Journal: Computational and Structural Biotechnology Journal

Article Title: Spatial domains identification in spatial transcriptomics using modality-aware and subspace-enhanced graph contrastive learning

doi: 10.1016/j.csbj.2024.10.029

Figure Lengend Snippet: GRAS4T accurately identified spatial domains in the HER2+ dataset. (a) Boxplot of ARI values across all sections of the HER2+ dataset for all compared methods. (b) The H&E image and manual annotation for the A1 section. (c) Spatial domains of HER2+ dataset in (b) detected by STAGATE, CCST, and GRAS4T.

Article Snippet: The efficacy of GRAS4T was further evaluated using the human HER2-positive breast tumor (HER2+) dataset , which used different spatial technologies (spatial transcriptomics) compared to the DLPFC dataset.

Techniques:

Journal: Cell reports

Article Title: A Mouse Homolog of a Human TP53 Germline Mutation Reveals a Lipolytic Activity of p53

doi: 10.1016/j.celrep.2019.12.074

Figure Lengend Snippet: (A) ADRB3 and p21 mRNA levels measured by RT-PCR (top) and p53 protein expression evaluated by immunoblotting in iWAT of the indicated p53 genotype (n = 4). Adipocytes differentiated from p53 178C/C SVF cells are shown as a positive control. p53 protein levels were quantified relative to GAPDH by densitometric scanning. (B) Immunoblots of total and phosphorylated HSL (Ser-660, p-HSL) in iWAT (n = 3). (C) SVFs isolated from iWAT of p53 178C/C mice were transduced with a non-specific (NS) or p53 shRNA lentivirus, differentiated into adipocytes, and analyzed by RT-PCR (n = 3). (D) The C/C adipocytes described in (C) were immunoblotted with antibodies, including those against phospho-PKA substrates (RRXS/T motif) and the PKAα catalytic subunit. Shown is one representative of 4 experiments with 3 biological replicates. (E) Glycerol release into culture medium by C/C adipocytes transduced with shRNA as an in vitro measure of lipolysis activity (n = 4). (F) Immunoblot of iWAT samples from the indicated p53 genotype mice after control saline, ADRB3 agonist CL316243 (CL), or ADRB3 antagonist SR59230A (SR) injection. (G) Effect of daily injections of control saline, CL, or SR for 5 days on body fat composition of mice (~15 weeks old), measured by NMR analyzer (n = 10–11). p53 R178 genotypes: wild-type ( R/R ), homozygous mutant ( C/C ), and null (−/−). Statistical difference by one-way ANOVA (A) and two-tailed unpaired t test (B, E, and G). Values are mean ± SEM. *p < 0.05. See also .

Article Snippet: Antibodies used in this study were as follows: ADRB3 and GAPDH (Thermo Fisher Scientific); α-tubulin (Sigma-Aldrich); p21 (CDKN1A, Millipore Sigma); PKAα cat (A-2, Santa Cruz Biotechnology); HSL, phospho-HSL (Ser660), phospho-PKA Substrate (RRXS*/T*) and p53 (1C12) (Cell Signaling Technology); TFAM, Total OXPHOS Rodent Antibody Cocktail and UCP1 (Abcam).

Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing, Western Blot, Positive Control, Isolation, Transduction, shRNA, In Vitro, Activity Assay, Control, Saline, Injection, Mutagenesis, Two Tailed Test

Journal: Cell reports

Article Title: A Mouse Homolog of a Human TP53 Germline Mutation Reveals a Lipolytic Activity of p53

doi: 10.1016/j.celrep.2019.12.074

Figure Lengend Snippet: KEY RESOURCE TABLE

Article Snippet: Antibodies used in this study were as follows: ADRB3 and GAPDH (Thermo Fisher Scientific); α-tubulin (Sigma-Aldrich); p21 (CDKN1A, Millipore Sigma); PKAα cat (A-2, Santa Cruz Biotechnology); HSL, phospho-HSL (Ser660), phospho-PKA Substrate (RRXS*/T*) and p53 (1C12) (Cell Signaling Technology); TFAM, Total OXPHOS Rodent Antibody Cocktail and UCP1 (Abcam).

Techniques: Control, Recombinant, Staining, Luciferase, Reporter Assay, Mutagenesis, Cloning, shRNA, CRISPR, Plasmid Preparation, Software

Journal: PLoS ONE

Article Title: ADAM17 Silencing in Mouse Colon Carcinoma Cells: The Effect on Tumoricidal Cytokines and Angiogenesis

doi: 10.1371/journal.pone.0050791

Figure Lengend Snippet: ( A ) RT-PCR analysis of the expression of TGFα, EGFR and ErbB4 in MC38CEA cells in culture, in MC38CEA tumors and in positive controls: murine brain endothelial cells (MBE) and murine breast cancer cell line (4T1) and murine brain. cDNA from: M – mock-transfected cells, TM – mock-transfected tumors, S – ADAM17-silenced cells, TS – ADAM17-silenced tumors. Amplification of cDNA coding for elongation factor 2 (EF2) was performed as a control of samples' quality. ( B ) Analysis of the effect of exogenous EGF on 3 H-thymidine incorporation into MC38CEA or 4T1 cells (positive control). * – P <0.01 vs. control. ( C ) Quantitative RT-PCR analysis of ErbB2 and ErbB3 expression in ADAM17-silenced (S2, S3) and mock-transfected (M1, M2, M3) cell lines and in control cell lines, MBE and 4T1 as well as in murine tissues. ( D ) The levels of NRG-1 released to the medium from ADAM17-silenced (S2, S3) and mock-transfected (M1, M3) cells measured by ELISA. * – P <0.01 vs. M1 and M3. ( E ) Western blotting analysis of ErbB2 phosphorylation in ADAM17-silenced (S2, S3), mock-transfected (M1, M2, M3) and wild-type (wt) MC38CEA cells incubated in the serum-free medium alone or in the medium enriched in exogenous rmNRG-1β. GAPDH was used as a loading control. Quantification of Western blot signals is described in and is based on at least 3 independent experiments for a given cell line. * – P <0.01 vs. the same cell line incubated w/o rmNRG-1β; & – P <0.001 vs. M1. All data in panels A, B, C and D come from at least 3 independent experiments performed in duplicates.

Article Snippet: Membranes were probed with rabbit polyclonal anti-ErbB2 (phospho Y1248) antibody (Abcam) at 1 ∶1000 dilution and mouse monoclonal anti-GAPDH antibody clone 6C5 (Biodesign Int.) (0.5 µg/ml) and HRP-conjugated appropriate secondary antibodies.

Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing, Transfection, Amplification, Positive Control, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Western Blot, Incubation

Journal: The Journal of biological chemistry

Article Title: Heregulin activation of extracellular acidification in mammary carcinoma cells is associated with expression of HER2 and HER3.

doi: 10.1074/jbc.270.38.22608

Figure Lengend Snippet: FIG. 2. Expression of HER2 and HER3 in human mammary and ovarian carcinoma cells. Human mammary and ovarian carcinoma cell lines were tested for the presence of HER2 mRNA by Northern blot analysis and p185 by Western blot analysis. A, total RNA (20 mg) isolated from human mammary carcinoma cells (MDA-MB-453, MCF-7, and SK-BR-3), human ovarian tumor cells (SK-OV-3), and human kid- ney cells (293) was fractionated in a 1% agarose-formaldehyde gel. After transferring to a nylon membrane, the blot was hybridized with a biotinylated HER2 cDNA probe. The hybridized blot was incubated with a streptavidin-alkaline phosphatase conjugate followed by chemi- luminescent detection as described under “Materials and Methods.” B, the membrane fraction isolated from the same breast and ovarian cell lines were solublized and electrophoresed in SDS-polyacrylamide gels for Western blot analysis as described under “Materials and Methods.” The p185 bands were detected using monoclonal anti-HER2 antibody followed by horseradish peroxidase-labeled anti-IgG and chemilumines- cent detection. The faint p185 band for MCF-7 is invisible in the photograph, but it is present on the original film. C, Western blot analysis of the mammary and ovarian cell lines for HER3 using anti- HER3 peptide rabbit antibody was conducted as described for B. The p160 bands corresponding to HER3 were detected by chemiluminescent detection.

Article Snippet: Western Blot Analysis—Cells were analyzed for the expression of HER2 (p185), HER3 (p160), and HER4 (p180) using Western blotting with mouse monoclonal anti-NEU (HER2) SC08, rabbit anti-erbB-3 peptide (HER3) SC285, and rabbit anti-erbB-4 peptide (HER4) SC283 antibodies (Santa Cruz Biotechnology).

Techniques: Expressing, Northern Blot, Western Blot, Isolation, Transferring, Membrane, Incubation, Labeling

Journal: The Journal of biological chemistry

Article Title: Heregulin activation of extracellular acidification in mammary carcinoma cells is associated with expression of HER2 and HER3.

doi: 10.1074/jbc.270.38.22608

Figure Lengend Snippet: FIG. 3. RT-PCR detection of HER2, HER3, and HER4 mRNA expression in human mammary and ovarian carcinoma cells. Total RNA was isolated and RT-PCR was performed as described under “Materials and Methods.” A, the ethidium bromide-agarose gels of the PCR products using primers for HER2, HER3, and HER4 are shown here. The DNA size standard fX174/HaeIII is also included. B, PCR products from A were blotted to a nylon membrane, probed with the corresponding biotinylated probes, and detected as described under “Materials and Methods.”

Article Snippet: Western Blot Analysis—Cells were analyzed for the expression of HER2 (p185), HER3 (p160), and HER4 (p180) using Western blotting with mouse monoclonal anti-NEU (HER2) SC08, rabbit anti-erbB-3 peptide (HER3) SC285, and rabbit anti-erbB-4 peptide (HER4) SC283 antibodies (Santa Cruz Biotechnology).

Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing, Isolation, Membrane

Journal: The Journal of biological chemistry

Article Title: Heregulin activation of extracellular acidification in mammary carcinoma cells is associated with expression of HER2 and HER3.

doi: 10.1074/jbc.270.38.22608

Figure Lengend Snippet: FIG. 4. Overexpression of HER2 RNA in CHO-K1, 293-EBNA, and MCF-7 stably transfected with pCEP/HER2 expression plas- mid. Clonal cell lines stably transfected with pCEP/HER2 plasmid was isolated as described under “Materials and Methods.” A, Northern blot analysis of total RNA was performed using a biotinylated HER2 cDNA probe. SK-BR-3 RNA was included as a positive control for HER2 mRNA expression. B, Western blot analysis of membrane fractions of CHO-K1, 293-EBNA, and MCF-7 cells transfected with pCEP/HER2 was performed as described under “Materials and Methods.” The p185 bands were detected using monoclonal anti-HER2 antibody followed by horseradish peroxidase-labeled anti-IgG and chemiluminescent detec- tion. SK-BR-3 was included as a positive control.

Article Snippet: Western Blot Analysis—Cells were analyzed for the expression of HER2 (p185), HER3 (p160), and HER4 (p180) using Western blotting with mouse monoclonal anti-NEU (HER2) SC08, rabbit anti-erbB-3 peptide (HER3) SC285, and rabbit anti-erbB-4 peptide (HER4) SC283 antibodies (Santa Cruz Biotechnology).

Techniques: Over Expression, Stable Transfection, Transfection, Expressing, Plasmid Preparation, Isolation, Northern Blot, Positive Control, Western Blot, Membrane, Labeling

Journal: The Journal of biological chemistry

Article Title: Heregulin activation of extracellular acidification in mammary carcinoma cells is associated with expression of HER2 and HER3.

doi: 10.1074/jbc.270.38.22608

Figure Lengend Snippet: FIG. 5. Lack of acidification response by HER2-transfected CHO-K1 and 293 cells to heregulin-a. HER2-transfected CHO-K1 and 293 EBNA cells were prepared and loaded into Cytosensor® capsules as described under “Materials and Methods.” The cells were monitored in the Cytosensor® until acidification rates stabilized, and then media containing 100 ng/ml heregulin-a or 2 mg/ml anti-HER2 monoclonal antibody were switched into the fluid stream for 15 min. The cells treated with anti-HER2 were immediately exposed to 4 mg/ml anti-mouse IgG for an additional 15 min. A, CHO/HER2-H cells were exposed to heregulin (G) or media alone (E). B, CHO/HER2-H cells were exposed to anti-HER2 (G) or control IgG1 (E) followed by anti-IgG. Parental CHO-K1 cells were also exposed to anti-HER2 (f) or control IgG1 (M) followed by anti-IgG. C, 293/HER2-O cells were exposed to heregulin (G) or media alone (E). D, 293/HER2-O cells were exposed to anti-HER2 (f) or control IgG1 (M) followed by anti-IgG. Parental CHO-K1 cells were also exposed to anti-HER2 (f) or control IgG1 (M) followed by anti-IgG. Due to the presence of a high concentration of buffer salts in the anti-HER2 antibody preparation, the apparent acidification rates were depressed by approximately 12% any time the preparation was present in the chamber.

Article Snippet: Western Blot Analysis—Cells were analyzed for the expression of HER2 (p185), HER3 (p160), and HER4 (p180) using Western blotting with mouse monoclonal anti-NEU (HER2) SC08, rabbit anti-erbB-3 peptide (HER3) SC285, and rabbit anti-erbB-4 peptide (HER4) SC283 antibodies (Santa Cruz Biotechnology).

Techniques: Transfection, Capsules, Control, Concentration Assay

Journal: The Journal of biological chemistry

Article Title: Heregulin activation of extracellular acidification in mammary carcinoma cells is associated with expression of HER2 and HER3.

doi: 10.1074/jbc.270.38.22608

Figure Lengend Snippet: FIG. 6. Increased acidification response to heregulin-a in HER2-transfected MCF-7 cells. Cells were prepared and loaded into Cytosensor® capsules as described under “Materials and Methods.” A, the MCF/HER2-A cells were monitored in the Cytosensor® until acidi- fication rates stabilized, and then media containing 50 ng/ml heregu- lin-a (G) or media alone (E) were switched into the fluid stream for 15 min. B, parental MCF-7 cells were also tested for response to 50 ng/ml heregulin-a (G) or media alone (E) under the same conditions as in A.

Article Snippet: Western Blot Analysis—Cells were analyzed for the expression of HER2 (p185), HER3 (p160), and HER4 (p180) using Western blotting with mouse monoclonal anti-NEU (HER2) SC08, rabbit anti-erbB-3 peptide (HER3) SC285, and rabbit anti-erbB-4 peptide (HER4) SC283 antibodies (Santa Cruz Biotechnology).

Techniques: Transfection, Capsules

Journal: International Journal of Molecular Medicine

Article Title: Epigenetically altered miR-193a-3p promotes HER2 positive breast cancer aggressiveness by targeting GRB7

doi: 10.3892/ijmm.2019.4167

Figure Lengend Snippet: Expression of miR-193a-3p is decreased in HER2 positive breast cancer and is associated with tumor stage and grade. (A) RT-qPCR was conducted to test the expression of miR-193a-3p in 35 pairs of HER2 positive breast cancer tissues and adjacent tissues. (B) The expression of miR-193a-3p in HER2 positive breast cancer tissues at different stages (Stage 1, n=7; Stage 2, n=11; Stage 3, n=12; Stage 4, n=5). (C) The expression of miR-193a-3p in HER2 positive breast cancer tissues at different grades (Grade 1, n=14; Grade 2, n=12; Grade 3, n=9). (D) RT-qPCR was conducted to determine the expression of miR-193a-3p in normal human breast cells and HER2 positive breast cancer cells. ** P<0.01 and *** P<0.001, as indicated. RT-qPCR, reverse transcription-quantitative polymerase chain reaction; HER2, human epidermal growth factor receptor 2; miR, microRNA.

Article Snippet: The human HER2 positive breast cancer cell lines HCC-1954, 21MT1 and JimT1 and human normal breast cell line MCF-10A were bought from the American Type Culture Collection (Manassas, VA, USA).

Techniques: Expressing, Quantitative RT-PCR, Reverse Transcription, Real-time Polymerase Chain Reaction

Journal: International Journal of Molecular Medicine

Article Title: Epigenetically altered miR-193a-3p promotes HER2 positive breast cancer aggressiveness by targeting GRB7

doi: 10.3892/ijmm.2019.4167

Figure Lengend Snippet: Identification of DNA methylation leads to the downregulation of miR-193a-3p in breast cancer cells. (A) Quantitative polymerase chain reaction was carried out to determine the expression of miR-193a-3p in HER2 positive breast cancer cells following 4-days treatment with 5-Aza-dc. ** P<0.01 vs. DMSO. (B) Pyrosequencing analysis was conducted to analyze the percentage of methylation in the miR-193-3p promoter in HER2 positive breast cancer tissues at different stages (Stage 1, n=7; Stage 2, n=11; Stage 3, n=12; Stage 4, n=5). (C) Pyrosequencing analysis was conducted to analyze the percentage of methylation in the miR-193-3p promoter in HER2 positive breast cancer tissues at different grades (Grade 1, n=14; Grade 2, n=12; Grade 3, n=9). * P<0.05 and *** P<0.001, as indicated. HER2, human epidermal growth factor receptor 2; miR, microRNA.

Article Snippet: The human HER2 positive breast cancer cell lines HCC-1954, 21MT1 and JimT1 and human normal breast cell line MCF-10A were bought from the American Type Culture Collection (Manassas, VA, USA).

Techniques: DNA Methylation Assay, Real-time Polymerase Chain Reaction, Expressing, Methylation

Journal: International Journal of Molecular Medicine

Article Title: Epigenetically altered miR-193a-3p promotes HER2 positive breast cancer aggressiveness by targeting GRB7

doi: 10.3892/ijmm.2019.4167

Figure Lengend Snippet: Overexpression of miR-193a-3p inhibits proliferation, migration and invasion of HER2 positive breast cancer cells. (A) miR-193-3p mimics were transfected into 3 HER2 positive breast cancer cell lines and the expression of miR-193a-3p was significantly increased compared with the miR group. (B) Cell vitality was significantly downregulated by miR-193a-3p overexpression compared with the miR group. (C) The colony formation capacity was significantly repressed by miR-193a-3p overexpression compared with the miR group. (D) Cell invasion and migration abilities were significantly inhibited by miR-193a-3p overexpression compared with the miR group. * P<0.05 and ** P<0.01 vs. the miR group. OD, optical density; miR, microRNA; HER2, human epidermal growth factor receptor 2.

Article Snippet: The human HER2 positive breast cancer cell lines HCC-1954, 21MT1 and JimT1 and human normal breast cell line MCF-10A were bought from the American Type Culture Collection (Manassas, VA, USA).

Techniques: Over Expression, Migration, Transfection, Expressing

Journal: International Journal of Molecular Medicine

Article Title: Epigenetically altered miR-193a-3p promotes HER2 positive breast cancer aggressiveness by targeting GRB7

doi: 10.3892/ijmm.2019.4167

Figure Lengend Snippet: miR-193a-3p directly inhibits the expression of GRB7 through targeting its 3′-UTR. (A) Western blotting and (B) statistical analysis of the expression of GRB7, which was significantly reduced by overexpression of miR-193a-3p in 3 HER2 positive breast cancer cell lines. miR-193a-3p could directly target 3′-UTR of GRB7 demonstrated by a (C) Cell Counting Kit-8 and (D) colony formation assay. ** P<0.01 vs. the miR group. (E) GRB7 was significantly upregu-lated in 35 pairs of HER2 positive breast cancer tissues. *** P<0.001, as indicated. (F) RNA immunoprecipitation assay demonstrating the association of GRB7 with miR-193a-3p in HCC-1954 cells. ** P<0.01 vs. the miR group. miR, microRNA; HER2, human epidermal growth factor receptor 2; UTR, untranslated region; GRB7, growth factor receptor bound protein 7; NC, negative control; OD, optical density.

Article Snippet: The human HER2 positive breast cancer cell lines HCC-1954, 21MT1 and JimT1 and human normal breast cell line MCF-10A were bought from the American Type Culture Collection (Manassas, VA, USA).

Techniques: Expressing, Western Blot, Over Expression, Cell Counting, Colony Assay, RNA Immunoprecipitation, Negative Control

Journal: International Journal of Molecular Medicine

Article Title: Epigenetically altered miR-193a-3p promotes HER2 positive breast cancer aggressiveness by targeting GRB7

doi: 10.3892/ijmm.2019.4167

Figure Lengend Snippet: GRB7 overexpression abolishes the inhibitory effect of miR-193a-3p on the oncogenic capacity of breast cancer. (A) Western blotting and (B) statistical analysis of the overexpression of GRB7 which abolished the decrease of GRB7 due to overexpression of miR-193a-3p in HER2 positive breast cancer cells. * P<0.05 and ** P<0.01, as indicated. (C) Cell vitality was significantly increased. ** P<0.01. (D) The colony formation capacity was significantly promoted. ** P<0.01, as indicated. (E) Cell invasion and (F) migration abilities were significantly accelerated by GRB7 overexpression. * P<0.05 and ** P<0.01. GRB7, growth factor receptor bound protein 7; NC, negative control; OD, optical density; miR, microRNA.

Article Snippet: The human HER2 positive breast cancer cell lines HCC-1954, 21MT1 and JimT1 and human normal breast cell line MCF-10A were bought from the American Type Culture Collection (Manassas, VA, USA).

Techniques: Over Expression, Western Blot, Migration, Negative Control

Journal: International Journal of Molecular Medicine

Article Title: Epigenetically altered miR-193a-3p promotes HER2 positive breast cancer aggressiveness by targeting GRB7

doi: 10.3892/ijmm.2019.4167

Figure Lengend Snippet: miR-193a-3p may inhibit HER2 positive breast cancer through downregulating GRB7 and inactivating the ERK/FOXM1 signaling pathway. (A) Western blotting and (B) statistical analysis of the protein level of pERK 1/2 and (C) FOXM1 were significantly decreased by GRB7 overexpression in HCC-1954 cells overexpressing miR-193a-3p. * P<0.05 and ** P<0.01, as indicated. miR, microRNA; pERK, phosphorylated extracellular signal regulated kinase; FOXM1, forkhead box M1; HER2, human epidermal growth factor receptor 2; GRB7, growth factor receptor bound protein 7.

Article Snippet: The human HER2 positive breast cancer cell lines HCC-1954, 21MT1 and JimT1 and human normal breast cell line MCF-10A were bought from the American Type Culture Collection (Manassas, VA, USA).

Techniques: Western Blot, Over Expression

Journal: Cancers

Article Title: Hypoxia-Inducible Factor-1α, a Novel Molecular Target for a 2-Aminopyrrole Derivative: Biological and Molecular Modeling Study

doi: 10.3390/cancers18010115

Figure Lengend Snippet: Anti-proliferative effects of 2-ANPC in epithelial cancer cell lines. Changes in growth kinetics of HCC1806, MDA-MB-231, H1299, and PC-3 cells treated with 2-ANPC, PTX (positive control) and solvent DMSO (negative control). Cells (0.5 × 10 5 /mL) were seeded into the wells of an E-Plate L8 PET cassette and installed in the iCELLigence cell growth kinetics system (ACEA Biosciences, San Diego, CA, USA). Cells were allowed to attach and grow for the following 24 h. Afterwards, 2-ANPC, PTX, or DMSO was introduced into the cell culture. Cell proliferation index values were recorded every hour throughout the experiment. RTCA Software version 1.0 (ACEA Biosciences, Inc., San Diego, CA, USA) was used to analyze the data.

Article Snippet: Triple negative breast cancer HCC1806, MDA-MB-231, and 4T1; non-small cell lung carcinoma H1299; and prostate cancer PC-3 cell lines were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Positive Control, Solvent, Negative Control, Cell Culture, Software

Journal: Cancers

Article Title: Hypoxia-Inducible Factor-1α, a Novel Molecular Target for a 2-Aminopyrrole Derivative: Biological and Molecular Modeling Study

doi: 10.3390/cancers18010115

Figure Lengend Snippet: Pro-apoptotic activity of 2-ANPC in epithelial cancer cell lines. HCC1806, MDA-MB-231, H1299, and PC-3 cells were treated with 2-ANPC (10 µM) for 48 h and subjected to Western blot analysis to examine the expression of apoptotic markers, including cleaved PARP and cleaved caspase-3. Actin staining was used to show the comparable amounts of protein loaded into each sample. Representative Western blotting results from 3 independent experiments are shown. The figure on the right shows the quantification of HIF-1α expression in cells based on the mean pixel density. The uncropped original Western blotting images can be found in .Values are means ± SD, n = 3. (*— p < 0.01; one-way ANOVA).

Article Snippet: Triple negative breast cancer HCC1806, MDA-MB-231, and 4T1; non-small cell lung carcinoma H1299; and prostate cancer PC-3 cell lines were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Activity Assay, Western Blot, Expressing, Staining

Journal: Cancers

Article Title: Hypoxia-Inducible Factor-1α, a Novel Molecular Target for a 2-Aminopyrrole Derivative: Biological and Molecular Modeling Study

doi: 10.3390/cancers18010115

Figure Lengend Snippet: 2-ANPC decreases expression of HIF-1α in epithelial cancer cell lines by reducing its stability and promoting the proteasome-dependent degradation. ( A ) HCC1806, MDA-MB-231, H1299, and PC-3 cells were treated with 2-ANPC (10 µM) for 48 h and subjected to Western blotting analysis three times to examine the expression of HIF-1α. Actin staining was used to show comparable amounts of protein loaded into each sample. The figure on the right shows the quantification of HIF-1α expression in cells based on the mean pixel density. Values are means ± SD; n = 3. An asterisk indicates a significant difference compared to relevant controls ( p < 0.05; one-way ANOVA). ( B ) Changes in the relative expression level of HIF-1α mRNA in epithelial cancer cells (HCC1806, MDA-MB-231, H1299, PC-3) treated with 2-ANPC (10 µM—48 h), as determined by quantitative RT-PCR. As an internal control, glyceraldehyde-3-phosphate dehydrogenase ( GAPDH ) amplification was used. Data are presented as median ± standard deviation (SD). Significant differences at p < 0.05 (*), p < 0.001 (**) from n ≥ 3 using an unpaired Student’s t -test are presented. ns – non significant ( C ) To examine the impact of 2-ANPC on proteasome degradation of HIF-1α, PC-3 prostate cancer cells were treated with 2-ANPC in the presence of MG-132 (2.5 μM) for 6 h and were subjected to immunoblotting for HIF-1α and actin as a loading control. Representative Western blotting results from 3 independent experiments are shown. The figure on the right shows the quantification of HIF-1α expression in cells based on the mean pixel density. Values are means ± SD; n = 3. An asterisk indicates significant difference compared to relevant controls (*— p < 0.01; one-way ANOVA).

Article Snippet: Triple negative breast cancer HCC1806, MDA-MB-231, and 4T1; non-small cell lung carcinoma H1299; and prostate cancer PC-3 cell lines were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Expressing, Western Blot, Staining, Quantitative RT-PCR, Control, Amplification, Standard Deviation

Journal: Cancers

Article Title: Hypoxia-Inducible Factor-1α, a Novel Molecular Target for a 2-Aminopyrrole Derivative: Biological and Molecular Modeling Study

doi: 10.3390/cancers18010115

Figure Lengend Snippet: 2-ANPC decreases HIF-1α’s expression under hypoxic conditions. Effects of CoCl 2 treatment on HIF-1α expression on protein ( A , B ) and mRNA ( C ) levels. Cancer cells (HCC1806, MDA-MB-231, PC-3) were incubated in the absence (control) or in the presence of 100 μM CoCl 2 for 24 h. 2-ANPC (10 μM) was introduced into the cell cultures simultaneously with CoCl 2 . HIF-1α protein expression was measured by Western blotting ( A ), and HIF-1α mRNA expression was determined by RT-PCR ( C ). ( B ) Quantification of HIF-1α expression in cells based on the average pixel density. Values are presented as mean ± standard deviation; n = 3. Statistically significant differences between the respective groups are marked with an asterisk (*— p < 0.05; **— p < 0.01; one-way ANOVA).

Article Snippet: Triple negative breast cancer HCC1806, MDA-MB-231, and 4T1; non-small cell lung carcinoma H1299; and prostate cancer PC-3 cell lines were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Expressing, Incubation, Control, Western Blot, Reverse Transcription Polymerase Chain Reaction, Standard Deviation