Journal: Scientific Reports

Article Title: Hypoxia-inducible ERO1α promotes cancer progression through modulation of integrin-β1 modification and signalling in HCT116 colorectal cancer cells

doi: 10.1038/s41598-017-09976-7

Figure Lengend Snippet: ERO1A gene deletion in HCT116 cells. ( a ) The human ERO1A gene is located on chromosome 14. Exons are represented as boxes. The asterisk indicates the region targeted by the gRNA, and the arrows indicate the PCR primer set. ( b ) Two representative mutated alleles in the HCT116 cell line induced by CRISPR/Cas9, as detected using RT-PCR. ( c ) CRISPR/Cas9-induced deletion is shown using dashed lines; the red vertical bar indicates the site of the insertion sequence. In KO-1, the origin of the pBluescript backbone sequence (236 bp) was inserted in the gRNA target locus. KO-2 contained a 76-bp deletion and a 1-bp (G) insertion (flame-shift mutation). ( d ) Western blotting analysis of the expression of ERO1α and related proteins: comparison of ERO1α KO clones with WT and mock control. ( e ) Cell growth in each clone was assessed by counting cells at the indicated time points; data represent the means ± s.e.m. ( n = 6). The groups showed no statistically significant differences.

Article Snippet: The primary antibodies that were used are as follows: anti-ERO1α (Abnova), anti-integrin-β1, anti-HIF-1α (BD Biosciences), anti-ERO1β (Proteintech Group), anti-PDI, anti-ILK1, anti-Snail, anti-E-cadherin, anti-α-tubulin (Cell Signaling Technology), anti-PRDX4, anti-GAPDH, and anti-β-actin (Abcam).

Techniques: CRISPR, Reverse Transcription Polymerase Chain Reaction, Sequencing, Mutagenesis, Western Blot, Expressing, Comparison, Clone Assay, Control

Journal: Scientific Reports

Article Title: Hypoxia-inducible ERO1α promotes cancer progression through modulation of integrin-β1 modification and signalling in HCT116 colorectal cancer cells

doi: 10.1038/s41598-017-09976-7

Figure Lengend Snippet: Effect of ERO1α KO on xenograft tumour growth. ( a ) Tumour size was measured twice a week after subcutaneous injection of control and KO cells. ( b ) Tumour weight was measured after dissecting out the tumours; values shown are the means ± s.e.m. ( n = 8). * P < 0.05, n.s, not significant (Student’s t test). ( c ) Representative image of haematoxylin and eosin staining and immunohistochemical staining against ERO1α.

Article Snippet: The primary antibodies that were used are as follows: anti-ERO1α (Abnova), anti-integrin-β1, anti-HIF-1α (BD Biosciences), anti-ERO1β (Proteintech Group), anti-PDI, anti-ILK1, anti-Snail, anti-E-cadherin, anti-α-tubulin (Cell Signaling Technology), anti-PRDX4, anti-GAPDH, and anti-β-actin (Abcam).

Techniques: Injection, Control, Staining, Immunohistochemical staining

Journal: Scientific Reports

Article Title: Hypoxia-inducible ERO1α promotes cancer progression through modulation of integrin-β1 modification and signalling in HCT116 colorectal cancer cells

doi: 10.1038/s41598-017-09976-7

Figure Lengend Snippet: In vitro culture of WT and ERO1α cells under normoxia vs hypoxia. Cultures were incubated for 24, 48, and 72 h under hypoxia or normoxia. ( a ) Cell-proliferation curve of WT and KO clones under hypoxia, assessed by counting cells at the indicated times. Data are shown as the means ± s.e.m. ( n = 4). * P < 0.05, Student’s t test. ( b ) Representative light microscopy images of WT (left) and KO (right). The KO clone displays reduced stacking (piling up) and increased cell-cell integrity as compared with WT. ( c ) Western blotting analysis of oxidizing enzymes and cell adhesion molecules. Under hypoxia, ERO1α depletion impairs the maturation of integrin-β1. The lower integrin band (110 kDa) represents the β1 precursor, and the upper (approximately130 kDa) the mature form. All experiments were conducted at least three times and representative data is presented.

Article Snippet: The primary antibodies that were used are as follows: anti-ERO1α (Abnova), anti-integrin-β1, anti-HIF-1α (BD Biosciences), anti-ERO1β (Proteintech Group), anti-PDI, anti-ILK1, anti-Snail, anti-E-cadherin, anti-α-tubulin (Cell Signaling Technology), anti-PRDX4, anti-GAPDH, and anti-β-actin (Abcam).

Techniques: In Vitro, Incubation, Clone Assay, Light Microscopy, Western Blot

Journal: Scientific Reports

Article Title: Hypoxia-inducible ERO1α promotes cancer progression through modulation of integrin-β1 modification and signalling in HCT116 colorectal cancer cells

doi: 10.1038/s41598-017-09976-7

Figure Lengend Snippet: ERO1α deletion drastically reduces the cell-surface-expressed form of integrin-β1 under hypoxia. ( a ) Normoxic and hypoxic samples were incubated without or with Endo H or PNGase F. ERO1α depletion impaired Endo H-resistant N-glycosylation of integrin-β1 under hypoxia. Membrane integrin-β1 levels in HCT116 clones under normoxia and hypoxia were assessed by performing cell-surface biotinylation assays, flow cytometry, and immunofluorescence labelling of cells: ( b ) Cell-surface biotinylation assay performed on WT and the mock and KO clones under normoxia and hypoxia. Total lysates (whole) and the NeutrAvidin agarose-bound fraction (cell surface) were subjected to western blotting. ( c ) Flow cytometric analysis of integrin-β1 in WT and KO. Cells were collected and incubated with APC-conjugated anti-integrin-β1 antibody or isotype control antibody. FMI, Fluorescence mean intensity. ( d ) Immunocytochemical labelling of integrin-β1 (green) and PDI (red) in WT and KO cells under hypoxia. PDI was used as an ER marker protein. All experiments were conducted in duplicate or triplicate and repeated at least twice.

Article Snippet: The primary antibodies that were used are as follows: anti-ERO1α (Abnova), anti-integrin-β1, anti-HIF-1α (BD Biosciences), anti-ERO1β (Proteintech Group), anti-PDI, anti-ILK1, anti-Snail, anti-E-cadherin, anti-α-tubulin (Cell Signaling Technology), anti-PRDX4, anti-GAPDH, and anti-β-actin (Abcam).

Techniques: Incubation, Membrane, Clone Assay, Flow Cytometry, Immunofluorescence, Cell Surface Biotinylation Assay, Western Blot, Control, Fluorescence, Marker

Journal: Scientific Reports

Article Title: Hypoxia-inducible ERO1α promotes cancer progression through modulation of integrin-β1 modification and signalling in HCT116 colorectal cancer cells

doi: 10.1038/s41598-017-09976-7

Figure Lengend Snippet: ERO1α deletion affects cell motility and integrin- and EMT-related signalling molecules under hypoxia through integrin-β1 downregulation. ( a ) Wound-healing assays performed using the indicated cell clones and culture conditions. Results are given as the means ± s.e.m. ( n = 4); * P < 0.05, Student’s t test. The upper panels show the wound at baseline, the lower the wound after 24 h. Under hypoxia, the KO clone exhibited significantly decreased migration as compared with WT and with KO under normoxia. ( b ) Expression of integrin- and EMT-related molecules in WT and the mock and KO clones under normoxia and hypoxia.

Article Snippet: The primary antibodies that were used are as follows: anti-ERO1α (Abnova), anti-integrin-β1, anti-HIF-1α (BD Biosciences), anti-ERO1β (Proteintech Group), anti-PDI, anti-ILK1, anti-Snail, anti-E-cadherin, anti-α-tubulin (Cell Signaling Technology), anti-PRDX4, anti-GAPDH, and anti-β-actin (Abcam).

Techniques: Clone Assay, Migration, Expressing

Journal: Scientific Reports

Article Title: Hypoxia-inducible ERO1α promotes cancer progression through modulation of integrin-β1 modification and signalling in HCT116 colorectal cancer cells

doi: 10.1038/s41598-017-09976-7

Figure Lengend Snippet: ERO1α re-expression reverses the effect of ERO1α KO. ( a ) Western blotting to confirm ERO1α expression. Res: ERO1α-transfected KO clone. ( b ) Tumour size was measured twice a week after subcutaneous injection; n = 6, * P < 0.05 (Student’s t test). ( c ) Images of tumours at 28 days after inoculation with the indicated clones.

Article Snippet: The primary antibodies that were used are as follows: anti-ERO1α (Abnova), anti-integrin-β1, anti-HIF-1α (BD Biosciences), anti-ERO1β (Proteintech Group), anti-PDI, anti-ILK1, anti-Snail, anti-E-cadherin, anti-α-tubulin (Cell Signaling Technology), anti-PRDX4, anti-GAPDH, and anti-β-actin (Abcam).

Techniques: Expressing, Western Blot, Transfection, Injection, Clone Assay