EMBO/sd-character-level-ner huggingface dataset:
histidine depletion slows down nerfin-1 dedifferentiation via decreasing cellular growth A-E) Representative images of nerfin-1 clones in the adult VNC after 6 days of feeding on CDD or -his diet, scale bar = 100μm.Figure 3A-E) Representative images of nerfin-1 clones in the adult VNC after 6 days of feeding on CDD or -his diet, scale bar = 100μm. Histidine dietary withdrawal resulted in clones significantly altering the amount of cell death per clone quantified in (C) (n = 6, 7). Histidine dietary withdrawal resulted in clones containing significantly more differentiated Elav+ neurons (red) quantified in (D) (n = 16,17) and significantly fewer Mira+ NBs (blue), quantified in (E) (n = 12,22), F-G) Larval nerfin-1 clones consisted of significantly greater proportion of differentiated neurons (n=10,7) and significantly reduced proportion of NBs per clone upon his dietary depletion (25% his, n=9,11). Representative images of adult nerfin-1 (I-J', green ) and pros clones (K-L', green), scale bar = 5μm. In nerfin-1 clones, the size of the NBs (Ase+, blue) was significantly reduced upon 6 days of histidine dietary withdrawal, quantified in (H) (n=33,20). In pros clones, the size of the NBs (Ase+ blue), and cellular growth measured as the ratio of nucleolus (Fib, white)/nuclear (Ase, blue) volume was not significantly altered upon 6 days of histidine dietary withdrawal, quantified in (H) (n=9,8) Cellular growth measured as the ratio of nucleolus (Fib, white)/nuclear (Ase, blue) volume was also significantly reduced, quantified in (M) (n=33,20). Cellular growth measured as the ratio of nucleolus /nuclear volume was significantly reduced in larval nerfin-1 NBs after 6 days his dietary reduction (25% his compared to CDD, n=14,6) quantified in (N). In pros clones, the size of the NBs (Ase+ blue), and cellular growth measured as the ratio of nucleolus (Fib, white)/nuclear (Ase, blue) volume was not significantly altered upon 6 days of histidine dietary withdrawal, quantified in (O) (n=9,7). Panel (P) shows that the larval nerfin-1 neuron to NB reversion requires 15-fold increase in cellular volume (n=10) and pros GMC to NB reversion requires approximately 3.5-fold increase in cellular volume (n=31).
Figure 3A Cell morphology using high-power magnification (40x) of the cultured cell lines under a microscope in LCC2 model-2-control and 2C3 cells. Scale bar: 50 µm. B Cell morphology using high-power magnification (40x) of the cultured cell lines under a microscope in LCC9 model-9-control and 9C2 cells. Scale bar: 50 µm. C-D Protein expression of key EMT-TFs in ESRP1 knockdown cells (2C3 and 9C2) compared to endocrine resistant control cell lines (2-control and 9-control) using Western blotting analysis. E Protein expression of key EMT-TFs in ESRP1 knockdown cells (T-47D-kESRP1) and ESRP1-overexpressing MCF-7 cells compared to their control counterparts (T-47D-control and MCF-7-control, respectively).
Figure 2A slice through the average structure of the apo-state at a position 22 Å away from the middle slice shown in Fig. 1C. The intra-SR extension is boxed on the density in red. An enlarged view of a volume-rendered representation fitted with an atomic model (PDB: 5TB2) corresponding to the region boxed in (A). The red arrow points to a density between the bilayer leaflets; this density is at a different location from the one in Figure 1C, D. Representative SR vesicles with different distributions of inner-SR density: segregated, evenly distributed, empty, and empty with filaments. RyR1 particles are circled in red, areas circled in yellow indicate accumulation of density inside the SR. The fractions of vesicles that fall into each density category for each RyR1 sample (EDTA: n=64; 1 mM Ca2+: n = 32; 5 mM Ca2+: n=21, technical replicates). Blue corresponds to the fraction of SR lumen vesicles showing evenly distributed density, orange to segregated density, and grey to empty vesicles. Structures of RyR1 determined in presence of increasing Ca2+ concentration (1 mM and 5 mM), all with intra-SR extensions. Scale bars: 10 nm in (A) and (E), 20 nm in (C).
a, Schematic structure of wild-type or truncated (&Dgr) Atg16L1 protein.figf1a, Schematic structure of wild-type or truncated (∆) Atg16L1 protein. CCD, Coiled-coil domain; WD, WD40 domain. Here and below, suffix α or β indicates isoform α or β. b-d, MEFs were cultured in nutrient-rich medium (fed) or Hanks' buffer (starved) for 1 h. Total cell lysates were prepared, and blotted with the indicated antibodies (b). α, β, indicate Atg16L1 isoform α, β; ∆CCD indicates Atg16L1∆CCD; asterisk indicates a non-specific band. Atg16L1∆/∆ indicates mice expressing ∆Atg16L1. The number of endogenous LC3 or GFP-Atg5 dots was counted (c, d). The results shown are mean + s.d. (n > 20). e, Electron micrograph of starved MEFs. f, Degradation of long-lived proteins in MEFs. Wortmannin is an autophagy inhibitor. The results shown are mean1s.d. Statistical significance (P value) was determined by the Student's t-test.
| A proposed model where GPR15-C10ORF99 signalling initiates the first colonic Treg homing reaction in herbivorous amniotes.Figure 7A proposed model where GPR15-C10ORF99 signalling initiates the first colonic Treg homing reaction in herbivorous amniotes.
Figure 8(A-F) Coronal brain sections obtained from mice at 4-6 (A-C) or 20-23 (D-F) months of age were immunostained for NeuN. The number of NeuN-positive cells with neuronal morphology was determined in dentate gyrus (A, D), CA3 (B, E), and CA1 (C, F). n = 11 (A-C) or 18 (D-F) NTG; 11 (A-C) or 5 (D-F) CamKII-tTA; 7 (A-C) or 9 (D-F) hTau-WT(L32); and 14 (A-C) or 10 (D-F) hTau-A152T(L1) mice. *P < 0.05, **P < 0.01, ***P < 0.001 by one-way ANOVA and Tukey test. Values are means ± SEM.
Manipulating the effector repertoire and target range of the VpT6SS1-based platform.Figure 4Viability counts of V. natriegens (cyan), V. vulnificus (purple), A. jandaei ∆tssB (brown), E. coli (green), and S. enterica (gray) prey before (0 h) and after (4 h) co-incubation with VnatReg attackers (yellow) harboring the indicated plasmids, on media containing 3% NaCl at 28 °C. The left panels show prey survival when competing alone (single prey) against the attacker at a 4:1 (attacker:prey) ratio; the right panels show the results of a competition experiment in which all prey were mixed together with the attacker at a 10:1:1:1:1:1 (attacker:prey) ratio. Data are shown as the mean ± SD, n = 3 technical replicates. Statistical significance between samples at the 4 h timepoint by an unpaired, two-tailed Student's t-test is denoted above and is color coded to match the relevant samples. A significant difference was considered as P < 0.05.
Figure 4A, Genome browser images of ChIP-seq for Pol II and Ub-H2B before and after induction of ER stress, with and without USP22 knockdown, at ER stress response genes CHOP, ERP70, and GRP78. ATF4/Xbp1s binding sites are from publicly available datasets (GEO accessions GSE69304 and GSE49952). B, Metagene profiles for Pol II (upper panel) and Ub-H2B (middle panel) ChIP-seq for USP22-dependent, USP22-Bound genes. mRNA analyses of select Bound gene transcripts are presented (lower panel). Three independent experiments are represented as mean ± SEM, with significance measured by Student's t-test, *p<0.05, **p<0.01, ***p<0.005 C, Metagene profiles for Pol II (upper panel) and Ub-H2B (middle panel) ChIP-seq for USP22-dependent, SAGA-Unbound genes. Nonparametric Mann-Whitney analyses of 7 bins (grey shade) surrounding TSS (TSS) and the midpoint of the profiles (Body) in Thaps treated conditions are reported. mRNA analysis of select Unbound gene transcripts are presented (lower panel). Three independent experiments are represented as mean ± SEM, with significance measured by Student's t-test, **p<0.01, ***p<0.005.
Consequences of changes in the lysosomal cholesterol content on CMA activity.Figure 7(A) Degradation of long‐lived proteins in mouse fibroblasts (NIH3T3 cells) treated or not with 25 mM MBCD. Where indicated, 15 mM NH4Cl/100 μM leupeptin (NH4Cl+Leup) were added into the medium. Values are the mean+s.e. of three experiments with triplicate samples. (B) Effect of MBCD on total rates of protein degradation, measured as in (A), in wild‐type mouse fibroblasts and in fibroblasts stably RNA interfered for LAMP‐2A (LAMP‐2A(−)). The ratio of degradation in MBCD‐treated and untreated cells is shown for cells maintained with or without 10 mM 3‐methyladenine (3MA). (C) Proteolysis of [14C]GAPDH or a pool of [3H]labeled cytosolic proteins (pool proteins) by isolated rat liver lysosomes, treated or not with MBCD. Broken: lysosomes disrupted by a hypotonic shock before the incubation with the radiolabeled proteins. Values are expressed as percentage of the degradation by untreated lysosomes and are the mean+s.e. of three experiments with triplicate samples. (D) Lysosomal-mitochondrial fractions were isolated from mouse fibroblasts untreated or loaded with cholesterol (50 μM). Top: Distribution of LAMP‐2A after Triton X‐114 extraction and sucrose density gradient centrifugation. Bottom: Ability of intact or disrupted lysosomal-mitochondrial fractions to proteolize [14C]GAPDH. Values are expressed as percentage of the degradation in fractions from untreated cells and are the mean±s.e. of triplicate samples.
linc-MYH modulates INO80 complex composition by reducing incorporation of YY1 and WDR5 A, B Co-Immunoprecipitation of INO80 complex subunits from TA muscles of WT and linc-MYH KO mice followed by Western Blot analysis.Figure 6A, B Co-Immunoprecipitation of INO80 complex subunits from TA muscles of WT and linc-MYH KO mice followed by Western Blot analysis. (B) Statistical analysis of Co-IP/Western Blot data. The amount of RUVBL1 and RUVBL2 in the INO80 complex is not changed after deletion of linc-MYH, while the amounts of YY1, WDR5 and TFPT pulled down by INO80 increase (n = 3 WT / 3 linc-MYH KO animals; one-way ANOVA, multiple comparisons by Fishers LSD test, *p<0.05). Data are mean ± S.E.M. C-K Proximity ligation assays between INO80 and YY1 (C, D), INO80 and WDR5 (F, G), and INO80 and RuvBl2 (I, J) in proliferating MuSCs. Red signals indicate close proximity of INO80-V5 and the respective interacting protein. Nuclei were stained using DAPI (blue). Statistical evaluation of proximity ligation assays indicating increased interactions of INO80-V5 and YY1 (E, n = 3/3 animals, 3 independent wells per animal, two-tailed students t-test, *p<0.05) and of INO80-V5 to WDR5 (H, n = 2 (WT)/2 (KO) animals, 3 independent wells per animal, two-tailed students t-test, **p<0.01). No increase in proximity was detected between Ino80-V5 and Ruvbl2 (K, n = 2 (WT)/2 (KO) animals, 3 independent wells per animal, two-tailed students t-test, ns: not significant). Specificity of all analyzed signals was analyzed using single primary or single secondary antibodies in PLA assays. No signals were detected for these controls. Data are mean ± S.E.M.
Figure 2A. Schematic diagram of CDKL5 deletion mutants, deleting from the N-terminal (blue) or C-terminal (black) ends. All proteins were expressed with an N-terminal NLS and GFP tag. B. BrdU-sensitized U-2-OS (Flp-In T-Rex) cells stably expressing GFP-NLS, the GFP-NLS-CDKL5 deletion mutants shown in A, or full length (FL) GFP-NLS-CDKL5 were subjected to line micro-irradiation (355 nm) and time lapse imaging. Three independent experiments were performed, and one representative experiment is shown. Scale bar is 10 μm C. Schematic for fragments corresponding to the PAR dependent recruitment region in CDKL5 as identified in B. D. Same as in B except that the GFP-NLS tagged CDKL5 fragments indicated were examined. Scale bar is 10 μm E. Coomassie gel showing recombinant fragments of human CDKL5 fused to GST purified from bacterial lysates. GST and APLF were also purified as controls. F. Recombinant fragments of CDKL5 fused to GST (1.2, 2.5, 5, 10 µg), or GST, were dot-blotted on nitrocellulose membrane and then incubated with synthetic PAR. PAR binding was detected by far western blotting. APLF was used as positive control. One of three independent experiments is shown (G-H) .U-2-OS (Flp-In T-Rex) cells stably expressing CDKL5 were either mock treated or treated with 500 µM H2O2 for 30 min. Extracts were subjected to immunoprecipitation with antibodies against CDKL5 (G) or PAR (H) (or non-specific IgG as control). Precipitates, and input lysates, were analyzed by western blotting using the indicated antibodies. One of two independent experiments is shown
Drug combinations induce toxicity in patient-derived organoids and sensitize to Paclitaxel treatment A Ratio of toxicity and viability determined in pancreatic cancer-derived organoids from two patients ((left) PDO-42 and (right) PDO-48) treated as indicated for 3 (PDO-42) or 5 (PDO-48) days.Figure 7A Ratio of toxicity and viability determined in pancreatic cancer-derived organoids from two patients ((left) PDO-42 and (right) PDO-48) treated as indicated for 3 (PDO-42) or 5 (PDO-48) days. Significant differences are shown for the comparison between single and combined drug treatments as indicated (NEN 1.2 µM or 2.5 µM, Amitriptyline (Ami, A), Imipramine (Imi, I), Desipramine (Desi, D), each 30 µM, Clomipramine (Clomi, C) 20 µM). Data information: data are presented as mean (SD) (N=3) and were analyzed by a one-way ANOVA with Tukey post-hoc test.* p< 0.05; ** p< 0.01;*** p< 0.001; **** p < 0.0001. B Ratio of toxicity and viability determined in pancreatic cancer-derived organoids from two patients ((left) PDO-42 and (right) PDO-48) treated as indicated for 3 (PDO-42) or 5 (PDO-48) days. Significant differences are shown for the comparison between drug treatments as indicated to the control condition (NEN (N) 1.2 µM or 2.5 µM, Amitriptyline (A), Imipramine (I) 20 µM, Paclitaxel (Pacli), 2, 20 or 200nM). As references for sensitization effects to standard chemotherapy, the bars showing single Paclitaxel treatments using the indicated concentrations are highlighted in blue. Bars for controls (Ctrl), Paclitaxel (Pacli), NEN (N) alone and combined NEN+Paclitaxel treatment are identical in the individual graphs for comparison to combinations with the respective TCA drugs as indicated. Data information: data are presented as mean (SD) (N=3) and were analyzed by a one-way ANOVA with Tukey post-hoc test.* p< 0.05; ** p< 0.01;*** p< 0.001; **** p < 0.0001.
Parkin-dependent MDVs incorporate endogenous mitochondrial cargoRepresentative immunoblot of whole-cell lysates from U2OS:GFP and U2OS:GFP-parkin cells transfected with non-targeting siRNA or siRNA targeting Drp1 (siDrp1).Figure 3A Representative immunoblot of whole‐cell lysates from U2OS:GFP and U2OS:GFP‐parkin cells transfected with non‐targeting siRNA or siRNA targeting Drp1 (siDrp1). B U2OS:GFP−parkin cells transfected with siRNA targeting Drp1 were treated with DMSO (upper panels) or 25 μM antimycin A (anti A, lower panels) for 90 min, then fixed and immunostained against PDH (red) and TOM20 (blue). PDHE2/E3 bp−positive/TOM20−negative MDVs colocalizing with GFP−parkin (arrows) or not (circles) are indicated. Scale bars, 20 μm (first panels on left) and 2 μm. C Quantification of PDH−positive/TOM20−negative vesicles in U2OS:GFP and U2OS:GFP−parkin cells transfected with the indicated siRNA, treated with DMSO or antimycin A (anti A) as in (B); both the total number (white bars) and the number colocalizing with GFP−parkin (gray bars) are indicated. Bars represent the mean ± s.e.m. P−values are given first for GFP−/GFP−parkin−positive vesicles, then for total vesicle number (n = 48-85 cells in 2-3 experiments); ns, not significant; ***P < 0.001). D 5 μm‐long profile of the parkin‐positive vesicle and adjacent mitochondrial tubule depicted in the antimycin A‐treated cell from (B) (left). Increasing position values on the x‐axis of the fluorescence intensity plot (right) correspond to moving from the top to the bottom of the profile. *MDV indicated by an arrow in (B). E Quantification of the distance between the centres of GFP−parkin−negative (black bar) or −positive (gray bar) vesicles and the edge of the nearest mitochondrial tubule for the vesicles quantified in antimycin A−treated U2OS:GFP−parkin cells transfected with siDrp1 in (C). Error bars represent the mean ± s.e.m.; *P < 0.05 (obtained by Student's t−test). F U2OS:GFP‐parkin cells transfected with siRNA targeting Drp1 (siDrp1) were treated with 25 μM antimycin A for 90 min prior to fixation. Samples were immunostained against TOM20 (blue) and the indicated mitochondrial marker (red). TOM20‐negative MDVs containing the specified cargo and colocalizing with GFP‐parkin (arrows) or not (circles) are indicated. Arrowheads show lack of colocalization between GFP‐parkin and the indicated mitochondrial marker. Scale bars, 20 μm (first two columns of panels) and 2 μm (remaining columns of panels). G Quantification of TOM20−negative structures that stained positively for the indicated cargo in U2OS:GFP (GFP) and U2OS:GFP−parkin (GFP−parkin) cells, transfected with siRNA targeting Drp1, treated with antimycin A (anti A); both the total number (white bars) and the number colocalizing with GFP−parkin (gray bars) are indicated. Error bars represent the mean ± s.e.m. P−values are given first for GFP−/GFP−parkin−positive vesicles, then for total vesicle number (n = 24-59 cells in two experiments; ns, not significant; ***P < 0.001).
Model of the protein architecture of the endocytic coat.Figure 6Individual coat layers together with their constituents and their approximate distances from the plasma membrane are listed on the left. Legend to individual depicted proteins (human homologs in parentheses) is shown on the right. Regulators of actin polymerization Las17, Vrp1 and Bzz1, and endocytic pioneering proteins Ede1 and Syp1, which were shown to localize in membrane-proximal regions outside of the depicted endocytic coat (Mund et al, 2018), are not shown for clarity. Note that only few copies of individual coat proteins (presented at the endocytic site in dozens of copies; Picco et al, 2015; Sun et al, 2019) are shown. See the text for further details.
Figure 6(a) Doxycycline-inducible RAB2A-shRNA MCF10.DCIS.com cells were incubated on a thick layer of Matrigel for 4 days to allow spheroid formation in the absence (CTR) or presence (shRAB2A) of doxycycline. Cells were stimulated with 5 ng/ml EGF and 20 ng/ml HGF (EGF+HGF) or left starved (STV) for 9 days. Representative phase contrast light microscopy images are shown. Invasive activity of the cells was expressed by circularity (with a value of 1 representing perfect circularity = no invasion) of each acini. Bar, 100 µm. (b-d) MCF10.DCIS.com cells (104 cells) transfected with scrambled siRNA (siNC) or siRNA against RAB2A (siRAB2A) were plated onto 6-well dish. Representative phase contrast light microscopy images (b) and the distribution of cell numbers per cluster of the indicated cell number (c) are shown. Data are means ± SD of 10 fields (n > 60 cells/field) of independent 4 independent experiments. Bars, 100 μm. Efficacy of RAB2A gene silencing was verified by qPCR (d). Bar, 200 µm. (e) Doxycycline-inducible RAB2A-shRNA MCF10.DCIS.com cells were cultured in the absence (CTR) or presence (shRAB2A) of doxycycline for three days, and immunostained with antibodies indicated on the top (green) and DAPI (blue). (f, g) MCF-10AneoT (f) cells were transfected with scrambled siRNA (siNC) or siRNA against RAB2A, and immunostained with antibodies indicated on the top (green) and DAPI (blue). Bars, 10 μm. (f, g) MCF10-CA1a (g) cells were transfected with scrambled siRNA (siNC) or siRNA against RAB2A, and immunostained with antibodies indicated on the top (green) and DAPI (blue). Bars, 10 μm. (h) Total cell lysates from (e), (f), and (g) were immunoblotted by anti-E-cadherin, RAB2A, and anti-actin antibodies. ***, P < 0.001, NS, not significant.
Autophagy‐deficient renal tubules exhibit severe injury and dysfunction under hyperuricaemia.Figure 5(A) Plasmacreatinine and ureanitrogen in Atg5F/F and Atg5F/F;KAPmice treated with vehicle or UA+OA. The values displayed represent means±s.e. Statistically significant differences (*P0.05) are indicated. F/F: Atg5F/Fmice; F/F;KAP: Atg5F/F;KAPmice. (B, C) PAS‐stained renal cortical region of Atg5F/F and Atg5F/F;KAP mice treated with vehicle or UA+OA (n=4-7). Bars: 40 μm (B). Kidney injury score of Atg5F/F and Atg5F/F;KAP mice treated with vehicle or UA+OA (C). (D) Kidney cortexes from Atg5F/F and Atg5F/F;KAP mice treated with vehicle or UA+OA were subjected to immunohistocheminal analysis of Lamp1 and LC3. Green: LC3; magenta: Lamp1; blue: DAPI. F/F: Atg5F/F mice; F/F;KAP: Atg5F/F;KAP mice. Bar: 5 μm. (E) Electron micrographs of kidney proximal tubules in Atg5 F/F and Atg5F/F;KAP mice treated with UA+OA. Yellow arrowhead: autophagosome; asterisk: lysosome; Mt: mitochondrion. Bar: 1 μm. (F) Model of autophagic clearance of damaged lysosomes.Source data for this figure is available on the online supplementary information page.
Figure 1a) Schematic drawing of the lentiviral vectors used. LV indicates conventional integrating lentiviral vector and NILV indicates non-integrating lentiviral vector, generated by using packaging plasmid with deficient integrase. S/MAR indicates that the vector contains a scaffold matrix attachment region element and S/MARrev means that the element is positioned in the reverse orientation. LTR: Long terminal repeat; CMV prom: cytomegalovirus immediate early gene promoter; GFP: green fluorescent protein; 2A: viral self-cleaving T2A sequence; puro: puromycin. b) Histograms of GFP expression (x-axis) are presented from A549 cells transduced with the different viral vectors. c) Percentage of GFP positive cells from the experiment described in (b). . d) A PCR assay along with primers, as illustrated in Appendix Figure S2a, was used to detect formation of LTR junction and thereby circularization of the vector. The expected size of PCR product is 552 bp from a 1-LTR circle and 1029 bp from a 2-LTR circle. . e) Southern Blot analysis to detect integration events in LV- and NILV-S/MAR-transduced single cell clones with DNA probed against the viral genome. A 6.8 kb positive control from a linearized plasmid (lane 3) was included for indication of the size of DNA circles formed by non-integrated NILV-S/MAR vector. f-h) A padlock probe assay, as illustrated in Appendix Figure S4, was used to demonstrate persistence of proviral DNA either in a circular or integrated form. Representative images show DNA persistence in (f) LV-, (g) NILV- or (h) NILV-S/MAR-transduced cells. Each red dot (Cy3) represents a single DNA molecule and cell nuclei were stained with Hoechst 33342.
a, HeLa cells were treated with biotin-conjugated peptides (30 &mgrM, 3 h) and proteins bound to peptides were analysed by immunoblot with anti-GAPR-1.f2a, HeLa cells were treated with biotin-conjugated peptides (30 μM, 3 h) and proteins bound to peptides were analysed by immunoblot with anti-GAPR-1. b-T-B, biotin-Tat-beclin 1; b-T-S, biotin-Tat-scrambled. b, Immunoprecipitation of Flag-beclin 1 with GAPR-1-Myc in HeLa cells 24 h post-transfection. c, GFP-LC3-positive dots in GAPR-1 siRNA-transfected peptide-treated HeLa/GFP-LC3 cells (20 &amp;amp;mgr;M, 3 h) with or without 100 nM bafilomycin A1. Bars represent mean ± s.e.m. of triplicate samples (50-100 cells per sample). Similar results were observed in three independent experiments. NC, non-silencing control. d, Localization of beclin 1 and GM130 (a Golgi marker) in HeLa cells stably transduced with empty vector (left panel) or GAPR-1-Myc (middle panel) or transfected with GAPR-1 siRNA (right panel) and treated with peptide (20 μM, 1 h). Scale bar, 20 μm. e, Immunoblot of beclin 1 in post-nuclear supernatant (PNS) and Golgi-enriched fractions in HeLa cells stably transduced with empty vector or GAPR-1-Myc after peptide treatment (20 μM, 2 h). f, WIPI2 dots in cells in the experimental conditions shown in d. Bars represent mean ± s.e.m. for 100-150 cells. *P 0.05, ***P 0.001; t-test. †††P 0.001; two-way ANOVA for comparison of magnitude of changes between groups.