Journal: Virology Journal

Article Title: Replication kinetics of pathogenic Eurasian orthohantaviruses in human mesangial cells

doi: 10.1186/s12985-024-02517-5

Figure Lengend Snippet: Flow cytometry analysis of surface expression of orthohantaviral entry receptors. CIHGM-1 cells were analyzed for the presence of integrin α v β 3 , integrin β 1 , and CD55 on the cell surface. Plots shown are gated on the viable cell population according to scatter profile and the exclusion of Via-Probe™ Cell Viability Solution. Quadrant statistics (Q1-Q4) indicate the percentage of cells in the respective quadrant

Article Snippet: CIHGM-1 cells were washed with PBS, scraped, and stained with phycoerythrin (PE)-conjugated mouse anti-integrin α V β 3 antibody (clone LM609, Millipore) or PE-conjugated mouse anti-integrin β 1 antibody (clone P5D2, R&D Systems, Minneapolis, MN, USA) together with allophycocyanin (APC)-conjugated anti-CD55 (clone IA10, BD Pharmingen, NJ, USA).

Techniques: Flow Cytometry, Expressing

Journal: PLoS Pathogens

Article Title: The RGD-binding integrins αvβ6 and αvβ8 are receptors for mouse adenovirus-1 and -3 infection

doi: 10.1371/journal.ppat.1010083

Figure Lengend Snippet: (A) Flow cytometry profiles of B16, B16-mβ6, B16-mβ8, A549 and A549-hβ6 cells. Green and red histograms show β6 and β8 specific staining, respectively, and grey histograms show background staining using an isotype control. Numbers indicate MFI values of specific or control antibodies. (B, C) Virus binding and dependency on divalent ions. Detached mouse (B) and human cells (C) were incubated with control medium (not containing virus) or medium containing the indicated viruses for 1 h on ice, followed by washing and staining with primary rabbit anti-FK-M3 antibodies and secondary fluorescently labeled antibodies for flow cytometry analysis. Incubation/washing buffers were adjusted to contain either Mg 2+ /Ca 2+ , 1 mM each, 1/0.2 mM Mn 2+ /Ca 2+ , or EDTA 2.5 mM. (D) Virus-receptor antibody competition experiment. Detached cells were first incubated on ice with control medium or the indicated viruses, followed by washing and incubation with either the anti-αvβ6 antibody (CMT-93 and B16-mβ6 cells), or the αvβ8 antibody (M000216 cells). Subsequently the cells were stained with secondary fluorescently labeled antibodies for flow cytometry analysis. (E) The indicated mouse and human cells were infected with recombinant M1-/M3-IX-G, M2-ΔE1A-G and fiber chimeric H5-ΔE3B-CG-FK-M1/-FK-M3 viruses at an MOI of 3. Cells were harvested at the indicated six time points and GFP intensity (MFI) was determined by flow cytometry. Cellular autofluorescence of uninfected cells was included as 0 h infection time point. (F) For analysis of virus progeny, CMT-93, B16, B16-mβ6 and B16-mβ8 cells were infected with M1-/M3-IX-G using an MOI of 1.5. After 14 h, the cells were thoroughly washed, trypsinized and re-seeded. Virus-containing supernatant samples were collected 48 (d2) and 72 h (d3) pi and used for titration analyses. Based on the virus input, fold increases of progeny virus were calculated. For B16 cells no measurable levels of viruses were detected, which translated to a virus progeny production of less than a factor of 0.01, based on the sensitivity level of this assay. Data in (B) to (F) represent triplicates, shown as mean ± SEM. Asterisks indicate level of significance for comparison of indicated values (*, P <0.05; **, P <0.005; ***, P <0.0005).

Article Snippet: The A20M1 and A20M3 consist of aa residues 490–509 of the M1 fiber (Uniprot P19721), and 452–471 of the M3 fiber (GenBank: ACJ14524.1) sequence, respectively. sITGs were obtained from R&D systems (Bio-techne brand, USA), including mouse and human αvβ3 (7889-AV-050/3050-AV-050), αvβ5 (7706-AV-050/2528-AV-050), αvβ6 (7480-AV-050/3817-AV-050), αvβ8 (8314-AV-050/4135-AV-050), recombinantly expressed in and secreted from CHO cells (quality control shown in ).

Techniques: Flow Cytometry, Staining, Control, Virus, Binding Assay, Incubation, Labeling, Infection, Recombinant, Titration, Comparison

Journal: PLoS Pathogens

Article Title: The RGD-binding integrins αvβ6 and αvβ8 are receptors for mouse adenovirus-1 and -3 infection

doi: 10.1371/journal.ppat.1010083

Figure Lengend Snippet: (A) Virus binding interference in CMT-93 and M000216 cells by β6/-β8 function blocking antibodies. Detached cells were sequentially incubated for 1 h on ice with control antibody, or the anti-β6/-β8 antibodies, followed by incubation with control medium or medium containing the indicated viruses at an MOI of 4, the rabbit anti-FK-M3 antibodies, and finally the secondary PE-conjugate antibodies. Incubation and washing buffer contained either Mg 2+ /Ca 2+ , 1 mM each, or 1/0.2 mM Mn 2+ /Ca 2+ . (B-C) Virus infection interference in CMT-93 and M000216 cells by β6- and β8-specific antibodies. CMT-93 (B) and M000216 cells (C) were pre-incubated for 1 h on ice using 5-fold dilution series of the specific β6- or β8-integrin antibodies, respectively, starting with 800 ng/ml as highest concentration, followed by addition of the different indicated GFP-expressing viruses and transfer to 37°C for 48 h. An MOI of 1 was used for CMT-93 cells and MOI of 3 for M000216 cells in all experiments shown in this figure. GFP analysis was performed 48 h pi, and expression index was normalized to a control antibody. IC 50 values determined in this experiment are summarized in . (D, E) Infection blocking assays by sITGs. M1-IX-G virus was incubated for 1 h at RT with 5-fold serial dilutions of the indicated sITGs starting from 800 ng/ml to 6.4 ng/ml, followed by addition to CMT-93 cells (D) and M00216 cells (E) and cultivated and further processed as above. (F-H) Infection blocking assays by peptides. (F) The 20-mer peptides tested for virus infection inhibition included peptides A20FMDV2 derived from the VP1 coat protein of FMDV2, A20FMDV2-E containing a D to E mutation in the critical RGD motif, A20M1 and A20M3 derived from M1-/M3-FK, respectively, as compared to LAP-hTGFβ1, all containing the critical αvβ6/αvβ8-binding RGDLXX(L/I) motif. (G, H) Cells were pre-incubated on ice with 5-fold serial dilutions of peptides resulting in final concentrations from 5,000 to 0.32 nM. Subsequently, M1-IX-G virus was added to CMT-93 cells (G) or M000216 cells (H), followed by processing as described above. (I) Comparative flow cytometry profiles of αvβ8 expression in 3T6 cells. Blue and red show β8-specific staining in 3T6-sgNT and 3T6-sgItgβ8 cells, respectively, and grey histogram shows background staining of 3T6-sgItgβ8 cells using a matched isotype control. Numbers indicate MFI values of specific or control antibodies. (J, K) Transduction of 3T6-sgNT and 3T6-sgItgβ8 cells using M1-/M3-IX-G, the fiber-chimeric H5-ΔE3B-CG-FK-M1/-FK-M3 and control H5-ΔE3B-CG at an MOI of 9. Cells were processed as described in . (L) Comparative flow cytometry MFI αvβ6 expression values in control CMT-93-sgNT versus β6 integrin shRNA knock down CMT-93-sgItgβ6 cells. (M-O) Infection of control CMT-93-sgNT and CMT-93-sgItgβ6 cells using M1-IX-G (M), M3-IX-G (N) and H5-ΔE3B-CG (O) at an MOI of 1. Cells were processed as described above. Except for the representative flow cytometry histogram in (I), data represent triplicates, shown as mean ± SEM. Asterisks indicate level of significance for comparison of indicated values (*, P <0.05; **, P <0.005; ***, P <0.0005).

Article Snippet: The A20M1 and A20M3 consist of aa residues 490–509 of the M1 fiber (Uniprot P19721), and 452–471 of the M3 fiber (GenBank: ACJ14524.1) sequence, respectively. sITGs were obtained from R&D systems (Bio-techne brand, USA), including mouse and human αvβ3 (7889-AV-050/3050-AV-050), αvβ5 (7706-AV-050/2528-AV-050), αvβ6 (7480-AV-050/3817-AV-050), αvβ8 (8314-AV-050/4135-AV-050), recombinantly expressed in and secreted from CHO cells (quality control shown in ).

Techniques: Virus, Binding Assay, Blocking Assay, Incubation, Control, Infection, Concentration Assay, Expressing, Inhibition, Derivative Assay, Mutagenesis, Flow Cytometry, Staining, Transduction, shRNA, Knockdown, Comparison

Journal: PLoS Pathogens

Article Title: The RGD-binding integrins αvβ6 and αvβ8 are receptors for mouse adenovirus-1 and -3 infection

doi: 10.1371/journal.ppat.1010083

Figure Lengend Snippet: (A, B) Sensor chips containing immobilized biotinylated FK-M1 and FK-M3 were probed with mouse sITG αvβ6. Following consecutive analyte injections over 120 s, dissociation was monitored for 600 s (black). Sensorgrams were fitted with a 1:1 kinetic model (red). (C-E) FK saturation cell binding assays using cells with defined αvβ6/αvβ8 expression included FK-M1 binding to B16-mβ6 (C), FK-M3 binding to B16-mβ6 (D) and FK-M3 binding to B16-mβ8 (E). Parental B16 cells were included in order to subtract background levels when calculating equilibrium dissociation constant K D values by Scatchard plot analyses.

Article Snippet: The A20M1 and A20M3 consist of aa residues 490–509 of the M1 fiber (Uniprot P19721), and 452–471 of the M3 fiber (GenBank: ACJ14524.1) sequence, respectively. sITGs were obtained from R&D systems (Bio-techne brand, USA), including mouse and human αvβ3 (7889-AV-050/3050-AV-050), αvβ5 (7706-AV-050/2528-AV-050), αvβ6 (7480-AV-050/3817-AV-050), αvβ8 (8314-AV-050/4135-AV-050), recombinantly expressed in and secreted from CHO cells (quality control shown in ).

Techniques: Binding Assay, Expressing

Journal: PLoS Pathogens

Article Title: The RGD-binding integrins αvβ6 and αvβ8 are receptors for mouse adenovirus-1 and -3 infection

doi: 10.1371/journal.ppat.1010083

Figure Lengend Snippet: (A) Overview of αvβ6 complexed with the 20mer A20M3 peptide. αv (light green), β6 (pink) and A20M3 (grey) chains are shown as cartoon traces. Side chains of the RGD motif are shown as sticks and spheres. (B) Superposition of αvβ6 in surface representation in complex with A20M1 (yellow carbons), A20M3 (grey carbons) and A20FMDV2 (salmon carbons). The position of manganese ions (cyan spheres) shown superimposed were derived from the human αvβ6/TGFβ1 complex (PDB ID: 5ffo). Labeled aa refer to the A20M3 sequence. (C) Superposition of the αvβ6/A20M3 complex onto the αvβ8 structure (blue carbons). Note that the A20M3 peptide is not shown here. Residue numbering refers to the Uniprot entry Q9Z0T9 (mouse integrin subunit β6) and the A20M3 peptide sequence (Arg16).

Article Snippet: The A20M1 and A20M3 consist of aa residues 490–509 of the M1 fiber (Uniprot P19721), and 452–471 of the M3 fiber (GenBank: ACJ14524.1) sequence, respectively. sITGs were obtained from R&D systems (Bio-techne brand, USA), including mouse and human αvβ3 (7889-AV-050/3050-AV-050), αvβ5 (7706-AV-050/2528-AV-050), αvβ6 (7480-AV-050/3817-AV-050), αvβ8 (8314-AV-050/4135-AV-050), recombinantly expressed in and secreted from CHO cells (quality control shown in ).

Techniques: Derivative Assay, Labeling, Sequencing, Residue

Journal: PLoS Pathogens

Article Title: The RGD-binding integrins αvβ6 and αvβ8 are receptors for mouse adenovirus-1 and -3 infection

doi: 10.1371/journal.ppat.1010083

Figure Lengend Snippet: (A) Immunoblot analysis of mouse CMT-93 cells infected with M1 wt, and recombinant M1-ΔE1A-G and M1-IX-G viruses using an MOI of 3. Cell lysate samples were harvested at six time points and analyzed with the indicated rabbit antibodies raised against early E1A-M1, E1B-19K-M1, intermediate protein IX-M1, and the late hexon protein-M1, plus mouse antibodies against GFP and control actin. Staining with protein IX-specific antibodies revealed a weak band corresponding to processed IX-2A (Mr 14.1 kDa), and a major form corresponding to unprocessed IX-2A-GFP (Mr 41 kDa). Staining with GFP-specific antibodies revealed two major processing forms, corresponding to processed GFP (Mr 27 kDa), and the unprocessed IX-2A-GFP, respectively. Both of these stainings gave rise to additional individual protein forms (denoted by *). (B) Mouse CMT-93 cells and (C) human M000216 cells were infected with recombinant M1-/M3-IX-G, M2-ΔE1A-G and fiber-chimeric H5-ΔE3B-CG-FK-M1 and–FK-M3 viruses at an MOI of 3. Cells were harvested at the indicated six time points and GFP intensity (upper panel) and percent infected cells (lower panel) were determined by flow cytometry. Cellular autofluorescence of uninfected cells was included as 0 h infection time point. Data represent triplicates, shown as mean ± SEM.

Article Snippet: Integrin antibodies included human αv mAb Sc-9969 (Santa Cruz), human β1 mAb sc-59829 (Santa Cruz), hu β3 mAb AP3 (ATCC), human β5 mAb B5-IVF2 (provided by M. Hemler, Harvard Medical School, Boston, USA), human αvβ3 mAb 23C6 (sc-7312, Santa Cruz), human and mouse αvβ5 mAb ALULA (provided by D. Sheppard, UCSF [ ]), human and mouse αvβ6 mAb 10D5 (function blocking [ ], ab77906, Abcam), human and mouse αvβ8 mAb ADWA-11 (function blocking, provided by D. Sheppard, UCSF [ ]), mouse αv rAb RMV7 (14–00512, Affymetrix eBioscience), mouse β1 mAb (MAB2405, R&D systems, Bio-techne brand, USA) mouse β3 (rAb MAB41182, R&D systems, Bio-techne brand, USA).

Techniques: Western Blot, Infection, Recombinant, Control, Staining, Flow Cytometry

Journal: PLoS Pathogens

Article Title: The RGD-binding integrins αvβ6 and αvβ8 are receptors for mouse adenovirus-1 and -3 infection

doi: 10.1371/journal.ppat.1010083

Figure Lengend Snippet: (A-D) CMT-93 cells were incubated for 1 h on ice using 5-fold dilution series of the indicated FK proteins starting with 0.8 μg/ml as highest concentration, followed by addition of the different GFP-expressing viruses and transfer to 37°C for 48 h. The virus input amounted to an MOI of 1 and included M1-IX-G (A), M3-IX-G (B), H5-ΔE3B-CG-FK-M1 (C), M2-ΔE1A-G (D). GFP analysis was performed 48 h pi, and expression index was normalized to FK-H3 control protein. (E) M000216 cells were preincubated and processed as described for CMT-93 cells, except that M1-IX-G was used at an MOI of 3. (F) The M3-IX-G and H5-ΔE3B-CG-FK-M3 viruses were pre-incubated for 1 h at RT with serial 5-fold dilutions of the different antisera ranging from 1:1,250 to 1:781,250, followed by addition of the mixes to CMT-93 cells for 48 h at 37°C. The rabbit antisera tested were raised against recombinant FK-M3, FK-M2 and FK-H3, respectively. The virus input amounted to an MOI of 1, and samples were processed for analysis as described above. (G) To check for cross-neutralization of the rabbit anti-FK-M3 for M1, M1-IX-G was preincubated with serial dilutions of rabbit anti-FK-M3, -FK-M2 and -FK-H3 sera and further processed as described above. For all experiments, data represent triplicates, shown as mean ± SEM. For highest concentrations of FKs and anti-FK sera, asterisks indicate level of significance for comparison of indicated values (*, P <0.05; **, P <0.005; ***, P <0.0005); ns: not significant ( P >0.05).

Article Snippet: Integrin antibodies included human αv mAb Sc-9969 (Santa Cruz), human β1 mAb sc-59829 (Santa Cruz), hu β3 mAb AP3 (ATCC), human β5 mAb B5-IVF2 (provided by M. Hemler, Harvard Medical School, Boston, USA), human αvβ3 mAb 23C6 (sc-7312, Santa Cruz), human and mouse αvβ5 mAb ALULA (provided by D. Sheppard, UCSF [ ]), human and mouse αvβ6 mAb 10D5 (function blocking [ ], ab77906, Abcam), human and mouse αvβ8 mAb ADWA-11 (function blocking, provided by D. Sheppard, UCSF [ ]), mouse αv rAb RMV7 (14–00512, Affymetrix eBioscience), mouse β1 mAb (MAB2405, R&D systems, Bio-techne brand, USA) mouse β3 (rAb MAB41182, R&D systems, Bio-techne brand, USA).

Techniques: Incubation, Concentration Assay, Expressing, Virus, Control, Recombinant, Neutralization, Comparison

Journal: PLoS Pathogens

Article Title: The RGD-binding integrins αvβ6 and αvβ8 are receptors for mouse adenovirus-1 and -3 infection

doi: 10.1371/journal.ppat.1010083

Figure Lengend Snippet: (A) The green and brown histograms show cytofluorometric analysis of FLAG-tag expression levels in parental B16 and B16-mCAR cells, consisting of B16 cells stably expressing N-terminal FLAG-tagged mCAR, respectively. The grey histogram shows background staining of B16-mCAR cells using a matched isotype control. Numbers indicate MFI values of specific or control antibodies. (B) Parental B16 cells with known low, and CMT-93 cells with high sensitivity for H5-ΔE1-CG, plus B16-mCAR cells were infected with M1-IX-G, M2-ΔE1A-G, M3-IX-G and H5-ΔE1-CG using an MOI of 15 for both B16 cell types, and an MOI of 3 for CMT-93 cells. Cells were analyzed by flow cytometry 48 h pi. MFI values for GFP in red and blue histograms are from uninfected and infected cells, respectively.

Article Snippet: Integrin antibodies included human αv mAb Sc-9969 (Santa Cruz), human β1 mAb sc-59829 (Santa Cruz), hu β3 mAb AP3 (ATCC), human β5 mAb B5-IVF2 (provided by M. Hemler, Harvard Medical School, Boston, USA), human αvβ3 mAb 23C6 (sc-7312, Santa Cruz), human and mouse αvβ5 mAb ALULA (provided by D. Sheppard, UCSF [ ]), human and mouse αvβ6 mAb 10D5 (function blocking [ ], ab77906, Abcam), human and mouse αvβ8 mAb ADWA-11 (function blocking, provided by D. Sheppard, UCSF [ ]), mouse αv rAb RMV7 (14–00512, Affymetrix eBioscience), mouse β1 mAb (MAB2405, R&D systems, Bio-techne brand, USA) mouse β3 (rAb MAB41182, R&D systems, Bio-techne brand, USA).

Techniques: FLAG-tag, Expressing, Stable Transfection, Staining, Control, Infection, Flow Cytometry

Journal: PLoS Pathogens

Article Title: The RGD-binding integrins αvβ6 and αvβ8 are receptors for mouse adenovirus-1 and -3 infection

doi: 10.1371/journal.ppat.1010083

Figure Lengend Snippet: (A) Flow cytometry profiles of B16, B16-mβ6, B16-mβ8, A549 and A549-hβ6 cells. Green and red histograms show β6 and β8 specific staining, respectively, and grey histograms show background staining using an isotype control. Numbers indicate MFI values of specific or control antibodies. (B, C) Virus binding and dependency on divalent ions. Detached mouse (B) and human cells (C) were incubated with control medium (not containing virus) or medium containing the indicated viruses for 1 h on ice, followed by washing and staining with primary rabbit anti-FK-M3 antibodies and secondary fluorescently labeled antibodies for flow cytometry analysis. Incubation/washing buffers were adjusted to contain either Mg 2+ /Ca 2+ , 1 mM each, 1/0.2 mM Mn 2+ /Ca 2+ , or EDTA 2.5 mM. (D) Virus-receptor antibody competition experiment. Detached cells were first incubated on ice with control medium or the indicated viruses, followed by washing and incubation with either the anti-αvβ6 antibody (CMT-93 and B16-mβ6 cells), or the αvβ8 antibody (M000216 cells). Subsequently the cells were stained with secondary fluorescently labeled antibodies for flow cytometry analysis. (E) The indicated mouse and human cells were infected with recombinant M1-/M3-IX-G, M2-ΔE1A-G and fiber chimeric H5-ΔE3B-CG-FK-M1/-FK-M3 viruses at an MOI of 3. Cells were harvested at the indicated six time points and GFP intensity (MFI) was determined by flow cytometry. Cellular autofluorescence of uninfected cells was included as 0 h infection time point. (F) For analysis of virus progeny, CMT-93, B16, B16-mβ6 and B16-mβ8 cells were infected with M1-/M3-IX-G using an MOI of 1.5. After 14 h, the cells were thoroughly washed, trypsinized and re-seeded. Virus-containing supernatant samples were collected 48 (d2) and 72 h (d3) pi and used for titration analyses. Based on the virus input, fold increases of progeny virus were calculated. For B16 cells no measurable levels of viruses were detected, which translated to a virus progeny production of less than a factor of 0.01, based on the sensitivity level of this assay. Data in (B) to (F) represent triplicates, shown as mean ± SEM. Asterisks indicate level of significance for comparison of indicated values (*, P <0.05; **, P <0.005; ***, P <0.0005).

Article Snippet: Integrin antibodies included human αv mAb Sc-9969 (Santa Cruz), human β1 mAb sc-59829 (Santa Cruz), hu β3 mAb AP3 (ATCC), human β5 mAb B5-IVF2 (provided by M. Hemler, Harvard Medical School, Boston, USA), human αvβ3 mAb 23C6 (sc-7312, Santa Cruz), human and mouse αvβ5 mAb ALULA (provided by D. Sheppard, UCSF [ ]), human and mouse αvβ6 mAb 10D5 (function blocking [ ], ab77906, Abcam), human and mouse αvβ8 mAb ADWA-11 (function blocking, provided by D. Sheppard, UCSF [ ]), mouse αv rAb RMV7 (14–00512, Affymetrix eBioscience), mouse β1 mAb (MAB2405, R&D systems, Bio-techne brand, USA) mouse β3 (rAb MAB41182, R&D systems, Bio-techne brand, USA).

Techniques: Flow Cytometry, Staining, Control, Virus, Binding Assay, Incubation, Labeling, Infection, Recombinant, Titration, Comparison

Journal: PLoS Pathogens

Article Title: The RGD-binding integrins αvβ6 and αvβ8 are receptors for mouse adenovirus-1 and -3 infection

doi: 10.1371/journal.ppat.1010083

Figure Lengend Snippet: (A) Virus binding interference in CMT-93 and M000216 cells by β6/-β8 function blocking antibodies. Detached cells were sequentially incubated for 1 h on ice with control antibody, or the anti-β6/-β8 antibodies, followed by incubation with control medium or medium containing the indicated viruses at an MOI of 4, the rabbit anti-FK-M3 antibodies, and finally the secondary PE-conjugate antibodies. Incubation and washing buffer contained either Mg 2+ /Ca 2+ , 1 mM each, or 1/0.2 mM Mn 2+ /Ca 2+ . (B-C) Virus infection interference in CMT-93 and M000216 cells by β6- and β8-specific antibodies. CMT-93 (B) and M000216 cells (C) were pre-incubated for 1 h on ice using 5-fold dilution series of the specific β6- or β8-integrin antibodies, respectively, starting with 800 ng/ml as highest concentration, followed by addition of the different indicated GFP-expressing viruses and transfer to 37°C for 48 h. An MOI of 1 was used for CMT-93 cells and MOI of 3 for M000216 cells in all experiments shown in this figure. GFP analysis was performed 48 h pi, and expression index was normalized to a control antibody. IC 50 values determined in this experiment are summarized in . (D, E) Infection blocking assays by sITGs. M1-IX-G virus was incubated for 1 h at RT with 5-fold serial dilutions of the indicated sITGs starting from 800 ng/ml to 6.4 ng/ml, followed by addition to CMT-93 cells (D) and M00216 cells (E) and cultivated and further processed as above. (F-H) Infection blocking assays by peptides. (F) The 20-mer peptides tested for virus infection inhibition included peptides A20FMDV2 derived from the VP1 coat protein of FMDV2, A20FMDV2-E containing a D to E mutation in the critical RGD motif, A20M1 and A20M3 derived from M1-/M3-FK, respectively, as compared to LAP-hTGFβ1, all containing the critical αvβ6/αvβ8-binding RGDLXX(L/I) motif. (G, H) Cells were pre-incubated on ice with 5-fold serial dilutions of peptides resulting in final concentrations from 5,000 to 0.32 nM. Subsequently, M1-IX-G virus was added to CMT-93 cells (G) or M000216 cells (H), followed by processing as described above. (I) Comparative flow cytometry profiles of αvβ8 expression in 3T6 cells. Blue and red show β8-specific staining in 3T6-sgNT and 3T6-sgItgβ8 cells, respectively, and grey histogram shows background staining of 3T6-sgItgβ8 cells using a matched isotype control. Numbers indicate MFI values of specific or control antibodies. (J, K) Transduction of 3T6-sgNT and 3T6-sgItgβ8 cells using M1-/M3-IX-G, the fiber-chimeric H5-ΔE3B-CG-FK-M1/-FK-M3 and control H5-ΔE3B-CG at an MOI of 9. Cells were processed as described in . (L) Comparative flow cytometry MFI αvβ6 expression values in control CMT-93-sgNT versus β6 integrin shRNA knock down CMT-93-sgItgβ6 cells. (M-O) Infection of control CMT-93-sgNT and CMT-93-sgItgβ6 cells using M1-IX-G (M), M3-IX-G (N) and H5-ΔE3B-CG (O) at an MOI of 1. Cells were processed as described above. Except for the representative flow cytometry histogram in (I), data represent triplicates, shown as mean ± SEM. Asterisks indicate level of significance for comparison of indicated values (*, P <0.05; **, P <0.005; ***, P <0.0005).

Article Snippet: Integrin antibodies included human αv mAb Sc-9969 (Santa Cruz), human β1 mAb sc-59829 (Santa Cruz), hu β3 mAb AP3 (ATCC), human β5 mAb B5-IVF2 (provided by M. Hemler, Harvard Medical School, Boston, USA), human αvβ3 mAb 23C6 (sc-7312, Santa Cruz), human and mouse αvβ5 mAb ALULA (provided by D. Sheppard, UCSF [ ]), human and mouse αvβ6 mAb 10D5 (function blocking [ ], ab77906, Abcam), human and mouse αvβ8 mAb ADWA-11 (function blocking, provided by D. Sheppard, UCSF [ ]), mouse αv rAb RMV7 (14–00512, Affymetrix eBioscience), mouse β1 mAb (MAB2405, R&D systems, Bio-techne brand, USA) mouse β3 (rAb MAB41182, R&D systems, Bio-techne brand, USA).

Techniques: Virus, Binding Assay, Blocking Assay, Incubation, Control, Infection, Concentration Assay, Expressing, Inhibition, Derivative Assay, Mutagenesis, Flow Cytometry, Staining, Transduction, shRNA, Knockdown, Comparison

Journal: bioRxiv

Article Title: MODULATION OF COLLAGEN-BINDING INTEGRINS AFFECTS FIBROBLAST ACTIVATION AND INHIBITS FIBROSIS

doi: 10.1101/2025.05.14.653428

Figure Lengend Snippet: (A) β1 integrin is diminished in the liver of conditional knockout (cKO) animals carrying the albumin promoter to drive cre expression in mice homozygous for floxed β1 integrin (Alb-cKO) compared to control mice (CT) by western blotting. N=4 CT and 4 Alb-cKO replicates. **p<0.005. (B) Western blot analysis shows successful depletion of β1 integrin in hepatocytes isolated from Alb-cKO animals. Flow cytometry confirms a decrease in the percentage of cells expressing β1 integrin in Alb-cKO hepatocytes. The number of replicates for the groups is shown in the order presented in the graphs: N=12/10 for western blot and 6/6 for flow cytometry. *p<0.05, ****p<0.0001. (C) Sirius-red and trichrome staining suggest an increase in extracellular matrix in Alb-cKO liver sections. Bars represent 100μm. (D) mRNA expression of collagen I, III and IV as well as fibronectin was increased in Alb-cKO livers compared to CT. N=13/21 for collagen I, 10/10 for collagen III, 10/10 for collagen IV, 8/9 for fibronectin. *p<0.05. (E) Collagen is increased in the liver of Alb-cKO animals. Collagen content was evaluated using a biochemical method to quantify hydroxyproline followed by adjustment to collagen amount. N=18/21, ***p<0.001. (F) Collagen I is increased in Alb-cKO livers by western blotting. N=8/10. (G) An increase in collagen I is suggested by immunofluorescence staining. Bars represent 100μm. (H) Despite the increase in matrix, no evidence for liver-related blood laboratory abnormalities. N=31/40. Livers and blood was examined in 14-16-week-old animals, while hepatocytes were isolated by liver perfusion from 8-10 week-old animals and examined immediately. Data were analyzed using unpaired t-tests for all graphs presented in this figure. In the case of collagen IV mRNA expression Welch’s correction was applied because of the significant difference in the variances between CT and Alb-cKO. All graphs show CT to the left and Alb-cKO to the right.

Article Snippet: The following murine integrin pairs were purchased from R&D systems: α1β1 #8188-AB, α2β1 #7828-A2, α10β1 #7827-AB, α11β1 #7808-AB, α5β1 #7728-A5, αvβ1 # 7705-AV, αvβ3 #7889-AV, as well as human α11β1 #6357-AB.

Techniques: Knock-Out, Expressing, Control, Western Blot, Isolation, Flow Cytometry, Staining, Immunofluorescence

Journal: bioRxiv

Article Title: MODULATION OF COLLAGEN-BINDING INTEGRINS AFFECTS FIBROBLAST ACTIVATION AND INHIBITS FIBROSIS

doi: 10.1101/2025.05.14.653428

Figure Lengend Snippet: (A) Total TGF-β protein decreased when hepatocytes were cultured on collagen type I, compared to cells cultured on fibronectin or vitronectin. Freshly isolated hepatocytes were cultured in wells precoated with poly-L-lysine 0.01% or the matrix proteins at a concentration of 10μg/mL for 24 hours. Total TGF-β protein was evaluated in the medium and cells and corrected to protein content. N=11/6/11/13 replicates. *p<0.05. (B) Integrin subunits expression on the surface of freshly isolated hepatocytes was evaluated by flow cytometry. N= 3 experiments. (C-D) Knockdown of β1 or α11 integrin using siRNA in hepatocytes increased total TGF-β protein (C). Depletion was confirmed by western blotting (D). Isolated cells were cultured and treated with siRNA targeting integrin subunits that bind to collagen, fibronectin and vitronectin. TGF-β: N=36/36/21/25/33/25/25/25/25 replicates in the order of the bars, β1 depletion: N=2/2, α11 depletion N=4/2. (E) Structure of cyclic GLQGE. (F) GLQGE did not affect hepatocyte proliferation (ki67 staining, left graph) or apoptosis (annexinv-propidium-iodide staining, right graph). N=7/8/7/7 for proliferation and 7/8/8/8 for apoptosis. Cells were cultured 24 hours in the presence of 50μg/mL of the molecules, stained and evaluated by flow cytometry. (G) In hepatocytes, only GLQGE diminishes TGF-β mRNA and protein compared to control (CT). Hepatocytes cultured on vitronectin were treated with 50μg/mL of the peptides and evaluated 24 hours later. N=10/9/10/10. *p<0.05. (H) TGF-β protein in the Huh-7 hepatoma cell line was reduced after GLQGE or GLNGE treatment. Huh7 cells were cultured on vitronectin, and treated for 24 hours with 50μg/mL of the molecules. N= 5 experiments with 2-9 replicates per experiment. *p<0.05. (I) pFAK is increased after treatment of hepatocytes with GLQGE. Cells were cultured in suspension without FCS for 2 hours and treated with 50μg/mL of the peptides for 30 minutes. N=35/29/30/30. *p<0.05, ***p<0.0005. (J) Increased pFAK in hepatocytes does not require β3 integrin expression. β3 hepatocytes were obtained from global β3 knockout mice (deletion confirmed using flow cytometry in peripheral blood and shown) and compared to wildtype littermate controls. Cells isolated were treated as in I. N=4/5/5/5. **p<0.01. Comparisons were performed using unpaired t-tests.

Article Snippet: The following murine integrin pairs were purchased from R&D systems: α1β1 #8188-AB, α2β1 #7828-A2, α10β1 #7827-AB, α11β1 #7808-AB, α5β1 #7728-A5, αvβ1 # 7705-AV, αvβ3 #7889-AV, as well as human α11β1 #6357-AB.

Techniques: Cell Culture, Isolation, Concentration Assay, Expressing, Flow Cytometry, Knockdown, Western Blot, Staining, Control, Suspension, Knock-Out

Journal: bioRxiv

Article Title: MODULATION OF COLLAGEN-BINDING INTEGRINS AFFECTS FIBROBLAST ACTIVATION AND INHIBITS FIBROSIS

doi: 10.1101/2025.05.14.653428

Figure Lengend Snippet: (A) Fibrosis was induced in animals by a single intratracheal bleomycin instillation. Starting on day 11, subcutaneous injections were administered of either NaCl 0.9% (CT) or GLQGE, GLNGE or GLOGE (in NaCl 0.9%) at a dose of 1 mg/mouse/day. On day 21, the animals were euthanized. (B) GLQGE treatment diminished collagen accumulation in the lung compared to fibrotic mice receiving 0.9% NaCl, GLNGE or GLOGE. Lung lysates were evaluated biochemically. N=18/18/17/17/18 replicates in the order of the bars. *p<0.05, ***p<0.0005, ****p<0.0001. (C) Western blot analysis shows that GLQGE diminishes collagen type I in lungs of fibrotic GLQGE-treated animals compared to fibrotic NaCl-treated animals. N=4/3/3/4/4. *p<0.05, **p<0.01, ****p<0.0001. (D) GLQGE did not alter the levels of TGF-β compared to fibrotic controls. N=18/17/16/17/17. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. (E) αSMA protein was diminished by western blot analysis of the lungs in GLQGE-treated fibrotic animals compared to NaCl-injected fibrotic CT animals. N=4/4/4/4/4 replicates. *p<0.05, **p<0.005, ***p<0.001. (F) Circulating levels of LDH, reflecting cell turnover induced by injury, were elevated in all fibrotic groups, but GLQGE treatment did not diminish LDH. N=18/18/17/17/18 replicates. *p<0.05, **p<0.01, ***p<0.0005. All numerical data were evaluated by analysis of variance (ANOVA) and when significant, t-tests were performed.

Article Snippet: The following murine integrin pairs were purchased from R&D systems: α1β1 #8188-AB, α2β1 #7828-A2, α10β1 #7827-AB, α11β1 #7808-AB, α5β1 #7728-A5, αvβ1 # 7705-AV, αvβ3 #7889-AV, as well as human α11β1 #6357-AB.

Techniques: Western Blot, Injection

Journal: Matrix biology : journal of the International Society for Matrix Biology

Article Title: Integrin alpha6 maintains the structural integrity of the kidney collecting system

doi: 10.1016/j.matbio.2016.12.003

Figure Lengend Snippet: (A) Expression of integrin α6 in papillary lysates from a 3-day old Itga6flox/flox and Hoxb7cre:Itga6flox/flox mice was analyzed by Western blotting analysis. α6 is indicated by the arrowhead. (B–I) Morphology of H&E stained kidney sections from Itga6flox/flox and Hoxb7cre:Itga6flox/flox mice at 2 months (magnification 40×, scale bar =500um) (B, C), 6 months (magnification 40×, scale bar =500um), (D, E) and 10 months of age (F, G) (magnification 40×, scale bar =500um) and (H, I) (magnification 200×, scale bar =100um).

Article Snippet: The following antibodies were used in Western immunoblot analyses: integrin α3 (AB1920, Millipore, Temecula, CA, USA and AF2787, R&D Systems); integrin β1 (AB1952, Millipore); integrin β1 (MAB1997, Millipore); integrin β4 (AF4054, R&D Systems); integrin α6 (3750), phospho-AktThr308 (9275), phospho-AktSer473 (9271), total Akt (9272), phospho-p38Thr180/Tyr182 (9211) and total p38 (9212), phospho-ERK1/2Thr202/Tyr204 (9101), total ERK1/2 (9102), (all from Cell Signaling Technologies) and collagen I (MD Biosciences).

Techniques: Expressing, Western Blot, Staining

Journal: Matrix biology : journal of the International Society for Matrix Biology

Article Title: Integrin alpha6 maintains the structural integrity of the kidney collecting system

doi: 10.1016/j.matbio.2016.12.003

Figure Lengend Snippet: A) Expression of integrin β4 in papillary lysates from a 3-day old Itgb4flox/flox and Hoxb7cre:Itgb4flox/flox mice analyzed by Western blotting analysis. (B–I) Morphology of H&E stained kidney sections from Itgb4flox/flox and Hoxb7cre:Itgb4flox/flox mice at 2 months (magnification 40×, scale bar =500um) (B, C), 6 months (magnification 40×, scale bar =500um), (D, E) and 12 months of age (F, G) (magnification 40×, scale bar =500um) and (H, I) (magnification 200×, scale bar =100um).

Article Snippet: The following antibodies were used in Western immunoblot analyses: integrin α3 (AB1920, Millipore, Temecula, CA, USA and AF2787, R&D Systems); integrin β1 (AB1952, Millipore); integrin β1 (MAB1997, Millipore); integrin β4 (AF4054, R&D Systems); integrin α6 (3750), phospho-AktThr308 (9275), phospho-AktSer473 (9271), total Akt (9272), phospho-p38Thr180/Tyr182 (9211) and total p38 (9212), phospho-ERK1/2Thr202/Tyr204 (9101), total ERK1/2 (9102), (all from Cell Signaling Technologies) and collagen I (MD Biosciences).

Techniques: Expressing, Western Blot, Staining

Journal: Matrix biology : journal of the International Society for Matrix Biology

Article Title: Integrin alpha6 maintains the structural integrity of the kidney collecting system

doi: 10.1016/j.matbio.2016.12.003

Figure Lengend Snippet: (A) Surface expression of integrin α6, β1 and β4 subunits were determined on Itgα6f/f and Itgα6−/− CD cells by flow cytometry using R-phycoerythrin (PE) conjugated secondary antibodies. (B) Lysates from Itgα6f/f and Itgα6−/− CD cells (20 μg total protein/lane) were immunoblotted for integrin α3 subunits or β-actin (loading control). Adhesion (C), migration (D) and proliferation (E) on LM-511 were evaluated as described in the Methods. For migration and proliferation, 1 μg/ml LM-511 was used. Shown are mean measurements ±SEM of 4–6 independent experiments. (F) Itgα6f/f and Itgα6−/− CD cells were plated in serum-free medium on LM-511 (1 μg/ml). Cells were lysed at 30, 60 and 90 min after plating and lysates (20 μg total protein/lane) were analyzed by Western blot for levels of phosphorylated and total Akt, p38, and ERK1/2 or β-actin (loading control).

Article Snippet: The following antibodies were used in Western immunoblot analyses: integrin α3 (AB1920, Millipore, Temecula, CA, USA and AF2787, R&D Systems); integrin β1 (AB1952, Millipore); integrin β1 (MAB1997, Millipore); integrin β4 (AF4054, R&D Systems); integrin α6 (3750), phospho-AktThr308 (9275), phospho-AktSer473 (9271), total Akt (9272), phospho-p38Thr180/Tyr182 (9211) and total p38 (9212), phospho-ERK1/2Thr202/Tyr204 (9101), total ERK1/2 (9102), (all from Cell Signaling Technologies) and collagen I (MD Biosciences).

Techniques: Expressing, Flow Cytometry, Control, Migration, Western Blot

Journal: Matrix biology : journal of the International Society for Matrix Biology

Article Title: Integrin alpha6 maintains the structural integrity of the kidney collecting system

doi: 10.1016/j.matbio.2016.12.003

Figure Lengend Snippet: Adhesion (A), migration (B), proliferation (C), and replating assay (D) on LM-322 (1 μg/ml) were evaluated as described in the Methods and in Fig. 5. Shown are mean measurements ±SEM of 4–6 independent experiments; *p<0.01between Itgα6−/− and Itgα6f/f CD cells. (E) The shear stress for 50% detachment (τ50), which represents the mean cell adhesion strength, was determined for Itgα6−/− and Itgα6f/f CD cells on LM-332 and fibronectin (which is not a ligand of a6 containing integrin) coated cell culture plates. Shown are the averaged τ50 values of 3 independent experiments; *p<0.01between Itgα6−/− and Itgα6f/f CD cells.

Article Snippet: The following antibodies were used in Western immunoblot analyses: integrin α3 (AB1920, Millipore, Temecula, CA, USA and AF2787, R&D Systems); integrin β1 (AB1952, Millipore); integrin β1 (MAB1997, Millipore); integrin β4 (AF4054, R&D Systems); integrin α6 (3750), phospho-AktThr308 (9275), phospho-AktSer473 (9271), total Akt (9272), phospho-p38Thr180/Tyr182 (9211) and total p38 (9212), phospho-ERK1/2Thr202/Tyr204 (9101), total ERK1/2 (9102), (all from Cell Signaling Technologies) and collagen I (MD Biosciences).

Techniques: Migration, Shear, Cell Culture