Journal: Oncotarget

Article Title: PI3K/AKT/mTOR and sonic hedgehog pathways cooperate together to inhibit human pancreatic cancer stem cell characteristics and tumor growth

doi:

Figure Lengend Snippet: A. CD44 + CD24 + ESA + CSCs were isolated from human primary pancreatic tumors, and treated with NVP-BEZ-235 (0–5 μM) for 7 days. At the end of incubation period, spheroids were collected, reseeded and treated with NVP-BEZ-235 for another week to obtain secondary spheroids. *, @, &, #, and ** = significantly different from control, P < 0.05. B. Pancreatic CSCs were seeded in soft agar and treated with NVP-BEZ-235 (0–5 μM) for 21 days. At the end of incubation period, numbers of colonies were counted. *, &, %, and # = significantly different from control, P < 0.05. C. Pancreatic CSCs were treated with different concentrations of NVP-BEZ-235 (0–2 μM) and expression of phospho-Akt, Phospho-PI3K, Phospho-p70S6K, Phospho-GSK3Kβ, Phospho-4EBP1, total Akt, p70S6K, total GSK3Kβ, and total 4EBP1 was analyzed by Western blot analysis. β-Actin was used as a loading control. D. Pancreatic CSCs were treated with NVP-LDE-225 (0–15 μM) for 7 days. At the end of incubation period, spheroids were collected, reseeded and treated with NVP-LDE-225 for another week to obtain secondary spheroids. *, @, &, #, and ** = significantly different from control, P < 0.05. E. Pancreatic CSCs were seeded in soft agar and treated with NVP-LDE-225 (0–15 μM) for 21 days. At the end of incubation period, numbers of colonies were counted. *, &, %, and # = significantly different from control, P < 0.05. F. CSCs were transduced with Gli-responsive GFP/firefly luciferase viral particles (pGreen Fire1-Gli with EF1, System Biosciences), and treated with NVP-LDE-225 (0–10 μM) for 36 h. Gli reporter activity was measured by luciferase assay. G. Pancreatic CSCs were treated with NVP-LDE-225 (0–10 μM) for 48 h. The expression of Gli1 and Gli2 was measured by qRT-PCR. Data represent mean ± SD. * &, and % = significantly different from control, P < 0.05. H. Effect of NVP-LDE-225 on components of Shh pathway. Pancreatic CSCs were treated with different concentrations of NVP-LDE-225 (0–10 μM) for 48 h, and expressions of Gli1, Gli2, Patched1, Patched2, smoothened and SuFu were determined by Western blot analysis. β-Actin was used as a loading control.

Article Snippet: Human pancreatic CSCs (1 × 10 6 cells mixed with Matrigel, Becton Dickinson, Bedford, MA, in 75 μl total volume, 50:50 ratio) were injected subcutaneously into the flanks of 4–6 weeks old NOD/SCID IL2Rγ null mice.

Techniques: Isolation, Incubation, Control, Expressing, Western Blot, Transduction, Luciferase, Activity Assay, Quantitative RT-PCR

Journal: Oncotarget

Article Title: PI3K/AKT/mTOR and sonic hedgehog pathways cooperate together to inhibit human pancreatic cancer stem cell characteristics and tumor growth

doi:

Figure Lengend Snippet: A. Effects LDE-225, BEZ-235 and their combination on cell viability. Pancreatic CSCs were seeded in 96 well plates and treated with NVP-LDE-225 (5 μM), NVP-BEZ -235 (1 μM) and their combination for 48 h. Thereafter, cell viability was measured by XTT assay. Viable cells were quantified. B. Pancreatic CSCs were treated with NVP-LDE-225 (5 μM), NVP-BEZ -235 (1 μM) and their combination for 48 h. Thereafter, cell apoptosis was measured by PI staining. Data are representative of 3 independent experiments. C. Quantification of apoptotic cells. Data represent mean ( n = 4) ± S.D. *, #, and $ = significantly different from control, P < 0.05. D. Expression of cleaved caspase-3 and cleaved PARP in pancreatic CSCs treated with NVP-LDE-225 (5 μM), NVP-BEZ -235 (1 μM) and their combination. Cells were treated with these drug(s) for 48 h, and the expression of cleaved caspase-3 and PARP was analyzed by Western blot analysis. E. Pancreatic CSCS were treated with NVP-LDE-225, NVP-BEZ-235 and their combination for 48 h, thereafter, fresh media added to cells. After 10 days, colonies were observed by staining the plates with 0.5% crystal violet strain. F. Quantification of pancreatic CSCs colonies. Data represent mean ( n = 4) ± S.D. *, #, and $ = significantly different from control, P < 0.05. G. Effects of NVP-LDE-225, and/or NVP-BEZ -235 on spheroid formation by pancreatic CSC. Pancreatic CSCs were seeded in suspension and treated with NVP-LDE-225 (5 μM), NVP-BEZ -235 (1 μM) and their combination for 7 days. Spheroid images were obtained by light microscopy. H. qRT-PCR analysis of expression profile for Bcl-2, XIAP, BMI and CCND1 in pancreatic CSCs after treatment with NVP-LDE-225 (5 μM), NVP-BEZ -235 (1 μM) and their combination for 36 h. HK-GAPD was used as the endogenous normalization control. Data represent mean ( n = 4) ± S.D. *, #, and @ = significantly different from control, P < 0.05. I. Pancreatic CSCs were treated with NVP-LDE-225, NVP-BEZ-235 and their combination for 48 h, and the expression of Bcl2, Bcl-X L , cIAP1, cIAP2, Bim, cyclin D1, and β-actin (loading control) was measured by Western blot analysis. J. Pancreatic CSCs were transduced with Gli-responsive GFP/firefly luciferase viral particles (pGreen Fire1-Gli with EF1, System Biosciences). After transduction, culture medium was replaced and CSCs were treated with BEZ235 (1 μM) and/or LDE225 (5 μM) for 24 h. Gli reporter activity was measured as we described . K. Pancreatic CSCs were treated with NVP-LDE-225 (5 μM), NVP-BEZ-235 (1 μM) or their combination for 36 h. RNA was extracted and expressions of Gli1, Gli2, Patched1 (PTCH1), and Patched2 (PTCH2) were measured by qRT-PCR. Data represent mean ( n = 4) ± S.D. *, #, and @ = significantly different from control, P < 0.05.

Article Snippet: Human pancreatic CSCs (1 × 10 6 cells mixed with Matrigel, Becton Dickinson, Bedford, MA, in 75 μl total volume, 50:50 ratio) were injected subcutaneously into the flanks of 4–6 weeks old NOD/SCID IL2Rγ null mice.

Techniques: XTT Assay, Staining, Control, Expressing, Western Blot, Suspension, Light Microscopy, Quantitative RT-PCR, Transduction, Luciferase, Activity Assay

Journal: Oncotarget

Article Title: PI3K/AKT/mTOR and sonic hedgehog pathways cooperate together to inhibit human pancreatic cancer stem cell characteristics and tumor growth

doi:

Figure Lengend Snippet: A. Human pancreatic CSCs isolated from primary tumors were treated with NVP-LDE-225, NVP-BEZ-235 and their combination for 7 days to obtain primary spheroids. At the end of incubation period, spheroids were collected, reseeded and treated with NVP-LDE-225 and/or NVP-BEZ-235 for another week to obtain secondary spheroids. Secondary spheroids were collected, reseeded and treated with NVP-LDE-225 and/or NVP-BEZ-235 for another week to obtain tertiary spheroids. Cell viability in spheroids was measured by trypan blue assay at the end of 7, 14 and 21 days. Data represent mean ± SD. *, &, #, % and $ = significantly different from control, P < 0.05. B. Pancreatic CSCs isolated from Pan kras/p53 mice were treated with NVP-LDE-225, NVP-BEZ-235 and their combination for 7 days to obtain primary spheroids. At the end of incubation period, spheroids were collected, reseeded and treated with NVP-LDE-225 and/or NVP-BEZ-235 for another week to obtain secondary spheroids. Secondary spheroids were collected, reseeded and treated with NVP-LDE-225 and/or NVP-BEZ-235 for another week to obtain tertiary spheroids. Cell viability in spheroids was measured by trypan blue assay at the end of 7, 14 and 21 days. Data represent mean ± SD. *, &, #, % and $ = significantly different from control, P < 0.05. C. Pancreatic CSCs were treated with NVP-LDE-225, NVP-BEZ-235 and their combination for 36 h, and expressions of Nanog, Oct-4, c-Myc and Sox-2 were quantified by qRT-PCR. HK-GAPDH was used as the endogenous normalization control. Data represent mean ( n = 4) ± SD. * = significant difference from control, P < 0.05. $ = significant difference from control or single agent alone, P < 0.05. D. Protein expression of Nanog, c-Myc, Oct-4 and Sox-2. Pancreatic CSCs were treated with NVP-LDE-225, NVP-BEZ-235 and their combination for 48 h, and the expression of Nanog, c-Myc, Oct-4 and Sox-2 was determined by the Western blot analysis. β-actin was used as a loading control. E. and F. Immunohistochemical examination of Nanog, Oct-4, c-Myc, and Sox2 in pancreatic CSCs treated with NVP-LDE-225, NVP-BEZ-235 and their combination. Pancreatic CSCs were grown in suspension and treated with above mentioned drugs for 48 h. Spheroids formed by pancreatic CSCs were fixed and IHC was performed as described in Material and Methods.

Article Snippet: Human pancreatic CSCs (1 × 10 6 cells mixed with Matrigel, Becton Dickinson, Bedford, MA, in 75 μl total volume, 50:50 ratio) were injected subcutaneously into the flanks of 4–6 weeks old NOD/SCID IL2Rγ null mice.

Techniques: Isolation, Incubation, Control, Quantitative RT-PCR, Expressing, Western Blot, Immunohistochemical staining, Suspension

Journal: Oncotarget

Article Title: PI3K/AKT/mTOR and sonic hedgehog pathways cooperate together to inhibit human pancreatic cancer stem cell characteristics and tumor growth

doi:

Figure Lengend Snippet: A. Motility Assay. In vitro motility of pancreatic CSCs was observed by using scratch technique. CSCs were grown in monolayer, scratched and treated with NVP-LDE-225, NVP-BEZ-235 and their combination for 48 h. Data are representative of three independent experiments. B. Transwell migration assay. Pancreatic CSCs were plated in the top chamber of the transwell and treated with NVP-LDE-225, NVP-BEZ-235 and their combination for 24 h. cells migrated to the lower chambered were fixed with methanol, stained with crystal violet and counted. Data represent mean ( n = 4) ± S.D. * = significantly different from control, P < 0.05. $ = significantly different from control or single drug treatment, P < 0.05. C–E. Pancreatic CSCs were treated with NVP-LDE-225, NVP-BEZ-235 and their combination for 36 h. After incubation, the expression of snail, slug and Zeb1 was measured by qRT–PCR. Data represent mean ( n = 4) ± S.D. * = significantly different from control, P < 0.05. $ = significantly different from control or single drug treatment, P < 0.05.

Article Snippet: Human pancreatic CSCs (1 × 10 6 cells mixed with Matrigel, Becton Dickinson, Bedford, MA, in 75 μl total volume, 50:50 ratio) were injected subcutaneously into the flanks of 4–6 weeks old NOD/SCID IL2Rγ null mice.

Techniques: Motility Assay, In Vitro, Transwell Migration Assay, Staining, Control, Incubation, Expressing, Quantitative RT-PCR

Journal: Oncotarget

Article Title: PI3K/AKT/mTOR and sonic hedgehog pathways cooperate together to inhibit human pancreatic cancer stem cell characteristics and tumor growth

doi:

Figure Lengend Snippet: A. Pancreatic CSCs were treated with NVP-BEZ-235 (1 μM) for 36 h. RNA was isolated and miRNA array (Affimetrix gene Chip) analysis was performed. Expression of selected miRNAs. P -values and fold change are also shown. B. Bio-function analysis. C. Netwrok analysis. D. Pancreatic CSCs were treated with NVP-LDE-225 (5 μM) for 36 h. RNA was isolated and miRNA array (Affimetrix gene Chip) analysis was performed. Expression of selected miRNAs. P-values and fold change are also shown. E. Bio-function analysis. F. Netwrok analysis. G. Pancreatic CSCs were treated with NVP-BEZ-235 (1 μM) and NVP-LDE-225 (5 μM) for 36 h. RNA was isolated and miRNA array (Affimetrix gene Chip) analysis was performed. Left panel, Expression of selected miRNAs. P-values and fold change are also shown. Right panel, Heat Map of miRNA arrays from NVP-LDE-225- and/or NVP-BEZ-235- treated pancreatic CSCs. H. Bio-function analysis. I. Netwrok analysis.

Article Snippet: Human pancreatic CSCs (1 × 10 6 cells mixed with Matrigel, Becton Dickinson, Bedford, MA, in 75 μl total volume, 50:50 ratio) were injected subcutaneously into the flanks of 4–6 weeks old NOD/SCID IL2Rγ null mice.

Techniques: Isolation, Expressing

Journal: Oncotarget

Article Title: PI3K/AKT/mTOR and sonic hedgehog pathways cooperate together to inhibit human pancreatic cancer stem cell characteristics and tumor growth

doi:

Figure Lengend Snippet: A–C. Expression of Lin28a, Let7a and Kras. Pancreatic CSCs were treated with BEZ235 (1 μM) and/or LDE225 (5 μM) for 36 h. RNA was extracted and the expression of Lin28a, Let7a and Kras was measured by qRT-PCR. Data represent mean ( n = 4) ± S.D. * and $ = significantly different from control, P < 0.05.

Article Snippet: Human pancreatic CSCs (1 × 10 6 cells mixed with Matrigel, Becton Dickinson, Bedford, MA, in 75 μl total volume, 50:50 ratio) were injected subcutaneously into the flanks of 4–6 weeks old NOD/SCID IL2Rγ null mice.

Techniques: Expressing, Quantitative RT-PCR, Control

Journal: Cell reports

Article Title: Impaired islet function and normal exocrine enzyme secretion occur with low inter-regional variation in type 1 diabetes

doi: 10.1016/j.celrep.2024.114346

Figure Lengend Snippet:

Article Snippet: Human Pancreatic Lipase ELISA Kit , Novus Biologicals , Cat# NB016815.

Techniques: Recombinant, Enzyme-linked Immunosorbent Assay, Software, Microscopy

Journal: Journal for Immunotherapy of Cancer

Article Title: Intratumoral combination therapy with poly(I:C) and resiquimod synergistically triggers tumor-associated macrophages for effective systemic antitumoral immunity

doi: 10.1136/jitc-2021-002408

Figure Lengend Snippet: Toxicological and immunomodulatory evaluation in vitro of intracellular TLR agonists alone or combined using primary human macrophages. Macrophages were in vitro differentiated from purified monocytes stimulated with 25 ng/mL of recombinant human macrophage colony-stimulating factor for 6 days (M0); M1 and M2 polarized macrophages were obtained by stimulation with 100 ng/mL of lipopolysaccharide+50 ng/mL of interferon-γ or 20 ng/mL of IL-4, respectively, for 24 hours. (A) Cell viability (Alamar blue) of M0 macrophages exposed 24 hours to TLR agonists alone or in different combinations. concentrations used were 5, 10, 20 and 50 µg/mL, represented by color gradient from left to right. (B–D) cytokine secretion (ELISA) of (B) CXCL10, (C) CCL5, and (D) IL-10 by macrophages exposed for 24 hours to 5 µg/mL of TLR agonists or untreated M0 macrophages. In each panel, M1 and M2 polarized macrophages from the same individuals are shown as reference populations. Each dot corresponds to macrophages from each blood donor. (E) THP-1-Lucia cells for monitoring the NF-kB signal transduction pathway were exposed for 16 hours to pIC and/or R848 at indicated concentrations. Bars represent mean±SD, n=3. (F, G) Cytotoxic activity of TLR-treated macrophages toward human Panc1 cancer cells stained with CellTrace. Each dot corresponds to macrophages from each blood donor. Bars represent mean±SEM. Statistical comparison was performed using one-way analysis of variance followed by Tukey’s multiple comparison test. Statistically significant differences are represented as follows: *p<0.05, **p<0.01, ***p<0.001, and ****p<0.001. IL, interleukin; NF-kB, nuclear factor-kappa B; ns, non-significant; pIC, poly(I:C); R837, imiquimod; R848, resiquimod; TLR, toll-like receptor.

Article Snippet: Human pancreatic carcinoma cell line PANC1 (ATCC), murine fibrosarcoma cell line MN/MCA1, and murine lung adenocarcinoma cell line CMT167 (ECACC) were cultured in DMEM (Lonza) with 10% FBS and 0.5% penicillin/streptomycin (Gibco) at 37°C and 5% CO 2 .

Techniques: In Vitro, Purification, Recombinant, Enzyme-linked Immunosorbent Assay, Transduction, Activity Assay, Staining, Comparison

Journal: Glycobiology

Article Title: DNA methylation and histone H3-K9 modifications contribute to MUC17 expression

doi: 10.1093/glycob/cwq155

Figure Lengend Snippet: Expression of the MU17 gene examined by quantitative RT–PCR and immunohistochemistry. (A) Quantitative RT–PCR results in 10 cell lines. Bars, gene expression levels relative to those in NCI-H292 cells. HPAFII, AsPC1 and LS174T cells showed a high mRNA expression level in MUC17 gene, whereas MCF-7, MDA-MB-453, NCI-H292, A427, PANC1, BxPC-3 and Caco2 cells had no or low expression. (B) MUC17 immunoreactivity in 10 cancer cell lines. MUC17 expression was consistent with the results of quantitative RT–PCR. (C) Quantitative RT–PCR results before and after treatment with 5-AzadC, TSA and 5AzadC/TSA in combination in cells with little or no MUC17 expression. After drug treatments, these cells showed significant restoration of MUC17 expression. *P < 0.05, **P < 0.005, when compared with nontreated cells; Student's t-test; n.s., not significant.

Article Snippet: Cells and treatment Human pancreatic carcinoma cell lines HPAFII, BxPC-3, PANC1 and AsPC-1, human breast cancer cell lines MCF-7 and MDA-MB-453, human lung cancer cell lines NCI-H292 and A427 and human colon adenocarcinoma cell lines Caco2 and LS174T were obtained from the American Type Culture Collection.

Techniques: Expressing, Quantitative RT-PCR, Immunohistochemistry, Gene Expression

Journal: JCI Insight

Article Title: VentX expression in tumor-associated macrophages promotes phagocytosis and immunity against pancreatic cancers

doi: 10.1172/jci.insight.137088

Figure Lengend Snippet: (A) VentX-TAMs promotes M2 to M1 transition of PDA TAMs. En block PDA tumor tissues were incubated with autologous TAMs transfected with GFP-VentX or GFP control. After 5-day incubation, the tumor endogenous TAMs were isolated, and the percentage of M1- and M2-like TAMs was determined by FACS analysis of respective cell surface markers, CD80 and CD163. (B) The effect of the incubation on Treg differentiation was determined by FACS analysis of percentage of CD4+CD25+Foxp3+ cells. Data represent the mean ± SD from 4 independent experiments. *P < 0.05, paired Student’s t test. (C) The effect of the incubation on proliferation (top) and activation (bottom) of CD8+ TIL cells was determined by FACS analysis of CD8 marker and IFN-γ expression, respectively. (D) Effects of VentX-TAM incubation on differentiation of PDA and infiltrating immune cells, as revealed by immunohistochemical staining. Representative images are shown. Anti-CD163, -CD8, and -vimentin antibodies were used for the immune staining, as indicated. Scale bar: 100 μm.

Article Snippet: Briefly, blood CD8 cells of pancreatic patients were enriched by the Easysep human CD8 enrichment kit (StemCell Technologies, catalog 19053) following the manufacturer’s instructions.

Techniques: Blocking Assay, Incubation, Transfection, Control, Isolation, Activation Assay, Marker, Expressing, Immunohistochemical staining, Staining