Journal: eLife

Article Title: TGF-β uses a novel mode of receptor activation to phosphorylate SMAD1/5 and induce epithelial-to-mesenchymal transition

doi: 10.7554/eLife.31756

Figure Lengend Snippet: ( A ) In vitro kinase assays using the kinase domains of ACVR1, BMPR1A, and TGFBR1 at 200, 100, 50, 25 ng with recombinant SMAD1 (S1) or SMAD2 (S2) as substrates. Top panels, autoradiograph; bottom panels, Coomassie-stained gel. ( B ) Incorporation of 32 P into SMAD1 and SMAD2 catalyzed by ACVR1 and TGFBR1 using different specific activities of [γ− 32 P]-ATP. A constant amount of [γ− 32 P]-ATP was added into the kinase reaction with either 200 or 50 µM cold ATP. Top panels, autoradiograph; bottom panels, Coomassie-stained gel. Numbers underneath indicate the fold changes relative to the 32 P incorporation in SMAD1 (upper) or SMAD2 (lower) catalyzed by TGFBR1 using 200 µM cold ATP. The phosphorylation of SMAD1 and 2 by ACVR1 and TGFBR1 was dependent on the specific activity of the [γ− 32 P]-ATP, whilst the apparent phosphorylation of SMAD1 by TGFBR1 is not, suggesting that it is non-specific. ( C ) Mapping ACVR1 phosphorylation sites on SMAD1. Full length SMAD1 phosphorylated by ACVR1 was digested with trypsin. Peptides were resolved by reverse phase HPLC (left panel). The C-terminal peptide of SMAD1 existed in three different phosphorylation states (peptides a, b , and c ); the three subsequent peaks are tryptic miscleavage products. The phosphorylation sites in the peptides were mapped using solid phase Edman sequencing (panels labeled a , b and c ). The deduced phosphorylation sites in the SSVS motif in the individual peptides are shown in red.

Article Snippet: Recombinant intracellular domains of ACVR1, BMPR1A and TGFBR1 which were expressed in insect cells were purchased from Carna Biosciences Inc (Japan; see Key Resources Table).

Techniques: In Vitro, Recombinant, Autoradiography, Staining, Phospho-proteomics, Activity Assay, Sequencing, Labeling

Journal: eLife

Article Title: TGF-β uses a novel mode of receptor activation to phosphorylate SMAD1/5 and induce epithelial-to-mesenchymal transition

doi: 10.7554/eLife.31756

Figure Lengend Snippet: ( A ) The kinase domains of TGFBR1 and ACVR1 were analyzed alone or together in an in vitro kinase reaction. SB-505124 and LDN-193189 were included as shown to inhibit the activity of TGFBR1 and ACVR1, respectively. The autoradiograph is shown in the top panel, with the Coomassie-stained gel below as a loading control. ( B ) Schematic to show the domain organization of the Opto receptors. In Opto-TGFBR1* and Opto-ACVR1, the kinase domains of TGFBR1 and ACVR1 are fused to the light-sensitive LOV domain. At the N-terminus there is a myristylation domain (indicated by the red zig zag). At the C-terminus there is an HA tag. The kinase domain of TGFBR1 contains the activating mutation T204D. These Opto receptors dimerize in the presence of blue light. ( C ) NIH-3T3 cells were untransfected or transfected with FLAG-SMAD1 together with either Opto-TGFBR1*, Opto-ACVR1 or both receptors together. Post-transfection, cells were either kept in the dark or exposed to blue light for 1 hr. Whole cell extracts were western blotted using antibodies against pSMAD1/5 (which detects endogenous and FLAG-tagged pSMAD1/5), SMAD1 (which detects endogenous and FLAG-SMAD1), HA (to detect the Opto receptors) and Tubulin as a loading control. ( D ) NIH-3T3 cells were untransfected or transfected with FLAG-SMAD1 together with either Opto-TGFBR1*, Opto-ACVR1 or both receptors together. Post-transfection, cells were either kept in the dark or exposed to blue light for 1 hr. The inductions were performed in the absence or presence of 0.5 µM LDN-193189 or 50 µM SB-505124 as indicated. Whole cell extracts were blotted as in ( C ). ( E ) The experimental set up was as in ( D ) except that GFP-SMAD3 was used instead of FLAG-SMAD1 to assess the activity of Opto-TGFBR1*. ( F ) As in ( C ), except that an ACVR1 mutant in which all the threonines and serines of the GS domain were mutated to valine or alanine respectively, was also assayed. ( G ) As in ( F ), except that GFP-SMAD3 was used instead of FLAG-SMAD1. Note that in all cases the 1 hr induction with blue light led to reduced levels of the transfected receptors and substrates.

Article Snippet: Recombinant intracellular domains of ACVR1, BMPR1A and TGFBR1 which were expressed in insect cells were purchased from Carna Biosciences Inc (Japan; see Key Resources Table).

Techniques: In Vitro, Activity Assay, Autoradiography, Staining, Control, Mutagenesis, Transfection, Western Blot

Journal: eLife

Article Title: TGF-β uses a novel mode of receptor activation to phosphorylate SMAD1/5 and induce epithelial-to-mesenchymal transition

doi: 10.7554/eLife.31756

Figure Lengend Snippet: ( A ) NIH-3T3 cells were untransfected or transfected with FLAG-SMAD1 together with either Opto-TGFBR1*, Opto-ACVR1 or both receptors together, or a kinase-dead version of Opto-TGFBR1 (Opto-TGFBR1-KR) alone or together with Opto-ACVR1. Post transfection, cells were either kept in the dark or exposed to blue light for 1 hr. Whole cell extracts were western blotted using antibodies against pSMAD1/5 (which detects endogenous and FLAG-tagged pSMAD1/5), SMAD1 (which detects endogenous and FLAG-SMAD1), HA (to detect the Opto receptors) and Tubulin as a loading control. ( B ) The experimental set up was as in ( A ) except that GFP-SMAD3 was used instead of FLAG-SMAD1 to assess the activity of Opto-TGFBR1* and Opto-TGFBR1-KR. The band marked with an asterisk is a background band.

Article Snippet: Recombinant intracellular domains of ACVR1, BMPR1A and TGFBR1 which were expressed in insect cells were purchased from Carna Biosciences Inc (Japan; see Key Resources Table).

Techniques: Transfection, Western Blot, Control, Activity Assay

Journal: eLife

Article Title: TGF-β uses a novel mode of receptor activation to phosphorylate SMAD1/5 and induce epithelial-to-mesenchymal transition

doi: 10.7554/eLife.31756

Figure Lengend Snippet: ( A ) NMuMG cells were transfected with siRNAs against ID1 or NT control, then left untreated or treated with TGF-β for 24 hr. Cells were imaged after indirect IF with antibodies against TJP1 and CDH1 or a merge of the two with DAPI in blue. All indirect IF images are maximum intensity projections of a z-stack in each channel. ( B ) Western blots to show knockdown efficiency of the ID1 siRNA. NMuMG cells were treated with TGF-β (T) or BMP4 (B) for 1 hr. ( C ) The model shows combinatorial signaling by TGF-β utilizing complexes containing two different type I receptors. Type II receptors are shown in blue, TGFBR1 in orange and ACVR1 in green as in . P denotes phosphorylation. S1/5, SMAD1/5; S2/3, SMAD2/3; S4, SMAD4. The question mark indicates that we do not yet know the function of the mixed R-SMAD complexes in the physiological responses. For discussion, see text.

Article Snippet: Recombinant intracellular domains of ACVR1, BMPR1A and TGFBR1 which were expressed in insect cells were purchased from Carna Biosciences Inc (Japan; see Key Resources Table).

Techniques: Transfection, Control, Western Blot, Knockdown, Phospho-proteomics

Journal: The Journal of Clinical Investigation

Article Title: ALK-transformed mature T lymphocytes restore early thymus progenitor features

doi: 10.1172/JCI134990

Figure Lengend Snippet: (A) Expression by qRT-PCR analysis of NPM-ALK mRNA in the transformed CD4+ T cells (CD4-NPM/ALK+ lane shows the mean from 9 independent cell lines) and 3 positive control NPM-ALK+ ALCL cell lines: KARPAS-299, SU-DHL-1, and COST.CD4+ T cells preactivated with CD3/CD28 antibody-coated beads were used as negative controls (preactivated CD4). MLNS1 was used as an internal control. Relative NPM-ALK expression was expressed as the 2–ΔCt relative to MLN51. Data represent mean ± SEM. *P < 0.05, **P < 0.001, ***P < 0.001; unpaired 2-tailed Student’s t test with Welch’s correction. (B) Suppressive effect of the ALK inhibitor crizotinib (500 nmol/L) on ALK and STAT3 phosphorylation in transformed CD4+ T cells and control NPM-ALK+ KARPAS-299 cells. The GAPDH protein served as an internal control to ensure equal loading. Blots from 1 representative experiment are shown.

Article Snippet: The human NPM-ALK + ALCL cell line COST was established in the laboratory ( 40 ) and KARPAS-299 and SU-DHL-1 NPM-ALK + ALCL cell lines were obtained from DSMZ (German Collection of Microorganisms and Cell Culture, Braunschweig, Germany).

Techniques: Expressing, Quantitative RT-PCR, Transformation Assay, Positive Control, Control, Phospho-proteomics

Journal: The Journal of Clinical Investigation

Article Title: ALK-transformed mature T lymphocytes restore early thymus progenitor features

doi: 10.1172/JCI134990

Figure Lengend Snippet: (A) We used publicly available methylation data sets (30) generated from different developmental T cell stages (multipotent ETPs [CD34+/CD1a–; n = 2]; T cell–committed progenitors [CD34+/CD1a+; n = 1]; pre-TCR T cells [n = 2]; TCR-expressing CD4+/CD8+ double-positive T cells [DP-TCR+, n = 2]; and single positive [SP] CD8+ or CD4+ cells [SP-CD4+; n = 2 or SP-CD8+; n = 2]) to identify a cluster of 510 DMRs available to discriminate each different stage of T cell differentiation in the thymus. (B) Hierarchical clustering dendrogram using a cluster of 510 DMRs revealed that NPM-ALK–transformed CD4+ T cells were distant to the healthy CD4+ lymphocyte profile and clustered with primary NPM-ALK+ ALCL biopsies. Heatmaps also showed a similarity of NPM-ALK+ cells (NPM-ALK–transformed CD4+ T cells and primary patient–derived NPM-ALK+ ALCL) with CD34+/CD1a– cells corresponding to the ETP stage.

Article Snippet: The human NPM-ALK + ALCL cell line COST was established in the laboratory ( 40 ) and KARPAS-299 and SU-DHL-1 NPM-ALK + ALCL cell lines were obtained from DSMZ (German Collection of Microorganisms and Cell Culture, Braunschweig, Germany).

Techniques: Methylation, Generated, Expressing, Cell Differentiation, Transformation Assay, Derivative Assay

Journal: The Journal of Clinical Investigation

Article Title: ALK-transformed mature T lymphocytes restore early thymus progenitor features

doi: 10.1172/JCI134990

Figure Lengend Snippet: Two hundred and forty-three among the 510 DMRs within NPM-ALK–transformed CD4+ T cells (CD4+/NPM-ALK+), primary patient–derived NPM-ALK+ ALCL cells, and CD34+/CD1a– cells corresponding to the ETP stage. Venn diagram reveals that the ETP and both the NPM-ALK+ tumor cell entities (CD4+/NPM-ALK+ lymphoma cells and primary NPM-ALK+ ALCLs) share 38 DMRs with similar expression patterns.

Article Snippet: The human NPM-ALK + ALCL cell line COST was established in the laboratory ( 40 ) and KARPAS-299 and SU-DHL-1 NPM-ALK + ALCL cell lines were obtained from DSMZ (German Collection of Microorganisms and Cell Culture, Braunschweig, Germany).

Techniques: Transformation Assay, Derivative Assay, Expressing

Journal: The Journal of Clinical Investigation

Article Title: ALK-transformed mature T lymphocytes restore early thymus progenitor features

doi: 10.1172/JCI134990

Figure Lengend Snippet: mRNA expression profiles from several cell populations isolated ex vivo from the neonatal human thymus defining in vivo maturation stages, multipotent ETPs (CD34+/CD1a–/CD7–; n = 3), late thymic precursor (CD34+/CD1a–/CD7+; n = 3), T cell–committed progenitors (CD34+/CD1a–/CD7+; n = 3), CD3–/CD4+ immature single-positive (ISP) (ISP-CD4+, n = 4), CD4+/CD8+ double-positive TCR– cells (DP-TCR–; n = 3), and TCR-expressing CD4+/CD8+ double-positive T cells (DP-TCR+, n = 3) were integrated with our previous findings from the gene expression array data of 55 primary NPM-ALK+ ALCL samples (NPM-ALK+ ALCL) (35) and RNA-Seq data from the NPM-ALK–transformed CD4+ T cells (CD4+/NPM-ALK+; n = 9). NPM-ALK CD4+/NPM-ALK+ cells were distant to the healthy CD4+ lymphocyte but close to NPM-ALK+ ALCL. Moreover, NPM-ALK+ cells (NPM-ALK–transformed CD4+ T cells and primary patient–derived NPM-ALK+ ALCL) showed a similarity with the ETP stage.

Article Snippet: The human NPM-ALK + ALCL cell line COST was established in the laboratory ( 40 ) and KARPAS-299 and SU-DHL-1 NPM-ALK + ALCL cell lines were obtained from DSMZ (German Collection of Microorganisms and Cell Culture, Braunschweig, Germany).

Techniques: Expressing, Isolation, Ex Vivo, In Vivo, Gene Expression, RNA Sequencing, Transformation Assay, Derivative Assay

Journal: The Journal of Clinical Investigation

Article Title: ALK-transformed mature T lymphocytes restore early thymus progenitor features

doi: 10.1172/JCI134990

Figure Lengend Snippet: (A) Quantitative RT-PCR analysis of ALK and EPAS1 mRNA was performed in primary patient–derived NPM-ALK+ ALCL cells (n = 29). Relative mRNA expression was expressed as the 2–ΔΔCt relative to MLN51, S5, ABL, GAPDH, S14, or RPL0 genes for normalization and compared with preactivated healthy CD4+ lymphocytes (n = 5). Data represent mean ± SEM. ***P < 0.001; unpaired 2-tailed Student’s t test. (B) Quantitative RT-PCR analysis of EPAS1 mRNA expression in NPM-ALK+ lymphoma cell lines, COST, KARPAS-299 (KARPAS), and SU-DHL1, treated for 72 hours or not (PBS) with crizotinib or transfected with either an irrelevant siRNA as the negative control (si-CTL) or a siRNA targeting ALK mRNA (si-ALK) or STAT3 (si-STAT3). Relative EPAS1 mRNA expression was expressed as the 2–ΔCt relative to MLN51. Data represent mean ± SEM from 3 independent experiments. *P < 0.05, ***P < 0.001; unpaired 2-tailed Student’s t test with Welch’s correction. (C) Western blotting analysis of HIF2A expression (top) in NPM-ALK+ COST, KARPAS-299, and SU-DHL1 cells treated with crizotinib (crizo) or not (PBS), transfected by si-CTL, si-ALK, or si-STAT3. The GAPDH protein (bottom) served as an internal control to ensure equal loading. Results from 1 representative experiment are shown.

Article Snippet: The human NPM-ALK + ALCL cell line COST was established in the laboratory ( 40 ) and KARPAS-299 and SU-DHL-1 NPM-ALK + ALCL cell lines were obtained from DSMZ (German Collection of Microorganisms and Cell Culture, Braunschweig, Germany).

Techniques: Quantitative RT-PCR, Derivative Assay, Expressing, Transfection, Negative Control, Western Blot, Control

Journal: The Journal of Clinical Investigation

Article Title: ALK-transformed mature T lymphocytes restore early thymus progenitor features

doi: 10.1172/JCI134990

Figure Lengend Snippet: (A) Expression by qRT-PCR analysis of NPM-ALK mRNA in the transformed CD4+ T cells (CD4-NPM/ALK+ lane shows the mean from 9 independent cell lines) and 3 positive control NPM-ALK+ ALCL cell lines: KARPAS-299, SU-DHL-1, and COST.CD4+ T cells preactivated with CD3/CD28 antibody-coated beads were used as negative controls (preactivated CD4). MLNS1 was used as an internal control. Relative NPM-ALK expression was expressed as the 2–ΔCt relative to MLN51. Data represent mean ± SEM. *P < 0.05, **P < 0.001, ***P < 0.001; unpaired 2-tailed Student’s t test with Welch’s correction. (B) Suppressive effect of the ALK inhibitor crizotinib (500 nmol/L) on ALK and STAT3 phosphorylation in transformed CD4+ T cells and control NPM-ALK+ KARPAS-299 cells. The GAPDH protein served as an internal control to ensure equal loading. Blots from 1 representative experiment are shown.

Article Snippet: Immunodetections were performed with antibodies directed against NPM-ALK at 1/5000 (Cell Signaling, catalog 3633), phospho-ALK at 1/1000 (Cell Signaling, catalog CS14678S), STAT3 at 1/1000 (Cell Signaling, catalog CS9132), P-STAT3 at 1/1000 (Cell Signaling, catalog CS9131), HIF2α at 1/1000 (Bethyl, catalog MAB374), NANOG at 1/1000 (Cell Signaling, catalog CST 4893S; mouse mAb 1E6C4), SOX2 at 1/1000 (Cell Signaling, catalog CST 4900S; mouse mAb L1D6A2), OCT4 at 1/500 (StemGent, catalog 09-0023 rabbit polyclonal), β-tubulin at 1/5000 (MilliporeSigma, catalog T4026; mouse mAb TUB 2.1), or GAPDH at 1/10,000 (Millipore, catalog MAB374).

Techniques: Expressing, Quantitative RT-PCR, Transformation Assay, Positive Control, Control, Phospho-proteomics

Journal: The Journal of Clinical Investigation

Article Title: ALK-transformed mature T lymphocytes restore early thymus progenitor features

doi: 10.1172/JCI134990

Figure Lengend Snippet: Normal CD4+ T cells prestimulated with CD3/CD28 antibody-coated beads were transduced with NPM-ALK and 40 days later flow cytometry analysis was performed to detect expression of T cell markers stained with an anti-ALK, -CD3, -CD4, -CD30, and -TCRαβ antibodies (NPM-ALK+ CD4+ T cells in green and prestimulated healthy CD4+ T cells in red) or IgG as control (blue). Preactivated human healthy CD4+ T cells were used as controls. Data are representative of the mean ± SEM from the 9 independent cell lines.

Article Snippet: Immunodetections were performed with antibodies directed against NPM-ALK at 1/5000 (Cell Signaling, catalog 3633), phospho-ALK at 1/1000 (Cell Signaling, catalog CS14678S), STAT3 at 1/1000 (Cell Signaling, catalog CS9132), P-STAT3 at 1/1000 (Cell Signaling, catalog CS9131), HIF2α at 1/1000 (Bethyl, catalog MAB374), NANOG at 1/1000 (Cell Signaling, catalog CST 4893S; mouse mAb 1E6C4), SOX2 at 1/1000 (Cell Signaling, catalog CST 4900S; mouse mAb L1D6A2), OCT4 at 1/500 (StemGent, catalog 09-0023 rabbit polyclonal), β-tubulin at 1/5000 (MilliporeSigma, catalog T4026; mouse mAb TUB 2.1), or GAPDH at 1/10,000 (Millipore, catalog MAB374).

Techniques: Transduction, Flow Cytometry, Expressing, Staining, Control

Journal: The Journal of Clinical Investigation

Article Title: ALK-transformed mature T lymphocytes restore early thymus progenitor features

doi: 10.1172/JCI134990

Figure Lengend Snippet: (A) NPM-ALK–transformed CD4+ T cells were injected subcutaneously into the left or right flank of NSG mice (n = 2). Representative image of tumor-bearing mice. (B) Tumor volume was evaluated over time by caliper measurements and reported as mean ± SEM (line). (C) Representative images of H&E staining and immunohistochemical staining for expression of NPM-ALK, CD3, CD4, and CD30 (original magnification ×20 or ×40; inset ×80).

Article Snippet: Immunodetections were performed with antibodies directed against NPM-ALK at 1/5000 (Cell Signaling, catalog 3633), phospho-ALK at 1/1000 (Cell Signaling, catalog CS14678S), STAT3 at 1/1000 (Cell Signaling, catalog CS9132), P-STAT3 at 1/1000 (Cell Signaling, catalog CS9131), HIF2α at 1/1000 (Bethyl, catalog MAB374), NANOG at 1/1000 (Cell Signaling, catalog CST 4893S; mouse mAb 1E6C4), SOX2 at 1/1000 (Cell Signaling, catalog CST 4900S; mouse mAb L1D6A2), OCT4 at 1/500 (StemGent, catalog 09-0023 rabbit polyclonal), β-tubulin at 1/5000 (MilliporeSigma, catalog T4026; mouse mAb TUB 2.1), or GAPDH at 1/10,000 (Millipore, catalog MAB374).

Techniques: Transformation Assay, Injection, Staining, Immunohistochemical staining, Expressing

Journal: The Journal of Clinical Investigation

Article Title: ALK-transformed mature T lymphocytes restore early thymus progenitor features

doi: 10.1172/JCI134990

Figure Lengend Snippet: Experimental metastasis assays were performed in NSG mice using NPM-ALK–transformed CD4+ T cells (n = 7) or PBS (n = 3) with injection through the mouse tail vein. (A) Representative image of cutaneous metastasis-bearing mice. Metastatic burden was assessed at 39 days after injection. (B–I) Representative H&E images. Mice presented skin nodules without dermis and subcutis hyperplasia (A–C) and spleen hyperplasia (D). A lymphomatous infiltration invaded the liver (E) and spleen (F). The neoplastic cells included a predominant population of small- to medium-sized neoplastic cells with irregular nuclei (G). Many cells are fried-egg cells with pale cytoplasms and centrally located nuclei (H, arrowheads) and cells with ring-like nuclei (I, arrowheads). Histological analysis with anti-ALK antibody showed a strong ALK staining in the large lymphoma cells as compared with the small variants (J and K). Small cells are often localized around blood vessels (L). Original magnifications ×5, ×20, or ×40).

Article Snippet: Immunodetections were performed with antibodies directed against NPM-ALK at 1/5000 (Cell Signaling, catalog 3633), phospho-ALK at 1/1000 (Cell Signaling, catalog CS14678S), STAT3 at 1/1000 (Cell Signaling, catalog CS9132), P-STAT3 at 1/1000 (Cell Signaling, catalog CS9131), HIF2α at 1/1000 (Bethyl, catalog MAB374), NANOG at 1/1000 (Cell Signaling, catalog CST 4893S; mouse mAb 1E6C4), SOX2 at 1/1000 (Cell Signaling, catalog CST 4900S; mouse mAb L1D6A2), OCT4 at 1/500 (StemGent, catalog 09-0023 rabbit polyclonal), β-tubulin at 1/5000 (MilliporeSigma, catalog T4026; mouse mAb TUB 2.1), or GAPDH at 1/10,000 (Millipore, catalog MAB374).

Techniques: Transformation Assay, Injection, Staining

Journal: The Journal of Clinical Investigation

Article Title: ALK-transformed mature T lymphocytes restore early thymus progenitor features

doi: 10.1172/JCI134990

Figure Lengend Snippet: (A) We used publicly available methylation data sets (30) generated from different developmental T cell stages (multipotent ETPs [CD34+/CD1a–; n = 2]; T cell–committed progenitors [CD34+/CD1a+; n = 1]; pre-TCR T cells [n = 2]; TCR-expressing CD4+/CD8+ double-positive T cells [DP-TCR+, n = 2]; and single positive [SP] CD8+ or CD4+ cells [SP-CD4+; n = 2 or SP-CD8+; n = 2]) to identify a cluster of 510 DMRs available to discriminate each different stage of T cell differentiation in the thymus. (B) Hierarchical clustering dendrogram using a cluster of 510 DMRs revealed that NPM-ALK–transformed CD4+ T cells were distant to the healthy CD4+ lymphocyte profile and clustered with primary NPM-ALK+ ALCL biopsies. Heatmaps also showed a similarity of NPM-ALK+ cells (NPM-ALK–transformed CD4+ T cells and primary patient–derived NPM-ALK+ ALCL) with CD34+/CD1a– cells corresponding to the ETP stage.

Article Snippet: Immunodetections were performed with antibodies directed against NPM-ALK at 1/5000 (Cell Signaling, catalog 3633), phospho-ALK at 1/1000 (Cell Signaling, catalog CS14678S), STAT3 at 1/1000 (Cell Signaling, catalog CS9132), P-STAT3 at 1/1000 (Cell Signaling, catalog CS9131), HIF2α at 1/1000 (Bethyl, catalog MAB374), NANOG at 1/1000 (Cell Signaling, catalog CST 4893S; mouse mAb 1E6C4), SOX2 at 1/1000 (Cell Signaling, catalog CST 4900S; mouse mAb L1D6A2), OCT4 at 1/500 (StemGent, catalog 09-0023 rabbit polyclonal), β-tubulin at 1/5000 (MilliporeSigma, catalog T4026; mouse mAb TUB 2.1), or GAPDH at 1/10,000 (Millipore, catalog MAB374).

Techniques: Methylation, Generated, Expressing, Cell Differentiation, Transformation Assay, Derivative Assay

Journal: The Journal of Clinical Investigation

Article Title: ALK-transformed mature T lymphocytes restore early thymus progenitor features

doi: 10.1172/JCI134990

Figure Lengend Snippet: Two hundred and forty-three among the 510 DMRs within NPM-ALK–transformed CD4+ T cells (CD4+/NPM-ALK+), primary patient–derived NPM-ALK+ ALCL cells, and CD34+/CD1a– cells corresponding to the ETP stage. Venn diagram reveals that the ETP and both the NPM-ALK+ tumor cell entities (CD4+/NPM-ALK+ lymphoma cells and primary NPM-ALK+ ALCLs) share 38 DMRs with similar expression patterns.

Article Snippet: Immunodetections were performed with antibodies directed against NPM-ALK at 1/5000 (Cell Signaling, catalog 3633), phospho-ALK at 1/1000 (Cell Signaling, catalog CS14678S), STAT3 at 1/1000 (Cell Signaling, catalog CS9132), P-STAT3 at 1/1000 (Cell Signaling, catalog CS9131), HIF2α at 1/1000 (Bethyl, catalog MAB374), NANOG at 1/1000 (Cell Signaling, catalog CST 4893S; mouse mAb 1E6C4), SOX2 at 1/1000 (Cell Signaling, catalog CST 4900S; mouse mAb L1D6A2), OCT4 at 1/500 (StemGent, catalog 09-0023 rabbit polyclonal), β-tubulin at 1/5000 (MilliporeSigma, catalog T4026; mouse mAb TUB 2.1), or GAPDH at 1/10,000 (Millipore, catalog MAB374).

Techniques: Transformation Assay, Derivative Assay, Expressing

Journal: The Journal of Clinical Investigation

Article Title: ALK-transformed mature T lymphocytes restore early thymus progenitor features

doi: 10.1172/JCI134990

Figure Lengend Snippet: mRNA expression profiles from several cell populations isolated ex vivo from the neonatal human thymus defining in vivo maturation stages, multipotent ETPs (CD34+/CD1a–/CD7–; n = 3), late thymic precursor (CD34+/CD1a–/CD7+; n = 3), T cell–committed progenitors (CD34+/CD1a–/CD7+; n = 3), CD3–/CD4+ immature single-positive (ISP) (ISP-CD4+, n = 4), CD4+/CD8+ double-positive TCR– cells (DP-TCR–; n = 3), and TCR-expressing CD4+/CD8+ double-positive T cells (DP-TCR+, n = 3) were integrated with our previous findings from the gene expression array data of 55 primary NPM-ALK+ ALCL samples (NPM-ALK+ ALCL) (35) and RNA-Seq data from the NPM-ALK–transformed CD4+ T cells (CD4+/NPM-ALK+; n = 9). NPM-ALK CD4+/NPM-ALK+ cells were distant to the healthy CD4+ lymphocyte but close to NPM-ALK+ ALCL. Moreover, NPM-ALK+ cells (NPM-ALK–transformed CD4+ T cells and primary patient–derived NPM-ALK+ ALCL) showed a similarity with the ETP stage.

Article Snippet: Immunodetections were performed with antibodies directed against NPM-ALK at 1/5000 (Cell Signaling, catalog 3633), phospho-ALK at 1/1000 (Cell Signaling, catalog CS14678S), STAT3 at 1/1000 (Cell Signaling, catalog CS9132), P-STAT3 at 1/1000 (Cell Signaling, catalog CS9131), HIF2α at 1/1000 (Bethyl, catalog MAB374), NANOG at 1/1000 (Cell Signaling, catalog CST 4893S; mouse mAb 1E6C4), SOX2 at 1/1000 (Cell Signaling, catalog CST 4900S; mouse mAb L1D6A2), OCT4 at 1/500 (StemGent, catalog 09-0023 rabbit polyclonal), β-tubulin at 1/5000 (MilliporeSigma, catalog T4026; mouse mAb TUB 2.1), or GAPDH at 1/10,000 (Millipore, catalog MAB374).

Techniques: Expressing, Isolation, Ex Vivo, In Vivo, Gene Expression, RNA Sequencing, Transformation Assay, Derivative Assay

Journal: The Journal of Clinical Investigation

Article Title: ALK-transformed mature T lymphocytes restore early thymus progenitor features

doi: 10.1172/JCI134990

Figure Lengend Snippet: (A) Quantitative RT-PCR analysis of ALK and EPAS1 mRNA was performed in primary patient–derived NPM-ALK+ ALCL cells (n = 29). Relative mRNA expression was expressed as the 2–ΔΔCt relative to MLN51, S5, ABL, GAPDH, S14, or RPL0 genes for normalization and compared with preactivated healthy CD4+ lymphocytes (n = 5). Data represent mean ± SEM. ***P < 0.001; unpaired 2-tailed Student’s t test. (B) Quantitative RT-PCR analysis of EPAS1 mRNA expression in NPM-ALK+ lymphoma cell lines, COST, KARPAS-299 (KARPAS), and SU-DHL1, treated for 72 hours or not (PBS) with crizotinib or transfected with either an irrelevant siRNA as the negative control (si-CTL) or a siRNA targeting ALK mRNA (si-ALK) or STAT3 (si-STAT3). Relative EPAS1 mRNA expression was expressed as the 2–ΔCt relative to MLN51. Data represent mean ± SEM from 3 independent experiments. *P < 0.05, ***P < 0.001; unpaired 2-tailed Student’s t test with Welch’s correction. (C) Western blotting analysis of HIF2A expression (top) in NPM-ALK+ COST, KARPAS-299, and SU-DHL1 cells treated with crizotinib (crizo) or not (PBS), transfected by si-CTL, si-ALK, or si-STAT3. The GAPDH protein (bottom) served as an internal control to ensure equal loading. Results from 1 representative experiment are shown.

Article Snippet: Immunodetections were performed with antibodies directed against NPM-ALK at 1/5000 (Cell Signaling, catalog 3633), phospho-ALK at 1/1000 (Cell Signaling, catalog CS14678S), STAT3 at 1/1000 (Cell Signaling, catalog CS9132), P-STAT3 at 1/1000 (Cell Signaling, catalog CS9131), HIF2α at 1/1000 (Bethyl, catalog MAB374), NANOG at 1/1000 (Cell Signaling, catalog CST 4893S; mouse mAb 1E6C4), SOX2 at 1/1000 (Cell Signaling, catalog CST 4900S; mouse mAb L1D6A2), OCT4 at 1/500 (StemGent, catalog 09-0023 rabbit polyclonal), β-tubulin at 1/5000 (MilliporeSigma, catalog T4026; mouse mAb TUB 2.1), or GAPDH at 1/10,000 (Millipore, catalog MAB374).

Techniques: Quantitative RT-PCR, Derivative Assay, Expressing, Transfection, Negative Control, Western Blot, Control

Journal: iScience

Article Title: DAB2IP suppresses invadopodia formation through destabilizing ALK by interacting with USP10 in breast cancer

doi: 10.1016/j.isci.2023.107606

Figure Lengend Snippet:

Article Snippet: Mouse anti-ALK , Proteintech , Cat# 60321-1-Ig, RRID: AB_2881432.

Techniques: Control, Virus, Recombinant, Magnetic Beads, CCK-8 Assay, Phospho-proteomics, Ab Array, Mutagenesis, Protease Inhibitor, Bicinchoninic Acid Protein Assay, Expressing, CRISPR, Knock-Out, Software