Journal: Cancer medicine

Article Title: Hypoxia and Hypoxia-Reoxygenation Potentiate Helicobacter pylori Infection and Gastric Epithelial Cell Proliferation.

doi: 10.1002/cam4.70860

Figure Lengend Snippet: FIGURE 1 | Hypoxia induces CEACAM6. Western blotting of whole cell lysates of AGS (a), MKN45 (b), and HFE145 (c) cells kept at normoxia or hypoxia (=H, 3% O2) showing levels of HIF1α and CEACAM6 at 6, 12, and 24 h. α-tubulin was used as loading control. Bar graphs indicated a significant increase in CEACAM6 level at 12-h time point. (d) A representative (n = 3) immunofluorescence micrograph showing elevated levels of HIF1α (green) and CEACAM6 (red) in AGS cells at 12 h of hypoxia (3% O2) exposure. Objective used = 40×, scale bar = 50 μm. (e) A representative (n = 3) immunofluorescence micrograph of human metastatic GC biopsy tissue sample showing the status of HIF1α (red) and CEACAM6 (green). Nuclei were stained for DAPI (blue). Tissues were sectioned at 5 μm thickness. Images were captured using 20× objective and scale bars = 50 μm. Graphs = mean ± SEM. Statistical significance was determined using two-way ANOVA followed by Tukey's post hoc analysis (n = 3). *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

Article Snippet: Human CEACAM6- pdKCR- neo construct, pCMV6- Xl5- HIF1α (Origene Technologies, MD, USA), human NOX4 (courtesy: Karl- Heinz Krause, Addgene plasmid #69352) overexpression plasmids and empty vectors were used in this study [36, 37].

Techniques: Western Blot, Control, Immunofluorescence, Staining

Journal: Cancer medicine

Article Title: Hypoxia and Hypoxia-Reoxygenation Potentiate Helicobacter pylori Infection and Gastric Epithelial Cell Proliferation.

doi: 10.1002/cam4.70860

Figure Lengend Snippet: FIGURE 2 | HIF1α upregulates CEACAM6 in hypoxic GECs. (a) Knockdown of HIF1α decreased hypoxia-induced CEACAM6 protein level in AGS cells as detected by western blot. Whole cell lysates of HIF1α shRNA expressing as well as control shRNA-expressing stable AGS cells, kept under normoxic and hypoxic conditions for 12 h, showed decreased levels of HIF1α as well as CEACAM6. Bar graphs showed a significant reduction in CEACAM6:α-tubulin ratio in normoxia as well as hypoxia. (b) Representative micrographs showing CEACAM6 upregulation in HIF1α stably expressing AGS cells at 12 h hypoxia. The nuclei were stained with DAPI. Scale bar = 20 μm, =60×. Bar graph indicated significant changes in the mean fluorescence intensity. Hypoxia = 3% O2. All graphical data indicated mean ± SEM. Two-way ANOVA was applied to determine statistical sig- nificance, and the results were corrected for multiple comparisons using Tukey's post hoc analysis. n = 3, *p < 0.05; ****p < 0.0001.

Article Snippet: Human CEACAM6- pdKCR- neo construct, pCMV6- Xl5- HIF1α (Origene Technologies, MD, USA), human NOX4 (courtesy: Karl- Heinz Krause, Addgene plasmid #69352) overexpression plasmids and empty vectors were used in this study [36, 37].

Techniques: Knockdown, Western Blot, shRNA, Expressing, Control, Stable Transfection, Staining, Fluorescence

Journal: Cancer medicine

Article Title: Hypoxia and Hypoxia-Reoxygenation Potentiate Helicobacter pylori Infection and Gastric Epithelial Cell Proliferation.

doi: 10.1002/cam4.70860

Figure Lengend Snippet: FIGURE 3 | Hypoxia and HIF1α upregulate CEACAM6 and NOX4 in hypoxic GECs, gastritis as well as GC samples. (a) Immunofluorescence microscopy images showing enhanced ROS production in AGS cells under hypoxia. The mean fluorescence intensity (MFI) graph denoted the signif- icant difference in ROS generation between normoxia and hypoxia. Graphical data indicated mean ± SEM. Paired t-test was performed to determine statistical significance. n = 3, **p < 0.01. (b) Correlation analysis performed in GEPIA2 showed a positive Pearson correlation coefficient between HIF1α and NOX4 gene expression normalized to TUBA4A (p = 0, R = 0.64). (c) Box plot representation of NOX4 expression in STAD tumor and normal samples (red = tumor, gray = normal) showed a significant NOX4 upregulation in tumor samples (num[T] = 408, num[N] = 36, p value cutoff was 0.01). (d) Pathological stage plot showed a high association of NOX4 with the four stages of STAD tumor (F value = 5.67, Pr(>F) = 0.000832). (e) GEPIA2 correlation analysis showed a positive Pearson correlation coefficient between CEACAM6 and NOX4 (p = 7.3e-05, R = 0.19). (f) Immunofluorescence micrographs of human gastritis biopsy tissue sample showing the status of HIF1α (green), CEACAM6 (red), and NOX4 (red) (n = 3). (g) Human met- astatic GC biopsy tissue and their paired normal tissues (n = 3) showing enhanced expression of HIF1α (green), CEACAM6 (red), and NOX4 (red) in GC samples. Nuclei were stained for DAPI (blue). Graphical representation showing significant changes in the levels of HIF1α, CEACAM6, and NOX4 are present in Figure S3. Tissues were sectioned at 5 μm thickness. Images were captured using 20× objective and scale bars = 50 μm. In panel c, *indicates significance.

Article Snippet: Human CEACAM6- pdKCR- neo construct, pCMV6- Xl5- HIF1α (Origene Technologies, MD, USA), human NOX4 (courtesy: Karl- Heinz Krause, Addgene plasmid #69352) overexpression plasmids and empty vectors were used in this study [36, 37].

Techniques: Immunofluorescence, Microscopy, Fluorescence, Gene Expression, Expressing, Staining

Journal: Cancer medicine

Article Title: Hypoxia and Hypoxia-Reoxygenation Potentiate Helicobacter pylori Infection and Gastric Epithelial Cell Proliferation.

doi: 10.1002/cam4.70860

Figure Lengend Snippet: FIGURE 7 | CEACAM6-expressing GECs activate the CEACAM6-NOX4 cascade, upregulate ROS generation, and gain significantly more prolif- erative ability after being exposed to hypoxia-reoxygenation and H. pylori infection. (a) Western blot analysis showed that levels of CagA, CEACAM6, and NOX4 were significantly increased (graphical representations are shown in Figure S7a–c) in AGS cells upon CEACAM6 stable overexpres- sion and hypoxia-reoxygenation before infection with H. pylori. (b) Fluorescence micrographs and graphical data (n = 3) demonstrated enhanced ROS production in hypoxia and reoxygenation-exposed and H. pylori-infected CEACAM6 overexpressing AGS cells when compared with the empty vector-expressing stable cells with similar treatments. For ROS detection, cells were incubated with 1 μM DCFDA for 1 h after infection. Nuclei were stained with DAPI. (c) Graphical representation of cellular proliferation assessed by MTT assay (n = 3) showed significantly increased proliferation of H. pylori-infected cells with CEACAM6 overexpression as well as hypoxia-reoxygenation when compared with the empty vector-expressing as well as normoxia-exposed cells. Objective used—60×, scale bars representing 25 μm. Graphs = mean ± SEM. Statistical significance was determined using two-way ANOVA followed by Tukey's post hoc analysis (n = 3). *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. In the Figure, Hp = H. pylori. (d) The summary figure depicting the regulation of ROS signaling events in H. pylori-infected normoxic and hypoxic GECs. H. pylori infection of GECs leads to the accumulation of HIF1α in normoxia, but the effect is far more enhanced in hypoxic cells due to the increased level of HIF1 and HIF1-driven CEACAM6 upregulation. CEACAM6 facilitates H. pylori adhesion and upregulates ROS via enhanced NOX4 generation. Enhanced HIF1-CEACAM6-NOX4 signaling has a proliferative effect on GECs. Numbers indicate the sequence of events.

Article Snippet: Human CEACAM6- pdKCR- neo construct, pCMV6- Xl5- HIF1α (Origene Technologies, MD, USA), human NOX4 (courtesy: Karl- Heinz Krause, Addgene plasmid #69352) overexpression plasmids and empty vectors were used in this study [36, 37].

Techniques: Expressing, Infection, Western Blot, Fluorescence, Plasmid Preparation, Incubation, Staining, MTT Assay, Over Expression, Sequencing

Journal: International Journal of Molecular Sciences

Article Title: Energy Metabolism and Stemness and the Role of Lauric Acid in Reversing 5-Fluorouracil Resistance in Colorectal Cancer Cells

doi: 10.3390/ijms26020664

Figure Lengend Snippet: Stemness of 5FU-resistant cell lines derived from two CRC cell lines. 5FU-resistant HT29R and CT26R cell lines were established from HT29 and CT26 cells, respectively, by continuous treatment with low-dose 5FU (IC 5 ) for 50 passages. ( A ) Apoptosis. ( B ) 5FU (5 μg/mL for 48 h). ( C ) Mitophagy. Scale bar: 50 μm. Right panel: semi-quantification of fluorescence images. ( D ) Expression of stemness-associated genes HIF1α and ME1. Right panel: semi-quantification of RT-PCR signals. ( E ) Sphere areas. ( F ) Expression of the naïve/prime transition-associated genes PRODH and LIN28a . Right panel: semi-quantification of RT-PCR signals. Error bars: standard deviation of three independent trials. Asterisk, p < 0.05. Statistical differences were calculated using ordinary ANOVA with Bonferroni correction. CRC, colorectal cancer; 5FU, 5-fluorouracil; ACTB, β-actin; RT-PCR, reverse transcription polymerase chain reaction; PRODH, proline dehydrogenase; HIF1A, hypoxia-inducible 1α; ME1, cytosolic NADPH dehydrogenase 1 (malic enzyme 1); ANOVA, analysis of variance.

Article Snippet: HIF1α , Human , #A43658 , Thermo Fisher, Tokyo, Japan , .

Techniques: Derivative Assay, Fluorescence, Expressing, Reverse Transcription Polymerase Chain Reaction, Standard Deviation, Reverse Transcription, Polymerase Chain Reaction

Journal: International Journal of Molecular Sciences

Article Title: Energy Metabolism and Stemness and the Role of Lauric Acid in Reversing 5-Fluorouracil Resistance in Colorectal Cancer Cells

doi: 10.3390/ijms26020664

Figure Lengend Snippet: Effect of lauric acid (LAA) on stemness in 5FU-resistant CRC cell lines. The 5FU-resistant cell lines HT29R and CT26R were treated with 5FU (10 μg/mL) with or without LAA (40 μg/mL) for 40 h. ( A ) Cell growth. ( B ) 4HNE. ( C ) Apoptosis. ( D ) ATP. ( E ) Lactate levels in the culture medium. ( F , G ) Protein levels of HIF1α ( F ) and ME1 ( G ). ( H ) Sphere areas. Error bars: standard deviation of three independent trials. Asterisk, p < 0.05. Statistical differences were calculated using ordinary ANOVA with Bonferroni correction. CRC, colorectal cancer; 5FU, 5-fluorouracil; LAA, lauric acid; 4HNE, 4-hydroxynonenal; HIF1α, hypoxia inducible 1α; ME1, cytosolic NADPH dehydrogenase 1 (malic enzyme 1); ANOVA, analysis of variance.

Article Snippet: HIF1α , Human , #A43658 , Thermo Fisher, Tokyo, Japan , .

Techniques: Standard Deviation

Journal: International Journal of Molecular Sciences

Article Title: Energy Metabolism and Stemness and the Role of Lauric Acid in Reversing 5-Fluorouracil Resistance in Colorectal Cancer Cells

doi: 10.3390/ijms26020664

Figure Lengend Snippet: RT-PCR primers and ELISA kits.

Article Snippet: HIF1α , Human , #A43658 , Thermo Fisher, Tokyo, Japan , .

Techniques: Enzyme-linked Immunosorbent Assay, Bioassay