EMBO/SourceData huggingface dataset:
figf2(a) Flow cytometry of T cells transduced with lentivirus expressing GFP and nontargeting control shRNA (Scr) or Lamp2a mRNA-specific shRNA (shL2A) (left), and immunoblot analysis of LAMP-2A in sorted GFP+ T cells (right). Numbers above bracketed lines (left) indicate percent GFP+ cells.(b) Enzyme-linked immunosorbent assay of IL-2 and interferon-γ (IFN-γ) in TH1 cells transduced as in a and then left unstimulated (Rest) or stimulated for 24 h with anti-CD3 and anti-CD28 (Act). *P = 0.038 and **P = 0.031 (t-test).(c) Proliferation in TH1 cells transduced as in a and left unstimulated or stimulated for 24 h as in b, assessed by incorporation of the thymidine analog BrdU and presented as absorbance at 495 nm (A495). *P = 0.013 (t-test).(d) Quantification of annexin V-positive (AnnV+) cells among resting or stimulated TH1 cells transduced as in a.
figf3(a) Immunoblot analysis of Itch and RCAN1 in lysosomal fractions isolated from resting cells TH1 cells and TH1 cells activated for 24 h with anti-CD3 and anti-CD28; LAMP-1 serves as a control.(b) Immunoblot analysis of Itch and RCAN1 in lysosomal fractions of TH1 cells activated for 24 h as in a with (+) or without (−) inhibition of lysosomal proteases during the final 3 h with NH4Cl and leupeptin (Lys inh); LAMP-1 serves as a control.(c) Immunoblot analysis of Itch and RCAN1 in resting TH1 cells and TH1 cells activated for 24 h as in a in the presence (NAc) or absence (Veh) of N-acetylcysteine.(d) Immunoblot analysis of lysates of TH1 cells transfected with GFP-Itch or GFP-mu-Itch (top) or with Myc-RCAN1 or Myc-mu-RCAN1 (bottom) and cotransfected with a GFP expression plasmid (to assess transfection efficiency), then activated for 24 h as in a.(e,f) Proliferation (e) and IL-2 production (f) of TH1 cells transfected with plasmid expressing GFP (Ctrl) or plasmids as in c and left unstimulated (Rest) or activated for 48 h (e) or 24 h (f) as in a (assessed as in Fig. 2c (e) or Fig. 2b (f)). *P = 0.019 and **P = 0.0034 (e) or *P = 0.046 and **P = 0.044 (f) (t test). Data are representative of four (a,b), two (c) or three (d) independent experiments or are from three independent experiments (e,f; mean and s.e.m.).
figf4(a) Immunoblot analysis of LAMP-2A in CD4+ T cells (left) and liver, kidneys, thymus and lungs (right) of L2A-cKO mice (KO) and their control littermates (Ctrl).(b) Quantitative PCR analysis of Lamp2a, Lamp2b and Lamp2c mRNA in T cells from L2A-cKO mice (right); results are presented relative to those of their control littermates (left), set as 1.(c) Immunoblot analysis of the autophagy related protein LC3 in its cytosolic form (LC3-I) and its autophagosome membrane-associated form (LC3-II) in total lysates of CD4+ T cells obtained from L2A-cKO mice or their control littermates and then left unstimulated (Rest) or activated for 24 h with anti-CD3 and anti-CD218 (Act), cultured for the final 4 h in the presence (+) or absence (−) of NH4Cl and leupeptin (NH4Cl+Leup).(d) Spleens from an L2A-cKO mouse and its control littermate (left), and flow cytometry of peripheral T cells from such mice (right). Numbers in quadrants (right) indicate percent CD4−CD8+ T cells (top left) or CD4+CD8− T cells (bottom right).(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).(g) Immunoblot analysis of LAMP-2A, Itch and RCAN1 in total lysates of CD4+ T cells obtained from L2A-cKO mice (n = 3 (KO1, KO2 and KO3)) and their control littermates (n = 3) and activated for 24 h as in e,f. *, nonspecific band.(h) Quantitative PCR analysis of Rcan1 and Itch mRNA in CD4+ T cells isolated from L2A-cKO mice and their control littermates and activated for 24 h as in e,f; results are presented relative to those of resting cells.(i,j) Recall responses to OVA(323-339) in draining lymph nodes isolated from L2A-cKO mice and their control littermates after immunization with OVA(323-339), assessed as proliferation (i) and IL-2 production (j) (as in e,f). *P = 0.0054 (i) or 0.0052 (j) (t-test).(k,l) Recall responses to L. monocytogenes in CD4+ T cells isolated from L2A-cKO mice and their control littermates (n = 6 per group) previously uninfected (UI) or previously infected with L. monocytogenes (Inf), assessed as IFN-γ production (k) and proliferation (l) (as in e,f). *P = 0.032 (k) or 0.02 (l) (t-test).(m) L. monocytogenes in spleen and liver of L2A-cKO mice and their control littermates (n = 3 per genotype) immunized with L. monocytogenes and then reinfected with a high dose of L. monocytogenes. CFU, colony-forming units. *P = 0.008 and **P = 0.001 (t-test).
figf5(a) Immunoblot analysis of LAMP-2A in CD4+ T cells obtained from 4-month-old or 22-month old mice, then left resting or stimulated for 24 h with anti-CD3 plus anti-CD28.(b) Immunoblot analysis of RCAN1 and Itch in CD4+ T cells obtained from 4-month-old or 22-month old mice (n = 3 per group), then stimulated for 24 h as in a (left), and quantification of protein in 22-month old mice, presented relative to that of 4-month-old mice, set as 100%. *P = 0.044 (t-test).(c) Immunoblot analysis of LAMP-2A in total lysates of naive and memory CD4+ T cells left resting or activated for 24 h as in a.(d) Immunoblot analysis (bottom) of LAMP-2A in total lysates of naive and memory CD4+ T cells obtained from 4- or 22-month-old mice and left resting (R) or activated for 24 h as in a (A); above, quantification from results below (n = 3 mice per group). *P = 0.009 (t-test).(e) Electrophoretic mobility-shift assay of the binding of NFAT (N) and free probe (FP) in nuclear extracts of CD4+ T cells isolated from 3-, 12- or 22-month-old mice and activated for 4 h as in a.(f) ROS production in CD4+ T cells isolated from 4- or 22-month-old mice (n = 3 per group) and left resting or activated for 12 h as in a, assessed by flow cytometry and presented as the mean fluorescence intensity (MFI) of a fluorogenic probe for oxidative stress (CellROX Green).(g-i) Immunoblot analysis of LAMP-2A (g) and analysis of the proliferation (h) and IL-2 production (i) of TH1 cells obtained from 4- or 22-month-old mice and transduced with retrovirus expressing GFP alone (RV-GFP) or human LAMP-2A and GFP (RV-LAMP2A), sorted by GFP expression, cultured for 2 weeks and then stimulated for 24-48 h as in a (assessed as in Fig. 2c (h) or Fig. 2b (i)). *P 0.05 (analysis of variance with Tukey's post-test).(j) Immunoblot analysis of LAMP-2A in total lysates of CD4+ T cells obtained from two human donors (A and B) and left resting or activated for 24 or 48 h as in a (above lanes).(k) Real-time PCR analysis of LAMP2A mRNA in naive and memory CD4+ T cells obtained from young human donors (26 ± 2 years of age; n = 3) or old human donors (72 ± 6 years of age; n = 6) and activated for 24 h as in a; results were calculated by the change-in-cycle-threshold method (2−ΔCt) relative to those of the gene encoding β-actin. *P = 0.047 and **P = 0.0033 (Mann-Whitney).
Figure 1(B) The degree of polymerisation of Z α1-antitrypsin after 72 hours at 37°C, as determined by an end-point immunoassay using the 2C1 monoclonal antibody, in varying concentrations of compound (shown in panel A). Modification of the phenyl and indole heterocycle of GSK425 (pIC50 6.5) resulted in an ~100 fold increase in potency and the discovery of the 2-oxindole GSK716 (pIC50 8.3). Data presented as mean ± SD, n = 2 (GSK425) and n = 25 (GSK716).(C) GSK716 binds to Z α1-antitrypsin with a high affinity mean pKD of 8.5 ± 0.12 (n = 18) as determined by a competition binding assay with a fluorescently labelled derivative. There was a 50-fold lower affinity for plasma-purified wild-type M α1-antitrypsin, with a mean pKD of 6.8 ± 0.18 (n = 10). Data presented as mean ± SD.(D) The compound bound to monomeric but not polymeric Z α1-antitrypsin (Z α1-AT) as reported by fluorescence polarisation of an Alexa-488-labelled variant of GSK716.(E) Representative curves reporting the interaction of different concentrations of GSK716 with Z α1-antitrypsin based on changes in intrinsic tryptophan fluorescence. Based on the concentration dependence (inset), the second-order rate constant of association was found to be 4.1 x 104 M-1 s-1. (F) The association of GSK716 with M α1-antitrypsin, giving a second-order rate constant of 2.1 x 102 M-1 s-1.
Figure 2 (G) CHO-K1 Tet-On cells expressing Z α1-antitrypsin were induced with doxycycline (0.5μg/ml) for 48 h. Cells were incubated with 10μM GSK716 (or 0.1% v/v DMSO for the control) during the induction. Culture media containing either the experimental compound or DMSO were changed every 24 h. After the induction, cells were labelled for 10 minutes with 35S Met/Cys and chased at the indicated times. Culture media were collected and cells lysed in 1% v/v NP-40 buffer. Intracellular fractions and culture media from cells expressing Z α1-antitrypsin were immunoprecipitated either with a mAb against total α1-antitrypsin (3C11) or with a polymer-specific mAb (2C1). Samples were resolved by 4-12% w/v acrylamide SDS-PAGE and detected by autoradiography. (H) The graphs show the effect of GSK716 on intracellular and extracellular Z α1-antitrypsin (mean±SEM, n=2)
Figure 4(B) M (above) and Z (below) α1-antitrypsin, at a concentration of 0.2mg/ml in PBS+5% v/v glycerol, were heated at a range of temperatures between 48°C-65°C for 4 hours in the presence and absence of GSK716, as indicated. The oligomerisation state was determined by non-denaturing PAGE electrophoresis; the lane denoted 'M' contains the unheated monomeric control.(C) M and Z α1-antitrypsin were subjected to equilibrium unfolding into different concentrations of guanidine hydrochloride (gdnhcl) in the presence and absence of GSK716, with bis-ANS dye added to report the presence of the unfolded intermediate. The normalised fluorescence intensity data were fitted with an equation describing a three-state unfolding curve. Values shown are the mean of three independent experiments and the error bars represent ±SEM.(D) Top panel, the association of GSK716 induced a marked quenching and blue-shift of the α1-antitrypsin intrinsic tryptophan fluorescence spectrum with respect to unbound protein (inset graph). The association of 10µM GSK716 with four α1-antitrypsin variants that vary in their propensity to polymerise, ranging from inert (M), to mild (S), moderate (Baghdad denoted 'B') and severe (Z). Representative progress curves of the change in intrinsic tryptophan fluorescence at 330nm for 0.2mg/ml protein are shown. Bottom panel, the half-time of association calculated from three such independent experiments (error bars are ±SEM) show a correspondence with the polymerisation propensity of the four variants. The change in Z α1-antitrypsin fluorescence was faster than the dead-time of the apparatus (~10s) and scaling was with reference to unbound intensity.
Figure 5(B) M α1-antitrypsin was incubated with different molar ratios of chymotrypsin and resolved by SDS-PAGE. The position of covalent α1-antitrypsin-chymotrypsin complex (cpx), native (nat) and cleaved (clv) is shown.(C) The inhibitory activity of α1-antichymotrypsin against chymotrypsin (ach+chym), neuroserpin against trypsin (neus+tryp), and antithrombin against thrombin (atiii+thrombin) was determined in the presence and absence of GSK716. Error bars reflect ±SD of two independent experiments.
Figure 6Z α1-antitrypsin transgenic animals were dosed with 10, 30 or 100 mg/kg GSK716 three times a day. Total concentrations of GSK716 were determined by LC-MS/MS (Table 2). (A) 100mg/kg GSK716 resulted in a mean 7-fold increase in circulating monomeric Z α1-antitrypsin levels demonstrating robust target engagement in the liver. The observed steady state total and free compound levels of GSK716 in the transgenic Z α1-antitrypsin mouse were well-predicted by the in silico PK model built on: (1) in vitro metabolic clearance data, (2) plasma protein binding data, (3) in vivo PK data from wild type mice and (4) a term comprising a 5μM circulating sink for drug with an affinity of 1.5 nM, representing the Z α1-antitrypsin within blood. The target free drug concentration was selected based on the observed potency in the in vitro secretion assays in which the total drug approximates to the free drug in the assay. Interestingly, 30mg/kg and 10mg/kg groups also gave significant, dose-dependent increases in circulating Z α1-antitrypsin despite free concentrations being below the cellular EC50 for secretion for much or all of the dosing period. Significance at p<0.05 by a student's t test is denoted by *, shown as pairwise comparisons (each animal is compared with itself, treated vs pre-treatment) and between groups (compound treated vs vehicle), with n= 7, 8 and 8 per group for day 6 dose level 10, 30 and 100mg/Kg respectively; day 15, n=10; day 21, n=5. Data presented as mean ± SD.
Figure 1A. Flow cytometry of cells isolated from heart tissue when scaled against TMRM staining and also FSC and SSC parameters of flow cytometry.B. Cardiac troponin I (cTnI) and α-Sarcomeric actinin (α-SA) staining of the TMRM++ population (CMs) and Vimentin staining of the TMRM+ population (NCs). The scale bar represents 100 µm.D. EGFP expression in iPSC-like colonies derived from CMs or NCs isolated from triple transgenic mice.E. EGFP expression combined with immunofluorescence staining of SSEA-1 and DAPI of iPSC-like colonies derived from CMs isolated from triple transgenic mice.F. Morphological changes of isolated CMs and NCs at different time points before AP positive colony formation at day 6. The scale bar represents 100 µm.
Figure 2A. Gene profiling of CM and NC at D0-D6 as shown clustered into 8 categories. Red indicates up-regulated genes while green indicates down-regulated genes (n=3, n=2 for NC-D6).B. Expression patterns of all genes per cluster by grey lines. Black line shows the mean value.D. Correlative analysis representing the relationships between CM and NC reprogramming specifically at days 2, 4 and 6. Red indicates closely related groups, while green indicates significantly different groups.
Figure 3A. RNA expression ratio (D2-to-D0) of candidates selected from day 2 microarray data.C. Ki67, α-MHC, and DAPI staining and quantification of CMs treated with control (Ctrl), or combinations of a FoxM1 up-regulation adenovirus (F), Id1 up-regulation adenovirus (I), Hmgb2 up-regulation adenovirus (H) or a small molecule Jnk3 inhibitor (Ji).D. Ki67, α-MHC, and DAPI staining and quantification of CMs treated with Ctrl or combination of FI with Jnk3-shRNAs in place of the small molecule (Ji). The arrow head indicates Ki67+/α-MHC+ proliferated CMs.E. Morphology and quantification of FIJs-treated CMs undergoing cytokinesis by H3P and Aurora kinase B (AURKB) staining.G. RNA expression ratio of Oct4, FoxM1, Id1, or Jnk3 in doxycycline-injected versus control adult CMs isolated from reprogrammable mice.H. Immunofluorescence of H3P and cTnI and quantification of the heart tissue sections from control or doxycycline injected mice.
Figure 4B. The size of hearts isolated from Ctrl or FIJs-treated mice; heart/body weight ratio of Ctrl or FIJs-treated mice.C. H3P, cTnI, and DAPI staining and quantification of heart tissues in Ctrl or FIJs-treated mice. The arrowhead indicates H3P+/cTnI+ proliferated CMs and the scale bar represents 50 µm.E. Immunofluorescence of H3P and cTnI and quantification of the heart tissue sections from adeno-Ctrl or adeno-FIJs injected mice.F. Quantification of BrdU+/EGFP+ population % in isolated CMs from adeno-Ctrl or adeno-FIJs injected M/Z mice by flow cytometry.
Figure 5B. RNA expressional changes of FoxM1, Id1, and Jnk3 after adeno-Ctrl or adeno-FIJs treatment after MI.C. Immunofluorescence staining of BrdU or H3P and cTnI or EGFP and quantification in the heart tissues after MI. The arrow head indicates cTnI+/BrdU+, cTnI+/H3P+ or EGFP+/BrdU+, EGFP+/H3P+proliferated CMs. The scale bar represents 50 µm.D. Ejection fraction and fraction shortening percentage in Ctrl or FIJs-treated mice 21 days post-MI.E. Catheterization in Ctrl or FIJs-treated mice 21 days post-MI under baseline or inferior vena cava occlusion condition.F. Trichrome staining and quantification showing percentage of fibrotic tissue (blue) in Ctrl or FIJs-treated mice 21 days after MI.
Figure 6A. RNA expression of mitosis checkpoint genes in CMs with FoxM1 or Id1 overexpression, Jnk3-shRNA, or combined treatment.B. Transcriptional expression of cyclin-dependent kinases in FoxM1 or Id1 up-regulated, Jnk3 down-regulated, or combined treated CMs.C. Real-time RT-PCR analysis of CDK inhibitor expression in CMs with FoxM1 or Id1 overexpression, Jnk3-shRNA or combined treatment.
Figure 2(B) Spontaneous EPSCs were recorded from striatal MSNs (wt=6; R6/2 ACSF=5; R6/2 chol-high=5) at a holding potential of -70 mV.(C-D) Average amplitude (C) and average frequency (D) of EPSCs from wt, R6/2 ACSF, and R6/2 chol-high MSNs. Data information: The data are shown as scatterplot graphs with means±standard error. Each dot corresponds to the value of each cell recorded Statistics: one-way ANOVA with Newman-Keuls post-hoc test (*p<0.05; **p<0.01; ***p<0.001; ****p<0.0001).(E-G) Number of excitatory synapses per volume of striatum by using FIB/SEM followed by 3D reconstruction. (E) Representative tissue block of striatum (10 µm x 15 µm x 10 µm) used for the 3D analysis. (F) Representative FIB-SEM segmentation and reconstruction of excitatory synapses (yellow) in 200 serial sections of striatum for a total volume of 500 µm3, of wt, R6/2, R6/2 ACSF, and R6/2 chol-high mice. (G) Density of excitatory synapses in at least 1500 μm3 of striatal tissue from wt, R6/2, R6/2 ACSF, and R6/2 chol-high mice (N=3 animals/group). Data information: The data are shown as scatterplot graphs with means±standard error. Each dot corresponds to the value of , the number of synapses counted/µm3 in different blocks of striatal tissue (G) Statistics: one-way ANOVA with Newman-Keuls post-hoc test (*p<0.05; **p<0.01; ***p<0.001; ****p<0.0001).(H-I) TEM images (H) and 2D profile (I) of pre-synaptic terminal sections contain SVs and mitochondria (m). Docked vesicles are indicated by a white arrowhead (H) and black circle (I) and are defined as a vesicle with its center located within 20 nm from the presynaptic membrane. Scale bar: 100 nm.(J, K) Quantification showing the total SVs/µm2 (J) and the docked SVs/µm (K) in R6/2, R6/2 ACSF, and wt striatal synapses (N=3 animals/group). N=60 excitatory synapses/group were counted. Data information: The data are shown as scatterplot graphs with means±standard error. Each dot corresponds to the number of total SVs/µm2 (J) and docked SVs/µm of active zone (K) counted in each synapse. Statistics: one-way ANOVA with Newman-Keuls post-hoc test (*p<0.05; **p<0.01; ***p<0.001; ****p<0.0001).
Figure 3(A) Spontaneous IPSCs were recorded from striatal MSNs (wt=6; R6/2 ACSF=5; R6/2 chol-high=5) at a holding potential of 0 mV.(B-C) Average amplitude (B) and average frequency (C) of IPSCs from wt, R6/2 ACSF, and R6/2 chol-high MSNs. Data information: The data are shown as scatterplot graphs with means±standard error. Each dot corresponds to the value of each cell recorded (B, C) Statistics: one-way ANOVA with Newman-Keuls post-hoc test (**p<0.01; ***p<0.001).(D-E) Evaluation of the number of inhibitory synapses per volume of striatum by using FIB/SEM followed by 3D reconstruction. (D) Representative FIB-SEM segmentation and reconstruction of inhibitory synapses (green) in 200 serial sections of striatum for a total volume of 500 µm3 in wt, R6/2, R6/2 ACSF, and R6/2 chol-high mice. (E) Density of inhibitory synapses in at least 2000 µm3 of striatal tissue of wt, R6/2, R6/2 ACSF, and R6/2 chol-high mice (N=3 mice/ group). Data information: The data E) are shown as scatterplot graphs with means±standard error. Each dot corresponds to the number of synapses counted /µm3 in different blocks of tissue for each group of animals (N=3 animals/group) (E). Statistics: one-way ANOVA with Newman-Keuls post-hoc test (**p<0.01; ***p<0.001).
Figure 4(B) 24S-OHC level measured by mass spectrometry in the infused and contralateral striatum of wt, R6/2‐ACSF, and R6/2 chol-high mice at 12 weeks of age after a 4-week striatal infusion of cholesterol (N=4/group). All values are expressed as % above the mean of wt, and these data are shown as scatterplots with means±standard error. Each dot corresponds to the value obtained from each animal.(C-E) Lanosterol (C), lathosterol (D), and desmosterol (E) level measured by mass spectrometry in the infused and contralateral striatum of wt, R6/2 ACSF, and R6/2 chol-high mice at 12 weeks of age after 4-week striatal infusion of cholesterol (N=4/group). All values are expressed as % above the mean of wt, and these data are shown as scatterplots with means±standard error. Each dot corresponds to the value obtained from each animal.(F-I) Nuclear (white arrows) and perinuclear (yellow arrows) localization of endogenous SREBP2 in the striatum of wt, R6/2 and R6/2-chol mice. Representative confocal image (F and H) and relative quantification (G and I) of SREBP2 (red) in wt and R6/2 mice and in the infused and contralateral striatum of R6/2 chol-high mice (N=4). Hoechst (Ho, blue) was used to counterstain nuclei. Scale bar in F and H: 10 µm. Graphs in (G and I) represents the intensity of SREBP2 normalized on nuclei (%). Data are shown as scatterplots with means±standard error. Each dot corresponds to the value obtained from each image. Statistics: Student's t-test (**p<0.01; ****p<0.0001).J, K Representative high-magnification confocal images of immunostaining against SREBP2 (red) and NeuN (J) or GFAP (K) (green) on coronal sections of brains from R6/2 chol-high mice. A cell in the infused striatum, positive for NeuN and GFAP respectively, is shown. Hoechst (Ho, blue) was used to counterstain nuclei. Scale bars: 1 µm.
Figure 6Representative confocal images showing p62 (A, red) in the infused and contralateral striatum on brain coronal sections from wt, R6/2 ACSF, and R6/2 chol-high mice (N=3/group). Hoechst (Ho, blue) was used to counterstain nuclei. Quantification of dots for p62 (B) in the contralateral and infused striatum of R6/2 ACSF and R6/2 chol-high mice. 20 images from 3 sections in the middle of the striatum for each animal were acquired and analyzed. The data in (B are shown as scatterplots with means±standard error, and each dot corresponds to the value obtained from each image. Statistics: one‐way ANOVA followed by Newman-Keuls multiple comparison tests (***p<0.001; ****p<0.0001). Scale bars in (A 10 µm.Representative confocal images showing LAMP1 (C, green) in the infused and contralateral striatum on brain coronal sections from wt, R6/2 ACSF, and R6/2 chol-high mice (N=3/group). Hoechst (Ho, blue) was used to counterstain nuclei. Quantification of density for LAMP1 (D) in the contralateral and infused striatum of R6/2 ACSF and R6/2 chol-high mice. 20 images from 3 sections in the middle of the striatum for each animal were acquired and analyzed. The data in D) are shown as scatterplots with means±standard error, and each dot corresponds to the value obtained from each image. Statistics: one‐way ANOVA followed by Newman-Keuls multiple comparison tests (***p<0.001; ****p<0.0001). Scale bars in C): 10 µm.
Figure 1Gene expression analyzed by quantitative PCR in brown adipocytes that were fed with or without 1mM asparagine as in (A). n=3 independent experiments. Data information: Data represent means±SEM. *P<0.05; **P<0.01; unpaired two-tailed Student's t test.Preadipocytes bearing the shRNA targeting Pgc1α were induced differentiation with or without 1mM asparagine. Protein expression was analyzed on day 6. n=3 biological replicates.Brown preadipocytes were infected by lentiviral shRNAs against Asns. Cells were differentiated in culture media with or without 1mM asparagine. Western blot was performed on day 6.