Journal: Blood Advances

Article Title: Targeting CD70 in cutaneous T-cell lymphoma using an antibody-drug conjugate in patient-derived xenograft models

doi: 10.1182/bloodadvances.2021005714

Figure Lengend Snippet: Frequencies of CD70 expression in biopsies from patients with systemic or cutaneous T-cell lymphomas

Article Snippet: We found that in 4 CTCL cell lines tested (HH, MJ, Hut78, and H9), more than 99% of CD4 + cells expressed CD70, whereas, in T-cell leukemia cell lines (CCRF-CEM, HPB-ALL, PF-382, and Jurkat), only 2∼23% of cells, expressed CD70 ( ).

Techniques: Expressing

Journal: Antioxidants

Article Title: Exploring the Leukemogenic Potential of GATA-1 S , the Shorter Isoform of GATA-1: Novel Insights into Mechanisms Hampering Respiratory Chain Complex II Activity and Limiting Oxidative Phosphorylation Efficiency

doi: 10.3390/antiox10101603

Figure Lengend Snippet: Western blot analysis for SDHC expression levels in total protein extracts obtained from K562 cells over-expressing GATA-1 FL and GATA-1 S isoforms and from a mock control. ( a ) Representative image of three independent experiments showing the presence of two SDHC-positive protein bands on the membrane possibly representing different SDHC isoforms. ( b ) Densitometric analysis of Western blot results showing total SDHC levels markedly increased only in K562 cells over-expressing the GATA-1 S isoform. For each sample, band intensities of the two SDHC signals, taken as a whole, were quantified from three independent experiments and normalized to α-actin used as a loading control. ( c ) Schematic representation of the alternative splicing mechanism generating SDHC variants (ASVs). Solid boxes and bars indicate the deleted exons and the corresponding protein domains, respectively. ( d ) Quantitative real-time PCR analysis of SDHC mRNA variants in cells over-expressing GATA-1 isoforms and in a mock control. mRNA expression levels were normalized against GAPDH. Results showed increased total SDHC transcript levels in cells over-expressing GATA-1 S , thus confirming western blot analysis. Moreover, transcript-specific amplification revealed that SDHC abnormal expression in these cells was mostly due to the Δ5 ASV transcript. All data represent the mean ± SD of three independent experiments. Statistical analysis was performed by one-way ANOVA, followed by Dunnett’s multiple comparisons test, where appropriate. Differences were considered significant when p < 0.05 and highly significant when p < 0.0001. * p < 0.05, ** p < 0.0001 versus mock control.

Article Snippet: The human K562 erythroleukemia cell line obtained from European Collection of Authenticated Cell Cultures (EACC #89122407) was grown in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS) plus 4 mM glutamine, 10 U/mL penicillin, and 10 mg/mL streptomycin (purchased from Gibco, Thermo Fisher Scientific, Inc., Waltham, MA, USA) at 37 °C in a humidified 5% CO 2 − containing atmosphere.

Techniques: Western Blot, Expressing, Control, Membrane, Alternative Splicing, Real-time Polymerase Chain Reaction, Amplification

Journal: Antioxidants

Article Title: Exploring the Leukemogenic Potential of GATA-1 S , the Shorter Isoform of GATA-1: Novel Insights into Mechanisms Hampering Respiratory Chain Complex II Activity and Limiting Oxidative Phosphorylation Efficiency

doi: 10.3390/antiox10101603

Figure Lengend Snippet: GATA-1 S knockdown experiments: ( a ) western blot analysis (10% SDS-page gel) of endogenous levels of GATA-1 isoforms and SDHC after K562 transfection with a custom GATA-1 S small interfering RNA (GATA-1 S siRNA) at final concentration of 50 and 100 nM. ( b ) Densitometric analysis of western blot results of GATA-1 S silenced protein. ( c ) Densitometric analysis of western blot results for SDHC after specific GATA-1 S siRNA transfection. ( d ) Quantitative real-time PCR analysis of SDHC mRNA variants in K562 cells previously transfected with two doses of specific GATA-1 S siRNA. mRNA expression levels were normalized against GAPDH and relative to negative control siRNA transfected cells. Results showed decreased total SDHC transcript levels in cells knocked down for GATA-1 S , thus confirming western blot analysis. In addition, transcript-specific amplification revealed a more significant dose-dependent reduction for the Δ5 ASV isoform of SDHC following GATA-1 S silencing. All data represent the mean ± SD of three independent experiments. Statistical analysis was performed by one-way ANOVA, followed by Dunnett’s multiple comparisons test, where appropriate. Differences were considered significant when p < 0.05 and highly significant when p < 0.0001. * p < 0.05, ** p < 0.0001 versus negative control; # p < 0.05 versus lower dose of siRNA transfection.

Article Snippet: The human K562 erythroleukemia cell line obtained from European Collection of Authenticated Cell Cultures (EACC #89122407) was grown in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS) plus 4 mM glutamine, 10 U/mL penicillin, and 10 mg/mL streptomycin (purchased from Gibco, Thermo Fisher Scientific, Inc., Waltham, MA, USA) at 37 °C in a humidified 5% CO 2 − containing atmosphere.

Techniques: Knockdown, Western Blot, SDS Page, Transfection, Small Interfering RNA, Concentration Assay, Real-time Polymerase Chain Reaction, Expressing, Negative Control, Amplification

Journal: Antioxidants

Article Title: Exploring the Leukemogenic Potential of GATA-1 S , the Shorter Isoform of GATA-1: Novel Insights into Mechanisms Hampering Respiratory Chain Complex II Activity and Limiting Oxidative Phosphorylation Efficiency

doi: 10.3390/antiox10101603

Figure Lengend Snippet: Enzymatic activity of succinate CoQ oxidoreductase (SQR) in K562 cells over expressing GATA-1 isoforms. ( a ) SQR activity detected on total cell lysates is expressed as OD absorbance/min/mg total protein. Data represent mean ± SD from three independent experiments. Differences were considered significant when * p < 0.05 and highly significant when ** p < 0.0001 versus mock control; ( b , c ) Schematic representation of complex II disassembly induced by over-expression of the SDHC Δ5 variant lacking the heme binding site with impaired SQR activity and increased O 2 − production . (Created with BioRender.com, accessed on 6 August 2021).

Article Snippet: The human K562 erythroleukemia cell line obtained from European Collection of Authenticated Cell Cultures (EACC #89122407) was grown in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS) plus 4 mM glutamine, 10 U/mL penicillin, and 10 mg/mL streptomycin (purchased from Gibco, Thermo Fisher Scientific, Inc., Waltham, MA, USA) at 37 °C in a humidified 5% CO 2 − containing atmosphere.

Techniques: Activity Assay, Expressing, Control, Over Expression, Variant Assay, Binding Assay

Journal: Antioxidants

Article Title: Exploring the Leukemogenic Potential of GATA-1 S , the Shorter Isoform of GATA-1: Novel Insights into Mechanisms Hampering Respiratory Chain Complex II Activity and Limiting Oxidative Phosphorylation Efficiency

doi: 10.3390/antiox10101603

Figure Lengend Snippet: ( a – d ). Mitochondrial respiration rates measured by a Seahorse XFe assay in K562 cells overexpressing GATA-1 isoforms. ( a ) Schematic description of the experimental procedure; ( b , c ) variations in extracellular acidification (ECAR) and oxygen consumption rate (OCR, pmol/min/ng/mL) that were found increased in cells over-expressing GATA-1 S and decreased in response to GATA-1 FL over-expression as compared to the mock control. ( d ) Relationship between ECAR and OCR in intact cells under basal conditions. Data are presented as mean ± SEM. ( e – i ). Mitochondrial respiration rates measured by a Seahorse XFe assay in K562 cells overexpressing GATA-1 isoforms. Evaluation of basal ( e ) and maximal ( f ) respiration rates, proton leak ( g ), ATP production ( h ) and spare respiratory capacity ( i ) showing that GATA-1 FL over-expression is accompanied by reduced proton leak and ATP production. Conversely, over-expression of GATA-1 S is associated with higher respiration rate, enhanced proton leak, ATP production and spare respiratory capacity. These data are suggestive of GATA-1 S cells mainly being dependent on mitochondrial oxidative processes with respect to the full-length isoform. Data represent mean ± SD from three independent experiments. Differences were considered significant when p < 0.05 and highly significant when p < 0.0001. # p < 0.05, ## p < 0.0001 GATA-1 FL versus GATA-1 S , * p < 0.05, ** p < 0.0001 versus mock control.

Article Snippet: The human K562 erythroleukemia cell line obtained from European Collection of Authenticated Cell Cultures (EACC #89122407) was grown in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS) plus 4 mM glutamine, 10 U/mL penicillin, and 10 mg/mL streptomycin (purchased from Gibco, Thermo Fisher Scientific, Inc., Waltham, MA, USA) at 37 °C in a humidified 5% CO 2 − containing atmosphere.

Techniques: Expressing, Over Expression, Control

Journal: Biochemistry and Biophysics Reports

Article Title: Tetracyclines downregulate the production of LPS-induced cytokines and chemokines in THP-1 cells via ERK, p38, and nuclear factor-κB signaling pathways

doi: 10.1016/j.bbrep.2015.11.003

Figure Lengend Snippet: (A) Time-dependent changes in cytokine and chemokine production in LPS-stimulated THP-1 cells. After LPS (10 μg/ml) treatment for 15, 30, 60, or 120 min without any agents and with minocycline (50 μg/ml), tigecycline (50 μg/ml), or doxycycline (50 μg/ml), cytokines and chemokines were measured using Multi Plex according to the manufacturer’s protocols. * p <0.05 compared with LPS only at 60 min. ** p <0.05 compared with LPS only at 120 min. Mino: minocycline, Tige: tigecycline, Doxy: doxycycline. (B) The rate of cytokine and chemokine production in the THP-1 cell line compared to the production of cytokines and chemokines by LPS stimulation without tetracyclines. After LPS treatment (10 μg/ml) for 30, 60, 120 or 240 min without any agents and with minocycline (50 μg/ml), tigecycline (50 μg/ml), or doxycycline (50 mg/ml), cytokines and chemokines were measured with Multi Plex.

Article Snippet: The THP-1 human monocytic leukemia cell line was purchased from RIKEN Cell Bank (Wako, Japan).

Techniques:

Journal: Biochemistry and Biophysics Reports

Article Title: Tetracyclines downregulate the production of LPS-induced cytokines and chemokines in THP-1 cells via ERK, p38, and nuclear factor-κB signaling pathways

doi: 10.1016/j.bbrep.2015.11.003

Figure Lengend Snippet: Effects of minocycline, doxycycline, and tigecycline on the modulation of NF-κB, phospho-NF-κB, IKKα/β, phospho-IKKα/β, IκBα, and phospho-IκBα in LPS-stimulated THP-1 cells. THP-1 cells were incubated without or with 10 μg/ml LPS, or with LPS plus minocycline (50 μg/ml), doxycycline (50 μg/ml), or tigecycline (50 μg/ml) for 30, 60, or 120 min. NF-κB, phospho-NF-κB, IKKα/β, phospho-IKKα/β, IκBα, and phospho-IκBα were assessed with Western blotting.

Article Snippet: The THP-1 human monocytic leukemia cell line was purchased from RIKEN Cell Bank (Wako, Japan).

Techniques: Incubation, Western Blot

Journal: Biochemistry and Biophysics Reports

Article Title: Tetracyclines downregulate the production of LPS-induced cytokines and chemokines in THP-1 cells via ERK, p38, and nuclear factor-κB signaling pathways

doi: 10.1016/j.bbrep.2015.11.003

Figure Lengend Snippet: Effects of tetracyclines (minocycline, doxycycline, and tigecycline) on the activation of phospho-ERK1/2 and phospho-p38 in LPS-stimulated THP-1 cells. THP-1 cells were incubated without or with 10 μg/ml LPS, or with LPS plus minocycline (50 μg/ml), doxycycline (50 μg/ml), or tigecycline (50 μg/ml) for 30 or 60 min. Phospho-p38 and phospho-ERK1/2 were assessed with Western blotting.

Article Snippet: The THP-1 human monocytic leukemia cell line was purchased from RIKEN Cell Bank (Wako, Japan).

Techniques: Activation Assay, Incubation, Western Blot

Journal: Biochemistry and Biophysics Reports

Article Title: Tetracyclines downregulate the production of LPS-induced cytokines and chemokines in THP-1 cells via ERK, p38, and nuclear factor-κB signaling pathways

doi: 10.1016/j.bbrep.2015.11.003

Figure Lengend Snippet: SB203580, U0126 and BAY11-7082 suppressed TNF-α and IL-8 production in LPS-stimulated THP-1 cells on treatment with or without tetracyclines. THP-1 cells were pre-incubated by SB203580 (10 μM), U0126 (5 μM) and BAY11-7082 (5 μM) for 30 min, followed treatment without or with LPS (10 μg/ml), or with LPS (10 μg/ml) plus minocycline (50 μg/ml), doxycycline (50 μg/ml), or tigecycline (50 μg/ml) for 60 min. TNF-α were measured with ELISA. * p <0.05, ** p <0.01, *** p <0.001 compared to the measurement without the inhibitor in the same group. Abbreviation; SB: SB203580, U: U0126, BAY: BAY11-7082.

Article Snippet: The THP-1 human monocytic leukemia cell line was purchased from RIKEN Cell Bank (Wako, Japan).

Techniques: Incubation, Enzyme-linked Immunosorbent Assay

Journal: Cancer discovery

Article Title: A gain-of-function p53 mutant oncogene promotes cell fate plasticity and myeloid leukemia through the pluripotency factor Foxh1

doi: 10.1158/2159-8290.CD-18-1391

Figure Lengend Snippet: (A) The ratio of sh.p53 BFP+ cells compared to sh.Ren BFP+ cells over time in leukemic lines. (B) CD11b expression in the shRNA BFP+ containing cells (n = 3) and (C) Annexin V shown by FACS 7 days after infection. Data presented as mean ± s.e.m (D) Relative expression of TP53 7 days post infection with pRS sh.TP53 in human KY821 cells. (E) CFU in human AML cells with sh.Ren or sh.TP53. Data presented as mean ± s.e.m (n = 3) (F) hCD16 expression by FACS in pRS Ctrl or pRS TP53 cells from growing in CFU assay (G) Percentage of hCD45 in the peripheral blood of mice 5 weeks post transplant in shCntr and shCntr moribund mice (black), shTP53 (blue) and shTP53 moribund mice (orange). Data are represented as mean ± s.e.m (n=5 and n=5) (H) Kaplan-Meier curve from NSG mice transplanted with equal numbers of KY821 cells transduced with wither shCntr or shTP53 (n=5 and n=5). (I) Western blot for human TP53 in cells before injection and in sorted hCD45 cells from the peripheral blood of 3 independent moribund shTP53 mice. *p <0.05, **p <0.005 ***p <0.0005. B. Kruskal-Wallis 1-way ANOVA. C, D, F, J, unpaired t-test.

Article Snippet: Cell lines The human leukemia line KY821 (JCRB cell bank) was grown in RPMI 1640 and 20% FBS (Gibco).

Techniques: Expressing, shRNA, Infection, Colony-forming Unit Assay, Transduction, Western Blot, Injection