EMBO/sd-character-level-ner huggingface dataset:
A) Snapshots and tracks of pxDsRed signal in WT, Miro1KO, Miro2KO and DKO MEFs following live imaging at 1.5 seconds per frame for two minutes. B) Quantification of median net displacement of individual peroxisomes (pxDsRed signal) for WT, Miro1KO, Miro2KO and DKO MEFs (n=42 cells over six independent experiments. Two different MEFs lines were used for each genotype). Data information: For multiple comparisons in B) a one-way ANOVA with Newman-Keuls post-hoc test was used to test for statistical significance.
A) Gastrocnemiusmuscle weight in young and old control and Mfn2KO mice (n=6 mice per group).
G. Binding kinetics of the UVR8HY5C44 chimera pre-monomerized by UV-B versus the COP1 WD40 domain obtained by GCI experiments. Sensorgrams of UVR8HY5C44 injected are shown in red, with their respective 1:1 binding model fits in black. The following amounts were typically used: ligand - COP1 (2000 pg/mm2); analyte - UVR8HY5C44 +UV-B (highest concentration 2 μM). ka = association rate constant, kd = disassociation rate constant, Kd = dissociation constant.
ILP-TNF/Mel/SM significantly reduced infiltration by CD3+CD4+Foxp3- T cells whilst (G) increasing infiltrations by CD3+CD4+Foxp3+ T regulatory cells (H).
L. Graph showing quantification of extravasated 70kDa Dextran from small intestine tissue of Casp8WT/MLKLko and Casp8ECko/MLKLko mice at 30 days after tamoxifen treatment (n= 7 WT/MLKLko, 6 ECko/MLKLko; two tailed unpaired Student's t-test). Data information: All data is shown as mean ± SEM. ns: not significant.
C. RAB13 protein levels were measured by quantitative Western blot and normalized to total α-Tubulin or GAPDH levels (for representative blot, see Figure 5E). Relative levels in RAB13 PMO-treated compared to control are shown. No significant differences were detected by Kruskal-Wallis test with Dunn's multiple comparisons test. N=3-8. Bars: mean ± SD.
C Sequence alignment of human Atg8 protein homologs. The alignment was obtained by Clustal W. Black and grey backgrounds represent degree of similarity. Closed circles indicate specific residues of GABARAP involved in the ALFY‐LIR peptide interaction.
(H) In vitro translation of 100 ng of luciferase mRNA with different 5' UTR lengths in the presence (orange columns) or absence (grey column) of 0.5 µM (PR) 20 (n=3). Data represent mean values ± SD (*, P<0.05; t-test).
Venn diagram showing the rescue of H2Bac levels by comparing differentially regulated peaks before (TAU VEH vs. WT VEH) and after (TAU MOL vs. TAU VEH) the treatment for all peaks with an FDR <0.001. About 95% of the peaks were not deregulated after CSP-TTK21 treatment
(A) A549 and IMR90 cells were transfected with XIAP‐specific or control siRNAs. Cell lysates were subjected to western blot analysis with the indicated antibodies. The data are representative of two biological replicates. The ratio of LCII/LC3I to actin is presented in Supplementary Figure S2A.
(C) Pex11-GFP pexophagy of WT cells containing either empty plasmid (WT) or GAL1-mRFP-ATG36 grown 18 h in oleate medium (0) then switched to oleate medium containing 2% galactose and harvested at 3, 6 or 22 h as indicated.
A. qRT-PCR analysis showing average TBP-normalized expression of GATA6 in the indicated cells. Data information: data are presented as mean values + SD for six replicates (n=6). The p-value (Student's t-test) is indicated, **<0.01, ***<0.005 and ns=non-significant
A, B. Venn diagram showing the number of hyper-DMRs that overlap between idap1-2 and rdd (A) and between idap2-1 and rdd (B). Box plots show the methylation level of each class of DMRs. Dark horizontal line, median; edges of boxes, 25th (bottom) and 75th (top) percentiles; whiskers, minimum and maximum percentage of DNA methylation. Asterisks indicate significant differences between the wild-type and the indicated mutants (*p < 10-15, Mann-Whitney U test).
C. MCF-7 cells were transfected with control vector or FLAG-TRIM46 for 48 h. Whole-cell lysates were prepared, and immunoprecipitation was performed with anti-FLAG followed by immunoblotting with antibodies against indicated proteins (top). Immunoprecipitation assays in MCF-7 cells with anti-TRIM46 followed by immunoblotting with antibodies against the indicated proteins (bottom).
A Epifluorescence stereo microscopy of inguinal mammary gland from a 5‐day‐old Wnt4::Cre; mT/mG female (n = 7). Scale bars: 0.5 mm and 0.1 mm (inset).
B. Concentration-response relationships of iodine-induced intracellular Ca2+ increase in HEK293 cells expressing WT or mutant TRPA1 channels. Data are presented as mean ± s.e.m. n ≥ 8 for each construct at each concentration. The smooth curves are fits to the Hill equation.
C-H. Immunoblots of subcellular fractions from control HeLa cells or HeLa cells treated with the indicated si-RNA and then exposed to hypoxia for 5 h. PNS: post nuclear supernatant; Cyto: cytosol; Mito (crude): crude mitochondrial fraction containing mitochondria and MAMs.
Changed metabolic pathways in blood of NMD (F) patients. Data information: Top 10 pathways with ≥ 10% of detected metabolites per pathway are shown. metab., metabolism.
A. Representative electron micrographs showing a small/clear vesicle (SV, arrow), dense-core vesicles (DCVs, arrowheads) and a clathrin-coated pit (hatched arrow) in DRG neurons.
A) futsch genomic region (X1,408,435 - 1,454,00) and corresponding browser tracks from miCLIP-seq experiments in S2R+ cells. futsch transcripts contain two m6A peaks in its 5´ UTR, as indicated.
Immunofluorescence analysis of Chr X-FISH (green), Chr Y-FISH (red), and SYCP3 (white) was performed in WT and XUsp26+XUsp26-YY spermatocytes (I). The arrows indicate the Y chromosome and the arrowheads indicate the X chromosome. Nuclei were stained with DAPI (blue).
Confocal fluorescence immunocytochemistry images of a single cultured mouse ciliated ependymal cell. Cilia were visualized using an antibody for acetylated alpha tubulin (green). Cwh43 immunoreactivity was visualized using a specific anti-Cwh43 antibody (red). Scale bar (left column) is approximately 4 µm. Images in the column on the right represent a Z-stack reconstruction of confocal images showing localization of Cwh43 immunoreactivity in cilia of a mouse ependymal cell. Scale bar (right column) is approximately 5 µm.
(A, B) NADPH levels (A) and NOX (B) level was measured in WT and MCK-KO muscles at rest and after a bout of low-intensity exercise for 50 min (n = 6, means ± SEM, * p < 0.05, two-tailed student's t-test and two-way ANOVA followed by Bonferroni post-hoc testing).
K: The number of primary basal dendrites is significantly reduced in NexCre cTKO animals compared to LM in sCA1 (Mann-Whitney test, *p = 0.0232).
Exposure of the catalytic Cys431 is parkin as assessed by reaction with a UbVS probe. The different stages of activation using parkin, parkin:pUb, pParkin:pUb and pParkin:pUb in combination with an isopeptide linked UbcH7-Ub conjugate are indicated above each gel. Following addition of UbVS, samples were taken at the times indicated (0-10 minutes) and visualized by SDS-PAGE.   Relative percentages of pParkin and the pParkin-Ub adduct as a function of time. Intensity percentages were calculated as a function of total intensity of pParkin -Ub, parkin and UbVS/pUb bands. Error bars represent standard deviation from the average for duplicate measurements.  Exposure of the catalytic Cys431 is R0RBR parkin as assessed by reaction with a UbVS probe. The different stages of activation using R0RBR, R0RBR:pUb, R0RBR:pUb:pUbl and R0RBR:pUb:pUbl in combination with an isopeptide linked UbcH7-Ub conjugate are indicated above each gel. Following addition of UbVS, samples were taken at the times indicated (0-60 minutes) and visualized by SDS-PAGE   Relative percentages of R0RBR and the R0RBR-Ub adduct as a function of time. Intensity percentages were calculated as a function of total intensity of the R0RBR-Ub, R0RBR and UbVS/pUb/pUbl bands.
D. A graphic model of mitochondrial stress sensor OMA1-coordinated glycolysis and mitochondrial oxidative phosphorylation in colorectal carcinogenesis and development. Hypoxia-induced OMA1 activation leads to the degradation of mitochondrial respiratory chain complexes (MRCC) components and OPA1, and results in cristae remodeling, all of which results in the inhibition of mitochondrial electron transport chain (ETC) and the generation of mitochondrial ROS. Elevated ROS can stabilize HIF1a, thereby promoting glycolysis. Thus, OMA1 promotes the Warburg effect by coordinating glycolysis and oxidative phosphorylation to facilitate colorectal cancer development.
Mass spectrometry analysis of BubR1 IPs from the indicated cell lines. Relative protein quantification values (MaxLFQ, log10) are plotted across conditions. Data from analysis of 3 technical repeats of BubR1 purifications with standard deviation indicated.
The engraftment of MOLM-13 after FST knockout was detected by flow cytometry of human CD45 and mouse CD45.1 positive cells in recipient mouse BM aspiration at week 2 post transplantation.
Whole cell phospholipidome of WT and Opa1Crispr MEFs treated with NT (non-targeting), Tamm41 or Pgs1 siRNAs. Data represent mean ± SD of five independent experiments; *p < 0.05, *** p < 0.001, ****p < 0.0001, ns; not significant.
(A) Competitive ELISA. ACE2-Fc can compete with ACE2-Fc-Biotin for Spike 1-674 binding in a dose-dependent manner. The competition effects of ACE2-Fc on ACE2-Spike protein binding was normalized to PBS control. Error bars represent the standard deviation (SD), n=2. Statistical analysis was performed by unpaired two tail t-test. **P < 0.01, ***P < 0.001.
(E) GBS strain K79 traversal of HBMEC monolayer was significantly decreased in CysLT1 knockdown HBMEC compare to control.
A. qRT-PCR analysis of NDIME (P = 0.0051) and MEF2C (P = 0.0062) in VPA-treated mice and saline-treated mice; n = 9 per group.
B. Representative visualization of cell tracks in the various clusters identified in Figure 6A. For visualization purposes, only cells from the control group are used.
A, Gene expression analysis of THEM6/c8orf55 in normal and tumoural prostate tissues according to the PRAD TCGA dataset (n=489). Data information: Center line corresponds to median of data, top and bottom of box correspond to 75th and 25th percentile, respectively. Whiskers extend to adjacent values (minimum and maximum data points not considered outliers). Statistical analysis: pairwise ANOVA. Data reproducibility: n = 489 tumours.
(C) Scheme of DNA fiber tract analysis in shWRNIP1 cells. Cells were transfected with an empty vector or a plasmid expressing a wild-type human RAD51, and 48 h thereafter labelled with IdU and treated or not with 4 mM HU.(D) Representative IdU tract length distributions in shWRNIP1 cells or shWRNIP1 cells expressing exogenous wild-type RAD51 after HU exposure. Median tract lengths are given in parentheses. See Tables S1 and S2 for details on the data sets and statistical test. Representative DNA fiber images are given. Scale bars, 10 µm. Western blot shows the expression of the RAD51 protein in shWRNIP1 cells. The membrane was probed with an anti-RAD51. LAMIN B1 was used as a loading control.
The schematic of CAF-1 body-mediated HIV-1 latency. Detailed information for the schematic was indicated in discussion.
D. Pie chart showing distinct categories of RNA species bound to hTrmt13 identified by CLIP-seq.
b. d-g, LDH release and immunoblots for caspase-1 and caspase-11 from BMDMs infected for 16 h or transfected with LPS in presence or absence of 3-methyladenine (3-MA).
B NHEJ repair assay using GFP expression-based reporter system described in Fig 5I (mean ± SEM of 3 independent experiments. See Fig EV5B for representative FACS dot plots).
(B) Model fit to the target concentration curve (mean interpolation of plasma serine over 24 hours) with standard deviation indicated in gray.
The image sequence shows formation of a vacuolar whorl that becomes a cytosolic whorl. Scale bar: 2 µm.
A NAD+ levels in limb muscles of 20-month-old WT (n=7), mdx (n=5) and mdx/CD38-/- (n=6) mice. Data information: Each dot of the graphs represents a mouse. 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.
Expression of CSAG2 decreased p53 ac-K382 levels and downregulated the expression of downstream p53 target genes in H460 cells. Cells were treated by 20 μM etoposide with 0.4 μM TSA for the indicated times. Cells were then harvested and blotted for the indicated proteins. Quantitation of expression levels is shown G) as the mean ± SD, n=3 biological replicates. Ac-p53 K382 levels normalized to total p53, whereas all other proteins were normalized to GAPDH.
A-C Relative mtDNA level (A), TMRM (B) and MitoSox (C) following treatment for 5 days with dsRNA against the genes indicated.
B Representative images of immunofluorescence detection of MAO-A/B and αSMA in LAM lung lesions, nuclei stained with DAPI (merged).
Average calcium responses of AWB::GCaMP3 worms exposed to 1-undecene at indicated concentrations. Each worm was recorded for 180 s. Worms were under stimulus between 11 s-130 s, window shown in grey, and stimulus withdrawal beyond 130 s. Shaded regions around the curves represent SEM. n = 3. Two dashed box represents 10 s time window after stimulus addition and stimulus removal respectively.
(d-e) Scatter plots of BTMs enriched (p>0.05) in blood-culture positive samples in Nepal (y-axis) versus Oxford (x-axis) for typhoid fever (d) and paratyphoid fever (e). For further details on BTMs refer to reference (Chaussabel et al., 2014).
A) Scheme of the protocol. ESCs were treated with 10µM KU60019 for 1.5 hours before sequential addition of IdU and CldU for 20 minutes each. DNA was treated and visualised as previously described.
(C) FRET efficiencies for donor- (Alexa488-) labeled WAVE1 variants (c=7.5nM, PWCA=blue, WCA=red, P_CA=grey) as a function of LatB-stabilized, quencher- (Atto540Q-) labeled actin monomers concentration. Lines are fits to a single site binding curve.
D: Scheme of the experimental protocol used for co-culture experiments with glomerular endothelial cells (GEnC) and podocytes.
F Northern blot analyses of mitochondrial transcripts of RKO cells treated with DMSO (RKO, "IMT1 -"), IMT1 (RKO "+ IMT1") and IMT1-resistant cells (Res, "IMT1 +"). n=3 independent experiments (Repl. 1, 2 and 3) were probed for COX2, ATP6, 16S and 12S mtRNA; the nuclear-encoded 18S RNA is shown as loading control.
B Hsf1 activation-attenuation cycle. In unstressed cells Hsf1 is in monomer-dimer equilibrium, occasionally trimerizing depending on the local concentration. Trimeric Hsf1 either binds to HSE-promoter DNA driving heat shock gene transcription by releasing paused RNA polymerase or is disassembled immediately by Hsp70/DnaJB1 action (not shown for clarity). Hsp70 binds dynamically to the TAD of DNA-bound Hsf1 trimers attenuating transcriptional activity and bind to its HR-B proximal binding site, disassembling Hsf1 trimers and thus removing it from heat shock promoters. Release of Hsf1 from Hsp70 restarts this cycle. Upon heat shock Hsf1 trimerizes at elevated rates due to its thermosensory function, integrating over temperature and time at elevated temperatures. Simultaneously, Hsp70/DnaJB1-mediated attenuation and disassembly is slowed down, due to binding of Hsp70 to misfolded and aggregated proteins. Both parts of the cycle shift the Hsf1 pool rapidly to the DNA bound active state, accelerating heat shock gene transcription. Elevating Hsp70 concentrations successively attenuate transcriptional activity of Hsf1 and disassemble Hsf1 trimers.
(B-C) Coimmunoprecipitation of Ams1 (B) and Ape4 (C) with Nbr1. Endogenously TAP-tagged Nbr1 was immunoprecipitated (IP) using IgG Sepharose beads. Cell lysates and immunoprecipitates were examined by immunoblotting (IB).
D Pearson correlation coefficient among three independent biological replicates of the SFB-TAP results and the BioID2 labeling experiments. Box limits represent 25th percentile and 75th percentile; horizontal line represents median. Whiskers display min. to max. values.
Expression of PID-4::mTagRFP-T H) together with 3xFLAG::GFP::ZNFX-1, a Z granule marker, in a pid-2 mutant background. The indicated dashed boxes reflect zoom-ins on specific nuclei to better visualize the granules, and their overlaps. One L4 gonad is shown for each animal. Scale bar: 25 µm.
B: Graph of the ratio of the median IC50 of the entire panel to that of each cell line generated from a published screen of 445 agents in 62 sarcoma cell lines (Teicher et al., 2015). The rhabdoid tumor cell line, G401 (red), appears on the left side of the graph indicating this cell line is more sensitive to mithramycin.
C) Representative immunofluorescence images showing the different sub-population of infected fPHH: hGK (red), Dapi (blue) and phalloidin (cyan). Parasites are stained with PfGAPDH (green). Images taken on the Leica High Content at 20x magnification and then cropped in Adobe Photoshop.
(a) Purified autolysosomes were treated with trypsin. The trypsin-treated (stripped) autolysosomes were incubated with rat brain cytosol and reaction mixture, and then analysed by western blotting. HC, heavy chain.
(a) GFP-LC3 puncta were visualized by fluorescent microscopy of tissue from each type of mouse in fed state, and the numbers of GFP-LC3 puncta counted. Scale bars, 20 μm. *P0.05, **P0.01; Student's t-test.
F. Addition of AKT inhibitor MK2206 (AKTi) abolishes Rab25-dependent glycogen storage. (a) p < 0.05 versus shRNA control and (b) p < 0.01 SF + AKTi versus SF.
(G) K437R‐Beclin 1 reduces the activation of Vps34. Beclin 1‐silenced HeLa cells stably expressing GFP‐HeLa were rescued with WT‐ or K437R‐Beclin 1, followed by stimulation with 1 h for 1 h. GFP‐WIPI1 dots were visualized by confocal microscopy.
C. WISH for runx1/c-myb in control injected embryos (upper left), evi1 morphants (upper right) or respectively evi1 MO with myr-AKT injected embryos with HS at 14 hpf (lower right) and evi1 MO with UAS-myr-AKT injected transgenic Tg(fli.1:Gal4FFubs3; UAS:RFP)rk embryos.D. Quantitation of results from (C). Shown are the percentages of embryos with normal or decreased runx1/c-myb expression for each condition.
B Ubiquitin conjugation assay of UbcH7 or its mutants catalyzed by GST-tagged PknG at 37°C for 30 min. Reaction products were immunoblotted using antibodies against UbcH7 (top panel), Ub (middle panel), and the GST tag on PknG (bottom panel).
D-F. Percentage of viable CD4+ βTcR+ (D), CD8+ βTcR+ (E) or CD4+CD25+ (F) cells among all epidermal cells analyzed by flow cytometry. Results from 5 experiments with ears pooled from at least 3 mice per genotype are shown. Representative flow cytometry scatter plots are shown in Appendix Fig S1A-C.
Correlative light and electron microcopy of F-actin (B) and YAP (C) on a liver tissue section at 1.8 d post PH. YAP was detected by immunogold-labelling, F-actin by fluorescence staining with phalloidin. Indicated BC regions with high (c', c'') or low (c''') F-actin levels are shown as magnifications. Arrows indicate gold particles.
(R, S) Scanning electron microscopy images of cilia (R) and quantification of the percentage of ciliated cells (S, n = 9 mice) in the mouse tracheal epithelium. To quantify the percentage of ciliated cells (S), >200 cells from six fields were analyzed for each mouse. Scale bar, 5 µm.
CRH-Cre::Ai9 reporter male mice fed on chow or 4 weeks on HFD were used to examine c-Fos expression in the PVH in 24hr fasting or refed after 24hr fasting. A) Representative images showing CRH neurons (red in the first colomn), c-Fos expression (green in the second colomn), merged of red and green (the third colomn) and the amplied pictures for the boxed areas in the merged images (the forth colomn). The experimental conditions for each group (fasting verus refed, chow versus HFD) are labelled on the left. Scale bar=200µm.
(D) Venn diagram depicting the number of genes upregulated (up, LFC > 1 and P ≤ 0.05) and downregulation (dn, LFC < -1 and P ≤ 0.05) by DEX in normoxia and hypoxia after 24h.
(C) Dissociation of deacylated tRNA from the E site by the fluorescence change of tRNAfMet(Flu) upon rapidly mixing of 80S 2C with buffer (grey), or with ternary complexes eEF1A-GTP-[14C]Val-tRNAVal in the presence of eEF2 and eEF3 (green), eEF2 (red), eEF3 (blue), or eEF2 and eEF3 without the ternary complex (magenta) in a stopped-flow apparatus. Each trace is an average of 5-7 individual time courses and normalized at 1 for the fluorescence at the start of the reaction.
Tumor cell killing in vitro by the HSVtk/GCV approach. Glioblastoma cells stably transduced with RGD4C/AAVP-Grp78-HSVtk or RGD4C/AAVP-CMV-HSVtk were treated with either GCV (10 μM) or TMZ (100 μM for LN229 and SNB19, 60 μM for U87) or combination of both GCV and TMZ. Cells were stained with MitoSOX and analyzed by FACS at day 4 post-treatment. Data shown are representative of three experiments, n=3. Two-way ANOVA with Tukey's multiple comparison test (GraphPad Prism 6) was used for data analysis.
G. Migration speed of RAB13 knockdown cells re-expressing GFP-RAB13 or GFP-RAB13 with a frameshift (fs) mutation at the beginning of the RAB13 coding region. 28-52 cells were analyzed. Bars: mean ± s.e.m.. Similar results were obtained in two additional independent experiments. Re-expressing cells were identified through GFP fluorescence and cells with similar, low GFP signal were tracked. All cells are expressing cherry-NLS for accurate tracking.
(C) Wild-type and PS1−/− hippocampal neurons (12 d) were scraped, pelleted, and treated with 10 mU EndoH, followed by Western blot detection of APP, TLN, PS1, nicastrin (NCT), synaptophysin, and ductin. TLN levels are increased almost fourfold in PS1−/− neurons. No EndoH-sensitive TLN was detected in PS1−/− neurons, indicating a complete maturation. APP-CTF shows a dramatic accumulation (44-fold; mean ± SEM, n = 3).
Western blot analysis of Flag-RNF8 denaturing-IP in HEK293 cells showing the ubiquitination pattern of RNF8-WT and RNF8-RING* variant, under siRNA-mediated luciferase (siLuc) or p97-depleted conditions (sip97).
(E) Colocalization of α-synuclein inclusions (n = 41-73) to lysosomes were measured in randomly chosen fields. The experiment was repeated three times. Data represent mean ± s.e.m. Statistical analysis was performed with one-way ANOVA (post-hoc Tukey's test). *p<0.01.
(D) The average DNA methylation levels at selected consensus transcription factor-binding motifs (gray: CD4+ naïve T cells; blue: WT Treg cells; red: Tet2/3 DKO Treg cells). Standard error is denoted in transparency of the corresponding colors.
(D) NAD+ levels without or with NMN addition in 9 months old WT and p32cKO heart lysates (n = 3). Error bars are presented as mean ± SD. Student's t-test was performed on WT vs p32cKO, **p < 0.002.
MDM were treated with 5μM ETO or cGAMP for 18h or 100ng/ml LPS for 2h. Cells were stained and analysed for IRF3 translocation into nucleus. Scale bars: 10μm.
(G) The expression of RUNX2 in BMSCs from Cdc20f/f mice and Sp7-Cre;Cdc20f/f mice after 7 days osteogenic differentiation treated with negative control or p65si by western blot analyses.
(A) The overall structure of the scFv FD20 (cartoon) in complex with SARS-CoV-2 RBD (green cartoon in semi-transparent surface). VH and VL denote heavy and light chain variable domains, respectively. The VH CDRs and VL CDRs are colored as indicated. The RBD structure resembles a highchair that has a 'seat' region and a short 'backrest' region.
C. Illustration of 405nm laser-photoactivated regions in monopolar spindles (blue circles, left). The effect of photoactivation and region selected for kymograph generation (dashed white rectangle, right). Scale bar, 10 µm. D. Corresponding kymograph profiles of the photoactivated regions in bipolar and monopolar spindles used for quantification of the flux rates (red dotted lines highlight MT-flux slopes). Scale bars, 30 sec.
A, Representative images of scaf1-/- and scaf1+/+ fish fed with the indicated diets.
Lyzs-Cre and p38γ/δLyzs-KO mice were fed a ND or a high-fat diet (HFD) for 10 weeks. (A) Body weight measured at the indicated times during HFD treatment.