Circle colors indicate normalized enrichment scores (NES) and sizes indicate the number of observed sites in each substrate phospho-set within each mouse model. Kinases enriched in at least one model are shown.
(D, E) HeLa cells were transfected with the indicated siRNAs. Forty-eight h after transfection, the cells were re-transfected with LAMP1-RFP (red) for 24 h. And then the cells were incubated with Earle's Balanced Salt Solution (starvation) for 2 h. The cells were stained with anti-LC3 antibody (green) and DAPI (blue). Cells were visualized using a confocal microscopy. Regions within the dotted boxes are magnified in the insets. Scale bar, 5 µm. The quantification data of the colocalization of LC3 and LAMP1 is shown in Figure 7E. Data from three independent experiments represented as means ± S.E.M., ns, not significantly different; **, p<0.01, one-way ANOVA.
K, L. Carfilzomib shrinks tumor through modulating tumor microenvironment. EG7 cells were inoculated in wild-type or LyzM-cre;lsl-dTA to allow tumor xenograft to reach a volume of around 80 mm3. Mice were randomized for treatment (n=5) by saline or Carfilzomib for two weeks. The xenografts were dissected to photograph (K) or weigh (L) after treatment. Data are shown as mean ±SD and n indicates the number of biological replicates. *p < 0.05, ***p < 0.001 (student's t-test).
B Atomic model of the H2A.B-NCP in disc view (left) and gyre view (right). The nucleosome dyad is indicated with Φ. The superhelical turns of H2A.B nucleosomal DNA are designated as superhelical locations SHL -5 through SHL +5.
(B) UCSC genome browser view of normalized TT-seq coverage on the plus and minus strand at the 2.8 Mbp gene desert surrounding MYC (hg38; chr8:127,042,406-129,779,109; (Kent et al., 2002)) in fourteen cancer cell lines. eRNAs represent the union from all cell lines. Genome-wide association studies (GWAS) risk loci contain GWAS single nucleotide polymorphisms (SNPs) for prostate cancer (PrCa), acute lymphocytic leukemia (ALL), breast cancer (BrCa), colorectal cancer (CRC), and glioblastoma (GBM) including SNPs in linkage disequilibrium (r2 ≥ 0.8). TT-seq coverage is displayed between 8-250 to allow for better visualization of the eRNA signal surrounding MYC. Dashed rectangle is shown at full scale on the right. Note: TT-seq reads were mapped against the hg38 reference genome and we cannot rule out the existence of genomic rearrangements (e.g. translocations) affecting the shown region in the used cancer cell line clones.
B RT-qPCR quantification of selected drought marker genes in mock- and ABA-treated Col-0 (WT) and two independent pDOG1shDOG1::LUC transgenic lines. Error bars represent standard deviation and *, ** and *** represent t-test P-values of < 0.05, < 0.01 and < 0.001, respectively.
Representative Ca2+‑currents (Ica) and IHC plasma membrane capacitance increments (ΔCm) of a wild‑type control, transduced, and non‑transduced Otof-/- IHC in response to a 20 ms depolarization pulse at maximum Ca2+‑current potentials (typically -14 mV)