EMBO/sd-character-level-ner huggingface dataset:
Growth rate and replication dynamics in BJ cells grown in a folate-free medium with and without nucleoside supplementation.Figure 1Population doublings (PDs) determined in BJ cells cultured with and without folate for 28 days. Example of a single combed DNA molecule labeled with IdU (green) and CldU (red), showing replication from three adjacent origins. Horizontal white arrows indicate fork orientation. Representative examples of single combed DNA molecules from control cells and cells grown for 7 days in a folate-free medium. Fork rate (Kb/min) distribution. Light blue bars: BJ cells (n = 126); black bars: BJ cells that were cultured for 7 days in a folate-free medium (n = 131); blue bars: BJ cells cultured for 7 days in a folate-free medium and supplemented with A, G, C, and T nucleosides for the last 48 h of the experiment (n = 138).Box plot summarizing the fork rate distribution (Kb/min) of three independent experiments. Control (n = 360); −folate (n = 372); −folate + AGCT (n = 361). Fork distance (Kb) distribution. The color code is as in (D). Control (n = 72); −folate (n = 71); −folate + AGCT (n = 75).Box plot summarizing the fork distance distribution (Kb) of three independent experiments. Control (n = 212); −folate (n = 215); −folate + AGCT (n = 209).
Figure 7(A) Western blotting analysis of mouse tissues for the expression of Jfk and Ing5. (B and C) Total proteins or RNAs were prepared in MEFs or liver tissues from wild-type or JfkKO mice for western blotting analysis (B) or qPCR measurement (C), respectively. Error bars represent mean ± SEM (n = 6, *p < 0.05, paired two-tailed Student's t-test). (D) The measurement of AMPK activity and the rate of fatty acid β-oxidation of wild-type or JfkKO mice. Cellular extracts prepared from MEFs or liver tissues were subjected to AMPK activity assays by ELISA. Mitochondria were isolated from MEFs or liver tissues, and the rate of fatty acid β-oxidation was quantified by spectrophotometric detection for ferricyanide-trapped reduced products. Error bars represent mean ± SEM (n = 6, *p < 0.05, paired two-tailed Student's t-test). Wild-type or JfkKO mice were fed with HFD for 11 weeks prior to injection of adenovirally-delivered vector control, shIng5, or shAmpkα1 via tail vein. Seven days after injection, cellular extracts prepared from liver tissues were subjected to western blotting analysis or measurement for hepatic TG and NEFA content (E). Asterisk (*) represents significant comparison to wild-type mice, octothorpe (#) represents significant comparison to JfkKO mice. Error bars represent mean ± SEM (n = 6, *p < 0.05, #p < 0.05, one-way ANOVA with Tukey's HSD test). H&E and Oil Red O staining of liver sections are shown (F). Scale bar, 100 µm. (G) Analysis of public dataset (GSE135251) for JFK and AMPKα1 mRNA expression in liver tissues from healthy controls (n = 10) and NAFLD patients (n = 206). Whiskers indicate upper or lower quartile, and lines inside the box represent median (*p < 0.05, welch two sample t-test). (H) The proposed model of the SCFJFK-ING5 axis in the development of obesity and metabolic syndrome.
Figure 5A Overall view of the central cavity of the β subunit showing insertion of the C-terminal tail of Ykt6. PTAR1 is omitted for clarity. The Cys194-linked geranylgeranyl and Cys195-linked farnesyl moieties are shown in orange and purple, respectively. PPi, inorganic pyrophosphate. B Electron density of the Cys194-linked geranylgeranyl and Cys195-linked farnesyl moieties (blue mesh; simulated-annealing Fo-Fc omit map contoured at 3σ level). Detailed views of the geranylgeranyl moiety in the GGTase-III­-product complex (C) Residues of the β subunit that form the lipid substrate binding pocket are shown. Detailed views of the geranylgeranyl moiety in the GGPP in the GGTase-III-GGPP complex (D). Residues of the β subunit that form the lipid substrate binding pocket are shown. E Surface representation of the central cavity of the β subunit showing a tunnel formed near the active site. The Cys195-linked farnesyl moiety is anchored into the hydrophobic tunnel. F Detailed view of the Cys195-linked farnesyl group bound in the hydrophobic tunnel. Residues of the β subunit that line the hydrophobic tunnel are shown. G Superposition of the β subunits of FTase (blue), GGTase-I (gray), and GGTase-III (cyan). In FTase and GGTase-I, the hydrophobic tunnel is blocked by the side chain of Leu103 and Ile50, respectively. The corresponding position is replaced by Gly49 in GGTase-III. H Geranylgeranylation activity of WT GGTase-III and tunnel mutants (100 nM each). Cys195-farnesyl Ykt6ΔAIM (1 μM) and 3H-GGPP (1 μM) were used as substrates (mean ± SEM, n = 3). I GGPP binding to GGTase-III. WT GGTase-III and tunnel mutants (1 μM each) were incubated with 3H-GGPP (2 μM) at 4ºC for 10 min. After desalting, enzyme-bound 3H-GGPP was quantified by scintillation counting (mean ± SEM, n = 3). J Alignment of the helix 3 sequences of RabGGTβ orthologues and human FTβ and GGT-Iβ. The position of the conserved glycine residue (Gly49 in human) is highlighted in gray.
| The relationship between endothelial kugeln and blood flow A MIP of the cerebral vessels of a 4dpf embryo before exsanguination (grey LUT; inverted).Figure 4MIP of the cerebral vessels of a 4dpf embryo before exsanguination (grey LUT; inverted). MIP of the same embryo after exsanguination, which did not alter kugel size. Time-lapse imaging of an embryo with transiently halted cardiac contraction (using Tricaine). Despite absent blood flow kugeln still changed shape (grey arrowhead), retained shape (white arrowhead) or protruded and retracted (black arrowhead; time post cessation of flow is indicated on micrographs; 3dpf; grey inverted LUT). Time-lapse of an embryo with transiently halted cardiac contraction as in (C) showed that kugel diameter still oscillates (time post cessation of flow). Dextran microangiography filled perfused vessels with dextran (arrowhead), while dextran was not observed to enter kugeln (unfilled arrowhead). Inhibition of cardiac contraction by tnnt2a morpholino (MO) knockdown statistically significantly reduced kugel number per embryo (****p<0.0001; control n=20 embryos 5.10 ± 1.47 (mean ± s.e.m.), tnnt2a MO=18 embryos 0.06 ± 0.06 (mean ± s.e.m.); 3dpf; 3 experimental repeats; Mann-Whitney U test).
Fig.Figure 3This figure compares conformation of two dipeptides in the ribosome nascent peptide tunnel:A. Phe-Leu peptideB. Pro-Pro peptide.
CLU silencing increases cabazitaxel efficacy.Figure 8(A) Cabazitaxel IC50 calculation using WST1 assay in PC3 cells after transfection with siSCR or siCLU (left), and SCR or OGX-011 (right) followed by 72h of cabazitaxel treatment. Percentage of surviving cells was calculated relative to control. Error bar represent mean ± SEM, n=3. Inset: Western blot was performed to verify CLU knockdown in cells. (B) Mitotic cells and cells in mitotic catastrophe were counted using DAPI in PC-3 cells after SCR or OGX-011 transfection followed by different concentration of cabazitaxel as indicated (>1000 counts) Error bar represent mean ± SEM.,*p<0.05, ***p<0.001, ****p<0.0001 by Student's t-test.( caba 1.5 nM p=0.00115 ; 3 nM p=0.0105 ; 4.5 nM p=1.93x10-7, ; 6.25 nM p=8.9x10-9; 12.5 nM p=6.8x10-9; 25 nM p=1.2x10-9). (C) Clonogenic assay in PC-3 cells after SCR or OGX-011 transfection followed by treatment with different concentrations of cabazitaxel as indicated. The number of survival clones was assessed after 15 days using crystal violet assay. Error bars represent mean ± SEM n=3,*p<0.05, **p<0.01 by Mann-Whitney (0 nM caba p=0.039; 0.5 nM p=0.0079; 2 nM p=0.0079)
Figure 2A, B Oil Red O staining of livers from WT and KO males after 25 weeks on NC or HFD, with quantification of relative stain intensity (n=3, 3 images each). Scale bar = 100 μm. Data information: values shown are mean ±SD. * = p < 0.05; ** = p < 0.01; *** = p < 0.001; **** = p < 0.0001 (two-way ANOVA with multiple comparisons). C Transcript levels for genes involved in lipid metabolism and gluconeogenesis in livers of WT and KO mice after 25 weeks on NC or HFD. Stearoyl-Coenzyme A desaturase (Scd1), fatty acid transporter (CD36), glycerol-3-phosphate acyltransferase (Gpat), carnitine palmitoyltransferase I (Cpt1), peroxisome proliferator-activated gamma (PPARγ), pyruvate dehydrogenase acetyl-transferase (Pdk4), PPARγ coactivator 1 alpha (PGC1α), diacylglycerol O-acyltransferase (Dgat), and facilitated glucose transporter (Glut4). (males, n=4-5). Transcript levels were normalized to 18S. Data information: values shown are mean ±SD. * = p < 0.05; ** = p < 0.01; *** = p < 0.001; **** = p < 0.0001 (two-way ANOVA with multiple comparisons). D-F Serum levels of free fatty acids (FAA), insulin, and leptin in WT and KO after 25 weeks on NC or HFD (males, n=8-10). Data information: values shown are mean ±SD. * = p < 0.05; ** = p < 0.01; *** = p < 0.001; **** = p < 0.0001 (two-way ANOVA with multiple comparisons). Glucose tolerance test (GTT) after 25 weeks on NC or HFD. GTT was performed following an overnight fast Data information: values shown are mean ±SD. * = p < 0.05; ** = p < 0.01; *** = p < 0.001; **** = p < 0.0001 (two-way ANOVA with multiple comparisons). Insulin tolerance test (ITT) after 25 weeks on NC or HFD. , ITT following a three hour fast (males, n=6). Data information: values shown are mean ±SD. * = p < 0.05; ** = p < 0.01; *** = p < 0.001; **** = p < 0.0001 (two-way ANOVA with multiple comparisons).
HVGs tend to be small and to be targeted by a higher number of TFs A.Figure 4A. Distribution of the gene length (in bp) for LVGs (green) and HVGs (blue). The distribution of average (black) and 95% confidence interval (dotted grey) for the thousand random sets is also represented. B. Distribution of the number of introns for LVG (green) and HVG (blue). The distribution of average (black) and 95% confidence interval (dotted grey) for the thousand random sets is also represented. C. Distribution of the number of TFs targeting a gene, based on the DAP-seq available dataset, for LVG (green) and HVG (blue). The distribution of average (black) and 95% confidence interval (dotted grey) for the thousand random sets is also represented.
Figure 5A Scheme of stress granule (SG) recruitment assay in semi-permeabilized cells. B Reduced SG association of TDP-43 by 12D mutations or in vitro phosphorylation. Bar, 20 µm. C Quantification of TDP-43-MBP-His6 mean fluorescence intensity in SGs normalized to Wt ± SEM of three independent experimental replicates (n=3) (≥ 10 cells; ≥ 46 SGs each). **p < 0.0021 and ***p < 0.0002 by one-way ANOVA with Dunnett´s multiple comparison test to Wt. D SG recruitment of TDP-43 variants in intact HeLa cells in absence of endogenous TDP-43. After TDP-43 silencing and expression of myc-TDP-43 Wt, 12D and 12A variants, SGs were induced by H2O2 treatment and SG recruitment of TDP-43 was monitored by TDP-43 and TIA1 immunostaining. For clarity, signals were converted to grey values in the individual channels (upper two rows). In the merge (lower row), nuclei were stained in DAPI (turquoise), TDP-43 (green) and TIA-1 (magenta). Bar, 25 µm. E Quantification of TDP-43 in SGs versus cytoplasm ± SD of two independent experimental replicates (n=2) (≥ 62 cells; ≥ 234 SGs each). ***p < 0.0002 by Krustal-Wallis test with Dunn´s multiple comparison test to Wt. F SG recruitment of different TDP-43-NLSmut variants in intact HeLa cells in the absence of endogenous TDP-43. After TDP-43 silencing and expression of NLSmut Wt, 12D and 12A variants, SGs were induced by H2O2 treatment and SG recruitment of TDP-43 was monitored by TDP-43 and G3BP1 immunostaining. For clarity, signals were converted to grey values in the individual channels (upper two rows). In the merge (lower row), nuclei were stained in DAPI (turquoise), TDP-43 (green) and G3BP1 (red). Bar, 40 µm. G Quantification of TDP-43-NLS mutants in SGs versus band around SGs of two independent replicates ± SD. ***p < 0.0002 by Kruskal-Wallis test with Dunn´s multiple comparison test to Wt (≥ 56 cells; ≥ 315 SGs per condition). H Recruitment of TDP-43 into arsenite-induced nuclear bodies (NBs) in HeLa cells. After TDP-43 silencing and expression myc-TDP-43 Wt, 12D and 12A, NBs were induced by sodium arsenite treatment and NB recruitment of TDP-43 was monitored by TDP-43 immunostaining. Bar, 20 µm. I Percentage of cells with TDP-43 in NBs ± SD of three independent experimental replicates (n=3). *p < 0.0332 by Kruskal-Wallis test with Dunn´s multiple comparison test to Wt. J Intensity profiles (right) of TDP-43 Wt, 12D and 12A variants (green) along white lines (left). Bar, 10 µm.
Lurbinectedin primes Ubiquitination and degradation of RNAPII:(a) Western blot showing RNAPII across the panel of; A549 and NCI-H460 NSCLC cells; IMR-90 HFL cells; NCI-H146, NCI-H82, DMS-53, NCI-H510A, NCI-H526 and SHP-77 SCLC cells before (-) and after (+) treatment with lurbinectedin.Figure 4(a) Western blot showing RNAPII across the panel of; A549 and NCI-H460 NSCLC cells; IMR-90 HFL cells; NCI-H146, NCI-H82, DMS-53, NCI-H510A, NCI-H526 and SHP-77 SCLC cells before (-) and after (+) treatment with lurbinectedin. α-Tubulin (α-Tub) is shown as loading control; lower panel, Histogram showing the average of RNAPII before (dark blue) and after (light Blue) lurbinectedin treatment in arbitrary units (a.u.) normalized against α-Tub. Data is presented as Mean ± SEM as determined by Two Way RM ANOVA and Šídák's multiple comparisons test (n=3 biological replicas). ****P ≤0.0001. (B Immunofluorescence of RNAPII (red) before (Ctrl) and after lurbinectedin treatment. DAPI (blue) was used to stain nuclei; scale bar is 65 μm. C) Immunofluorescence of RNAPII (red) before (Ctrl) and after lurbinectedin treatment. Data is presented as Mean ± SEM. ****p ≤0.0001*P ≤ 0.00332, **P ≤ 0.0021, as determined by Student's t-test (n = 3 biological replicates). (D) Affinity purification of Bio-lur fractions containing elongating RNAPII Ser2 (lane 4). Input (lanes 1-2) represents 10% of the starting material. Respective molecular weights are shown on the right (kDa). (E) Upper panel: GST-pulldown of ubiquitinated proteins showing hypo- (IIA), -hyper (II0) phosphorylated RNAPII (lanes 4-5 upper panel) and Ser2 Phosphorylated RNAPII (Ser2P) (lanes 4-5 lower panel) after lurbinectedin treatment. Empty beads (Mock) (lane 3) as a negative control; Lower Panel: GST-pulldown of ubiquitinated proteins Ser2P) (lanes 4-5). Representative smear of successful ubiquitinated proteins pulldown (Lanes 4 and 5) as compared to input (lanes 1 and 2). Empty beads (Mock) (lane 3) as a negative control. Each Western blot is representative of three independent experiments. (F) Density profile of annotated genes for RNAPII-Ser2 P ChIP seq. Lines representing ChIP seq profile at 0h (blue) and 4h (green) after lurbinectedin treatment -10.0 and +10.0 kb to TSS coordinates (P=0.005476). Decrease in density between 0h and 4h is highlighted in yellow.
Metabolic stress suppresses the PSR through AMPK.Figure 4A, B NIH3T3 reporter cells were cultured overnight in DMEM with and without leucine (A) or 4.5 g/l glucose (B). Following heat shock at 43°C for 30 min and recovery for 5 h, GFP induction was quantitated by flow cytometry. C, D Nuclear proteins were extracted from reporter cells with and without nutrient deprivations overnight for the ELISA-based DNA binding assay (mean ± SD, n = 3, Student's t-test, *P < 0.05, **P < 0.01, ***P < 0.001). E, F Following nutrient deprivations overnight, the reporter cells were heat-shocked at 43°C for 30 min and recovered overnight. Individual proteins were detected by immunoblotting. G, H Reporter cells were subjected to nutrient starvation for the indicated time. AMPKα Thr172 and HSF1 Ser121 phosphorylation was measured by immunoblotting. G, H Reporter cells were subjected to nutrient starvation for the indicated time. AMPKα Thr172 and HSF1 Ser121 phosphorylation was measured by immunoblotting. I, J HEK293T cells were sequentially transfected with control or AMPKα1/2-targeting siRNAs and dual HSF1 reporter plasmids. 24 h later, transfected cells were starved overnight before reporter activities were measured (mean ± SD, n = 6, Student's t-test, n.s., not significant, **P < 0.01, ***P < 0.001). I, J HEK293T cells were sequentially transfected with control or AMPKα1/2-targeting siRNAs and dual HSF1 reporter plasmids. 24 h later, transfected cells were starved overnight before reporter activities were measured (mean ± SD, n = 6, Student's t-test, n.s., not significant, **P < 0.01, ***P < 0.001). K, L FLAG-HSF1WT or FLAG-HSF1S121A plasmids were co-transfected with the dual HSF1 reporter plasmids into HSF1-deficient HEK293T cells for 2 days, followed by overnight starvation (mean ± SD, n = 5, Student's t-test, n.s., not significant, *P < 0.05, **P < 0.01).
Microtubules (MTs) promote LUZP1 inhibition of myosin phosphatase.Figure 6Box plots with dot density plots showing the ratio of the apical area/basal area in co-cultures of Venus-LUZP1-expressing LUZP1 knockout (REV) and LUZP1 knockout (LUZP1 KO) Eph4 cells; 2 μM nocodazole treatment for 30 min partially reversed apical constriction of REV cells [REV, 0.65 ± 0.16 (control) vs. 0.90 ± 0.18 (nocodazole) vs. 0.64 ± 0.16 (washout); KO, 1.30 ± 0.17 (control) vs. 1.07 ± 0.13 (nocodazole) vs. 1.32 ± 0.19 (washout)]. **p < 0.01 (Kruskal-Wallis test followed by Steel-Dwass test). The solid lines represent the medians and the boxes represent the inter-quartile ranges. The error bars extending from the box represents the data within 1.5 times of the interquartile range. Representative confocal micrographs of co-cultures of LUZP1-expressing wild-type (WT) and LUZP1 KO Eph4 cell treated with 2 μM nocodazole for 30 min. Nocodazole treatment partially reversed the difference in di-phosphorylated MLC (ppMLC) levels within circumferential rings (CRs) between WT and LUZP1 KO cells. Scale bar, 10 μm.   Bar plots with dot density plots showing that ppMLC levels within CRs were significantly down-regulated in WT Eph4 cells after nocodazole treatment. Importantly, ppMLC levels in LUZP1 KO Eph4 cells were unchanged after nocodazole treatment [WT, 21.43 ± 6.96 arbitrary units (a.u.) (control) vs. 17.67 ± 5.40 a.u. (nocodazole) vs. 20.84 ± 7.19 a.u. (washout); KO, 8.74 ± 1.71 a.u. (control) vs. 8.67 ± 1.89 a.u. (nocodazole) vs. 7.96 ± 2.35 a.u. (washout)]. n = 3. **p < 0.01 (Kruskal-Wallis test followed by Steel-Dwass test). Bars and error bars represent the mean ± standard deviation (SD).   In vitro MLC phosphorylation assay using 1 μg MTs in addition to 25 ng GST-MLC, 4 ng GST-ROCK1 catalytic domain, 1 mM ATP, 1 μg GST-protein phosphatase 1c β/δ (PP1c β/δ), and 0-5 μg GST-LUZP1. Quantification of the relative ppMLC/MLC ratio to the control showed that MTs promote LUZP1-mediated inhibition of PP1c β/δ [1.00 (1st-lane, control) vs. 1.42 ± 0.59 (2nd-lane) vs. 1.72 ± 0.76 (3rd-lane) vs. 1.99 ± 0.56 (4th-lane) vs. 1.14 ± 0.37 (5th-lane) vs. 2.87 ± 1.51 (6th-lane) vs. 2.74 ± 1.19 (7th-lane) vs. 2.50 ± 0.88 (8th-lane)]. n = 6. *p < 0.05 [Kruskal-Wallis test followed by Steel test (compared with 1st lane)]. Bars and error bars represent the mean ± SD. A schematic drawing of the relationships among MTs, ppMLC, LUZP1, and myosin phosphatase at TJ-associated CRs to promote apical constriction.
Figure 6A LC-MS/MS quantification of MIAA in plasma from C57BL/6 female mice with or without Tsc2-deficient 105K tumors grown. The asterisks indicate a significant difference with two-sided t-test (P = 8x10-3; replicates/condition n = 5). Average values are indicated with lilac-colored lines. B Inhibition of Tsc2-deficient 105K tumor growth with different monotherapies, as indicated in the inset. Asterisks indicate significant reductions relative to vehicle (two-way ANOVA; clorgyline P = 0.015, loratadine P = 0.018, and rasagiline P = 0.049). Each data point represents the mean and SEM. C Further reduction of Tsc2-deficient 105K tumor growth by rapamycin combined with clorgyline or loratadine, relative to rapamycin alone. Asterisks indicate significant reductions relative to rapamycin alone (two-way ANOVA; clorgyline P = 0.035, loratadine P = 0.045). Each data point represents the mean and SEM. D Tsc2-deficient 105K tumor weight (g) changes at the end of the rapamycin and rapamycin-combination assays. Asterisks indicate a significant reduction relative to control, as determined by a one-sided t-test (loratadine P = 0.009 and rapamycin P = 3x10-5). Average weight values are indicated with lilac-colored lines. E Inhibition of Tsc2-deficient 105K tumor growth with Maoa expression depletion using shRNAs. Asterisks indicate significant reductions relative to control pLKO (two-way ANOVA; P = 1x10-3). Each data point represents the mean and SEM. F Inhibition of Tsc2-deficient 105K tumor growth with Hrh1 expression depletion. Asterisk indicates significant reduction relative to control pLKO (two-way ANOVA; P = 0.035) . Each data point represents the mean and SEM. G Inhibition of Tsc2-deficient 105K tumor growth with administration of α-methyl-DL-histidine. Asterisk indicates significant reduction relative to vehicle (two-way ANOVA; P = 0.028). Each data point represents the mean and SEM. H Inhibition of Tsc2-deficient 105K tumor growth by the administration of a low L-histidine concentration mouse diet (0.07% versus 0.49%). Asterisks indicate significant reduction relative to 0.49% L-histidine diet (two-way ANOVA; P = 0.001). Each data point represents the mean and SEM. I Increase in DLCO over a year in patients treated with rapamycin plus loratadine, relative to those treated with rapamycin alone. Significant difference determined by a two-sided Mann-Whitney test (P = 0.045; number (n) of samples are indicated). Average values are indicated with lilac-colored lines.
a, b, THP1 cells were stimulated with rotenone (10 &mgrM), TTFA (10 &mgrM) or antimycin (40 &mgrg ml−1) for 6 h and then stained with Mitotracker green and Mitotracker deep red (a) or MitoSOX (b) for 30 min and analysed by flow cytometry.f1a, b, THP1 cells were stimulated with rotenone (10 µM), TTFA (10 µM) or antimycin (40 µg ml−1) for 6 h and then stained with Mitotracker green and Mitotracker deep red (a) or MitoSOX (b) for 30 min and analysed by flow cytometry. c, THP1 cells were stimulated for 6 h with the indicated amounts of rotenone, TTFA or antimycin. Supernatants (SN) and cell extracts (input) were analysed by western blotting as indicated. d, LPS-primed bone-marrow-derived macrophages (BMDMs) from wild-type (WT), Nlrp3 or Ipaf deficient mice were stimulated for 6 h with MSU (150 µg ml−1), rotenone (10 µM for THP1 cells and 40 µM for BMDMs) or antimycin (40 µg ml−1 for THP1 cells and 10 µg ml−1 for BMDMs). The release of caspase-1 (western blot) was then determined. Data shown are representative of three independent experiments.
HIV-mediated autophagy induction and nuclear translocation of TFEB is inhibited by HIV Nef.Fig 8(A) Macrophages were infected with HIVNL(AD8) or HIVNL(AD8) ΔNef. At 1, 3, 5, 7, and 10 days post-infection cells were harvested, lysed and analyzed for endogenous LC3B, SQSTM1, and ACTB by Western blotting. Left, representative blots are shown. Right, densitometric analysis of immunoblots from independent donors presented as means ± s.e.m., n = 4. (B) Macrophages were infected with HIVNL(AD8) or HIVNL(AD8) ΔNef. At 1, 3, 5, 7, and 10 days post-infection cells were harvested, lysed, fractionated for cytoplasmic and nuclear content, and analyzed for TFEB, ACTB and H3 histone by Western blotting. Left, representative blots are shown. Right, densitometric analysis of immunoblots from independent donors presented as means ± s.e.m., n = 4.