EMBO/soda-nel huggingface dataset:
Chemical modulation of chaperone-mediated autophagy by retinoic acid derivatives (2013)
figf7

(a) Immunoblot for the indicated proteins in homogenates (Hom) and lysosomes (Lys) isolated from cells untreated (None) or treated for 12 h with 20 μM of the indicated compounds. (b) mRNA levels of LAMP-2A in control mouse fibroblasts (left) or RARα(−) cells (right) treated with AR7 or paraquat (PQ) as in a; n = 4–5. Values are presented as fold change in mRNA relative to untreated cells. (c) Cellular viability of control (left) or LAMP-2A(−) (right) fibroblasts exposed to 2 mM or 0.5 mM paraquat, respectively, and treated with the indicated compounds for 12 h before or after the paraquat treatment; n = 3. (d) Viability of mouse fibroblasts transfected with the indicated concentrations of a plasmid encoding α-synuclein and left untreated (None) or treated with 1 mM paraquat alone or in the presence of 20 μM AR7; n = 3. In b–d, all values are mean ± s.e.m. Differences with cells that are untreated (marked with asterisk) or those treated only with paraquat (§) were significant for P 0.001. Full-length blots are shown in Supplementary Figure 21. (e) Immunoblot for α-synuclein (α-syn) in the same cells as in d. Top, higher-exposure blot to highlight oligomeric (oligo) species. Asterisk denotes nonspecific band. M, monomer; MW, molecular weight.
The N-end rule ubiquitin ligase UBR2 mediates NLRP1B inflammasome activation by anthrax lethal toxin (2019)
Figure 1

Figure 1. The N-end rule pathway controls LT-induced NLRP1B inflammasome activation.

A, B Effects of basic and bulky hydrophobic amino acids on LT-induced pyroptosis and caspase-1 activation in RAWRA cells. Cells were pre-treated with 10 mM indicated amino acids prior to LT stimulation. Cell viability was measured by using the ATP assay and data shown are mean values ± SD from three replicates (A). Mock, DMEM medium. Cell supernatants were subjected to anti-caspase-1 immunoblotting and the lysates were blotted with the anti-tubulin antibody (B). p10, mature caspase-1; p45, caspase-1 precursor.

C, D Effects of basic and bulky hydrophobic amino acids on LT-induced RFP-ASC specks formation in RAWRA cells. Fluorescence images were taken on a confocal microscopy (C). The percentages of cells showing RFP-ASC specks (mean values ± SD from three replicates) are in (D).

E Effects of basic and bulky hydrophobic amino acids on LT-induced caspase-1 activation in Tlr4-/- iBMDMs. Cells were treated as that in (A). Cell supernatants were subjected to anti-caspase-1 immunoblotting and the lysates were blotted with the anti-tubulin or anti-MEK3 antibody.
The N-end rule ubiquitin ligase UBR2 mediates NLRP1B inflammasome activation by anthrax lethal toxin (2019)
Figure 2

Figure 2. The ubiquitin ligase UBR2 is required for LT-induced NLRP1B activation.

A RFP-ASC specks formation assay of the effect of Ubr2 knockdown on LT-induced inflammasome activation. RAWRA cells were transfected with a control siRNA or Ubr2-targeting siRNA mixtures for 60 h followed by LT treatment. The numbers in the merged panel are the percentages of the cells showing RFP-ASC specks.

B Effect of Ubr2 stable knockdown on caspase-1 activation in RAWRA cells. Cells stably expressing a control or Ubr2-targeting shRNA were incubated with WT LT (+) or its E687C mutant (-) for 3 h.

C Effects of Ubr2 knockdown (siRNA #05) on LT-induced NLRP1B inflammasome activation reconstituted in 293T cells. Cells were treated with WT LT (+) or its E687C mutant (-).

D, E Effect of Ubr2 knockout on NLRP1B or NAIP2-NLRC4 inflammasome activation. WT and Ubr2-/- iBMDMs were treated with LT or BsaK for 3 h. Cell viability was measured by using the ATP assay and data shown are mean values ± SD from three replicates (D).
The N-end rule ubiquitin ligase UBR2 mediates NLRP1B inflammasome activation by anthrax lethal toxin (2019)
Figure 3

Figure 3. UBE2O functions together with UBR2 to mediate NLRP1B inflammasome activation.

A Immunoblotting of caspase-1 activation in Ubrs-knockdown BMDMs derived from the 129/Sv mice. Cells were transfected with the indicated siRNA for 60 h, and then treated with WT LT (+) or its E687C mutant (-) for 3 h.

B-D Immunoblotting of caspase-1 activation in E2s-knockdown BMDMs derived from the 129/Sv mice. Additional Ube2o siRNAs were used to confirm the inhibitory effect of Ube2o knockdown on LT-induced caspase-1 activation (C). Ube2o knockdown efficiency was measured by qPCR and are shown as mean values ± SD from three replicates.

E RFP-ASC specks formation assay of the effect of Ube2o knockdown (siRNA Ube2o-01) on LT-induced inflammasome activation in RAWRA cells. The percentages of cells showing RFP-ASC specks (mean values ± SD from three replicates) are in (D).

F Co-immunoprecipitation interaction of UBE2O and UBR2. Myc-UBE2O and Flag-UBR2 were co-expressed in 293T cells and cell lysates were subjected to anti-Flag immunoprecipitation followed by immunoblotting analyses as shown.
The N-end rule ubiquitin ligase UBR2 mediates NLRP1B inflammasome activation by anthrax lethal toxin (2019)
Figure 4

Figure 4. LT cleavage of NLRP1B triggers N-end rule-mediated degradation of NLRP1B for inflammasome activation.

A Domain structure of NLRP1B. The LT cleavage site and the FIIND auto-cleavage site are marked.

B Effect of LT treatment on NLRP1B protein level. 293T cells were transfected with Flag-NLRP1B expression plasmid for 24 h, and cells were then treated with WT LT (+) or its E687C mutant (-) for 4 h. Shown is the anti-Flag immunoblot of the total cell lysates.

C RFP-ASC specks formation in 293T cells expressing an indicated NLRP1B variant. Cells were treated with LT for 3 h. Scale bar, 20 μm. 1-983 and 984-C (residues 984 to the C terminus) are the FIIND-mediated autocleaved fragments of NLRP1B.

D, E Effect of MG132 and Leu on LT-induced NLRP1B degradation. 293T cells expressing NLRP1B (1-983)-HA were treated with LT and MG132 for 8 h. 10 mM Leu was added to cells 30 min prior to LT treatment. Shown are the anti-HA and anti-tubulin immunoblots of the total cell lysates.

F LT-induced ubiquitination of NLRP1B. 293T cells expressing NLRP1B (1-983)-HA were treated with LT and/or MG132 for 6 h. Cells were then harvested for anti-HA immunoprecipitation and subjected to immunoblotting analyses using indicated antibodies.

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IFNλ is a Potent Anti-Influenza Therapeutic without the Inflammatory Side Effects of IFNα Treatment (2016)
Figure 1

Figure 1. Therapeutic administration of IFNα and IFNλ differentially influences the outcome of IAV induced disease.

A. Relative antiviral activity of IFNα (circles) or IFNλ (triangles). AEC cultures were stimulated for 4hrs with stated IFN at specified concentrations (ng/ml) and induction of indicated ISGs was assessed by qPCR (data shown is representative of four independent experiments, n=3-4).

B, C. Mice were pretreated with equivalent doses of IFNα (1.45μg/50μl) or IFNλ (2.6μg/50μl) or Veh Ctrl (squares, 50μl PBS) 24hrs prior to infection with PR8; weight loss and survival was assessed throughout infection (B), and viral load (C) assessed at 4dpi (data shown is representative of two independent experiments, n=8-10 (B), n=3 (C)).

D, E. Mice were infected with PR8 and treated with equivalent doses of IFNα or IFNλ or Veh Ctrl at days 2, 4 and 5 post infection; survival and weight loss was monitored (D, data pooled from 4 independent experiments, n=12-29) and viral load assessed at 4dpi (E) (data representative of two independent experiments, n=3-5).

Data information: Significance assessed by Log-rank (Mantel-Cox) test (survival), 2-way ANOVA (weight loss) and Unpaired t tests (viral load). * indicates IFNα:Veh Ctrl, + indicates IFNλ:Veh Ctrl, and ° indicates IFNα:IFNλ. * P=0.0236, + P=0.0236 *** P<0.0001, +++ P<0.001 (B); * P= 0.012, + P=0.012 (C); * P=0.0443, + P=0.035, °° P=0.0015 (D); ** P=0.0081, ++ P=0.0066 (E). Symbols on the right of graphs indicate significance of whole curve. Graphs show mean ± SEM.
IFNλ is a Potent Anti-Influenza Therapeutic without the Inflammatory Side Effects of IFNα Treatment (2016)
Figure 2

Figure 2. IFNα treatment correlates with increased inflammation during IAV infection.

A, B. Mice were infected with PR8 and treated with IFNα (circles, 1.45μg/50μl), IFNλ (triangles, 2.6μg/50μl) or Veh Ctrl (squares) as previously stated. Concentrations of stated proinflammatory cytokines in BAL fluid was measured by multiplex cytokine assay (A) and flow cytometric quantification of pDCs and Inflammatory Monocytes in the lung was performed (B) (data shown is representative of two independent experiments, n=2-6).

C, D. Lung sections from control and infected mice treated as indicated, were stained by TUNEL for apoptotic cells at 6dpi. Quantification of TUNEL+ cells in whole lung slides by Icy Spot Detector (ICY-R3M2Y2) (C) (data shown is pooled from three independent experiments, n=3-8). Red arrowheads indicate TUNEL signal (D). Scale bar, 200 μM (data shown is representative of two independent experiments, n=3-4).

Data information: Significance assessed by 2-way ANOVA with Bonferroni post tests (where * denotes IFNα:Veh Ctrl, + indicates IFNλ:Veh Ctrl , and ° indicates IFNα:IFNλ). Symbols on the right of graphs indicate statistical significance of the whole curve. IL-6 whole curve: ** 0.0041, ° P=0.0144. IL-6 5 d.p.i.: ** P=0.001884, °° P=0.001645. IP-10 5 d.p.i.: ** P=0.004897, °° P=0.005354. MCP-1 5.d.p.i.: ** P=0.007473, °° P= 0.002003. Eotaxin whole curve: * 0.0235, ° P=0.0386. Eotaxin 5 d.p.i. *** P=0.000149, °° P=0.001975. Mip-1α 5 d.p.i.: °° P= 0.002921 (A). Plasmacytoid Dendritic Cells: * P=0.0211, °° P= 0.006965. Inflammatory monocytes ** P=0.007842, °° P=0.000895 (B). ** P=0.0011, ++ P=0.0051, °°° P=0.0005 (C). Graphs show mean ± SEM.
IFNλ is a Potent Anti-Influenza Therapeutic without the Inflammatory Side Effects of IFNα Treatment (2016)
Figure 3

Figure 3. IFNα, but not IFNλ treatment induces pulmonary cytokine secretion through activation of immune cells.

A, B. IL-6, IP-10 and MCP-1 concentrations were measured by multiplex cytokine assay in AEC culture supernatants (A) and Macrophage, pDC and cDC culture supernatants (B) at 24hrs post stimulation with IFNα4 (0.725ng/ml) or IFNλ2 (1.3ng/ml) or LPS (AEC only) (data shown is representative of two independent experiments, n=3-6).

C. BAL samples taken from mice treated with IFNα, IFNλ or Veh Ctrl at specified time points (data shown is representative of two independent experiments, n=5-6).

Data information: Significance assessed by Unpaired t tests where * denotes IFNα:Veh Ctrl and ° indicates IFNα:IFNλ. IFNλ:Veh Ctrl was not significant. IL6 pDC: *** P=0.0004, °°° P= 0.0005, IL6 cDC: * P=0.0102, ° P=0.0151. IP-10 macrophage: ** P=0.0033, °° P=0.0033, pDC: **** P<0.0001, °°°° P<0.0001, cDC: ** P= 0.0013, °° P=0.0013, MCP-1 macrophage: **** P<0.0001, °°° P=0.003 (B). IL-6 10hrs: * P=0.0112, ° P=0.0262, 18hrs: * P=0.0314, ° P=0.373. IP-10 10hrs: * P= 0.0261, ° P=0.0472. MCP-1 10hrs: ** P=0.0081, ° P=0.0206, 18hrs: ** P=0.0089, ° P=0.01 (C).
IFNλ is a Potent Anti-Influenza Therapeutic without the Inflammatory Side Effects of IFNα Treatment (2016)
Figure 4

Figure 4. Pathogenicity-related gene clusters are specifically induced by IFNα, not by IFNλ treatment.

Mice were treated with IFNα (1.45μg/50μl), IFNλ (2.6μg/50μl) or Veh Ctrl (50μl PBS), and whole lungs were taken at 18hrs post treatment for global analysis by Illumina.SingleColor.Mouse WG-6_V2_0_R0_1127 microarrays. Samples (n=5) were normalized to the median of the vehicle control group and filtered for a fold change of 1.5, yielding 553 genes differently regulated between treatments (One way ANOVA, P<0.01, Benjamini-Hochberg multiple test correction), of which 429 genes are upregulated. K means clustering revealed six gene clusters, one of which encompassed genes primarily induced by IFNα4 (A), while the remaining clusters contained genes upregulated by both IFNα4 and IFNλ2 (B). The two clusters of genes were analysed by Ingenuity Pathway Analysis (IPA) (C,D).
IFNλ is a Potent Anti-Influenza Therapeutic without the Inflammatory Side Effects of IFNα Treatment (2016)
Figure 5

Figure 5. IFNα, but not IFNλ treatment induces cytokine secretion from human immune cells.

A. Human AEC cultures were stimulated for 4hrs with IFNα (circles) or IFNλ (triangles) at specified concentrations then assessed for stated ISG induction by qPCR (data is representative of 2 independent experiments, n=3).

B, C. ISG induction in human PBMCs was assessed at 4 and 24hrs post IFNα (21 U/ml) or IFNλ (1.2 ng/ml) stimulation (B). PBMC proinflammatory cytokine secretion was measured by multiplex cytokine assay at 4 and 24hrs post stimulation with IFNα or IFNλ (C) (data shown is pooled from 6 independent donors).

Data information: Significance tested by 2-way ANOVA where * denotes IFNα:Veh Ctrl and ° indicates IFNα:IFNλ. IFNλ:Veh Ctrl was not significant. IRF7: ** P=0.0037, Rsad2 4hrs: ** P=0.0038, °° P=0.037, 24hrs: * P=0.0234. OAS1 4hrs: * P=0.0328, ° P=0.0358, 24hrs: **** P<0.0001 (B). IL-6: * P=0.0124, ** P=0.0021. MCP-1 4hrs: **** P<0.0001, °°° P=0.001, 24hrs: ** P=0.0046, °° P= 0.005. IP-10 4hrs: ** P=0.0033, °° P= 0.0024, 24hrs: ** P=0.0011, °°° P=0.001 (C).
ZEB1-mediated melanoma cell plasticity enhances resistance to MAPK inhibitors (2016)
Figure 1

Figure 1. High levels of ZEB1 expression are correlated with low MITF levels and are associated with inherent resistance to MAPKi in BRAFV600-mutated melanoma cell lines

(A) Pearson correlation between ZEB1 and MITF mRNA expression in 61 melanoma cell lines available through the CCLE. (B) ZEB1, ZEB2, TWIST1 and MITF expression in a panel of BRAFV600-mutated melanoma cells assessed by Western blot. GLO and C-09.10 cells are patient-derived short-term cultures. Actin was used as a loading control. (C) Quantitative PCR analyses of ZEB1, ZEB2, TWIST1 and MITF in the same panel of cell lines. mRNA expression levels are represented relatively to C-09.10 cells, in which the levels were arbitrarily fixed at 1 (n=3, mean ± SD). The dotted line separates ZEB1high (left) and ZEB1low (right) cell lines. (D) Tukey box plot of ZEB1, ZEB2, TWIST1, and MITF mRNA expression according to the IC50 of the drug (µM) administered (BRAFi/MEKi), in melanoma cell lines from the CCLE (n=28) (Student’s t-Test). High ZEB1, low ZEB2, and low MITF expression levels were correlated with BRAFi (PLX4720) and MEKi (AZD6244) resistance. PLX4720 is an analog of PLX4032. (E) IC50 values of PLX4032 (µM) in the panel of BRAFV600 melanoma cells as determined by ATP assay (n=3, mean ± SD). For SKMEL24 and WM793, IC50 was >8µM.
ZEB1-mediated melanoma cell plasticity enhances resistance to MAPK inhibitors (2016)
Figure 2

Figure 2. High ZEB1 and low MITF levels are associated with inherent resistance to MAPKi in BRAFV600-mutated melanoma tumors

(A) Pearson correlation between ZEB1 and MITF mRNA expression levels in 467 melanoma tumors from TCGA data set. (B) Representative pictures of ZEB1 and MITF immunostaining in primary melanomas. Scale bar = 40 µm. The aberrant activation of ZEB1 in melanomas is correlated with a MITFlow phenotype. (C) Representative pictures of ZEB1 immunostaining in BRAFV600 tumors from patients, classified in ZEB1 high, int and low subgroups, based on the intensity of ZEB1 staining and on the percentage of cells positive for ZEB1. Scale bar = 80 µm. (D) Pie charts representing the distribution of ZEB1 alone (upper part), or ZEB1 and TWIST1 (lower part) immunohistochemical staining in tumors according to their initial response to vemurafenib +/- cobimetinib treatment. ZEB1 +/- TWIST1 levels are higher in MAPKi primary resistant melanomas (initial non-responders) compared to tumors that initially respond to treatment (n=70, Fisher’s exact test). (E) Representative pictures of ZEB1 and MITF immunostainings, before and after vemurafenib treatment, in the tumor from patient 1, exhibiting primary resistance to BRAFi. Scale bar = 80 µm. Right: Magnification of MITFhigh and MITFlow clones in the resistant tumor under treatment. Arrows indicate endothelial and stromal cells that also show positive staining for ZEB1, besides tumor cells.
ZEB1-mediated melanoma cell plasticity enhances resistance to MAPK inhibitors (2016)
Figure 3

Figure 3. ZEB1 expression is activated in BRAFV600-mutated melanoma cell lines with acquired resistance to BRAFi and in biopsies from patients relapsing while under treatment

(A) PLX4032 IC50 (µM) of sensitive A375 and SKMEL5 and resistant (A375-R, SKMEL5-R) cell lines, as well as of GOKA and ESP cells, two BRAFi-resistant patient-derived short-term cultures, as determined by ATP assay (n=3, mean ± SD). For ESP, IC50 was >8µM. (B) Western blot analyses of ZEB1, TWIST1 and FRA1 in A375-R and SKMEL5-R versus the parental naive cells, and in GOKA and ESP cells. GAPDH was used as a loading control. (C) Quantitative PCR analyses of ZEB1 and MITF in A375-R and SKMEL5-R versus the parental naive cells, and in GOKA and ESP cells. mRNA expression levels are represented as arbitrary units (a.u). Statistical difference relative to sensitive A375 cells is shown (n=3, mean ± SD, Student’s t-test). (D) Representative pictures of ZEB1 and MITF immunostainings in tumors from patients 2, 3 and 4, before and after vemurafenib treatment. Scale bar = 40 µm. For ZEB1 staining in patient 4, the inset shows a magnification. Arrows point at stromal cells (s). All other cells positive for ZEB1 are tumor cells.
ZEB1-mediated melanoma cell plasticity enhances resistance to MAPK inhibitors (2016)
Figure 4

Figure 4. ZEB1 overexpression in A375 melanoma cells potentiates the conversion into a MITFlow/p75high stem-like tumor initiating phenotype, and promotes resistance to MAPKi (A) A375 cells were infected with retroviruses expressing ZEB1. Western blot analyses of ZEB1 and p75. GAPDH was used as a loading control. (B) Quantitative PCR analyses of MITF, p75, ABCB5 and JARID1B upon ZEB1 expression. mRNA expression levels are represented relatively to control cells, in which the levels were fixed at 1 (mean ± SD, n=3, Student’s t-test). (C) Soft agar colony formation assay upon ZEB1 expression. Scale bar = 200 µm. Histograms represent quantitative analyses (mean ± SD, n=3, Student’s t-test). (D) 2 x 106 Control or ZEB1-overexpressing A375 cells were injected subcutaneously into nude mice. The mean tumor volume for 5 mice is represented ± SEM (Student’s t-test). (E) Western blot analyses of ZEB1, MITF, P-ERK, and TWIST1 levels in control or ZEB1-expressing cells +/- 150 nM PLX4032 treatment for 24 h. GAPDH was used as a loading control. (F) FACS analyses of p75 cell surface expression upon ZEB1 overexpression, after 10 days treatment with or without 150 nM PLX4032. Bar chart representing the mean percentage of p75-high, int and low cells from 2 independent experiments (Fisher’s Exact Test). (G) Control or ZEB1-overexpressing A375 cells were transfected with control or p75-siRNA. p75 and MITF expression levels were was analyzed by quantitative PCR after 48 h. mRNA expression levels are represented relatively to control cells transfected with siRNA Control, in which the levels were fixed at 1 (mean ± SD, n=2). (H) Clonogenic assay +/- PLX4032 (150 nM), +/- GDC-0973 (5 nM) treatment for 10 days. The graphs represent the mean number of colonies (± SD) in 3 independent experiments (Student’s t-test). (I) Number of weeks of chronic exposure to PLX4032 before emergence of resistance in control or ZEB1-expressing cells (n=3, Student’s t-test).
ZEB1-mediated melanoma cell plasticity enhances resistance to MAPK inhibitors (2016)
Figure 5

Figure 5. ZEB1 overexpression in patient-derived ZEB1low/MITFhigh short-term culture cells promotes the conversion into a MITFlow/p75high stem-like phenotype, resistant to MAPKi

(A) C-09.10 short-term culture cells were infected with retroviruses expressing ZEB1. Western blot analyses of ZEB1 and p75. GAPDH was used as a loading control. (B) Quantitative PCR analyses of MITF and p75 upon ZEB1 expression. mRNA expression levels are represented relatively to control cells (mean ± SD, n=3, Student’s t-test). (C) Soft agar colony formation assay following ZEB1 expression. Scale bar = 200 µm. Histograms represent quantitative analyses (mean ± SD, n=3, Student’s t-test). (D) FACS analyses of p75 cell surface expression upon ZEB1 overexpression. Bar chart representing the mean percentage of p75-high, int, low and negative cells from 2 independent experiments (Fisher’s exact test). (E) Clonogenic assay +/- PLX4032 (100 nM) treatment for 10 days. The graph represents the mean number of colonies in 3 independent experiments (Student’s t-test).
ZEB1-mediated melanoma cell plasticity enhances resistance to MAPK inhibitors (2016)
Figure 6

Figure 6. ZEB1 knock-down in ZEB1high/MITFlow melanoma cells promotes the reversible conversion into a MITFhigh/p75low differentiated phenotype and inhibits tumor growth in vivo

(A) A375 cells were infected with retroviruses expressing a control or ZEB1-shRNA. Western blot analyses of ZEB1 and p75 upon ZEB1 knock-down. GAPDH was used as a loading control. High and low exposures (exp) for p75 are shown. (B) Quantitative PCR analyses of MITF, p75, ABCB5 and JARID1B upon ZEB1 knock-down. mRNA expression levels are represented relatively to shRNA control cells (mean ± SD, n=3, Student’s t-test). (C) FACS analyses of p75 expression upon ZEB1 knock-down. Bar chart representing the mean percentage of p75-high, int and low cells from 2 independent experiments (Fisher’s exact test). (D) Soft agar colony formation assay upon ZEB1 knock-down. Scale bar = 200 µm. Histograms represent quantitative analyses (mean ± SD, n=3, Student’s t-test). (E) 2 x 106 Control or ZEB1-shRNA A375 cells were injected subcutaneously into nude mice. The mean tumor volume for 5 mice is represented (± SEM). (Student’s t-test). (F) A375 cells were infected with an IPTG-inducible ZEB1-shRNA. Left panel: Western blot analyses of ZEB1 expression ± IPTG (100µM) treatment for 6 days. Actin was used as a loading control. Right panel: 2 x 106 IPTG-inducible shRNA-Control or shRNA-ZEB1 A375 cells were injected subcutaneously into nude mice. When the tumor reached 5 mm in diameter, ZEB1 expression was silenced by adding IPTG (10 mM) into the drinking water for 20 days. The mean tumor volume for 5 mice is represented (± SEM). (Student’s t-test). (G) SKMEL5 cells expressing an IPTG-inducible ZEB1-shRNA were treated with IPTG (100µM) for 10 days (+IPTG), then IPTG was removed (-IPTG) and ZEB1, MITF and p75 expression levels were analyzed by Western blot and/or quantitative PCR analyses. GAPDH was used as a protein loading control, mRNA expression levels are represented relatively to untreated cells, in which the levels were fixed at 1 (mean ± SD, n=2).