integrin subunit β6  (Bioss)

Journal: The Biochemical journal

Article Title: IFIT3 (Interferon Induced Protein with Tetratricopeptide Repeats 3) Modulates STAT1 Expression in small Extracellular Vesicles

doi: 10.1042/BCJ20210580

Figure Lengend Snippet: (A) Immunoblotting (IB) analysis of total cell lysates (TCL) from PC3 cells containing the β6 integrin subunit, PC3-WT not treated with siRNA (−), PC3 cells treated with non-silencing siRNA (NS), or the β6 integrin subunit siRNA (D2) (30 µg). TCL were separated using 12.5% SDS–PAGE. TCL were examined for the expression of the β6 integrin subunit (left panel, non-reducing SDS–PAGE), IFIT3 and STAT1 (right panel, reducing SDS–PAGE). TSG101 (left panel, non-reducing SDS–PAGE) and Actin (right panel, reducing SDS–PAGE) were used as protein loading controls. (B) IB analysis of TCL from PC3-WT cells and PC3-CRISPR clones devoid of the β6 integrin subunit, β6KO C5 and C7 (30 µg). TCL were separated using 12.5% SDS–PAGE. TCL were examined for the expression of the β6 integrin subunit (left panel, non-reducing SDS–PAGE), IFIT3 and STAT1 (right panel, reducing SDS–PAGE). The Actin membrane (right panel, reducing SDS–PAGE) was stripped to visualize IFIT3. Actin (left panel, non-reducing SDS–PAGE), and (right panel, reducing SDS–PAGE) was used as a protein loading control.

Article Snippet: The following Abs were used for IB analyses: mouse monoclonal Abs against the human β6 integrin subunit (6.2A1) [ 34 ], CD9 (Santa Cruz, sc-13118), CD63 (Abcam, ab8219), CD81 (Abcam, ab23505), STAT1 (Santa Cruz, sc-271661) and IFIT3 (Santa Cruz, sc-393512); rabbit polyclonal Abs against Actin (Sigma–Aldrich, A2066) and calnexin (Cell signaling, 2433S); rabbit monoclonal TSG101 (Abcam, ab125011); and goat-affinity purified polyclonal Abs against the β6 integrin subunit (R&D Systems, AF2389).

Techniques: Western Blot, SDS Page, Expressing, CRISPR, Clone Assay, Membrane, Control

Journal: The Biochemical journal

Article Title: IFIT3 (Interferon Induced Protein with Tetratricopeptide Repeats 3) Modulates STAT1 Expression in small Extracellular Vesicles

doi: 10.1042/BCJ20210580

Figure Lengend Snippet: (A) IB analysis of sEV fractions (F1-F10) isolated via iodixanol density gradients and derived from PC3 wild-type (PC3-WT) cells containing the β6 integrin subunit, (PC3-WT). PC3-WT TCL (20 µg), PC3-WT (PC3-WT 100K) sEV lysate (10 µg) and PC3-WT sEV fraction lysates were separated using 12.5% SDS–PAGE. Comparable volumes of each sEV fraction were loaded (30 µl). The 10 consecutive sEV fractions have densities of 1.103, 1.117,1.134,1.151, 1.169, 1.186, 1.193, 1.193, 1.200, and 1.207 g/ml, respectively. Expression of the β6 integrin subunit, CD63, CD81 (left panel, non-reducing SDS–PAGE) as well as STAT1, IFIT3 and CD9 (right panel, reducing SDS–PAGE) was analyzed in sEV fractions 1 to 10. The STAT1 membrane (right panel, reducing SDS–PAGE) was stripped to visualize calnexin (CNX). CNX (right panel, reducing SDS–PAGE) in PC3-β6KO C7, PC3-WT TCL, PC3-WT 100K as well as 10 consecutive PC3-WT derived sEV fractions was analyzed. PC3-WT 100K lysate was the input for the density gradient. (B) IB analysis of PC3-β6KO C7 derived sEV fractions isolated via density gradients. Total lysates (TCL 20 µg) from PC3-WT cells containing the β6 integrin subunit, as well as the PC3-CRISPR clone devoid of the β6 integrin subunit, PC3-β6KO C7, and PC3-β6KO C7 sEV fraction lysates were separated using 12.5% SDS–PAGE. Comparable volumes of each sEV fraction were loaded (30 µl). The 10 consecutive sEV fractions have densities of 1.075, 1.099, 1.123, 1.134, 1.151, 1.165, 1.169, 1.182, 1.186, and 1.193 g/ml, respectively. Expression of the β6 integrin, CD63, CD81 (left panel, non-reducing SDS–PAGE), STAT1, IFIT3 and CD9 (right panel, reducing SDS–PAGE) was analyzed in sEV fractions 1 to 10. The STAT1 membrane (right panel, reducing SDS–PAGE) was stripped to visualize calnexin (CNX). CNX (right panel, reducing SDS–PAGE) in the PC3-CRISPR clone lacking the β6 integrin subunit, PC3-β6KO C7 TCL and PC3-β6KO C7 derived sEV fractions was analyzed. A and B, TCL = total cell lysates.

Article Snippet: The following Abs were used for IB analyses: mouse monoclonal Abs against the human β6 integrin subunit (6.2A1) [ 34 ], CD9 (Santa Cruz, sc-13118), CD63 (Abcam, ab8219), CD81 (Abcam, ab23505), STAT1 (Santa Cruz, sc-271661) and IFIT3 (Santa Cruz, sc-393512); rabbit polyclonal Abs against Actin (Sigma–Aldrich, A2066) and calnexin (Cell signaling, 2433S); rabbit monoclonal TSG101 (Abcam, ab125011); and goat-affinity purified polyclonal Abs against the β6 integrin subunit (R&D Systems, AF2389).

Techniques: Isolation, Derivative Assay, SDS Page, Expressing, Membrane, CRISPR

Journal: The Biochemical journal

Article Title: IFIT3 (Interferon Induced Protein with Tetratricopeptide Repeats 3) Modulates STAT1 Expression in small Extracellular Vesicles

doi: 10.1042/BCJ20210580

Figure Lengend Snippet: (A) Nanoparticle tracking analysis (NTA) measurement of size distribution and concentration of sEV fractions 2 to 5 derived from cells containing the β6 integrin subunit (PC3-WT). (B) NTA measurement of size distribution and concentration of sEV fractions 2 to 5 derived from the PC3-CRISPR clone devoid of the β6 integrin subunit, PC3-β6KO C7.

Article Snippet: The following Abs were used for IB analyses: mouse monoclonal Abs against the human β6 integrin subunit (6.2A1) [ 34 ], CD9 (Santa Cruz, sc-13118), CD63 (Abcam, ab8219), CD81 (Abcam, ab23505), STAT1 (Santa Cruz, sc-271661) and IFIT3 (Santa Cruz, sc-393512); rabbit polyclonal Abs against Actin (Sigma–Aldrich, A2066) and calnexin (Cell signaling, 2433S); rabbit monoclonal TSG101 (Abcam, ab125011); and goat-affinity purified polyclonal Abs against the β6 integrin subunit (R&D Systems, AF2389).

Techniques: Concentration Assay, Derivative Assay, CRISPR

Journal: The Biochemical journal

Article Title: IFIT3 (Interferon Induced Protein with Tetratricopeptide Repeats 3) Modulates STAT1 Expression in small Extracellular Vesicles

doi: 10.1042/BCJ20210580

Figure Lengend Snippet: (A) NTA measurement of size distribution and concentration of density gradient-isolated sEVs (pooled fractions 2 to 5) derived from cells containing the β6 integrin subunit, PC3-CRISPR control and the PC3-CRISPR clones devoid of the β6 integrin subunit, β6KO C5 and β6KO C7. (B) Transmission electron microscopy analysis of density gradient-isolated sEVs (pooled fractions 2 to 5) derived from PC3-CRISPR control and β6KO C5 cells. Scale bar = 100 nm.

Article Snippet: The following Abs were used for IB analyses: mouse monoclonal Abs against the human β6 integrin subunit (6.2A1) [ 34 ], CD9 (Santa Cruz, sc-13118), CD63 (Abcam, ab8219), CD81 (Abcam, ab23505), STAT1 (Santa Cruz, sc-271661) and IFIT3 (Santa Cruz, sc-393512); rabbit polyclonal Abs against Actin (Sigma–Aldrich, A2066) and calnexin (Cell signaling, 2433S); rabbit monoclonal TSG101 (Abcam, ab125011); and goat-affinity purified polyclonal Abs against the β6 integrin subunit (R&D Systems, AF2389).

Techniques: Concentration Assay, Isolation, Derivative Assay, CRISPR, Control, Clone Assay, Transmission Assay, Electron Microscopy

Journal: The Biochemical journal

Article Title: IFIT3 (Interferon Induced Protein with Tetratricopeptide Repeats 3) Modulates STAT1 Expression in small Extracellular Vesicles

doi: 10.1042/BCJ20210580

Figure Lengend Snippet: (A) IB analysis of PC3-WT and the PC3-CRISPR clones devoid of IFIT3: PC3-IFIT3KO C13 and PC3-IFIT3KO C15 TCL (total cell lysates) (30 µg). TCL were separated using 12.5% SDS–PAGE and examined for the expression of STAT1 and IFIT3 (reducing SDS–PAGE). The IFIT3 membrane (reducing SDS–PAGE) was stripped to visualize Actin. Actin (reducing SDS–PAGE) was used as a protein loading control. Dark and light exposures shown (Top and bottom, respectively). (B) IB analysis of cells expressing the β6 integrin subunit, PC3-CRISPR control cell-derived sEV fractions isolated via density gradients. PC3-CRISPR control TCL (20 µg), PC3-CRISPR control (CRISPR control 100K) sEV lysate (10 µg), and PC3-CRISPR control sEV fraction lysates were separated by using 12.5% SDS–PAGE. Comparable volumes of each sEV fraction were loaded (30 µl). The 10 consecutive sEV fractions have a density of 1.099, 1.113,1.123,1.137, 1.148, 1.162, 1.179, 1.186, 1.193, and 1.210 g/ml, respectively. Expression of the β6 integrin subunit, CD63, CD81 (left panel, non-reducing SDS–PAGE), STAT1, IFIT3, TSG101 and CD9 (right panel, reducing SDS–PAGE) was analyzed in sEV fractions 1 to 10. IFIT3 (right panel, reducing SDS–PAGE) and STAT1 membranes (right panel, reducing SDS–PAGE) were stripped to visualize TSG101 and calnexin (CNX), respectively. CNX expression (right panel, reducing SDS–PAGE) in PC3-CRISPR control TCL, CRISPR control 100K as well as 10 consecutive PC3-CRISPR control cell-derived sEV fractions is shown. CRISPR control 100K sEV lysate was used as input for the density gradient. (C) IB analysis of sEV fractions derived from a PC3-CRISPR clone devoid of IFIT3, designated as PC3-IFIT3KO C13, and isolated via density gradients. PC3-IFIT3KO C13 TCL (20 µg), PC3-IFIT3KO C13 (IFIT3KO C13 100K) sEV lysate (10 µg) and PC3-IFIT3KO C13 sEV fraction lysates were separated using 12.5% SDS–PAGE. Comparable volumes of each sEV fraction were loaded (30 µl). The 10 consecutive sEV fractions have a density of 1.106, 1.110,1.130,1.134, 1.155, 1.169, 1.175, 1.182, 1.182, and 1.189 g/ml, respectively. Expression of the β6 integrin subunit, CD63 and CD81 (left panel, non-reducing SDS–PAGE) is analyzed in sEV fractions 1 to 10. The β6 integrin subunit membrane (left panel, reducing SDS–PAGE) was stripped to visualize calnexin (CNX). CNX expression (left panel, non-reducing SDS–PAGE) in PC3-IFIT3KO C13 TCL, PC3-WT 100K, IFIT3 C13 100K and 10 consecutive PC3-IFIT3KO C13 cell-derived sEV fractions is shown. Expression of STAT1, TSG101 and CD9 (right panel, reducing SDS–PAGE) is analyzed. IFIT3KO C13 100K was the input for the density gradient.

Article Snippet: The following Abs were used for IB analyses: mouse monoclonal Abs against the human β6 integrin subunit (6.2A1) [ 34 ], CD9 (Santa Cruz, sc-13118), CD63 (Abcam, ab8219), CD81 (Abcam, ab23505), STAT1 (Santa Cruz, sc-271661) and IFIT3 (Santa Cruz, sc-393512); rabbit polyclonal Abs against Actin (Sigma–Aldrich, A2066) and calnexin (Cell signaling, 2433S); rabbit monoclonal TSG101 (Abcam, ab125011); and goat-affinity purified polyclonal Abs against the β6 integrin subunit (R&D Systems, AF2389).

Techniques: CRISPR, Clone Assay, SDS Page, Expressing, Membrane, Control, Derivative Assay, Isolation

Journal: The Biochemical journal

Article Title: IFIT3 (Interferon Induced Protein with Tetratricopeptide Repeats 3) Modulates STAT1 Expression in small Extracellular Vesicles

doi: 10.1042/BCJ20210580

Figure Lengend Snippet: (A) IB analysis of PC3-CRISPR control cells and PC3-CRISPR clones devoid of STAT1, PC3-STAT1KO C21 as well as PC3-STAT1KO C1C5 TCL (total cell lysates) (30 µg). TCL were separated using 12.5% SDS–PAGE. TCL were examined for the expression of STAT1, IFIT3, and Actin (reducing SDS–PAGE). The IFIT3 membrane (reducing SDS–PAGE) was stripped to visualize Actin. Actin was used as a protein loading control. (B) NTA measurement of size distribution and concentration of PC3-STAT1KO C21 derived sEV fractions 2-5. (C) IB analysis of sEV fractions derived from a PC3-CRISPR clone lacking STAT1, designated as PC3-STAT1KO C21, and isolated via density gradients. PC3-β6KO C7, PC3-STAT1KO C21 TCL (20 µg), PC3-STAT1KO C21 (STAT1KO C21 100K) sEV lysate (10 µg) and PC3-STAT1KO C21 sEV fraction lysates were separated by using 12.5% SDS–PAGE. Comparable volumes of each sEV fraction were loaded (30 µl). The 10 consecutive sEV fractions have a density of 1.106, 1.123,1.127,1.144 1.165, 1.169,1.186, 1.193, 1.200, and 1.207 g/ml, respectively. Expression of STAT1, IFIT3, TSG101, CD9 and CD81 (reducing SDS–PAGE) was analyzed in sEV fractions 1 to 10. The IFIT3, STAT1 and CD9 membranes (reducing SDS–PAGE) were stripped to visualize TSG101, calnexin (CNX), and CD81 respectively. CNX expression (reducing SDS–PAGE) was analyzed in PC3-CRISPR clone devoid of the β6 integrin subunit, PC3-β6KO C7 as well as PC3-CRISPR clone lacking STAT1, PC3-STAT1KO C21 TCL, STAT1KO C21 100K and 10 consecutive PC3-STAT1KO C21 derived sEV fractions. PC3-STAT1KO C21 100K sEV lysate was used as input for the density gradient.

Article Snippet: The following Abs were used for IB analyses: mouse monoclonal Abs against the human β6 integrin subunit (6.2A1) [ 34 ], CD9 (Santa Cruz, sc-13118), CD63 (Abcam, ab8219), CD81 (Abcam, ab23505), STAT1 (Santa Cruz, sc-271661) and IFIT3 (Santa Cruz, sc-393512); rabbit polyclonal Abs against Actin (Sigma–Aldrich, A2066) and calnexin (Cell signaling, 2433S); rabbit monoclonal TSG101 (Abcam, ab125011); and goat-affinity purified polyclonal Abs against the β6 integrin subunit (R&D Systems, AF2389).

Techniques: CRISPR, Control, Clone Assay, SDS Page, Expressing, Membrane, Concentration Assay, Derivative Assay, Isolation

Journal: Cancer Cell International

Article Title: Targeting of 3D oral cancer spheroids by αVβ6 integrin using near-infrared peptide-conjugated IRDye 680

doi: 10.1186/s12935-024-03417-y

Figure Lengend Snippet: Analysis of αVβ6 expression. ( A ) ITGB6 proteomic expression profile in normal tissue and primary tumor (data from UALCAN) ( n = 71–108; *** p < 0.001; Two sample t test). ( B ) RT-qPCR analysis of ITGB6 mRNA expression; β-actin was used as housekeeping gene. The (αVβ6+) cell line HT-29 was used as reference ( n = 3–8; *** p < 0.001; One-way ANOVA followed by Tukey’s assay). ( C - D ) β6 integrin subunit protein expression assessed by Western-Blot; tubulin was used as a loading control ( n = 3–7; *** p < 0.001; One way ANOVA followed by Tukey’s assay). Data are presented as mean ± SEM

Article Snippet: 40 µg of protein lysate was loaded to 7.5% non-reducing SDS PAGE and after 150 min of migration and 90 min of blotting, PVDF membrane was saturated with 5% (w/v) solution of non-fat powered milk in TBST (Tris buffer solution with 0.1% Tween-20) for 1 h. Membrane was incubated overnight at 4 °C with either 1:100 mouse anti-human β6 integrin subunit antibody (Merck Millipore corp, USA, 407,317) or 1:1000 mouse anti-α-tubulin antibody (Santa Cruz Biotechnology, 23,948), followed by incubation with 1:2000 anti-mouse IgG HRP-conjugated secondary antibody (Cell signaling technology, 7076 S) for 1 h at room temperature.

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Control

Journal: Cancer Cell International

Article Title: Targeting of 3D oral cancer spheroids by αVβ6 integrin using near-infrared peptide-conjugated IRDye 680

doi: 10.1186/s12935-024-03417-y

Figure Lengend Snippet: Multicellular tumor spheroid characterization. ( A ) Typical cryosection images of H10 and H5M5 spheroids after KI-67 immunohistochemistry staining (scale bar = 150 μm). ( B ) Typical immunofluorescence images of H10 and H5M5 spheroids cryosections stained with antibodies against αVβ6 integrin, cytokeratin 19, E-cadherin, vimentin, and fibronectin at day 4 post-seeding. Proteins of interest are in red and nuclei are in bleu after counterstaining with Vectashield-DAPI (scale bar = 100 μm at x40 and 40 μm at x100). ( C ) Relative quantification of immunofluorescence markers expression using ImageJ software ( n = 3–4; * p < 0.05, ** p < 0.01; Two sample t test). Data are presented as mean ± SEM

Article Snippet: 40 µg of protein lysate was loaded to 7.5% non-reducing SDS PAGE and after 150 min of migration and 90 min of blotting, PVDF membrane was saturated with 5% (w/v) solution of non-fat powered milk in TBST (Tris buffer solution with 0.1% Tween-20) for 1 h. Membrane was incubated overnight at 4 °C with either 1:100 mouse anti-human β6 integrin subunit antibody (Merck Millipore corp, USA, 407,317) or 1:1000 mouse anti-α-tubulin antibody (Santa Cruz Biotechnology, 23,948), followed by incubation with 1:2000 anti-mouse IgG HRP-conjugated secondary antibody (Cell signaling technology, 7076 S) for 1 h at room temperature.

Techniques: Immunohistochemistry, Staining, Immunofluorescence, Expressing, Software