Journal: Cell Reports Medicine

Article Title: Haploinsufficiency of NFKBIA reshapes the epigenome antipodal to the IDH mutation and imparts disease fate in diffuse gliomas

doi: 10.1016/j.xcrm.2023.101082

Figure Lengend Snippet:

Article Snippet: For near-complete knockdown of NFKBIA , G418-resistant primary human astrocytes transduced to stably express wildtype IDH1 or mutant IDH1- ( R132H ) were transfected with Accell human NFKBIA small interfering (si)RNA or non-targeting control siRNA (Dharmacon), at 20nM concentration using lipofectamine 2000 reagent (Invitrogen) at 1:1 ratio for 48 h. For complete clustered regularly interspaced short palindromic repeats (CRISPR) knockout of NFKBIA , astrocytes were transfected with IκBα Double Nickase Plasmid (h) (sc-400034-NIC, Santa Cruz)—consisting of a pair of plasmids each encoding a D10A mutated Cas9 nuclease and a target-specific 20-nucleotide (nt) guide RNA (gRNA)—using plasmid transfection medium (sc-108062) and Ultra-Cruz transfection reagent (sc-395739) at 1:1.5 ratio and were incubated for 72 h. After incubation, cells were screened for GFP-positivity to select for successfully transfected cells.

Techniques: Plasmid Preparation, Recombinant, Transfection, Activation Assay, Methylation, Marker, DNA Methylation Assay, Sequencing, Empire Assay, Software

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: Nature

Article Title: Hedgehog is an early and late mediator of pancreatic cancer tumorigenesis

doi: 10.1038/nature02009

Figure Lengend Snippet: Cyclopamine treatment blocks tumour formation of human pancreatic adenocarcinoma cells after transplantation into nude mice. a, Schematic indicating sites of tumour cell and cyclopamine/vehicle injections. b, Isolated tumours derived from control or cyclopamine-treated L3.6sl and Panc 05.04 adenocarcinoma cells. Cyclopamine/vehicle injections were initiated either after palpable tumours had formed (delayed) or simultaneously with injection of tumour cells (concurrent). All pictures are shown at the same magnification. c, Weight of isolated tumours. Untreated control tumours of each cell line were adjusted to 1 to allow comparison of relative change in tumour mass. For delayed cyclopamine/vehicle injections, the values are: BxPC3, control 1 (n = 6), cyclopamine 1.1 (n = 5); Panc 05.04, control 1 (n = 5), cyclopamine 0.48 (n = 4); L3.6sl, control 1 (n = 5), cyclopamine 0.39 (n = 4). The values for concurrent cyclopamine/vehicle injections are: L3.6sl, control 1 (n = 4), cyclopamine 0.16 (n = 4). Error bars indicate standard deviation. Double asterisks, P < 0.01. d–h, Histological analysis of the effect of cyclopamine treatment on L3.6sl-derived tumours. d, e, Haematoxylin/eosin staining of sections through the peripheral tumour regions. f, g, TUNEL staining of apoptotic cells in control (f) and cyclopamine-treated (g) tumours. h, Quantification of TUNEL-positive cells in control (blue) and cyclopamine-treated (red) tumours. Error bars indicate s.e.m. Double asterisks, P < 0.01.

Article Snippet: BxPC3 and all the Panc cell lines were grown in RPMI medium (Gibco) supplemented with 10% fetal bovine serum (Gibco), l -glutamine and penicillin/streptomycin; medium for Panc cell lines was also supplemented with insulin–transferrin–selenium (Gibco).

Techniques: Transplantation Assay, Isolation, Derivative Assay, Control, Injection, Comparison, Standard Deviation, Staining, TUNEL Assay

Journal: bioRxiv

Article Title: SHIP2 controls matrix mineralization by regulation of the RhoA/ROCK pathway and remodeling of the actin cytoskeleton

doi: 10.1101/2022.10.30.514432

Figure Lengend Snippet: Immunoblots confirming the absence of SHIP2 protein in independent ATDC5 (a) and Saos-2 clones (b). Wild-type cells plus two clones of ATDC5 (c) and SaOs-2 (d) genetically-depleted for SHIP2 differentiated and then stained with Alizarin Red S. ATDC5 (e) or SaOs-2 (f) cells differentiated in presence of DMSO (vehicle) or SHIP2 inhibitor AS1949490 (0-10 μM) and stained for mineralization with Alizarin Red S. Histograms represent the quantification of the cell staining after extraction. Data is representative of 3 individual experiments (3 wells per experiment). * p <0.05, ** p <0.01, *** p <0.001.

Article Snippet: ATDC5 and SaOS-2 cells were transfected with the mouse or human SHIP2 double nickase plasmid (Santa Cruz, sc-421138-NIC (mouse), sc-401622-NIC (human)) using the UltraCruz transfection reagent and the Plasmid transfection medium (Santa Cruz) following manufacturer’s instructions.

Techniques: Western Blot, Clone Assay, Staining, Extraction

Journal: bioRxiv

Article Title: SHIP2 controls matrix mineralization by regulation of the RhoA/ROCK pathway and remodeling of the actin cytoskeleton

doi: 10.1101/2022.10.30.514432

Figure Lengend Snippet: (a) Differentiated ATDC5 were stained for proteoglycans using Alcian Blue. Histograms represent the quantification of the cell staining after extraction. (b) Relative expression of hypertrophy and terminal differentiation markers (c) and matrix related genes in ATDC5-neg#1 cells. (d) Relative expression of osteoblast differentiation markers in SaOs-2 neg#1 cells. Histograms represent the difference in expression compared to WT cells (b, c, d). Data is representative of 3 individual experiments (3 wells per experiment). *p<0.05, **p<0.01, ***p<0.001. WT vs SHIP2-neg.

Article Snippet: ATDC5 and SaOS-2 cells were transfected with the mouse or human SHIP2 double nickase plasmid (Santa Cruz, sc-421138-NIC (mouse), sc-401622-NIC (human)) using the UltraCruz transfection reagent and the Plasmid transfection medium (Santa Cruz) following manufacturer’s instructions.

Techniques: Staining, Extraction, Expressing

Journal: bioRxiv

Article Title: SHIP2 controls matrix mineralization by regulation of the RhoA/ROCK pathway and remodeling of the actin cytoskeleton

doi: 10.1101/2022.10.30.514432

Figure Lengend Snippet: (a) Immuno-detection and quantification of MMP13 protein and (b) detection of MMP13 activity by collagen zymography in conditioned media of ATDC5 WT and SHIP2-negative ATDC5 cells. Data is representative of two individual experiments.

Article Snippet: ATDC5 and SaOS-2 cells were transfected with the mouse or human SHIP2 double nickase plasmid (Santa Cruz, sc-421138-NIC (mouse), sc-401622-NIC (human)) using the UltraCruz transfection reagent and the Plasmid transfection medium (Santa Cruz) following manufacturer’s instructions.

Techniques: Activity Assay, Zymography

Journal: bioRxiv

Article Title: SHIP2 controls matrix mineralization by regulation of the RhoA/ROCK pathway and remodeling of the actin cytoskeleton

doi: 10.1101/2022.10.30.514432

Figure Lengend Snippet: (a) ATDC5 cells differentiated in presence of DMSO (VEH) or 1.5 μM of AS1949490 (top panels) and SHIP2-negative ATDC5 cells (bottom panels) were immunostained for PI(3,4,5)P3. Arrows indicate focal adhesion-like structures. (b) Immunodetection of Akt and pAKT (S473) protein in SHIP2-negative ATDC5 cells. Scale bars represent 0.01mm.

Article Snippet: ATDC5 and SaOS-2 cells were transfected with the mouse or human SHIP2 double nickase plasmid (Santa Cruz, sc-421138-NIC (mouse), sc-401622-NIC (human)) using the UltraCruz transfection reagent and the Plasmid transfection medium (Santa Cruz) following manufacturer’s instructions.

Techniques: Immunodetection

Journal: bioRxiv

Article Title: SHIP2 controls matrix mineralization by regulation of the RhoA/ROCK pathway and remodeling of the actin cytoskeleton

doi: 10.1101/2022.10.30.514432

Figure Lengend Snippet: (a) Wild-type and SHIP2-negative ATDC5 (top panels) and SaOs-2 (bottom panels) cells immunostained for vinculin. Arrows indicate focal adhesion-like structures. Table shows the number of focal adhesions per cell and their surface. (b) Cell adhesion assay showing increased ATDC5 and SaOs-2 attachment 30 min after seeding cells on fibronectin * p <0.05, ** p <0.01, *** p <0.001 for wild-type vs SHIP2-deleted cells. Scale bars represent 0.01 mm.

Article Snippet: ATDC5 and SaOS-2 cells were transfected with the mouse or human SHIP2 double nickase plasmid (Santa Cruz, sc-421138-NIC (mouse), sc-401622-NIC (human)) using the UltraCruz transfection reagent and the Plasmid transfection medium (Santa Cruz) following manufacturer’s instructions.

Techniques: Cell Adhesion Assay

Journal: bioRxiv

Article Title: SHIP2 controls matrix mineralization by regulation of the RhoA/ROCK pathway and remodeling of the actin cytoskeleton

doi: 10.1101/2022.10.30.514432

Figure Lengend Snippet: (a) Alizarin Red staining of ATDC5 and SaOs-2 WT cells vs SHIP2-negative cells differentiated in presence of 0.3μM of the actin polymerization inhibitor, cytochalasin D. (b) Alizarin Red staining of ATDC5 and SaOs-2 WT cells vs SHIP2-negative ATDC5 cells differentiated in presence of 15 μM of the ROCK pathway inhibitor, Y-27632. (c) Alcian Blue staining of ATDC5 WT cells vs SHIP2-negative cells differentiated in presence of 15 μM of the ROCK pathway inhibitor, Y-27632. Histograms represent the quantification of the cell staining after extraction. *,‡ p <0.05, **,‡‡ p <0.01, ***,‡‡‡ p <0.001. * WT vs SHIP2-neg. ‡ vehicle vs treatment.

Article Snippet: ATDC5 and SaOS-2 cells were transfected with the mouse or human SHIP2 double nickase plasmid (Santa Cruz, sc-421138-NIC (mouse), sc-401622-NIC (human)) using the UltraCruz transfection reagent and the Plasmid transfection medium (Santa Cruz) following manufacturer’s instructions.

Techniques: Staining, Extraction

Journal: bioRxiv

Article Title: SHIP2 controls matrix mineralization by regulation of the RhoA/ROCK pathway and remodeling of the actin cytoskeleton

doi: 10.1101/2022.10.30.514432

Figure Lengend Snippet: In the absence of SHIP2, PI(3,4,5)P3 accumulates, activates ROCK which stimulates the production of ECM through the phosphorylation of Sox9 in chondrocytes. Increased ROCK activity also leads to stabilization of the actin cytoskeleton and inhibition of MV budding and ECM mineralization.

Article Snippet: ATDC5 and SaOS-2 cells were transfected with the mouse or human SHIP2 double nickase plasmid (Santa Cruz, sc-421138-NIC (mouse), sc-401622-NIC (human)) using the UltraCruz transfection reagent and the Plasmid transfection medium (Santa Cruz) following manufacturer’s instructions.

Techniques: Phospho-proteomics, Activity Assay, Inhibition

Journal: International Journal of Molecular Sciences

Article Title: IL11 Activates Pancreatic Stellate Cells and Causes Pancreatic Inflammation, Fibrosis and Atrophy in a Mouse Model of Pancreatitis

doi: 10.3390/ijms23073549

Figure Lengend Snippet: IL11 induces pancreatic stellate cell activation and invasion. ( a ) t-distributed stochastic neighbour embedding (tSNE) plots showing scRNA-seq expression of Il11ra1 , Il6ra and Il-6st ( gp130 ) in mouse pancreatic tissue. Cell clusters were identified using the Tabula Muris web tool ( https://tabula-muris.ds.czbiohub.org/ (accessed on 21 February 2022)). . Black arrows indicate pancreatic stellate cells (PSCs). ( b ) Representative immunostaining images of gp130, IL11RA, IL6RA in PSCs. Cells were counterstained with DAPI. Scale bars: 50 µm. ( c ) Western blot analysis of phosphorylated and total ERK and STAT3 and αSMA in lysates from PSC treated with IL11 (10 ng/mL) or IL 6 (10 ng/mL) across the indicated time-points (0 to 24 h). GAPDH serves as a loading control. ( d ) ELISA-based quantification of secreted MMP2 levels in PSC supernatants. ( e , f ) Representative immunofluorescence images and quantification of αSMA +ve cells, Collagen I intensity/area and EdU +ve cells at baseline and after 24 h treatment with either recombinant human TGFβ1 (5 ng/mL), IL11 (5 ng/mL), bFGF (10 ng/mL), CTGF (50 ng/mL), PDGF (200 ng/mL) or EDN1 (250 ng/mL). Cells were counterstained with DAPI. Scale bar: 200 µm. ( g ) Matrigel invasion capacity of PSCs was determined at baseline and after 24 h treatment with PDGF (20 ng/mL) or with increasing concentrations of IL11 (5–20 ng/mL). Scale bars: 150 µm. AU: Arbitrary Unit. Data are represented as mean ± SD in panel ( d ) and median and whiskers extending from minimum to maximum values in panels ( f , g ). p values were determined by one-way ANOVA with Dunnet’s correction. BL: baseline.

Article Snippet: Primary human pancreatic stellate cells (PSCs; ScienCell, Carlsbad, CA, USA; Cat #3830, Lot #14289) were isolated from human pancreas healthy samples and were maintained in Stellate cell medium (SteCM, Cat #5301, ScienCell, Carlsbad, CA, USA) supplemented with stellate cell growth supplement(ScienCell, Carlsbad, CA, USA Cat #5352), 2% fetal bovine serum (ScienCell, Carlsbad, CA, USA; Cat #0010) and 1% Penicillin-streptomycin (ScienCell, Carlsbad, CA, USA Cat #0503), in an incubator at 37 °C and with an atmosphere of 5% CO 2 .

Techniques: Activation Assay, Expressing, Immunostaining, Western Blot, Control, Enzyme-linked Immunosorbent Assay, Immunofluorescence, Recombinant

Journal: International Journal of Molecular Sciences

Article Title: IL11 Activates Pancreatic Stellate Cells and Causes Pancreatic Inflammation, Fibrosis and Atrophy in a Mouse Model of Pancreatitis

doi: 10.3390/ijms23073549

Figure Lengend Snippet: Autocrine IL11 signalling is important downstream of several pancreatitis factors. ( a ) ELISA of secreted IL11 from PSCs after 24 h treatment with recombinant human TGFβ1 (5 ng/mL), bFGF (10 ng/mL), CTGF (10 ng/mL), PDGF (200 ng/mL), EDN1 (250 ng/mL). ( b , c ) Representative immunofluorescence images and quantification of αSMA +ve cells and collagen I immunostaining of PSCs treated with IgG or the neutralizing IL11RA antibody (X209, 24 h) and profibrotic cytokines listed in panel ( a ). Cells were counterstained with DAPI. Scale bars: 200 µm. ( d ) ELISA of secreted MMP2 and ( e ) Sirius Red quantification of secreted collagen in the culture supernatant of PSCs treated as depicted in panel ( c ). ( f ) Western blot analysis of phosphorylated and total ERK and STAT3, and αSMA in lysates of PSCs treated with various profibrotic stimuli with either IgG or X209 (2 µg/mL, 24 h). ( g ) Matrigel invasion capacity of PSCs treated with IgG or X209 (2 µg/mL) and PDGF (20 ng/mL). Scale bars: 150 µm. AU: arbitrary unit. Data are represented as mean ± SD in ( a , d , e , g ) or as median and whiskers extending from minimum to maximum values in panel ( b ). p values were determined by one-way ANOVA with Dunnett’s correction in panel a , two-way ANOVA (Sidak’s correction) in panels ( b , d , e ) and by Student’s t test in ( g ). BL: baseline.

Article Snippet: Primary human pancreatic stellate cells (PSCs; ScienCell, Carlsbad, CA, USA; Cat #3830, Lot #14289) were isolated from human pancreas healthy samples and were maintained in Stellate cell medium (SteCM, Cat #5301, ScienCell, Carlsbad, CA, USA) supplemented with stellate cell growth supplement(ScienCell, Carlsbad, CA, USA Cat #5352), 2% fetal bovine serum (ScienCell, Carlsbad, CA, USA; Cat #0010) and 1% Penicillin-streptomycin (ScienCell, Carlsbad, CA, USA Cat #0503), in an incubator at 37 °C and with an atmosphere of 5% CO 2 .

Techniques: Enzyme-linked Immunosorbent Assay, Recombinant, Immunofluorescence, Immunostaining, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: IL11 Activates Pancreatic Stellate Cells and Causes Pancreatic Inflammation, Fibrosis and Atrophy in a Mouse Model of Pancreatitis

doi: 10.3390/ijms23073549

Figure Lengend Snippet: IL11 drives pancreatic stellate cell activation via ERK signalling and post-transcriptional effects. ( a ) RNA expression of IL11 , ACTA2 , COL1A1 and TIMP1 in PSCs treated with recombinant human TGFβ1 or IL11 (5 ng/mL; 24 h). ( b ) Representative immunofluorescence images and quantification of αSMA +ve cells, Collagen I intensity/area and EdU +ve cells at baseline and after 24 h treatment with IL11 (5 ng/mL) and ERK inhibitor U0126 (10 µM). Data are represented as mean ± SD in panel ( a ) and as median and whiskers extending from minimum to maximum values in panel ( b ). Scale bars: 100 µm. p values were determined by one way ANOVA (Dunnet’s correction) in ( a ) and one way ANOVA (Tukey’s correction) in ( b ). BL: baseline.

Article Snippet: Primary human pancreatic stellate cells (PSCs; ScienCell, Carlsbad, CA, USA; Cat #3830, Lot #14289) were isolated from human pancreas healthy samples and were maintained in Stellate cell medium (SteCM, Cat #5301, ScienCell, Carlsbad, CA, USA) supplemented with stellate cell growth supplement(ScienCell, Carlsbad, CA, USA Cat #5352), 2% fetal bovine serum (ScienCell, Carlsbad, CA, USA; Cat #0010) and 1% Penicillin-streptomycin (ScienCell, Carlsbad, CA, USA Cat #0503), in an incubator at 37 °C and with an atmosphere of 5% CO 2 .

Techniques: Activation Assay, RNA Expression, Recombinant, Immunofluorescence

Journal: International Journal of Molecular Sciences

Article Title: IL11 Activates Pancreatic Stellate Cells and Causes Pancreatic Inflammation, Fibrosis and Atrophy in a Mouse Model of Pancreatitis

doi: 10.3390/ijms23073549

Figure Lengend Snippet: IL11RA antibody treatment reduces pancreatic fibrosis in a mouse model of pancreatitis. ( a ) Schematic of the induction of pancreatitis in wildtype C57BL/6 mice by pancreatic duct ligation (PDL) and Western blot analysis of IL11 and fibronectin (FN1) expression in pancreatic lysates post-sham or 14 days post-PDL surgery (n = 3/group). ( b ) Schematic of the administration timepoints of neutralizing IL11RA antibody (X209) or IgG control antibody treatment in the PDL model. ( c ) Gross pancreas anatomy and the tissue weights of the ligated splenic lobe in X209 or IgG treated mice. ( d ) Hematoxylin and eosin staining of pancreatic sections from the healthy or ligated splenic lobes of X209 or IgG treated mice. Scale bars: 100 µm. ( e ) Masson’s trichrome staining images and collagen quantification of fibrosis in the ligated splenic lobes of X209 or IgG treated mice (n = 3–4). Scale bars: 1000 µm. AU: arbitrary unit. ( f ) Collagen I immunostaining of the ligated splenic lobes of X209 or IgG treated mice. Scale bars: 50 µm. Data shown as median and whiskers extending from minimum to maximum values. p values were determined by Student’s t -test in ( c ) and one way ANOVA (Tukey’s correction) in ( e , f ).

Article Snippet: Primary human pancreatic stellate cells (PSCs; ScienCell, Carlsbad, CA, USA; Cat #3830, Lot #14289) were isolated from human pancreas healthy samples and were maintained in Stellate cell medium (SteCM, Cat #5301, ScienCell, Carlsbad, CA, USA) supplemented with stellate cell growth supplement(ScienCell, Carlsbad, CA, USA Cat #5352), 2% fetal bovine serum (ScienCell, Carlsbad, CA, USA; Cat #0010) and 1% Penicillin-streptomycin (ScienCell, Carlsbad, CA, USA Cat #0503), in an incubator at 37 °C and with an atmosphere of 5% CO 2 .

Techniques: Ligation, Western Blot, Expressing, Control, Staining, Immunostaining

Journal: International Journal of Molecular Sciences

Article Title: IL11 Activates Pancreatic Stellate Cells and Causes Pancreatic Inflammation, Fibrosis and Atrophy in a Mouse Model of Pancreatitis

doi: 10.3390/ijms23073549

Figure Lengend Snippet: IL11RA antibody treatment attenuates pancreatic inflammation and pathological signalling in a mouse model of pancreatitis. ( a ) Western blot analysis of IL11, IL6, IL1β, TNF, FN1, αSMA, cleaved (Clv.) and total caspase-3 and ( b ) phosphorylated and total protein levels of ERK, STAT3 and NF-kB in lysates from the ligated splenic lobes of X209 or IgG treated mice (n = 4/group). GAPDH served as loading control in ( a ). p values were determined by one way ANOVA (Tukey’s correction).

Article Snippet: Primary human pancreatic stellate cells (PSCs; ScienCell, Carlsbad, CA, USA; Cat #3830, Lot #14289) were isolated from human pancreas healthy samples and were maintained in Stellate cell medium (SteCM, Cat #5301, ScienCell, Carlsbad, CA, USA) supplemented with stellate cell growth supplement(ScienCell, Carlsbad, CA, USA Cat #5352), 2% fetal bovine serum (ScienCell, Carlsbad, CA, USA; Cat #0010) and 1% Penicillin-streptomycin (ScienCell, Carlsbad, CA, USA Cat #0503), in an incubator at 37 °C and with an atmosphere of 5% CO 2 .

Techniques: Western Blot, Control

Journal: Oncology Letters

Article Title: Anticancer effect of arsenite on cell migration, cell cycle and apoptosis in human pancreatic cancer cells

doi: 10.3892/ol.2016.4564

Figure Lengend Snippet: Effects of PDGF-BB on the migration of AsPC-1 and BxPC-3 pancreatic cancer cells, compared with normal PE cells. (A) AsPC-1, (B) BxPC-3 and (C) PE cells (5×104 cells/well) were treated with the indicated doses of arsenite for 24 h, prior to be seeded in the upper Boyden chamber. Following incubation for 16 h, cells were exposed to 30 ng/ml PDGF-BB for 36 h at 37°C. Cells were then fixed, stained and visualized under a microscope. The average number of migrated cells from five randomly selected fields on the lower surface of the membrane was counted. Data were obtained from ≥3 independent experiments. *P<0.05 vs. controls. Right panels show representative images of the migrated cells stained with clonogenic reagent. PDGF, platelet-derived growth factor; PE, pancreatic epithelial.

Article Snippet: Primary normal human pancreatic epithelial (PE) cells were purchased from DS Pharma Biomedical Co., Ltd. (Osaka, Japan) and maintained in CSC medium (Cell Systems Corporation, Kirkland, WA, USA).

Techniques: Migration, Incubation, Staining, Microscopy, Membrane, Derivative Assay

Journal: Oncology Letters

Article Title: Anticancer effect of arsenite on cell migration, cell cycle and apoptosis in human pancreatic cancer cells

doi: 10.3892/ol.2016.4564

Figure Lengend Snippet: Effects of arsenite on PARP cleavage in AsPC-1, BxPC-3 and PE cells. (A) AsPC-1, (B) BxPC-3 and (C) PE cells were exposed to arsenite (30 µM) for the indicated time periods. Protein extracts were then harvested and examined by western blotting using anti-PARP and anti-glyceraldehyde-3-phosphate dehydrogenase antibodies. Background-subtracted signal intensity of each protein band was normalized to GAPDH. Data are presented as the mean ± standard deviation of triplicate assay. *P<0.05. PARP, poly(adenosine diphosphate-ribose) polymerase; PE, pancreatic epithelial; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

Article Snippet: Primary normal human pancreatic epithelial (PE) cells were purchased from DS Pharma Biomedical Co., Ltd. (Osaka, Japan) and maintained in CSC medium (Cell Systems Corporation, Kirkland, WA, USA).

Techniques: Western Blot, Standard Deviation

Journal: Oncology Letters

Article Title: Anticancer effect of arsenite on cell migration, cell cycle and apoptosis in human pancreatic cancer cells

doi: 10.3892/ol.2016.4564

Figure Lengend Snippet: Effects of arsenite on the phosphorylation of p44/p42 MAPK and Akt in AsPC-1, BxPC-3 and PE cells. (A) AsPC-1, (B) BxPC-3 and (C) PE cells were exposed to arsenite (30 µM) and incubated with 30 ng/ml platelet-derived growth factor-BB for the indicated time periods. Protein extracts were then harvested and examined by western blotting using specific antibodies against phospho-p44/p42 MAPK, p44/p42 MAPK, phospho-Akt and Akt. Background-subtracted signal intensity of each protein band was normalized to Akt. Data are presented as the mean ± standard deviation of triplicate assay. *P<0.05 vs. cells without arsenite exposure. PE, pancreatic epithelial; PDGF, platelet-derived growth factor; phospho, phosphorylated; MAPK, mitogen-activated protein kinase; M, marker.

Article Snippet: Primary normal human pancreatic epithelial (PE) cells were purchased from DS Pharma Biomedical Co., Ltd. (Osaka, Japan) and maintained in CSC medium (Cell Systems Corporation, Kirkland, WA, USA).

Techniques: Phospho-proteomics, Incubation, Derivative Assay, Western Blot, Standard Deviation, Marker