Journal: Scientific Reports

Article Title: IL-15-Activated CD38 + HLA-DR + CD8 + T cells induce liver injury in cirrhosis via JAK/STAT5 and PI3K/mTOR pathways

doi: 10.1038/s41598-025-02693-6

Figure Lengend Snippet: The signal pathways involved in IL-15-induced innate cytotoxicity of CD38 + HLA-DR + CD8 + T cells. (A) 1 × 10 6 CD8 + T cells from healthy donors were stimulated with IL-15 (20 ng/mL) for 48 h and 72 h, after which phosphorylation of signaling proteins was assessed by flow cytometry for STAT5 ( n = 10), ERK1/2 ( n = 7) and mTOR ( n = 8). Representative dot plots and the summary data show the expression of signaling proteins in CD38 + HLA-DR + CD8 + T cells. (B) The percentage of CD38 + HLA-DR + CD8 + T cells was analyzed after inhibitors treatment ( n = 4). Representative dot plots from a single donor (left) and summary data (right) are presented. (C-G) The percentage NKG2D, FasL, perforin, and Granzyme B in CD38 + HLA-DR + CD8 + T cells were analyzed after inhibitors treatment ( n = 4). (H) CD8 + T cells from healthy donors were pre-treated with STAT5 inhibitor pimozide, MEK inhibitor PD98059, PI3K inhibitor LY294002 or mTOR inhibitor PP242, followed by IL-15 (20 ng/mL) stimulation for the next 72 h. Then, CD8 + T cells were co-cultured with CFSE-labeled K562 cells at a 10:1 E: T ratio and cytotoxicity against K562 cells evaluated ( n = 4). Representative dot plots and the summary data show expression of PI in the gate of CFSE + . (I) Schematic representation of cytokine-mediated crosstalk between T cells and liver cells in liver cirrhosis. The Wilcoxon matched-pairs signed rank test (A) was used for comparisons among groups. The one-way ANOVA (B , C , D-H) was used for comparisons between groups. Control group was indicated treatment only IL-15. ns, not significant, * p < 0.05, ** p < 0.01.

Article Snippet: CD8 + T cells (1 × 10 6 ) from healthy donors were pre-treated with inhibitors including STAT5 inhibitor pimozide, MEK inhibitor PD98059, PI3K inhibitor LY294002 or mTOR inhibitor PP242 (MedChemExpress, NJ, USA) and stimulated with IL-15 (20 ng/mL) (Thermo, MA, USA) for 72 h. CD8 + T cells were subsequently co-cultured with CFSE-labeled K562 cells at a 10:1 effector-to-target (E: T) ratio for 6 h, and cytotoxicity against K562 cells was assessed.

Techniques: Phospho-proteomics, Flow Cytometry, Expressing, Cell Culture, Labeling, Control

Journal: Scientific Reports

Article Title: IL-15-Activated CD38 + HLA-DR + CD8 + T cells induce liver injury in cirrhosis via JAK/STAT5 and PI3K/mTOR pathways

doi: 10.1038/s41598-025-02693-6

Figure Lengend Snippet: The signal pathways involved in IL-15-induced innate cytotoxicity of CD38 + HLA-DR + CD8 + T cells. (A) 1 × 10 6 CD8 + T cells from healthy donors were stimulated with IL-15 (20 ng/mL) for 48 h and 72 h, after which phosphorylation of signaling proteins was assessed by flow cytometry for STAT5 ( n = 10), ERK1/2 ( n = 7) and mTOR ( n = 8). Representative dot plots and the summary data show the expression of signaling proteins in CD38 + HLA-DR + CD8 + T cells. (B) The percentage of CD38 + HLA-DR + CD8 + T cells was analyzed after inhibitors treatment ( n = 4). Representative dot plots from a single donor (left) and summary data (right) are presented. (C-G) The percentage NKG2D, FasL, perforin, and Granzyme B in CD38 + HLA-DR + CD8 + T cells were analyzed after inhibitors treatment ( n = 4). (H) CD8 + T cells from healthy donors were pre-treated with STAT5 inhibitor pimozide, MEK inhibitor PD98059, PI3K inhibitor LY294002 or mTOR inhibitor PP242, followed by IL-15 (20 ng/mL) stimulation for the next 72 h. Then, CD8 + T cells were co-cultured with CFSE-labeled K562 cells at a 10:1 E: T ratio and cytotoxicity against K562 cells evaluated ( n = 4). Representative dot plots and the summary data show expression of PI in the gate of CFSE + . (I) Schematic representation of cytokine-mediated crosstalk between T cells and liver cells in liver cirrhosis. The Wilcoxon matched-pairs signed rank test (A) was used for comparisons among groups. The one-way ANOVA (B , C , D-H) was used for comparisons between groups. Control group was indicated treatment only IL-15. ns, not significant, * p < 0.05, ** p < 0.01.

Article Snippet: CD8 + T cells (1 × 10 6 ) from healthy donors were pre-treated with STAT5 inhibitor pimozide, PI3K inhibitor LY294002 or mTOR inhibitor PP242, followed by IL-15 (20 ng/mL) (Thermo, MA, USA) stimulation for the next 72 h. All the inhibitors were obtained from MedChemExpress (NJ, USA).

Techniques: Phospho-proteomics, Flow Cytometry, Expressing, Cell Culture, Labeling, Control

Journal: Nature

Article Title: Signalling input from divergent pathways subverts B cell transformation.

doi: 10.1038/s41586-020-2513-4

Figure Lengend Snippet: Fig. 1 | Segregation of STAT5 and ERK activation in human B-ALL. a, Correlation between ERK-pT202/Y204 and STAT5-pY694 levels in B-ALL cells (n = 23 independent biological samples; P = 0.001, two-tailed t-test; r = −0.657, Pearson’s r). b, Western blots of patient-derived B-ALL cells (n = 8) and Ph+ B-ALL cells before (n = 8) and after (n = 8) ponatinib treatment. c, Analysis of patient-derived Ph+ B-ALL cells treated with trametinib or ponatinib, showing the percentage of growth inhibition (means of three independent

Article Snippet: A STAT5 inhibitor (pimozide) was purchased from TOCRIS.

Techniques: Activation Assay, Two Tailed Test, Western Blot, Derivative Assay, Inhibition

Journal: Nature

Article Title: Signalling input from divergent pathways subverts B cell transformation.

doi: 10.1038/s41586-020-2513-4

Figure Lengend Snippet: Fig. 2 | STAT5–MYC and ERK–BCL6 signalling are incompatible and define distinct stages of B-cell development. a, Analysis of Hardy fractions B–F for MyceGFP/+ Bcl6mCherry/+ bone-marrow cells, including single-cell phosphoprotein analyses of the indicated fractions. b, Expression of eGFP and mCherry in MyceGFP/+ Bcl6mCherry/+ bone marrow cells in Hardy fractions B–F. c, Surface expression of Igκλ light chain (LC) on IL7-dependent pro-B cells expressing empty vector (EV), and upon doxycycline-inducible expression of Ig-HC or NRASG12D. Numbers in the top right corners denote the percentage of cells expressing Ig-LC. d, Expression of eGFP and mCherry in IL7-dependent MyceGFP/+ Bcl6mCherry/+ B-cell precursors induced to differentiate or transduced with

Article Snippet: A STAT5 inhibitor (pimozide) was purchased from TOCRIS.

Techniques: Expressing, Plasmid Preparation, Transduction

Journal: Nature

Article Title: Signalling input from divergent pathways subverts B cell transformation.

doi: 10.1038/s41586-020-2513-4

Figure Lengend Snippet: Fig. 3 | Concurrent oncogenic STAT5 and ERK activation subverts B-cell leukaemogenesis. a, Western blotting following doxycycline-induced expression of Stat5aCA or NRASG12D in IL7-dependent mouse pro-B cells. b, Fluorescence-activated cell sorting (FACS) plots and western blots of hIL2Rβ–TetO–NRASG12D mouse pro-B cells following the indicated treatments (doxycycline, 24 h; hIL2, 15 min, 50 ng μl−1). c, d, Colony formation (c; P = 1.9 × 10−5, two-tailed t-test) and viable cell counts (d; mean ± s.d.) of hIL2Rβ–TetO–NRASG12D mouse pro-B cells treated with doxycycline, hIL2 (10 ng μl−1), or a combination of both. a–d, Data from three independent experiments. For gel source data, see Supplementary Fig. 1.

Article Snippet: A STAT5 inhibitor (pimozide) was purchased from TOCRIS.

Techniques: Activation Assay, Western Blot, Expressing, Fluorescence, FACS, Two Tailed Test

Journal: Nature

Article Title: Signalling input from divergent pathways subverts B cell transformation.

doi: 10.1038/s41586-020-2513-4

Figure Lengend Snippet: Fig. 4 | Genetic deletion of alternative pathways triggers initiation of STAT5- and ERK-driven leukaemia. a, Western blots of BCR–ABL1 B-ALL cells upon Cre-mediated ablation of Mapk1 (Erk2). b, Western blots of NRASG12D B-ALL cells upon Cre-mediated deletion of Stat5. c, d, Enrichment or depletion of GFP+ Stat5fl/fl or Mapk1fl/fl BCR–ABL1 (c) or NRASG12D (d) B-ALL cells transduced with ERT2–GFP or Cre–ERT2–GFP. ERT2 is a mutant ligand-binding domain of the human oestrogen receptor that binds to 4-hydroxytamoxifen (4-OHT) or tamoxifen. e, f, Colony formation by Mapk1fl/fl BCR–ABL1 B-ALL cells (e) and Stat5fl/fl NRASG12D B-ALL cells (f) upon deletion of Mapk1 or Stat5, respectively. The values above each image show colony formation as a percentage of control values. g, h, Colony formation (percentage of control) of Mb1–Cre × LSL–BCR–ABL1 (g) and Mb1–Cre × LSL–KRASG12D (h) pro-B cells primed with trametinib (g; 1 nmol l−1, 10 days) and ruxolitinib (h; 10 nmol l−1, 10 days). i, j, Kaplan–Meier analyses (Mantel–Cox log-rank test) of recipient mice (n = 4 per group) bearing BCR–ABL1 (i) or NRASG12D-driven (j) B-ALL cells with or without deletion of Mapk1 (i; 24 h) or Stat5 (j; 24 h). a–h, Data shown as means ± s.d. (n = 3 independent experiments. c–h, Assessed by two-tailed t-test. For gel source data, see Supplementary Fig. 1.

Article Snippet: A STAT5 inhibitor (pimozide) was purchased from TOCRIS.

Techniques: Western Blot, Transduction, Mutagenesis, Ligand Binding Assay, Control, Two Tailed Test

Journal: Cells

Article Title: Thymic Stromal Lymphopoietin Interferes with the Apoptosis of Human Skin Mast Cells by a Dual Strategy Involving STAT5/Mcl-1 and JNK/Bcl-x L

doi: 10.3390/cells8080829

Figure Lengend Snippet: MC maintenance by TSLP critically depends on JNK and STAT5 activation. Impact of ( a , c ) STAT5 and ( b , d ) JNK perturbation on TSLP-promoted MC recovery (at 7.5 ng/mL) after 8 h, evaluated by the ratio of YoPro TM -1 positivity (corresponding to the percentage of early and late apoptotic/necrotic cells combined) in TSLP-treated versus untreated MCs (described in methods). ( a , b ) Interference by Accell ® -mediated RNAi (48 h prior to TSLP treatment); ( c , d ) interference by specific inhibitors (STAT5 inhibitor: pimozide, JNK inhibitor: SP600125). Top: the results represent the mean ± SEM of six independent experiments. Bottom: representative flow cytometry dot plots (specified in red is the percentage of early and late apoptotic/necrotic cells combined); w/o—without. The data were analyzed by paired t -test, ** p < 0.01, *** p < 0.001.

Article Snippet: For kinase inhibition studies, cells (at 5 × 10 5 cells/mL) were pre-incubated for 15 min, with pimozide (STAT5 inhibitor, 5 μM), SCH772984 (extracellular signal–regulated kinases (ERK)1/2 inhibitor, 5 μM) (both from Selleckchem, Houston, TX, USA), SB203580 (p38 inhibitor, 5 μM), or SP600125 (JNK inhibitor, 5 μM) (both from ApexBio, Houston, TX, USA), prior to the addition of TSLP.

Techniques: Activation Assay, Flow Cytometry

Journal: Cells

Article Title: Thymic Stromal Lymphopoietin Interferes with the Apoptosis of Human Skin Mast Cells by a Dual Strategy Involving STAT5/Mcl-1 and JNK/Bcl-x L

doi: 10.3390/cells8080829

Figure Lengend Snippet: STAT5 perturbation leads to Mcl-1 downregulation, while interference with JNK attenuates Bcl-x L expression. Impact of ( a – d ) STAT5 and ( e – h ) JNK perturbation on TSLP-triggered the expression (at 7.5 ng/mL) of ( a , b , e , f ) Mcl-1 and ( c , d , g , h ) Bcl-x L after 40 min, using ( a , c , e , g ) knockdown by RNAi (48 h prior to the experiment), and ( b , d , f , h ) pre-incubation with specific inhibitors (STAT5 inhibitor: pimozide, JNK inhibitor: SP600125), evaluated by RT-qPCR analysis (normalized to Cyclophilin B ). The results represent the mean ± SEM of five (RNAi) or six (inhibitors) independent experiments. The data were analyzed by the one-way Anova test with Tukey’s post-test for multiple comparisons, * p < 0.05, ** p < 0.01; ( i , j ) impact of specific inhibitors on the TSLP modulation of ( i ) Mcl-1 and ( j ) Bcl-x L by Western blot analysis (shown are representative Western blots out of two independent experiments). Densitometry units were normalized to the housekeeping protein.

Article Snippet: For kinase inhibition studies, cells (at 5 × 10 5 cells/mL) were pre-incubated for 15 min, with pimozide (STAT5 inhibitor, 5 μM), SCH772984 (extracellular signal–regulated kinases (ERK)1/2 inhibitor, 5 μM) (both from Selleckchem, Houston, TX, USA), SB203580 (p38 inhibitor, 5 μM), or SP600125 (JNK inhibitor, 5 μM) (both from ApexBio, Houston, TX, USA), prior to the addition of TSLP.

Techniques: Expressing, Knockdown, Incubation, Quantitative RT-PCR, Western Blot

Journal: Oncotarget

Article Title: FLT3-ITD induces expression of Pim kinases through STAT5 to confer resistance to the PI3K/Akt pathway inhibitors on leukemic cells by enhancing the mTORC1/Mcl-1 pathway

doi: 10.18632/oncotarget.22926

Figure Lengend Snippet: ( A ) 32D/TKD or 32D/ITD cells were lysed and subjected to Western blot analysis with antibodies against indicated proteins. Abbreviations: STAT5-PY, phospho-Y694-STAT5. ( B ) 32D/ITD cells were left untreated or treated for 6 h with 100 nM AC-220, 10 μM Pimozide, or 1 μM AZD1208, as indicated, and analyzed. ( C ) 32D/ITD cells were cultured for 48 h with or without 1 μM GDC-0941 (GDC), 3 μM MK-2206 (MK), or 1 μM AZD1208 (AZD), as indicated, and viable cell numbers were counted after trypan blue staining. The means of relative cell numbers, expressed as percentages of control cells without inhibitors, from triplicate measurements are shown with error bars indicating standard errors. ( D ) 32D/ITD cells were treated for 48 h with or without 3 μM GDC-0941 (GDC), 3 μM MK-2206 (MK), or 1 μM AZD1208 (AZD), as indicated, and analyzed for the cellular DNA content by flow cytometry. Percentages of apoptotic cells with sub-G1 DNA content are indicated. ( E ) 32D/ITD cells were treated for 48 h with or without 3 μM GDC-0941 (GDC) or 1 μM AZD1208 (AZD), as indicated, and analyzed for activation of Bax or Bak, as indicated, by flow cytometry. ( F ) 32D/ITD cells were treated for 48 h with or without 1 μM GDC-0941 (GDC) or 1 μM AZD1208 (AZD), as indicated, and analyzed for activation of Caspase-3 by flow cytometry. ( G ) 32D/TKD cells transduced with STAT5A1*6 or vector control cells (Cont.) were lysed and subjected to Western blot analysis with antibodies against indicated proteins. ( H ) 32D/TKD cells transduced with STAT5A1*6 or vector control cells (Cont.) were cultured for 48 h with or without 3 μM GDC-0941 (GDC) or 1 μM AZD1208 (AZD), as indicated, and analyzed. ( I ) MV4-11 cells were treated for 16 h with 50 nM AC-220, 10 μM pimozide, or 1 μM AZD1208, as indicated, and analyzed. HSP90 was used for a loading control. ( J ) MV4-11 cells were cultured for 48 h with indicated concentrations of GDC-0941 and AZD1208, and viable cell numbers were measured by using the Cell counting Kit-8. The means of relative viable cell numbers obtained from triplicate measurements are plotted with error bars indicating standard errors. Combination index (CI) values obtained by the method of Chou and Talalay are also indicated. ( K ) MV4-11 cells were cultured for 48 h with or without 1 μM GDC-0941 (GDC) or 3 μM AZD1208 (AZD), as indicated, and analyzed. ( L ) MV4-11 cells were cultured for 48 h with or without 1 μM GDC-0941 (GDC) or 1 μM AZD1208 (AZD), as indicated, for analysis for activation of Caspase-3. Percentages of cells with cleaved Caspase-3 are indicated. ( M ) MV4-11 cells were cultured for 48 h with both 2 μM GDC-0941 and 3 μM AZD1208 (GDC/AZD) with or without 100 μM Boc-d-FMK, as indicated, and analyzed.

Article Snippet: The STAT5 inhibitor pimozide (sc-203662) and antibodies against HSP90 (sc-13119) and murine/human Pim-2 (sc-13514) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Western Blot, Cell Culture, Staining, Control, Flow Cytometry, Activation Assay, Transduction, Plasmid Preparation, Cell Counting

Journal: Oncotarget

Article Title: FLT3-ITD induces expression of Pim kinases through STAT5 to confer resistance to the PI3K/Akt pathway inhibitors on leukemic cells by enhancing the mTORC1/Mcl-1 pathway

doi: 10.18632/oncotarget.22926

Figure Lengend Snippet: ( A ) 32D/ITD cells were cultured for 6 h with indicated concentrations of AZD1208 (AZD) and lysed. Cell lysates were run on duplicate gels and subjected to Western blot analysis with antibodies against indicated proteins. The results obtained from duplicate gels are shown above or below a thin horizontal line. Abbreviations: Akt-PT, phospho-T308-Akt; Akt-PS, phospho-S473-Akt; S6K-PT, phospho-T389-p70S6 kinase; 4EBP1-P, phospho-T37/46-4EBP1; 4EBP1-nonP, non-phospho-T46-4EBP1; STAT5-PY, phospho-Y694-STAT5; PRAS40-P, phospho-T246-PRAS40. ( B ) 32D/ITD cells were cultured for 6 h with indicated concentrations of GDC-0941 (GDC) in the presence or absence of 1 μM AZD1208 (AZD), as indicated, and analyzed as in A. ( C ) MV4-11 cells were cultured for 18 h with indicated concentrations of AZD1208 (AZD) and analyzed. 4EBP1-S65P: phospho-S65-4EBP1. ( D ) MV4-11 cells were cultured for 18 h with or without 0.25 μM GDC-0941 (GDC) and 1 μM AZD1208 (AZD), as indicated, and analyzed. ( E ) MV4-11 cells were cultured for 6 h with 0.25 μM GDC-0941 (GDC) or 1 μM AZD1208 (AZD), as indicated, and subjected to the cap-binding assay to analyze the eIF4E/eIF4G complex formation. Proteins affinity purified (AP) with m 7 -GTP-sepharose (m 7 -GTP) were subjected to Western blot analysis.

Article Snippet: The STAT5 inhibitor pimozide (sc-203662) and antibodies against HSP90 (sc-13119) and murine/human Pim-2 (sc-13514) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Cell Culture, Western Blot, Binding Assay, Affinity Purification

Journal: Oncotarget

Article Title: FLT3-ITD induces expression of Pim kinases through STAT5 to confer resistance to the PI3K/Akt pathway inhibitors on leukemic cells by enhancing the mTORC1/Mcl-1 pathway

doi: 10.18632/oncotarget.22926

Figure Lengend Snippet: ( A ) 32D/TKD cells overexpressing Pim-1 (Pim1) or vector control cells (C) were treated with or without 1 μM AZD1208 for 6 h, as indicated, and lysed for Western blot analysis with antibodies against indicated proteins. S6K-TP: phospho-T389-p70S6 kinase. ( B ) 32D/TKD cells overexpressing Pim-1 (Pim1) or vector control cells (Cont.) were cultured for 48 h with 0.4 μM GDC-0941 (GDC), 0.2 μM MK-2206 (MK) or 2 μM Pimozide (PMZ), as indicated. The means of relative cell numbers, expressed as percentages of control cells without inhibitors, from triplicate measurements are shown with error bars indicating standard errors. The asterisks indicate statistically significant differences determined by Student’s t -test ( * P < 0.05). ( C , D ) 32D/TKD cells overexpressing Pim-1 (Pim1) or vector control cells (Cont.) were cultured for 48 h with 3 μM GDC-0941 (GDC) or 2 μM MK-2206 (MK), as indicated, and analyzed for the cellular DNA content by flow cytometry. The means of percentages of apoptotic cells with sub-G1 DNA content are shown with error bars indicating standard errors. The asterisks indicate statistically significant differences determined by Student’s t -test ( * P < 0.05). ( E ) 32D/TKD cells overexpressing Pim-1 (Pim1) or vector control cells (Cont.) were cultured for 6 h with 1 μM MK-2206 (MK), 1 μM GDC-0941 (GDC), 50 nM gilteritinib (Gilt.), or 1 μM AZD1208 (AZD), as indicated. Cells were lysed and subjected to Western blot analysis with antibodies against indicated proteins. Abbreviations: Akt-PT, phospho-T308-Akt; PRAS40-P, phospho-T246-PRAS40; TSC2-S939-P, phospho-S939-TSC2; TSC2-T1462P, phospho-T1462-TSC2; mTOR-PS, phospho-S2481-mTOR; S6K-PT, phospho-T389-p70S6 kinase; 4EBP1-S65P, phospho-S65-4EBP1. ( F ) A schematic model of intracellular signaling mechanisms involving enhancement of the mTORC1/Mcl-1 pathway by Pim kinases expressed through STAT5 activation in regulation of survival and proliferation of FLT3-ITD-positive AML cells in response to the PI3K/Akt pathway inhibitors.

Article Snippet: The STAT5 inhibitor pimozide (sc-203662) and antibodies against HSP90 (sc-13119) and murine/human Pim-2 (sc-13514) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Plasmid Preparation, Control, Western Blot, Cell Culture, Flow Cytometry, Activation Assay

Journal: International Journal of Medical Sciences

Article Title: Estrogen receptor α/prolactin receptor bilateral crosstalk promotes bromocriptine resistance in prolactinomas

doi: 10.7150/ijms.51176

Figure Lengend Snippet: PRL exposure induces ERα phosphorylation via the JAK2-PI3K/Akt-MEK/ERK pathway in primary cultured HPA cells. ( A ) PRLR-knockdown abolishes PRL-induced JAK2-PI3K/Akt-MEK/ERK signaling. After overnight serum-starvation, HPA cells transiently transfected with control or PRLR-specific shRNA (shPRLR) were stimulated with PRL (20 ng/ml) for 4 h and the cells were harvested for western blotting using the indicated antibodies. ( B ) MMQ cells were treated with PRL (20 ng/ml) in the absence and presence of the JAK2 inhibitor AG-490 (50 μM), the PI3K inhibitor wortmannin (0.5 µM), the MEK inhibitor U0126 (10 µM) or the STAT5 inhibitor pimozide (5 µM). AG-490, wortmannin and U0126 partially blocked PRL-induced ERα phosphorylation, with the most profound effect in the AG-490 group; pimozide had no effect on PRL-induced ERα phosphorylation. PRL, prolactin; ERα, estrogen receptor α; HPA, human pituitary adenoma; PRLR, prolactin receptor; sh, short hairpin (RNA); CON. Control; Pim, pimozide; Wort, wortmannin.

Article Snippet: PRL, estradiol (E2), fulvestrant, the JAK2 inhibitor AG-490, the PI3K inhibitor Wortmannin, the ERK inhibitor U0126, and the STAT5 inhibitor pimozide were purchased from Selleck Chemicals.

Techniques: Phospho-proteomics, Cell Culture, Knockdown, Transfection, Control, shRNA, Western Blot

Journal: Lymphatics

Article Title: BCL2 Protein Progressively Declines during Robust CLL Clonal Expansion: Potential Impact on Venetoclax Clinical Efficacy and Insights on Mechanism

doi: 10.3390/lymphatics2020005

Figure Lengend Snippet: IL-15-triggered STAT5/PI-3K pathway reduces BCL2 mRNA within ODN (TLR9)-primed CLL cells. These experiments sought evidence for altered BCL2 mRNA levels after IL15 exposure to ODN-stimulated CLL cells. ODN priming was important because we earlier reported that 20 h ODN exposure elicited the NF-kB-dependent upregulation of IL15 receptors (CD122 and IL15Rα) above negligible baseline levels on resting CLL cells . Any evidence that short-term IL15 exposure resulted in dampened BCL2 mRNA might be explained by an IL15-fostered rise in BCL2 repressive miR (e.g., miR15a/miR16-1) . Our rationale for suspecting a critical role for IL15 cytokine in mediating BCL2 repression was based upon the following. First, IL15 signaling was necessary for a significant rise in p53 TF protein within CLL cells receiving ODN signals ( - ), as well as for significant CLL clonal expansion within ODN-stimulated cultures [ , , ]. Second, the IL15 boost in p53 TF (a direct transactivator of miR15a/miR16-1 synthesis) was detected in undivided CLL cells by at least day 3, even prior to IL15-driven divisions characterized by sustained/further elevated p53 TF protein ( , ). For these experiments, quiescent CLL cells were primed with ODN for 20 h and subsequently pulsed with IL-15 (or medium alone) for 4 h or 20–28 h intervals before total RNA was harvested and BCL2 and MCL1 mRNA quantified by specific q-PCR. ( A,B ) ΔCt values for ( A ) BCL2 mRNA and ( B ) MCL1 mRNA within IL15-pulsed or un-pulsed cultures at differing intervals after the IL15 pulse. A paired, 2-sided t-test was used to compare ΔCt values from primed cultures with/without IL15. ( C ) More intuitive fold-change values, calculated with the 2 −ΔΔCt method , better reveal the altered levels of BCL2 and MCL1 mRNA at 20–28 h after the IL15 pulse. Bars represent the mean ± SD of the diverse experiments, with overlaid symbols representing values from individual CLL. Statistical significance was determined by the non-parametric Mann–Whitney rank sum test. ( C ) Experiments with STAT5/PI-3K inhibitors examined whether IL15 activation of STAT5/PI-3K pathways is critical for IL15-triggered BCL2 mRNA repression, as earlier noted for IL15-facilitated growth of ODN-primed CLL cells . Specific inhibitors of PI-3K (LY294002), STAT5 (pimozide or STAT5 INH II), or vehicle alone were added to ODN-primed cultures 30 min prior to a 20 h pulse with IL-15. By q-PCR, the yield of BCL2 mRNA in IL15 pulsed cultures was compared to the yield in ODN-primed cultures that had been exposed to DMSO but not IL15. Consequently, IL15-induced fold change in BCL2 mRNA was determined. (Note: in cultures pulsed with DMSO alone, IL15-elicited fold change in BCL2 mRNA was 0.57 ± 0.04 (mean ± SD) for the diverse experiments). When pooling inhibitor results from these experiments, data from each CLL experiment was normalized by comparing IL15-induced fold change “with inhibitor” to IL15-induced fold change “with DMSO alone”. From the latter determinations, plotted values for % of IL15-elicited fold change without inhibitor” could be calculated. Bar blots display median/range values for the above determination noted with the diverse CLL evaluated, with overlaid symbols representing values for individual CLL evaluated. The dotted horizontal line represents the fact that, with this approach, all values for “IL15-induced fold change without inhibitor” are effectively normalized to 100%. Any rise in this relative percentage indicates that the inhibitor blocked the IL15-induced BCL2 mRNA decline. p values for statistical significance were determined using a non-parametric Mann–Whitney rank sum test.

Article Snippet: In other cultures involving inhibitors of STAT5 and PI-3K, LY294002 (Selleckchem), an inhibitor of PI3K p110α, β, and ɣ isoforms [ ], was used at 20 mM; STAT5 inhibitor, pimozide (also known as STAT Inhibitor III) (Calbiochem; CAS2062-78-4) and STAT5 Inhibitor II (CAS 285986-31-4) were used at doses near their reported IC50 values: pimozide (5 mM) [ ] and weaker STAT5 Inhibitor II (47 mM) [ ].

Techniques: MANN-WHITNEY, Activation Assay

Journal: International Journal of Medical Sciences

Article Title: Estrogen receptor α/prolactin receptor bilateral crosstalk promotes bromocriptine resistance in prolactinomas

doi: 10.7150/ijms.51176

Figure Lengend Snippet: Synergistic effects of PRL and E2 on the proliferation of MMQ/BRO cells and tumor growth in nude mice. ( A ) MMQ/BRO cells and ( B ) MMQ cells were treated with PRL (0, 5, 10 and 20 ng/ml) in the presence or absence of E2 (0, 0.625, 1.25, 2.5, 5, 10, 20 and 40 µM) for three days. Cell viability was assessed using the Cell Counting Kit 8 assay. ( C ) Representative image of xenograft tumors isolated from nude mice. Human prolactinoma tissue xenograft mice were treated with E2 (25 ng/mouse), rhPRL (10 µg/mouse) or E2 plus rhPRL every three days for a total of 10 injections (n=4 per group). ( D ) Tumor volume as calculated according to the formula 0.5 x length x width 2 . ( E ) HE (magnification, x40) and Ki67 (magnification, x10) staining of human prolactinoma tissue xenograft tumors in nude mice (from left to right). All data are presented as the mean ± SD. ** P <0.05. PRL, prolactin; E2, estradiol; CON, control; rh, recombinant human; ns, not significant; HE, hematoxylin and eosin.

Article Snippet: PRL, estradiol (E2), fulvestrant, the JAK2 inhibitor AG-490, the PI3K inhibitor Wortmannin, the ERK inhibitor U0126, and the STAT5 inhibitor pimozide were purchased from Selleck Chemicals.

Techniques: Cell Counting, Isolation, Staining, Control, Recombinant

Journal: International Journal of Medical Sciences

Article Title: Estrogen receptor α/prolactin receptor bilateral crosstalk promotes bromocriptine resistance in prolactinomas

doi: 10.7150/ijms.51176

Figure Lengend Snippet: PRL induces ERα activity, while E2 activates PRLR signaling. ( A ) Effects of E2, PRL or E2 plus PRL on pERα, ERα, PRLR and D2R protein levels in MMQ and MMQ/BRO cells. MMQ or MMQ/BRO cells were serum-starved for 24 h, and then cultured with E2 (10 µM) or PRL (20 ng/ml) for 4 h. E2 and PRL stimulation resulted in upregulated pERα and PRLR, but decreased D2R expression. ERα had no notable effects. ( B ) Luciferase reporter assay demonstrating the synergistic effects of E2 and PRL on ER-mediated transcription. MMQ and MMQ/BRO cells were transfected with ERE (estrogen Response element) -Luc, and then treated with PRL (20 ng/ml) in the absence and presence of E2 (10 µM) for 4 h (Student's t-test; ** P <0.05). ( C ) Protein expression levels of pERα, ERα and PRLR in primary cultured HPA cells transiently transfected with control or ERα-specific shRNA (shERα), as detected by western blotting. ERα-knockdown (10 µM, 4 h) abolished E2-induced PRLR upregulation in HPA cells. ( D ) Fulvestrant abolished E2-induced pERα and PRLR upregulation in HPA cells. Primary cultured HPA cells were treated with E2 (10 µM) in the absence and presence of fulvestrant (20 nM) for 24 h. The protein expression levels of pERα, ERα and PRLR were detected by western blotting. E2, estradiol; PRL, prolactin; ERα, estrogen receptor α; pERα, phosphorylated ERα; PRLR, prolactin receptor; D2R, dopamine D2 receptor; CON, control; FUL, fulvestrant; sh, short hairpin (RNA); HPA, human pituitary adenoma.

Article Snippet: PRL, estradiol (E2), fulvestrant, the JAK2 inhibitor AG-490, the PI3K inhibitor Wortmannin, the ERK inhibitor U0126, and the STAT5 inhibitor pimozide were purchased from Selleck Chemicals.

Techniques: Activity Assay, Cell Culture, Expressing, Luciferase, Reporter Assay, Transfection, Control, shRNA, Western Blot, Knockdown

Journal: International Journal of Medical Sciences

Article Title: Estrogen receptor α/prolactin receptor bilateral crosstalk promotes bromocriptine resistance in prolactinomas

doi: 10.7150/ijms.51176

Figure Lengend Snippet: PRL exposure induces ERα phosphorylation via the JAK2-PI3K/Akt-MEK/ERK pathway in primary cultured HPA cells. ( A ) PRLR-knockdown abolishes PRL-induced JAK2-PI3K/Akt-MEK/ERK signaling. After overnight serum-starvation, HPA cells transiently transfected with control or PRLR-specific shRNA (shPRLR) were stimulated with PRL (20 ng/ml) for 4 h and the cells were harvested for western blotting using the indicated antibodies. ( B ) MMQ cells were treated with PRL (20 ng/ml) in the absence and presence of the JAK2 inhibitor AG-490 (50 μM), the PI3K inhibitor wortmannin (0.5 µM), the MEK inhibitor U0126 (10 µM) or the STAT5 inhibitor pimozide (5 µM). AG-490, wortmannin and U0126 partially blocked PRL-induced ERα phosphorylation, with the most profound effect in the AG-490 group; pimozide had no effect on PRL-induced ERα phosphorylation. PRL, prolactin; ERα, estrogen receptor α; HPA, human pituitary adenoma; PRLR, prolactin receptor; sh, short hairpin (RNA); CON. Control; Pim, pimozide; Wort, wortmannin.

Article Snippet: PRL, estradiol (E2), fulvestrant, the JAK2 inhibitor AG-490, the PI3K inhibitor Wortmannin, the ERK inhibitor U0126, and the STAT5 inhibitor pimozide were purchased from Selleck Chemicals.

Techniques: Phospho-proteomics, Cell Culture, Knockdown, Transfection, Control, shRNA, Western Blot

Journal: International Journal of Medical Sciences

Article Title: Estrogen receptor α/prolactin receptor bilateral crosstalk promotes bromocriptine resistance in prolactinomas

doi: 10.7150/ijms.51176

Figure Lengend Snippet: ERα-knockdown significantly sensitizes cells to bromocriptine in HPA cells. ( A ) Cell survival rate of shRNA-ERα or Vector-transfected HPA cells after treatment with different concentrations of bromocriptine (0, 5, 10, 20 and 40 µM) in the presence of E2 (10 µM) plus PRL (20 ng/ml) for three days (Student's t-test; ** P <0.05). ( B ) CD133+/nestin+ HPA stem-like cells were transiently transfected with control or ERα-specific shRNA (shERα) and cultured with bromocriptine (25 µM) for five days. Primary tumorspheres were counted and images were captured after five days. The average number of tumorsphere was recorded as the mean ± S.E. ( ** P ≤ 0.05 vs. corresponding control cells). ( C ) Representative images of the tumor spheres. Sphere forming ability of HPA cells with ERα knockdown was significantly reduced, compared with that of vector and control group. sh, short hairpin (RNA); ERα, estrogen receptor α; HPA, human pituitary adenoma; E2, estradiol; PRL, prolactin; CON, control.

Article Snippet: PRL, estradiol (E2), fulvestrant, the JAK2 inhibitor AG-490, the PI3K inhibitor Wortmannin, the ERK inhibitor U0126, and the STAT5 inhibitor pimozide were purchased from Selleck Chemicals.

Techniques: Knockdown, shRNA, Plasmid Preparation, Transfection, Control, Cell Culture

Journal: International Journal of Medical Sciences

Article Title: Estrogen receptor α/prolactin receptor bilateral crosstalk promotes bromocriptine resistance in prolactinomas

doi: 10.7150/ijms.51176

Figure Lengend Snippet: PRL exposure induces ERα phosphorylation via the JAK2-PI3K/Akt-MEK/ERK pathway in primary cultured HPA cells. ( A ) PRLR-knockdown abolishes PRL-induced JAK2-PI3K/Akt-MEK/ERK signaling. After overnight serum-starvation, HPA cells transiently transfected with control or PRLR-specific shRNA (shPRLR) were stimulated with PRL (20 ng/ml) for 4 h and the cells were harvested for western blotting using the indicated antibodies. ( B ) MMQ cells were treated with PRL (20 ng/ml) in the absence and presence of the JAK2 inhibitor AG-490 (50 μM), the PI3K inhibitor wortmannin (0.5 µM), the MEK inhibitor U0126 (10 µM) or the STAT5 inhibitor pimozide (5 µM). AG-490, wortmannin and U0126 partially blocked PRL-induced ERα phosphorylation, with the most profound effect in the AG-490 group; pimozide had no effect on PRL-induced ERα phosphorylation. PRL, prolactin; ERα, estrogen receptor α; HPA, human pituitary adenoma; PRLR, prolactin receptor; sh, short hairpin (RNA); CON. Control; Pim, pimozide; Wort, wortmannin.

Article Snippet: PRL, estradiol (E2), fulvestrant, the JAK2 inhibitor AG-490, the PI3K inhibitor Wortmannin, the ERK inhibitor U0126, and the STAT5 inhibitor pimozide were purchased from Selleck Chemicals.

Techniques: Phospho-proteomics, Cell Culture, Knockdown, Transfection, Control, shRNA, Western Blot

Journal: International Journal of Medical Sciences

Article Title: Estrogen receptor α/prolactin receptor bilateral crosstalk promotes bromocriptine resistance in prolactinomas

doi: 10.7150/ijms.51176

Figure Lengend Snippet: PRL exposure induces ERα phosphorylation via the JAK2-PI3K/Akt-MEK/ERK pathway in primary cultured HPA cells. ( A ) PRLR-knockdown abolishes PRL-induced JAK2-PI3K/Akt-MEK/ERK signaling. After overnight serum-starvation, HPA cells transiently transfected with control or PRLR-specific shRNA (shPRLR) were stimulated with PRL (20 ng/ml) for 4 h and the cells were harvested for western blotting using the indicated antibodies. ( B ) MMQ cells were treated with PRL (20 ng/ml) in the absence and presence of the JAK2 inhibitor AG-490 (50 μM), the PI3K inhibitor wortmannin (0.5 µM), the MEK inhibitor U0126 (10 µM) or the STAT5 inhibitor pimozide (5 µM). AG-490, wortmannin and U0126 partially blocked PRL-induced ERα phosphorylation, with the most profound effect in the AG-490 group; pimozide had no effect on PRL-induced ERα phosphorylation. PRL, prolactin; ERα, estrogen receptor α; HPA, human pituitary adenoma; PRLR, prolactin receptor; sh, short hairpin (RNA); CON. Control; Pim, pimozide; Wort, wortmannin.

Article Snippet: PRL, estradiol (E2), fulvestrant, the JAK2 inhibitor AG-490, the PI3K inhibitor Wortmannin, the ERK inhibitor U0126, and the STAT5 inhibitor pimozide were purchased from Selleck Chemicals.

Techniques: Phospho-proteomics, Cell Culture, Knockdown, Transfection, Control, shRNA, Western Blot