Journal: BioMed Research International

Article Title: Cytokine Effects on Cell Viability and Death of Prostate Carcinoma Cells

doi: 10.1155/2014/536049

Figure Lengend Snippet: Western blot analysis of LNCaP protein expression. (a), (b), (c), and (d) LNCaP cells were treated with TNF- α (10 and 100 ng/mL) for 24 h or IL-13 (20 and 100 ng/mL) for 30 and 90 min and 24 h or LY-29004 (20 μ M) for 90 min or pretreated with LY-29004 (20 μ M), BAY-117082 (30 μ M), or SP600125 (30 μ M) for 1 h and then treated with IL-13 (20 and 100 ng/mL) for 90 min. Untreated cells for 30 and 90 min and 24 h were used as control (ctrl). Cell extracts were resolved by SDS-PAGE and analyzed by western blot with antibodies against Bid, Bad, Bax, Bcl-2, Cyclin D1, p-ERK 1/2, I κ B- α , c-IAP2, p-p38, p-Akt, Fas, FLIP L , p-JNK, and Bcl-X L . Reprobing with antibody against α -tubulin or β -actin was used as a loading and transfer marker.

Article Snippet: The following primary antibodies were used: monoclonal mouse anti-Bcl-X L 1 : 100 (sc-8392), monoclonal mouse anti-Bad 1 : 100 (sc-8044), polyclonal rabbit anti-Bid 1 : 100 (sc-11423), polyclonal rabbit anti-Bax 1 : 200 (sc-493), monoclonal mouse anti-FLIP L/S 1 : 200 (sc-5276), monoclonal mouse anti-I κ B α 1 : 100 (sc-1643), monoclonal mouse anti-p-JNK 1 : 200 (sc-6254), polyclonal goat anti-c-IAP1 1 : 200 (sc-1867), polyclonal rabbit anti-c-IAP2 1 : 200 (sc-7944), monoclonal mouse anti-caspase 3 1 : 100 (sc-7272) (all from Santa Cruz Biotechnology, Inc.), monoclonal mouse anti-p-Akt 1 : 200 (4051S), monoclonal mouse anti-p-p44/42 MAPK 1 : 200 (ERK 1/2; 9106S), monoclonal mouse anti-p-p38 1 : 200 (9216S) (all from Cell Signaling), monoclonal mouse anti-Fas 1 : 500 (Millipore, #05-201), monoclonal mouse anti-Bcl-2 1 : 20 (Cell Marque, clone: 124), monoclonal rabbit anti-cyclin-D1 1 : 10 (Cell Marque, clone: SP4), monoclonal mouse anti- β -actin 1 : 5000 (Sigma, A5441) and monoclonal mouse anti- α -tubulin 1 : 5000 (Sigma, T5168).

Techniques: Western Blot, Expressing, SDS Page, Marker

Journal: BioMed Research International

Article Title: Cytokine Effects on Cell Viability and Death of Prostate Carcinoma Cells

doi: 10.1155/2014/536049

Figure Lengend Snippet: Western blot analysis of LNCaP protein expression. (a) and (b) LNCaP cells were treated with TNF- α (10 and 100 ng/mL) for 24 h or IFN- γ (25 ng/mL) for 30, 90 min, and 24 h or pretreated with LY-29004 (20 μ M) for 1 h and then treated with IFN- γ (25 ng/mL) for 90 min. Untreated cells for 90 min and 24 h were used as controls (ctrl). (c) and (d) LNCaP cells were treated with IL-1 β (5 ng/mL) for 30, 90 min, and 24 h or pretreated with UO126 (30 μ M), BAY-117082 (30 μ M), or SP600125 (30 μ M) for 1 h and then treated with IL-1 β (5 ng/mL) for 90 min. Untreated cells for 90 min and 24 h were used as controls (ctrl). (e) and (f) LNCaP and PC-3 cells were treated with IL-1 β (5 ng/mL) or TNF- α (10 and 100 ng/mL) or SP600125 (30 μ M) or UO126 (30 μ M) for 72 h, or pretreated with SP600125 (30 μ M) or UO126 (30 μ M) for 1 h and then treated with IL-1 β (5 ng/mL) for 72 h. Untreated cells for 72 h were used as controls (ctrl). Cell extracts were resolved by SDS-PAGE and analyzed by western blot with antibodies against Bid, Bad, Bax, Bcl-2, Cyclin D1, p-ERK 1/2, p-p38, p-Akt, I κ B- α , c-IAP2, and caspase 3. Reprobing with antibody against α -tubulin or β -actin was used as a loading and transfer marker.

Article Snippet: The following primary antibodies were used: monoclonal mouse anti-Bcl-X L 1 : 100 (sc-8392), monoclonal mouse anti-Bad 1 : 100 (sc-8044), polyclonal rabbit anti-Bid 1 : 100 (sc-11423), polyclonal rabbit anti-Bax 1 : 200 (sc-493), monoclonal mouse anti-FLIP L/S 1 : 200 (sc-5276), monoclonal mouse anti-I κ B α 1 : 100 (sc-1643), monoclonal mouse anti-p-JNK 1 : 200 (sc-6254), polyclonal goat anti-c-IAP1 1 : 200 (sc-1867), polyclonal rabbit anti-c-IAP2 1 : 200 (sc-7944), monoclonal mouse anti-caspase 3 1 : 100 (sc-7272) (all from Santa Cruz Biotechnology, Inc.), monoclonal mouse anti-p-Akt 1 : 200 (4051S), monoclonal mouse anti-p-p44/42 MAPK 1 : 200 (ERK 1/2; 9106S), monoclonal mouse anti-p-p38 1 : 200 (9216S) (all from Cell Signaling), monoclonal mouse anti-Fas 1 : 500 (Millipore, #05-201), monoclonal mouse anti-Bcl-2 1 : 20 (Cell Marque, clone: 124), monoclonal rabbit anti-cyclin-D1 1 : 10 (Cell Marque, clone: SP4), monoclonal mouse anti- β -actin 1 : 5000 (Sigma, A5441) and monoclonal mouse anti- α -tubulin 1 : 5000 (Sigma, T5168).

Techniques: Western Blot, Expressing, SDS Page, Marker

Journal: iScience

Article Title: hGFAP -mediated GLI2 overexpression leads to early death and severe cerebellar malformations with rare tumor formation

doi: 10.1016/j.isci.2023.107501

Figure Lengend Snippet: Representative images and staining of giant cells, tumors and tumor-like structures (A–H) Representative H&E and Ki67 stains of a hG::p53 Fl/Fl ;Gli2N + animal ( Figures 4 A–4D). Giant cells were found near the piriform cortex ( Figure 4 B), near the hippocampus area and caudate putamen ( Figure 4 C), and the cerebellum ( Figure 4 D). Scale bar is 1 mm for images of the whole brain demonstrated on the left, 20 μm for H&E in B, C, and D and 50 μm for Ki67 staining. E: hG::p53 Fl/Fl ;Gli2N + mouse showed so-called giant cells, stained with Olig2 ( Figure 4 E, scale bar: 200μm) and Cyclin-D1 ( Figure 4 F, scale bar: 200 μm), both show expression. High-power images of Olig-2 expression giant cells ( Figure 4 G, scale bar: 20 and 50 μm) and Cyclin-D1 expressing giant cell ( Figure 4 H, scale bar: 20 and 50 μm) are demonstrated. (I–L) In one out of 23 hG::Gli2N + mice, a tumor in the cerebellum (scale bar: 1 mm) was observed and is illustrated here. Besides H&E staining ( Figure 3 J, scale bar: 20 and 50 μm) and Ki67 staining (Fig, 3K, scale bar: 20 and 50 μm), also Nmyc staining was applied ( Figure 3 L, scale bar: 20 and 50 μm). (M–O) The single of the 12 transgenic hG::NMYC Fl/+ ;Gli2N + mice with tumor-like structures is displayed (scale bar: 1 mm). Tumor-like structures demonstrated in the brain stem ( Figure 3 N) and in the subventricular zone (SVZ) ( Figure 3 O) are shown after H&E, Ki67, and Nmyc staining (scale bar: 20 μm H&E and 50 μm Ki67 and Nmyc). (P) Mouse body weights of hG::Gli2N + , hG::NMYC Fl/+ ;Gli2N + , hG::p53 Fl/Fl ;Gli2N + , and control mice ( p53 Fl/Fl ;Gli2N + , NMYC Fl/+ ;Gli2N + , and Gli2N + ) are displayed. For statistical analysis, a grouped analysis with multiple t test was applied compared with the sum of the control mice ( p ∗0.03 for of hG::NMYC Fl/+ ;Gli2N + mice). Data are shown as mean ± standard error of mean (SEM), and p values less than 0.05 were considered significant.

Article Snippet: Rabbit monoclonal anti-Cyclin D1 , Abcam , Cat# ab134175; RRID: AB_2750906.

Techniques: Staining, Expressing, Transgenic Assay

Journal: iScience

Article Title: hGFAP -mediated GLI2 overexpression leads to early death and severe cerebellar malformations with rare tumor formation

doi: 10.1016/j.isci.2023.107501

Figure Lengend Snippet:

Article Snippet: Rabbit monoclonal anti-Cyclin D1 , Abcam , Cat# ab134175; RRID: AB_2750906.

Techniques: Clone Assay, Recombinant, Plasmid Preparation, Software, Imaging

Journal: PLoS ONE

Article Title: Syndecan-1 deficiency promotes tumor growth in a murine model of colitis-induced colon carcinoma

doi: 10.1371/journal.pone.0174343

Figure Lengend Snippet: (A) Quantitative RT-PCR analysis revealed increased levels of Cyclin D1 (left), CCL-2 (middle) and Myc (right) in AOM-DSS induced colonic tumors derived from Sdc1-KO as compared to WT mice (n = 5). (B) Representative immunoreactive staining (brown) for Cyclin D1 in AOM-DSS induced colonic tumors (day 61) of WT and Sdc1-KO mice. Scale bar, 100 μm. (C) Quantification of average numbers of Cyclin D1 positive cells per high power field (X400) in ≥ 12 fields of each slide from 3 mice of each group. Error bars represent mean ± SE. *P < 0.05, ***P < 0.001 by Student’s t test. (D) Immunostaining for cMyc (brown) revealed increased levels of cMyc protein in AOM-DSS induced colonic tumors derived from Sdc1-KO vs. WT mice. Of note, cytoplasmic localization of cMyc was previously reported in several pathophysiological settings, including tumors of diverse origins (reviewed in ).

Article Snippet: Primary antibodies used were rat monoclonal anti-Sdc-1 (clone 281–2, Thermo Scientific), rabbit monoclonal anti-cyclin D1 (1:50, Thermo SCIENTIFIC), rabbit monoclonal anti-p-STAT3 (1:100, Cell Signaling), rat monoclonal anti-F4/80 (1:400, Serotec), mouse monoclonal anti-cMyc, rabbit polyclonal anti beta-catenin (1:400, Santa Cruz) and rabbit polyclonal anti-IL-6 (1:400, Abcam).

Techniques: Quantitative RT-PCR, Derivative Assay, Staining, Immunostaining