Journal: Life Science Alliance

Article Title: Hypoxia favors tumor growth in colorectal cancer in an integrin αDβ1/hemoglobin δ-dependent manner

doi: 10.26508/lsa.202402925

Figure Lengend Snippet: Integrin and erythroid cell genes expressed in Colo205 cells grown in hypoxia.

Article Snippet: The primers for Hbβ, Hbδ, and αD integrin were obtained from Origene.

Techniques:

Journal: Life Science Alliance

Article Title: Hypoxia favors tumor growth in colorectal cancer in an integrin αDβ1/hemoglobin δ-dependent manner

doi: 10.26508/lsa.202402925

Figure Lengend Snippet: (A) Cells were stained for integrin αD (green, rabbit polyclonal antibody), and β2 integrin (red) chains showed absence of β2. Integrin β1 staining (red) was strong and it co-distributed with αD staining. Scale bar 10 μm. (B) Cells were grown in normoxia, transferred to hypoxia, and stained at different time points for αD (red) and Hb (green, goat polyclonal). After 2 h under hypoxia, the cells showed increased staining for αD, but weak staining for Hb. After 9 h, Hb staining was strong and showed partial co-distribution with αD. After 24 h, cells expressed cup-like structures. Scale bar 10 μm. The last figure shows the integrin αD (red) and Hbδ (green) at 24 h under hypoxic conditions. The figure was obtained from the . Scale bar 10 μm. (C) Higher magnification of αD-mCherry and Hbδ-GFP- transfected cells grown under normoxia (above) and hypoxia (below). Scale bar 10 μm. Hbδ formed aggregates and there was more capping in hypoxia-grown cells. (D) Cells transfected with integrin β1-mCherry and Hbδ-GFP showed partial co-distribution under normoxia. The magnification is the same as in .

Article Snippet: The primers for Hbβ, Hbδ, and αD integrin were obtained from Origene.

Techniques: Staining, Transfection

Journal: Life Science Alliance

Article Title: Hypoxia favors tumor growth in colorectal cancer in an integrin αDβ1/hemoglobin δ-dependent manner

doi: 10.26508/lsa.202402925

Figure Lengend Snippet: (A) Blotting of β1 and β2 integrin polypeptides from Colo205 and U937 cell lysates. Colo205 cells contained only β1, whereas U937 cells contained both β1 and β2 integrin chains. (B) Blotting of integrin αD from a slab gel of immune precipitates obtained with control and integrin β1 antibodies (Santa Cruz and BD Biosciences) from Colo205 cells. (C) Immune precipitation with Hb antibody (Thermo Fisher Scientific) from cells grown under normoxia (N) and hypoxia (H), and visualization of integrin αD by blotting the gels with αD antibody. The integrin αD band was obtained from cells grown under hypoxia, but not from cells grown under normoxia. (D) Schematic structure of the αDβ1 integrin bound to the Hbδ tetramer (upper left), and predicted structure of the Hbδ tetramer bound to the integrin αD I-domain (upper right). The possible interacting peptide loops of Hbδ are below. (D, G) The sequence in site 1 is somewhat like the RGD sequence containing the amino acids (D, G) implicated in binding. The other important sequence is quite different. (E) Predicted structure of the Hbδ tetramer bound to the αD I-domain. The possible interacting peptide loops of Hbδ are below. (D, G) The sequence in site 1 is somewhat like the RGD sequence containing the amino acids (D, G) implicated in binding. The other important sequence is quite different. (F) Sequence of Hbδ used for cell transfections. The locations of the I-domain interacting sequences are highlighted in red and green, and the amino acids, which differ between Hbδ and Hbβ are highlighted in blue. (G) Sequence of Hbβ. Source data are available for this figure.

Article Snippet: The primers for Hbβ, Hbδ, and αD integrin were obtained from Origene.

Techniques: Control, Sequencing, Binding Assay, Transfection

Journal: Life Science Alliance

Article Title: Hypoxia favors tumor growth in colorectal cancer in an integrin αDβ1/hemoglobin δ-dependent manner

doi: 10.26508/lsa.202402925

Figure Lengend Snippet: (A) Adhesion of cells to Hb grown under hypoxia and normoxia. Cells grown under hypoxia strongly adhered to red blood cell Hb, whereas those grown under normoxia did not. (B) The αD and β1 integrin antibodies effectively blocked adhesion, whereas the β2 integrin-blocking antibody showed no effect. The RGD-containing peptide also blocked cell adhesion, whereas the control peptide did not. (C) Binding of cells to fibronectin. Soluble Hb (250 μg/ml) and αD antibodies significantly blocked adhesion of cells grown in hypoxia. (D) Oxygen consumption by cells grown under hypoxia was inhibited by the Hbδ/β monoclonal antibody and the Hbδ-derived peptide QLSELHCDKL (400 μM), but not by the corresponding Hbβ-derived peptide ATLSELHCDKL. (E) Proliferation of hypoxia-grown cells was efficiently blocked by RNAi down-regulation of either Hbδ or integrin αD, but not by RNAi of Hbβ. *** P < 0.001, **** P < 0.0001, ** P < 0.01, * P < 0.05.

Article Snippet: The primers for Hbβ, Hbδ, and αD integrin were obtained from Origene.

Techniques: Blocking Assay, Control, Binding Assay, Derivative Assay

Journal: Life Science Alliance

Article Title: Hypoxia favors tumor growth in colorectal cancer in an integrin αDβ1/hemoglobin δ-dependent manner

doi: 10.26508/lsa.202402925

Figure Lengend Snippet: (A) Non-transfected cells bound to red cell Hb. (B) αD-mCherry-transfected cells showed increased binding. Integrin β1 antibodies blocked binding. (C) The cells bound well to recombinant Hbδ, and the adhesion was inhibited by integrin β1 and αD antibodies, and the RGD motif-containing peptide. Average of three parallel results. The experiments were repeated with similar results.

Article Snippet: The primers for Hbβ, Hbδ, and αD integrin were obtained from Origene.

Techniques: Transfection, Binding Assay, Recombinant

Journal: Life Science Alliance

Article Title: Hypoxia favors tumor growth in colorectal cancer in an integrin αDβ1/hemoglobin δ-dependent manner

doi: 10.26508/lsa.202402925

Figure Lengend Snippet: (A) The integrin αD antibody inhibited oxygen consumption, whereas the integrin αX antibody showed no effect. (B) The integrin αD antibody blocked cell proliferation, whereas the integrin αX antibody did not. Average of three parallel measurements. The experiments were repeated with similar results.

Article Snippet: The primers for Hbβ, Hbδ, and αD integrin were obtained from Origene.

Techniques:

Journal: Life Science Alliance

Article Title: Hypoxia favors tumor growth in colorectal cancer in an integrin αDβ1/hemoglobin δ-dependent manner

doi: 10.26508/lsa.202402925

Figure Lengend Snippet: (A) A cancer stained with Hbδ antibody. The tumor tissue is in the middle and lower right corner. (B) An adjacent section from the same tumor stained with mouse IgG. (C) Staining of a cancer with Hbδ antibody. (D) Staining of a consecutive section with Hbα antibody shows no staining of the cancer but positivity of red cells in the vessels. (E) Staining of a cancer with Hbδ antibody shows positive staining of the tumor. (F) Staining of a consecutive section of the same tumor with integrin αD antibody demonstrates co-expression of Hbδ and αD. (G) Staining with integrin β1 antibody. The cancer cells were strongly positive, as were the normal tissue to the right. (H) Staining of with an integrin β2 antibody. The tumor remained negative, whereas the leukocytes in normal tissue were strongly positive. (I) Four images of Hbδ-stained samples in a tissue microarray, magnification 100x. (J) Hbδ staining, magnification 100x. (K) Four examples of αD staining of a tissue microarray, magnification 100x. (L) Integrin αD staining. Original magnification 100x.

Article Snippet: The primers for Hbβ, Hbδ, and αD integrin were obtained from Origene.

Techniques: Staining, Expressing, Microarray

Journal: Life Science Alliance

Article Title: Hypoxia favors tumor growth in colorectal cancer in an integrin αDβ1/hemoglobin δ-dependent manner

doi: 10.26508/lsa.202402925

Figure Lengend Snippet: (A) Whereas the epithelium remains negative, weak staining with anti-Hbδ is found in erythrocytes. (B) Anti-integrin αD stains mainly interstitial macrophages. Original magnification 100x.

Article Snippet: The primers for Hbβ, Hbδ, and αD integrin were obtained from Origene.

Techniques: Staining

Journal: Science Advances

Article Title: Phenotypic discovery and therapeutic evaluation of an ITGA3B1 -targeting antibody-drug conjugate for bladder cancer

doi: 10.1126/sciadv.ady0041

Figure Lengend Snippet: ( A ) ELISA assessing the binding specificity of 2E7 scFv-Fc binding to the ITGA3B1 heterodimer and its individual subunits ( ITGA3 and ITGB1 ), as well as to the structurally related integrin complex ITGA6B4 and its subunits ( ITGA6 and ITGB4 ). HuIgG and BSA were included as negative controls. Data represent the mean ± SD from three technical replicates. A 450 , absorbance at 450 nm. ( B ) Dose-dependent binding of 2E7 scFv-Fc to immobilized ITGA3 , ITGB1 , and ITGA3B1 , as determined by ELISA. ( C ) Flow cytometry analysis of HEK293 cells transiently transfected with ITGA3 , ITGB1 , or both. ( D ) Flow cytometry of MDA-MB-231 cells following siRNA knockdown of ITGA3 , ITGB1 , or both ITGA3 and ITGB1 . ( E ) SPR sensorgrams showing 2E7 scFv-Fc binding to recombinant ITGA3B1 in the presence of either divalent cations or EDTA. Data shown are representative of three independent experiments.

Article Snippet: Recombinant human integrin subunits ( ITGA3 , ITGA6 , ITGB1 , and ITGB4 ; OriGene Technologies, Rockville, MD) and heterodimers ( ITGA3B1 and ITGA6B4 ; Acrobiosystems, Newark, DE) were each coated onto 96-well Maxisorp plates (Thermo Fisher Scientific) at a concentration of 2 μg/ml in PBS and incubated overnight at 4°C.

Techniques: Enzyme-linked Immunosorbent Assay, Binding Assay, Flow Cytometry, Transfection, Knockdown, Recombinant

Journal: Biology

Article Title: Effect of Maternal Hyperglycemia on Fetal Pancreatic Islet Development.

doi: 10.3390/biology14060728

Figure Lengend Snippet: Figure 3. Islet of Langerhans basal membrane protein immunostaining. (A,C,E,G): Immunohis- tochemistry of pan-laminin, laminin alpha 1 and gamma 1 chains and alpha 3 integrin. Islet of Langerhans—dashed line. Brown staining indicates protein-of-interest staining. Scale bar: 20.0 µm. (B,D,F,H): Quantification of stained area related to total islet area for each analyzed protein. Data presented as mean ± standard error of the mean, * p < 0,02 and *** p < 0.0001 vs. control group by Student’s t-test.

Article Snippet: For protein detection, sections were incubated with specific primary rabbit antibodies: pan-laminin (ab1575; Abcam, diluted 1:500, Cambridge, UK), alpha 1 laminin (sc H-300 Santa Cruz H-300: SC-5582 diluted 1:50, Santa Cruz, CA, USA), gamma 1 laminin (sc5584; Santa Cruz, diluted 1:50), alpha 3 integrin (600-401 R13, Rockland diluted 1:200, Baltimore, MD, USA), anti-PCNA (ab2426 Abcam 1: 200), anti-glucagon (# A0565 1:350) and anti-insulin (# A0564 1:250).

Techniques: Membrane, Immunostaining, Staining, Control