rspo conditioned medium  (Proteintech)

Journal: bioRxiv

Article Title: Regulation of HDAC2-PDX1 by RNF125 defines pancreatic cancer development

doi: 10.1101/2020.01.06.896555

Figure Lengend Snippet: ( A ) left, LC-MS/MS analysis of cultured Mia PaCa-2 cells using RNF125RM as bait. right, Networks of nuclear and non-nuclear RNF125 interactors. ( B ) PDX1 transcript levels in Mia PaCa-2 cells transfected with indicated siRNAs. Results are means ± SEM in a representative experiment. ( C ) Validation of RNF125-HDAC2 interaction based on western analysis with the indicated antibodies in total lysates, flow-through (FT) and the FLAG-immunoprecipitated fraction of Mia PaCa-2 cells expressing empty vector (EV) or FLAG-tagged RNF125 RM . ( D ) Interaction of endogenous RNF125 with HDAC2 in Mia PaCa-2 and KPC cells based on western blotting with an HDAC2 antibody in input and indicated immunoprecipitation fractions, and with or without the proteasome inhibitor MG132. ( E ) Western blot of RNF125 in input and indicated immunoprecipitated fractions showing interaction of endogenous RNF125 with HDAC2 in the cytoplasm (Cyt) and nucleus (Nuc) of Mia PaCa-2 cells. ( F) HDAC2 staining of normal pancreas from WT and Rnf125 −/− mice. Scale bar in low magnification panels, 250μm and in inset, 100 μm. ( G ) HDAC2 staining of control and shRNF125 KPC tumors. Scale bar in low magnification panels, 400μm and in inset, 50 μm. ( H ) HDAC2 staining in RNF125 Low vs. RNF125 High human PDA. Scale bar in low magnification panels, 600 μm and in inset, 150μm. ( I ) K63 HDAC2 ubiquitination of as analyzed in HEK293T cells overexpressing HDAC2, HA-Ub, and different RNF125 levels. IP, HDAC2; immunoblot, anti K63-linked Ub antibody.

Article Snippet: Briefly, bulk orthotopic KPC tumors were minced and digested overnight with collagenase XI and dispase, washed, counted, embedded in growth factor-reduced (GFR) matrigel (Corning, 1 × 10 5 cells suspended in a 50μl matrigel dome), and cultured in murine complete medium [Advanced DMEM/F12 medium supplemented with HEPES (1x, Corning), Glutamax (1x, Gibco), penicillin/streptomycin (1x, Gibco), B27 (1x, Invitrogen), N-acetyl-L-cystenine (1mM, Sigma), A83-01 (500 nM, Sigma), RSPO-1 conditioned medium (10% v/v, Cultrex® Rspo1 produced by HEK293T cells, Trevigen), mNoggin (0.1 μg/ml, Peprotech), mouse epidermal growth factor (mEGF, 50 ng/ml), Gastrin-I (10nM, Peprotech), human fibroblast growth factor 10 (hFGF10, 100 nm/ml, Peprotech), and nicotinamide (10 mM, Sigma)].

Techniques: Liquid Chromatography with Mass Spectroscopy, Cell Culture, Transfection, Western Blot, Immunoprecipitation, Expressing, Plasmid Preparation, Staining

Journal: bioRxiv

Article Title: Regulation of HDAC2-PDX1 by RNF125 defines pancreatic cancer development

doi: 10.1101/2020.01.06.896555

Figure Lengend Snippet: ( A ) NR5A2 transcript levels in Mia PaCa-2 cells transfected with indicated siRNAs. Results represent mean ± SEM in a representative experiment. ( B ) Western blot analysis of HDAC2 stability in Mia PaCa-2 cells with and without RNF125 knockdown. Seventy-two hours after siRNA transfection, cells were treated with 20μM cycloheximide for indicated times. ( C ) Western blot analysis with indicated antibodies upon RNF125 knockdown in different cell lines. ( D, E ) K63 ubiquitination of endogenous HDAC2 as analyzed in Mia PaCa-2 (D) and KPC (E) cells expressing different levels of RNF125. IP, HDAC2; immunoblot, anti K63-linked Ub antibody. ( F ) K48 ubiquitination of HDAC2 as analyzed in HEK293T cells expressing HDAC2 and HA-Ub, under different RNF125 conditions. IP, HDAC2; immunoblot, anti K48-linked Ub antibody. ( G ) K63 ubiquitination of HDAC2 as analyzed in HEK293T cells expressing HDAC2, HA-Ub, and RNF125, in the presence or absence of K63R. IP, HDAC2; immunoblot, anti K63-linked Ub antibody. * P <0.05, ** P <0.01, *** P <0.005, **** P <0.001, NS – nonsignificant.

Article Snippet: Briefly, bulk orthotopic KPC tumors were minced and digested overnight with collagenase XI and dispase, washed, counted, embedded in growth factor-reduced (GFR) matrigel (Corning, 1 × 10 5 cells suspended in a 50μl matrigel dome), and cultured in murine complete medium [Advanced DMEM/F12 medium supplemented with HEPES (1x, Corning), Glutamax (1x, Gibco), penicillin/streptomycin (1x, Gibco), B27 (1x, Invitrogen), N-acetyl-L-cystenine (1mM, Sigma), A83-01 (500 nM, Sigma), RSPO-1 conditioned medium (10% v/v, Cultrex® Rspo1 produced by HEK293T cells, Trevigen), mNoggin (0.1 μg/ml, Peprotech), mouse epidermal growth factor (mEGF, 50 ng/ml), Gastrin-I (10nM, Peprotech), human fibroblast growth factor 10 (hFGF10, 100 nm/ml, Peprotech), and nicotinamide (10 mM, Sigma)].

Techniques: Transfection, Western Blot, Expressing