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

Journal: Journal of Clinical Investigation

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

doi: 10.1172/jci134990

Figure Lengend Snippet: Figure 1. NPM-ALK dependence of the transformed CD4+ T cells. (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: Transformation Assay, Expressing, Quantitative RT-PCR, Positive Control, Control, Phospho-proteomics

Journal: Journal of Clinical Investigation

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

doi: 10.1172/jci134990

Figure Lengend Snippet: Figure 5. DMRs revealed that NPM-ALK–transformed CD4+ T cells and primary NPM-ALK+ ALCL cells have a close similarity with the ETP. (A) We used publicly available methylation data sets (30) generated from different devel- opmental 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-pos- itive 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 similar- ity 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: Transformation Assay, Methylation, Generated, Expressing, Cell Differentiation, Derivative Assay

Journal: Journal of Clinical Investigation

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

doi: 10.1172/jci134990

Figure Lengend Snippet: Figure 6. DMRs of NPM-ALK–transformed CD4+ T cells and primary patient–derived NPM-ALK+ ALCL cells and the ETP. 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: Journal of Clinical Investigation

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

doi: 10.1172/jci134990

Figure Lengend Snippet: Figure 7. Transcriptional pattern links NPM-ALK–transformed CD4+ T cells and primary patient–derived NPM-ALK+ ALCL cells to ETP cells. 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+ imma- ture 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: Transformation Assay, Derivative Assay, Expressing, Isolation, Ex Vivo, In Vivo, Gene Expression, RNA Sequencing

Journal: Journal of Clinical Investigation

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

doi: 10.1172/jci134990

Figure Lengend Snippet: Figure 8. HIF2A, encoded by the EPAS1 gene, is strictly dependent on NPM-ALK activity and activation of the STAT3 key signal transduction pathways in lymphoma cells. (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 pre- activated 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 crizo- tinib 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: Activity Assay, Activation Assay, Transduction, Quantitative RT-PCR, Derivative Assay, Expressing, Transfection, Negative Control, Western Blot, Control

Journal: Nature Communications

Article Title: Oncogenic EML4-ALK assemblies suppress growth factor perception and modulate drug tolerance

doi: 10.1038/s41467-024-53451-7

Figure Lengend Snippet: A EGF stimulates EGFR and downstream RAS/ERK signaling. B ppERK levels in response to EGF (100 ng/mL) in the presence of 1 µM ALKi (orange) or DMSO (gray) in STE-1 and H3122 cancer cells. Data points represent mean of 1000–2800 cells for STE-1 and 800–3000 cells for H3122. Error bars = 95% CI. **** p < 0.0001, *** p = 0.0007, * p = 0.02 by one-sided T-test comparing ALKi vs DMSO. n = 3 biological replicates. C Representative images of ALKi-dependent potentiation of ERK response to EGF ( B ). D Fold-change increase over a range of EGF concentrations. Data represent ratio of mean ppERK from EGF-stimulated (15 min) vs unstimulated cells. Significance assessed using one-sided T-test, n = 3 biological replicates. E , F Representative single-cell (top) and average (bottom) ERK responses to EGF (50 ng/mL, 15 min) and ALKi (crizotinib, 1 μm, 2 h pretreatment) in two primary patient-derived cell lines, CUTO-8 and CUTO-9, that harbor EML4-ALK(V1) ( E ), or in cell lines driven by a constitutively active, full-length ALK mutant ( F ). Data points in ( E , F ) (bottom) represent mean ppERK normalized to mean ppERK of ALKi-treated cells for 900–2100 STE-1, 900–1500 CUTO-8, 200–300 CUTO-9, 200–600 Kelly cells and 1700–2700 SY5Y cells. Significance assessed by one-sided T-test, n = 3 biological replicates for STE-1, CUTO-8, Kelly, and SY5Y; n = 6 biological replicates for CUTO-9. G EML4-ALK(V1) (EML4-ALK-2A-H2B-miRFP) or a control construct (H2B-miRFP) were transiently expressed in lung epithelial Beas2B cells. H Time course of ppERK immunofluorescence levels in response to EGF stimulation (50 ng/mL). Data points represent mean ± SEM of 120–300 cells for untransfected Beas2B and 80–160 cells for Beas2B expressing EML4-ALK. ** p = 0.003, * p = 0.04, by one-sided T-test for ppERK in transfected vs untransfected cells, n = 3 biological replicates. I Dynamic range of ppERK in EML4-ALK-expressing Beas2B in response to EGF in the presence or absence of ALKi (1 µM) pretreatment. Significance assessed using one-sided T-test, n = 3 biological replicates.

Article Snippet: Primary antibodies used were: phospho-p44/42 MAPK (ERK1/2) (Thr202/Tyr204), Cell Signaling #4370; phospho-EGF Receptor (Tyr1068), Cell Signaling #3777; phospho-ALK receptor (Tyr 1507), Cell Signaling #14678; EGR1, Cell Signaling #4153.

Techniques: Derivative Assay, Mutagenesis, Control, Construct, Immunofluorescence, Expressing, Transfection

Journal: Nature Communications

Article Title: Oncogenic EML4-ALK assemblies suppress growth factor perception and modulate drug tolerance

doi: 10.1038/s41467-024-53451-7

Figure Lengend Snippet: A Time course of EGFR sensitization was obtained by pre-incubating cancer cells with 1 µM crizotinib for a variable period before stimulation with EGF (50 ng/mL) for 15 min, followed by fixation and immunostaining for ppERK. B ppERK induction as a function of ALKi pre-incubation time. Open circles: unstimulated; closed circles: EGF-stimulated. Data points represent mean of three biological replicates, each representing 1200–2700 cells for STE-1 and 2000–3300 cells for H3122. Error bars = 95% CI. C Pinpointing the location of EML4-ALK interaction with RTK/ERK signaling. D OptoFGFR and optoSOS permit optogenetic stimulation at successive nodes of the pathway. E Quantification of ppERK levels in response to optoFGFR or optoSOS in the presence (orange) or absence (gray) of ALKi (1 µM crizotinib). X -axis represents optoFGFR or optoSOS expression quartiles. Data points represent mean ± SEM of three biological replicates, each representing 200–1100 cells. Significance assessed using one-sided T-test, n = 3 biological replicates. Open circles: unstimulated; closed circles: light stimulated. F ALK-dependent suppression is observed only with optoFGFR, suggesting that suppression happens upstream of RAS but downstream of RTK activation. G Testing the role of ERK-dependent negative feedback on RTK suppression. Light stimulation of optoSOS drives elevated levels of ppERK during ALKi treatment and sustains any potential ERK-dependent negative feedback that would otherwise be lost during ALK inhibition. H STE-1 cells were treated with either ALKi or ALKi and optoSOS stimulation, and the response to EGF was assessed. I Predicted results and implications for ERK-dependent feedback. J optoSOS did not suppress ALKi-induced potentiation of EGF response, suggesting that ERK-dependent negative feedback does not account for EGFR suppression. Data points represent means of 150–900 cells per condition. Significance assessed using one-sided T-test, n = 3 biological replicates.

Article Snippet: Primary antibodies used were: phospho-p44/42 MAPK (ERK1/2) (Thr202/Tyr204), Cell Signaling #4370; phospho-EGF Receptor (Tyr1068), Cell Signaling #3777; phospho-ALK receptor (Tyr 1507), Cell Signaling #14678; EGR1, Cell Signaling #4153.

Techniques: Immunostaining, Incubation, Expressing, Activation Assay, Inhibition

Journal: Nature Communications

Article Title: Oncogenic EML4-ALK assemblies suppress growth factor perception and modulate drug tolerance

doi: 10.1038/s41467-024-53451-7

Figure Lengend Snippet: A Quantification of ppERK response to EGF (50 ng/mL) in Beas2B cells that were transfected with EML4-ALK (V1), EML4-ALK (∆TD), or kinase-dead EML4-ALK (K589M), or with an empty vector (EV). Data points represent mean ± SEM of three biological replicates, each representing 200–400 EV cells, 130-270-EML4-ALK (V1) cells, 55–160 EML4-ALK (∆TD) cells, or 260–450 EML4-ALK (K589M) cells. B Colocalization of EML4-ALK condensates with endogenously tagged GRB2 ( GRB2:mNG2 ). See Fig. for quantitation. Scale = 10 µm. C GRB2:mNG2 Beas2B cells were transiently transfected with EML4-ALK and stimulated with EGF (50 ng/mL) to visualize GRB2 translocation in the presence and absence of EML4-ALK (V1). D Impaired membrane translocation of GRB2 in the presence of EML4-ALK condensates. Time in mm:sec. See Supplementary Movie . E Line scan of GRB2 intensity distribution in the presence (red) or absence (gray) of EML4-ALK expression, as depicted in ( C ). F Quantitation of translocation of endogenous GRB2 or SOS1 in the presence (red) or absence (gray) of EML4-ALK. Boxplot indicates the median and upper/lower quartiles, and whiskers extend to 1.5*IQR. See Fig. for full quantitation. G GRB2 localization and translocation were visualized upon treatment with 1 µM ALKi and subsequent stimulation with EGF (50 ng/mL). Time in hh:mm. H Quantification of kinetics of GRB2 dissociation from condensates after ALKi treatment. I ALKi restores GRB2 and SOS translocation. Plot shows median translocation of endogenous adapters in cells expressing EML4-ALK(V1) represented as a fraction of translocation in the absence of EML4-ALK (V1). Data represent medians, error bars show 1st and 3rd quartiles of 1000 bootstrapped samples (distributions found in Fig. F, G). Significance assessed by one-sided bootstrap test for comparison of medians. See Fig. for underlying data and quantitation. J Immunoprecipitation of EGFR shows enhanced co-precipitation of GRB2 and SOS1 in the presence of both ALKi pretreatment and EGF in STE-1 cells. gray arrows: non-specific bands. K Densitometry quantification of three independent pulldowns. L Testing effect of GRB2 overexpression on ERK response. M Expression levels of GRB2-GFP or GFP analyzed in ( N , O ). N ppERK levels in the absence (open circles) or presence (closed circles) of EGF stimulation (50 ng/mL) as a function of expression levels of GFP or GRB2-GFP. Data represent mean ± SEM of three biological replicates, each representing the mean of 100–300 cells. O Absolute magnitude of ppERK increase for each expression bin from data shown in ( N ). Significance assessed by one-sided T-test, n = 3 biological replicates. P Fold-change of response calculated from data in ( N ). Q Conceptual model of how EML4-ALK suppresses transmembrane RTKs. EML4-ALK sequesters adapters like GRB2/SOS1 and prohibits their translocation to activated RTKs. ALK inhibition releases adapter sequestration and restores cellular response to RTK stimulation.

Article Snippet: Primary antibodies used were: phospho-p44/42 MAPK (ERK1/2) (Thr202/Tyr204), Cell Signaling #4370; phospho-EGF Receptor (Tyr1068), Cell Signaling #3777; phospho-ALK receptor (Tyr 1507), Cell Signaling #14678; EGR1, Cell Signaling #4153.

Techniques: Transfection, Plasmid Preparation, Quantitation Assay, Translocation Assay, Membrane, Expressing, Comparison, Immunoprecipitation, Over Expression, Inhibition

Journal: Nature Communications

Article Title: Oncogenic EML4-ALK assemblies suppress growth factor perception and modulate drug tolerance

doi: 10.1038/s41467-024-53451-7

Figure Lengend Snippet: A Three common oncogenic variants of EML4-ALK (Variants 1–3) share a common ALK fragment but differ in the lengths of the EML4 domain. B Expression of mCh-EML4-ALK(V1/2/3) in GRB2:mNG2 Beas2B cells showed condensation of each variant as well as the propensity of the condensates to colocalize with GRB2. Scale = 10 µm. C Quantification of puncta per cell for each variant. D Quantification of the percent of EML4-ALK puncta that overlap with GRB2 puncta in each cell. Boxplots in ( C , D ) show median and upper/lower quartile, and whiskers extend to 1.5*IQR. C , D Data points represent 18 (V1), 28 (V2), and 21 (V3) cells. E Translocation was quantified by identifying the cell edge and defining a 10 pixel ring into the cytoplasm (“edge”). The remaining cell pixels beyond this ring wire designated as the cell “core”. Membrane localization was defined as the ratio of mean edge fluorescence to mean core fluorescence. Translocation was defined as the difference in adapter membrane localization after 1.5 min of EGF stimulation vs pre-stimulation. F , G Quantitation of translocation of GRB2 ( F ) or SOS ( G ) for cells transfected with one of the 3 EML4-ALK variants or for neighboring untransfected cells (wt). Due to small variations in imaging plane between acquisitions, the absolute magnitude of translocation differed between variants and drug conditions (note the differences in untransfected Beas2B responses, which are equivalent conditions between panels). However, cells with or without EML4-ALK (black vs. red in the same panel) were imaged in the same field of view and thus can be compared directly. Data points represent individual cells. For ( F ), n = 57(WT)/40(V1), 170(WT)/42(V2), 24(WT)/34(V3) cells. For ( G ), n = 46(WT)/50(V1), 50(WT)/27(V2), 67(WT)/40(V3) cells. Boxplots show median and upper/lower quartile, and whiskers extend to 1.5*IQR. H Definition of the magnitude of translocation. I , J Comparison of translocation suppression of GRB2 ( F ) or SOS1 ( G ) for each of the three variants. Data represent median translocation suppression from resampling of 1000 bootstrapped samples. Error bars show lower and upper quartiles. Significance determined by either one-sided T-test (panels F , G ) or one-sided bootstrap test (panels I , J ). Data for Variant 1 in ( F – J ) is reproduced from Fig. . K Beas2B cells were transfected with EML4-ALK-2A-H2B-miRFP constructs for one of 3 EML4-ALK variants (V1, V2, V3), or with an H2B-iRFP control, and ppERK levels were assessed after stimulation with EGF (15 min, 50 ng/mL) in the presence or absence of ALKi (1 µM crizotinib, 2 h), through immunofluorescence. L Quantification of ppERK immunostaining after EGF stimulation of Beas2B transiently expressing EML4-ALK in the presence or absence of ALKi. Significance assessed by Hsu MCB test. n = 8 biological replicates. M ppERK response in the presence and absence of ALKi pretreatment. Data points represent the mean ppErk intensity of 20–60 cells. Significance assessed by one-sided T-test. n = 8 biological replicates. Gray bars in ( M ) are reproduced from ( L ) for direct comparison to between non-treated and ALKi-treated cells.

Article Snippet: Primary antibodies used were: phospho-p44/42 MAPK (ERK1/2) (Thr202/Tyr204), Cell Signaling #4370; phospho-EGF Receptor (Tyr1068), Cell Signaling #3777; phospho-ALK receptor (Tyr 1507), Cell Signaling #14678; EGR1, Cell Signaling #4153.

Techniques: Expressing, Variant Assay, Translocation Assay, Membrane, Fluorescence, Quantitation Assay, Transfection, Imaging, Comparison, Construct, Control, Immunofluorescence, Immunostaining

Journal: Nature Communications

Article Title: Oncogenic EML4-ALK assemblies suppress growth factor perception and modulate drug tolerance

doi: 10.1038/s41467-024-53451-7

Figure Lengend Snippet: A The ErkKTR reporter indicates ERK activity through nuclear-cytoplasmic translocation of a fluorescent protein. B Sensitivity of single cells (STE-1) to 15 min EGF stimulation. Plot shows fold change of ERK activity in single cells upon stimulation with the indicated amount of EGF in the presence or absence of ALKi (crizotinib, 1 µM). Boxplot shows median and upper/lower quartiles, and whiskers extend to 1.5*IQR. Significance tests indicate increased response above 0 ng/mL EGF. **** p < 0.0001 by Hsu multiple comparison with the best (MCB) test. n = 316, 384, 421, 285, 307, 256, 294, 421 cells respectively for DMSO and 473, 727, 536, 595, 541, 425, 439, 415 cells respectively for ALKi. See Supplementary Movie . C Live-cell imaging of STE-1 cells expressing ErkKTR in the presence or absence of ALKi (1 µM crizotinib). See Supplementary Movie . D Representative single-cell traces of cytoplasmic/nuclear ErkKTR intensity ratio from conditions shown in ( C ). E Quantification of ErkKTR activity in the presence of ALKi or its combination with EGFRi (1 µM erlotinib) or MMPi (10 µM marimastat). F Quantification of ERK activity pulses. Boxplot shows median and upper/lower quartiles, whiskers show 1.5*IQR. Significance assessed by Hsu MCB test. n = 177 (control), 198 (ALKi), 170 (ALKi/EGFRi), and 182 (ALKi/MMPi) single cells. G Percent of cells that exhibited any pulses over 22 h of imaging. Error bars indicate 95% CI. Significance assessed by the Hsu MCB test. n = 200 cells per condition. H Apoptotic cells secrete paracrine EGFR ligands to their neighbors. Paracrine signaling can be blocked by inhibiting either EGFR or the MMPs that mediate shedding of EGFR ligands from the surface of the sender cell. I ErkKTR activity pulses are primarily observed surrounding a dying cell during ALK inhibition but not in the absence of drug or in the added presence of EGFR or MMP inhibitors. J Definition of neighbors and non-neighbors of a death event. K Quantification of pulses per cell for each death event in neighbors or in equal number of randomly chosen subset of cells not near a death event (see “Methods” for more details), n = 91, 103, 149, 20 events for ALKi, ALKi/EGFRi, ALKi/MMPi and DMSO, respectively. Boxplot shows median and upper/lower quartiles, whiskers show 1.5*IQR. L Fraction of total neighbor vs random non-neighbor cells that show any pulsing. Cell numbers as in ( F ). Error bars show 95% CI. Significance in ( K , L ) determined by independent T-tests (within treatment conditions) or by ANOVA followed by the Hsu MCB test (across treatment conditions).

Article Snippet: Primary antibodies used were: phospho-p44/42 MAPK (ERK1/2) (Thr202/Tyr204), Cell Signaling #4370; phospho-EGF Receptor (Tyr1068), Cell Signaling #3777; phospho-ALK receptor (Tyr 1507), Cell Signaling #14678; EGR1, Cell Signaling #4153.

Techniques: Activity Assay, Translocation Assay, Comparison, Live Cell Imaging, Expressing, Control, Imaging, Inhibition

Journal: Nature Communications

Article Title: Oncogenic EML4-ALK assemblies suppress growth factor perception and modulate drug tolerance

doi: 10.1038/s41467-024-53451-7

Figure Lengend Snippet: A Signaling through EGFR activates RAS/ERK and stimulates transcription, including of the immediate early gene EGR1. B Quantification of single-cell IF of EGR1 in STE-1 cells under the conditions indicated. C Overlay of EGR1 expression at the 6 h time point in ( B ). D Testing whether restored perception of paracrine signals can promote survival during ALKi treatment. E Quantification of pulses per cell in cells that died ( D ) or survived ( S ) through 22 h of imaging in the conditions where ERK pulsing could be observed. Boxplot shows median and upper/lower quartiles, whiskers show 1.5*IQR. Significance assessed by one-sided T-tests. n = 88 ALKi(D), 110 ALK(S), 136 ALKi/MMPi(D), and 46 ALKi/MMPi(S) cells. F Caspase-3 activation was assessed using the NucView reporter after 24 h treatment with the indicated drugs. Data points show proportion of 2300–3500 STE-1 cells and 3500–4500 H3122 cells. Significance assessed by one-sided T-test, n = 3 biological replicates. G , H DAPI imaging (left) and cell counts (right) of cell survival after 17 days of the indicated treatments in both H3122 ( G ) and STE-1 ( H ) cell lines. Significance assessed using one-sided T-test. n = 3 (H3122) and n = 4 (STE1) biological replicates. I Summary of the effects of drug-induced RTK resensitization.

Article Snippet: Primary antibodies used were: phospho-p44/42 MAPK (ERK1/2) (Thr202/Tyr204), Cell Signaling #4370; phospho-EGF Receptor (Tyr1068), Cell Signaling #3777; phospho-ALK receptor (Tyr 1507), Cell Signaling #14678; EGR1, Cell Signaling #4153.

Techniques: Expressing, Imaging, Activation Assay

Journal: Journal of Advanced Research

Article Title: LncRNA NIPA1-SO confers atherosclerotic protection by suppressing the transmembrane protein NIPA1

doi: 10.1016/j.jare.2023.01.017

Figure Lengend Snippet: NIPA1-SO increases BMPR2 by reducing NIPA1 expression. ( A ) HUVECs, THP-1 cells, and HASMCs were transduced with NIPA1-SO -expressing lentiviral vectors or a control lentivirus (LV-Mock) (n = 5). Western blotting was used to assess BMPR2 protein levels in response to transduction. Data shown are normalized to the mock control samples. ( B ) Knockdown of NIPA1-SO in HUVECs, THP-1 cell, and HASMCs, using shRNA, was also used to assess the role of NIPA1-SO in BMPR2 expression via Western blot analysis (n = 5). Data were normalized to the control shRNA samples following normalization to β-actin. ( C ) To demonstrate that the regulatory effect of NIPA1-SO on BMPR2 is mediated by NIPA1, NIPA1 -targeting siRNA and overexpression of NIPA1-SO was used to assess BMPR2 expression using Western blotting (n = 5). Data for the treatment combinations shown were normalized to β-actin and the control sample (no NIPA1-SO overexpression or NIPA1 knockdown). ( D ) Protein levels of BMPR2 (brown staining) in atherosclerotic plaque tissues or normal tissue samples was assessed via immunohistochemistry (n = 5). Data shown are normalized to the values obtained from normal intimal tissue samples. ( E ) Immunofluroescence staining of atherosclerotic plaque tissue was performed to assess the expression of BMPR2 in different cell types. The left-most images show immunofluorescence staining with cell-type specific markers for endothelial cells (EC; CD34), monocytes (MΦ; LGALS3L) and vascular smooth muscle cells (VSMC; α-smooth muscle actin) with green fluorescence. The center images show corresponding BMPR2 immunofluorescence staining (red) in the three cell types. The images on the right show merged images, demonstrating regions of co-localization between the cell type markers and BMPR2 (as indicated by an arrow). In all immunofluorescence images, staining with DAPI (blue) was used to visualize cell nuclei. (A-E) * P < 0.05 by unpaired 2-tailed Student’s t -test, n = 5 per group. Data are expressed as the mean (±SD) fold differences between the cells transfected with NIPA1-SO -expressing lentivirus and cells transfected with the lentivirus vector in three independent experiments. * P < 0.05 by unpaired 2-tailed Student’s t -test. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: Protein extracts were separated using 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis and then subjected to Western blot analyses using rabbit polyclonal anti-NIPA1, -FUBP1, -BMPR2 antibodies (Proteintech Group), rabbit polyclonal anti-pSmad1/5/8 antibodies (Abcam) and rabbit polyclonal anti-ICAM-1, anti-VCAM-1, and anti-β-actin antibodies (Santa Cruz Biotechnologies).

Techniques: Expressing, Transduction, Control, Western Blot, Knockdown, shRNA, Over Expression, Staining, Immunohistochemistry, Immunofluorescence, Fluorescence, Transfection, Plasmid Preparation

Journal: Journal of Advanced Research

Article Title: LncRNA NIPA1-SO confers atherosclerotic protection by suppressing the transmembrane protein NIPA1

doi: 10.1016/j.jare.2023.01.017

Figure Lengend Snippet: NIPA1-SO , via NIPA1 signaling, alters expression of adhesion molecules by HUVECs, influencing THP-1 cell adhesion. (A to C) The effects of NIPA1-SO , NIPA1 and BMPR2 upon pSmad1/5/8 expression were examined via Western blotting and either lentiviral-mediated overexpression ( A ) or knockdown with NIPA1 siRNA ( B ) or BMPR2 siRNA ( C ). Data were normalized to β-actin and to control samples (n = 5). ( D ) VCAM1 and ICAM1 expression in response to overexpression of NIPA1-SO and/or siRNA-mediated knockdown of BMPR2 was examined via Western blotting. Data were normalized to β-actin and to control samples (n = 5). ( E ) Knockdown of NIPA1-SO and BMPR2 , both separately and in combination, was used to assess VCAM1 and ICAM1 expression via Western blotting (n = 5). Data are presented following normalization to β-actin and untreated control cells. ( F ) Monocyte adhesion in response to endothelial cell knockdown of NIPA1-SO , NIPA1 and BMPR2 was performed. Data are presented as a count of adhered cells per field of the acquired images (n = 5). ( G ) The effect of NIPA1-SO overexpression and BMPR2 knockdown upon monocyte adhesion was also assessed (n = 5). Data shown are a count of the number of adhered cells per image field. * P < 0.05 by unpaired 2-tailed Student’s t -test ( A to C ) or one-way ANOVA with Tukey’s post hoc tests ( D to G ).

Article Snippet: Protein extracts were separated using 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis and then subjected to Western blot analyses using rabbit polyclonal anti-NIPA1, -FUBP1, -BMPR2 antibodies (Proteintech Group), rabbit polyclonal anti-pSmad1/5/8 antibodies (Abcam) and rabbit polyclonal anti-ICAM-1, anti-VCAM-1, and anti-β-actin antibodies (Santa Cruz Biotechnologies).

Techniques: Expressing, Western Blot, Over Expression, Knockdown, Control

Journal: Journal of Advanced Research

Article Title: LncRNA NIPA1-SO confers atherosclerotic protection by suppressing the transmembrane protein NIPA1

doi: 10.1016/j.jare.2023.01.017

Figure Lengend Snippet: NIPA1-SO , via BMPR2, increases ABCA1 and ABCG1 levels in THP-1 cells. ( A and B ) Western blotting of the cholesterol efflux-mediators ABCA1 and ABCG1 ( A ) and intracellular cholesterol concentration ( B ), in human monocytes (THP-1 cells) in response to lentiviral overexpression of NIPA1-SO and BMPR2 siRNA knockdown (n = 3). ( C and D ) Western blotting of ABCA1 and ABCG1 ( C ) and intracellular cholesterol concentration ( D ), in human THP-1 cells with NIPA1-SO shRNA knockdown and BMPR2 siRNA transfection (n = 3). Data in ( A ) and ( C ) are presented following normalization to β-actin and untreated control cells. * P < 0.05 by one-way ANOVA with Tukey’s post hoc tests ( A and C ) or unpaired 2-tailed Student’s t -test ( B and D ).

Article Snippet: Protein extracts were separated using 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis and then subjected to Western blot analyses using rabbit polyclonal anti-NIPA1, -FUBP1, -BMPR2 antibodies (Proteintech Group), rabbit polyclonal anti-pSmad1/5/8 antibodies (Abcam) and rabbit polyclonal anti-ICAM-1, anti-VCAM-1, and anti-β-actin antibodies (Santa Cruz Biotechnologies).

Techniques: Western Blot, Concentration Assay, Over Expression, Knockdown, shRNA, Transfection, Control

Journal: Journal of Advanced Research

Article Title: LncRNA NIPA1-SO confers atherosclerotic protection by suppressing the transmembrane protein NIPA1

doi: 10.1016/j.jare.2023.01.017

Figure Lengend Snippet: NIPA1-SO and NIPA1 influence atherosclerotic plaque formation in mice in vivo . ( A ) En face analysis of aortas from LDLR -/- / NIPA1 +/+ mice injected with lentivirus to overexpress NIPA1-SO or a control lentivirus (LV-Mock) was performed to demonstrate if NIPA1-SO is involved in atherogenesis in vivo (n = 5). Tissue samples were stained with Oil Red O and the lesion area quantified as a percentage of total en face area. Immunohistochemistry analysis of lesions in LDLR -/- / NIPA1 +/+ mice overexpressing NIPA1-SO was performed to determine if the composition of atherosclerotic lesions in such mice differs from control mice. Data shown is the % of lesion area positive for Oil Red O (lipid) staining or Masson staining. ( B ) Aortic ring monocyte adhesion assays were performed on LDLR -/- / NIPA1 +/+ mice injected with NIPA1-SO lentiviral particles or a control lentivirus as described in C (n = 5). ( C ) En-face dissection and subsequent Oil Red O lipid staining and immunohistochemical analysis of LDLR -/- / NIPA1 +/+ and LDLR -/- / NIPA1 -/- mice fed an atherogenic Western diet for 12 weeks was performed (n = 5). En face aorta samples were stained with Oil Red O (red) to visualize atherosclerotic plaques. Lesion areas, as a percentage of total en face area, are shown. Further analysis of atherosclerotic lesions from the two mouse strains was also performed using Oil Red O staining (quantifying the level of lipid as percentage of the lesion area). Hematoxylin (blue) and eosin (pink) (H&E) staining was also performed to visualize the nuclei and cytoplasm/extracellular matrix (respectively). H&E staining was quantified as a percentage of total lesion area. Immunohistochemistry was used to stain for monocytes (using the marker LGALS3) and smooth muscle cells (using α-smooth muscle actin) as indicated by brown staining. The abundance of these cell types as a percentage of total lesion area is shown. Staining with Masson’s trichrome stain was used to determine the quantity of collagen, as a percentage of total lesion area. ( D ) Immunohistochemistry of LDLR -/- / NIPA1 +/+ and LDLR -/- / NIPA1 -/- mice was performed for BMPR2, pSmad1, ICAM1, VCAM1, ABCA1 and ABCG1, with brown staining indicating the presence of the protein of interest. For all immunohistochemistry experiments, the level of each protein, relative to the control ( LDLR -/- / NIPA1 +/+ ) mice, is presented (n = 5). ( E ) An aortic ring monocyte adhesion assay was performed using LDLR -/- / NIPA1 +/+ and LDLR -/- / NIPA1 -/- mouse aorta samples to determine if NIPA1 knock-out alters monocyte adhesion to endothelial cells (n = 5). The aortic rings were labeled with calcein-AM and incubated with Dil–labeled mouse peritoneal monocytes. Data presented are the total number of monocytes adhered to the aortic ring for the two groups. Data are presented as the total number of cells adhering to the aortic endothelium. * P < 0.05 by unpaired 2-tailed Student’s t -test. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: Protein extracts were separated using 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis and then subjected to Western blot analyses using rabbit polyclonal anti-NIPA1, -FUBP1, -BMPR2 antibodies (Proteintech Group), rabbit polyclonal anti-pSmad1/5/8 antibodies (Abcam) and rabbit polyclonal anti-ICAM-1, anti-VCAM-1, and anti-β-actin antibodies (Santa Cruz Biotechnologies).

Techniques: In Vivo, Injection, Control, Staining, Immunohistochemistry, Dissection, Immunohistochemical staining, Western Blot, Marker, Cell Adhesion Assay, Knock-Out, Labeling, Incubation

Journal: Journal of Advanced Research

Article Title: LncRNA NIPA1-SO confers atherosclerotic protection by suppressing the transmembrane protein NIPA1

doi: 10.1016/j.jare.2023.01.017

Figure Lengend Snippet: Schematic diagram of the signalling pathways regulated by NIPA1-SO . Our study has uncovered an athero-protective role of the lncRNA NIPA1-SO , which, by interacting with a single transcription factor, is capable of inhibiting monocyte adhesion and foam cell formation, two fundamental processes in atherosclerosis. The transcription factor FUBP1 negatively regulates NIPA1 expression; this inhibitory effect is increased by the interaction of NIPA1-SO with FUBP1, resulting in lower transcription of NIPA1. A reduction in NIPA1 protein results in increased BMPR2 protein due to a reduction in NIPA1-mediated BMPR2 endocytosis and degradation, leading to higher levels of Smad1/5/8 phosphorylation (pSmad1/5/8), which, through complexation with Smad4, inhibit transcription of the adhesion molecules VCAM1 and ICAM1, reducing monocyte adhesion to endothelial cells. Further, the pSmad1/5/8:Smad4 complex promotes ABCA1 and ABCG1 transcription, both of which promote cholesterol efflux via high-density lipoprotein (HDL) particles, thereby inhibiting foam cell formation.

Article Snippet: Protein extracts were separated using 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis and then subjected to Western blot analyses using rabbit polyclonal anti-NIPA1, -FUBP1, -BMPR2 antibodies (Proteintech Group), rabbit polyclonal anti-pSmad1/5/8 antibodies (Abcam) and rabbit polyclonal anti-ICAM-1, anti-VCAM-1, and anti-β-actin antibodies (Santa Cruz Biotechnologies).

Techniques: Expressing, Phospho-proteomics