Journal: bioRxiv

Article Title: Fibrin fragment E potentiates TGF-β-induced myofibroblast activation and recruitment

doi: 10.1101/829945

Figure Lengend Snippet: Interactions between soluble recombinant αVβ 3 (5-50 nM) and immobilized FnE (A) or FnDD (B) were determined by SPR. Immobilization was at 952 RU and 1251 RU respectively. (C) SPR sensorgrams of the interaction between FnE and integrin αVβ 3 fitted to a bivalent analyte model (i), a heterogeneous ligand model (ii) or to the 1:1 two state interaction model (iii). (D) Representative western blot and (E) quantification of α-SMA protein levels of HFL1 fibroblasts in the presence of the cyclic-RGD peptide EMD 66203 in combination with TGF-β (5 ng/ml) and/or FnE (2 nM). α-SMA was normalized against β-actin and expressed as compared to vehicle (DMSO) treated controls. (F) Chemotaxis and (G) net migration of HFL1 cells in presence of 5 µM cyclic-RGD peptide. Dashed grey lines indicate mean values for cells without addition of cyclic-RGD. (H) Integrin β3 and β5 protein levels in HFL1 cells after transfection with siRNA selectively targeting these integrins, as indicated. (I) α-SMA ( ACTA2 ) mRNA levels of siRNA transfected cells stimulated with TGF-β (5 ng/ml) with or without addition of FnE (2 nM) for 48 h. (J) Chemotaxis and (K) net migration of HFL1 cells treated with siRNA to suppress integrin β3 or β5 in response to a PDGF-BB (0-20 ng/ml) or FnE (0-2 nM) gradients. * p<0.05. Error bars represent standard error of the mean ( n ≥3).

Article Snippet: Primary antibodies used were mouse anti-α-SMA (clone 1A4, Sigma-Aldrich) 1:10 000, rabbit anti-β-actin (ab8227, Abcam) 1:5000, mouse anti-PARP1 (BD-bioscience) 1:1500, mouse anti-integrin β3 (Cell signaling) 1:500, rabbit anti-integrin β5 (Abnova) 1:250, all added in blocking buffer with 0.1% Tween-20.

Techniques: Recombinant, Western Blot, Chemotaxis Assay, Migration, Transfection

Journal: Oncotarget

Article Title: Overexpression of HE4 (human epididymis protein 4) enhances proliferation, invasion and metastasis of ovarian cancer

doi:

Figure Lengend Snippet: Quantitative real-time PCR for selected genes (IL1A, DUSP1, COL1A1 and ITGB5) found to be differentially expressed in gene microarrays. Both in ES-2 cells and Caov-3 cells, the relative mRNA expression of 4 genes was significantly higher in HE4 high expressed cells and lower in HE4 low expressed cells (panel A, compared with Mock cells, one-way ANOVA). ICC staining showed that integrin β5 was highly expressed in HE4-high expression cells, relatively low expressed in the mock cells (HE4-H-Mock and HE4-L-Mock) and sparsely expressed in HE4 low expression cells (panel B) Immunohistochemical staining in xenograft tumor tissues showed that integrin β5 was high expressed in all the HE4 highly expressed tumor tissues, relatively low expressed in the HE4-H-Mock tumors and totally not expressed in HE4-L tumors (100%, 80% and 0% in 3 groups, respectively, P=0.003, panel C). IHC staining of ovarian cancer sample showed that expressions of HE4 and integrin β5 were mainly on membrane and cytoplasm, their expressions were relatively lower in chemotherapy sensitive group than the resistant group, the correlation coefficient was 0.213 (Spearman correlation analysis, P = 0.042, panel D). Kaplan-Meier survival analysis (panel E) showed that high expressions of HE4 and integrin β5 were independent risk factors for overall survival (OS) and progression-free survival (PFS), log Rank P = 0.001, 0.001 for HE4 in OS and PFS, P = 0.029, 0.042 for integrin β5 in OS and PFS, respectively.

Article Snippet: Rabbit polyclonal anti-HE4 antibody and rabbit polyclonal anti-integrin β5 antibody were purchased from Abcam Company (USA).

Techniques: Real-time Polymerase Chain Reaction, Expressing, Staining, Immunohistochemical staining, Immunohistochemistry, Membrane

Journal: Oncotarget

Article Title: Overexpression of HE4 (human epididymis protein 4) enhances proliferation, invasion and metastasis of ovarian cancer

doi:

Figure Lengend Snippet: Comparison of clinical characteristics and immunohistochemical expression of HE4 and Integrin β5 in 92 cases of epithelial ovarian cancer

Article Snippet: Rabbit polyclonal anti-HE4 antibody and rabbit polyclonal anti-integrin β5 antibody were purchased from Abcam Company (USA).

Techniques: Comparison, Immunohistochemical staining, Expressing

Journal: Oncotarget

Article Title: Overexpression of HE4 (human epididymis protein 4) enhances proliferation, invasion and metastasis of ovarian cancer

doi:

Figure Lengend Snippet: Expression of HE4 and integrin β5 in chemotherapy sensitive group and resistant group of 92 cases of epithelial ovarian cancer

Article Snippet: Rabbit polyclonal anti-HE4 antibody and rabbit polyclonal anti-integrin β5 antibody were purchased from Abcam Company (USA).

Techniques: Expressing

Journal: bioRxiv

Article Title: Proteomic and functional analyses of the periodic membrane skeleton in neurons

doi: 10.1101/2020.12.23.424206

Figure Lengend Snippet: a-e Left: 3D STORM images of axon regions of cultured hippocampal neurons immunostained for αII-spectrin ( a ), tropomodulin 1 ( b ), tropomodulin 2 ( c ), dematin ( d ), and coronin 2B ( e ). Middle: One-dimensional (1D) autocorrelation functions of the imaged molecules for the axon region indicated by the dashed line in the left panels. Signals were projected to the longitudinal axis of the axon segment, and the 1D autocorrelation functions were calculated using the projected signals. Right: Average 1D autocorrelation functions of the imaged molecules over many randomly selected axon regions. αII-spectrin, dematin and coronin 2B were immunolabeled with their corresponding antibodies. Tropomodulin 1 and tropomodulin 2 were labeled using moderate expression of GFP-tagged proteins through low-titer lentiviral transfection, which were in turn immunolabeled using anti-GFP antibody. GFP-tagged tropomodulin 1 was expressed in tropomodulin 1 knockout neurons; GFP-tagged tropomodulin 2 was expressed in WT neurons. Scale bars: 1 μm. Colored scale bar indicates the z-coordinate information. f Average 1D auto-correlation amplitudes of the βII-spectrin distribution, indicating the degree of periodicity of the MPS, calculated from many axon segments of untreated neurons, neurons transfected with adenoviruses expressing αII-spectrin shRNA, α-adducin shRNA, ankyrin B shRNA, or coronin 2B shRNA, and neurons cultured from tropomodulin 1, tropomodulin 2 or dematin knockout mice. The average 1D auto-correlation amplitude was defined as the difference between the first peak (at ∼190 nm) and the average of the two first valleys (at ∼95 nm and ∼285 nm, respectively) of the average 1D auto-correlation function . g Domain organization of βII-spectrin. h-j Left: 3D STORM image of axon regions of βII-spectrin knockout neurons transfected with plasmid expressing GFP-tagged full length βII-spectrin ( h ), βII-spectrin-ΔCH mutant ( i ) or βII-spectrin-ΔPH mutant ( j ). The proteins were visualized by immunostained with anti-GFP antibody. Scale bars: 1 μm. Right: average 1D autocorrelation functions of the imaged molecules indicated in the left panels. k Average 1D auto-correlation amplitudes for the distribution of GFP-tagged full length βII-spectrin and the distributions of the two GFP-tagged βII-spectrin truncation mutants, as described in ( h-j ), calculated from many GFP-positive axon segments. Data are mean ± s.e.m. ( n = 3 biological replicates for each condition; for each biological replicate, 50-100 axonal regions were examined). * indicates p < 0.05 and ** indicates p < 0.005 (unpaired Student’s t-test).

Article Snippet: The following primary antibodies were used in this study: guinea pig anti-MAP2 antibody (Synaptic Systems, 188004), rabbit anti-MAP2 antibody (Synaptic Systems, 188002), mouse anti-αII spectrin antibody (Biolegend, 803201), mouse anti-βII spectrin antibody (BD Biosciences, 612563), mouse anti-dematin antibody (Santa Cruz Biotechnology, sc-135881), rabbit anti-coronin 2B antibody (Novus Biologicals, NBP 1-85567), mouse anti-tubulin antibody (Santa Cruz Biotechnology, sc-5286), rabbit anti-Tau antibody (Synaptic Systems, 314002), mouse anti-K v 1.2 channel antibody (Neuromab, 75-008), rabbit anti-neurofascin antibody (Neuromab, 75-172), rabbit anti-NrCAM (Abcam, ab24344), goat anti-CHL1 antibody (R&D systems, AF2147), rabbit anti-NCAM1 antibody (EMD Millipore, AB5032), mouse anti-ankyrin G antibody (Santa Cruz Biotechnology, sc-12719), mouse anti-bassoon antibody (Enzo, ADI-VAM-PS003-F), rabbit anti-homer antibody (Synaptic Systems, 160003), rabbit anti-protein 4.1B antibody (LifeSpan BioSciences, LS-C83790-100), rabbit anti-L1CAM antibody (ABclonal, A8555), rat anti-L1CAM antibody (R&D Systems, MAB5674), rabbit anti-Myh10 (N-terminus) antibody (GeneTex, GTX133378), abbit anti-Myh9 (N-terminus) antibody (GeneTex, GTX101751), rabbit anti-Myh10 (C-terminus) antibody (Biolegend, 909901), rabbit anti-GFP antibody (Thermo Fisher Scientific, A11122). rabbit anti-β-actin antibody (Proteintech, 20536-1-AP).

Techniques: Cell Culture, Immunolabeling, Labeling, Expressing, Transfection, Knock-Out, shRNA, Plasmid Preparation, Mutagenesis

Journal: bioRxiv

Article Title: Proteomic and functional analyses of the periodic membrane skeleton in neurons

doi: 10.1101/2020.12.23.424206

Figure Lengend Snippet: a Average fluorescence intensities of αII-spectrin, α-adducin, ankyrin B, and coronin 2B in the axons of neurons transfected with adenoviruses expressing scrambled (control) shRNA or the shRNA against the corresponding protein, showing the knockdown of αII-spectrin, α-adducin, ankyrin B, and coronin 2B. Data are mean ± s.e.m. m ( n = 3 biological replicates; 10-20 imaged regions were examined per condition). ** indicates p < 0.005 (unpaired student’s t-test). b 3D STORM images of βII-spectrin in axon segments of neurons treated with control shRNA, αII-spectrin shRNA, α-adducin shRNA, ankyrin B shRNA, coronin 2B shRNA, and of neurons cultured from dematin, tropomodulin 1, and tropomodulin 2 knockout mice. Scale bars: 1 µm

Article Snippet: The following primary antibodies were used in this study: guinea pig anti-MAP2 antibody (Synaptic Systems, 188004), rabbit anti-MAP2 antibody (Synaptic Systems, 188002), mouse anti-αII spectrin antibody (Biolegend, 803201), mouse anti-βII spectrin antibody (BD Biosciences, 612563), mouse anti-dematin antibody (Santa Cruz Biotechnology, sc-135881), rabbit anti-coronin 2B antibody (Novus Biologicals, NBP 1-85567), mouse anti-tubulin antibody (Santa Cruz Biotechnology, sc-5286), rabbit anti-Tau antibody (Synaptic Systems, 314002), mouse anti-K v 1.2 channel antibody (Neuromab, 75-008), rabbit anti-neurofascin antibody (Neuromab, 75-172), rabbit anti-NrCAM (Abcam, ab24344), goat anti-CHL1 antibody (R&D systems, AF2147), rabbit anti-NCAM1 antibody (EMD Millipore, AB5032), mouse anti-ankyrin G antibody (Santa Cruz Biotechnology, sc-12719), mouse anti-bassoon antibody (Enzo, ADI-VAM-PS003-F), rabbit anti-homer antibody (Synaptic Systems, 160003), rabbit anti-protein 4.1B antibody (LifeSpan BioSciences, LS-C83790-100), rabbit anti-L1CAM antibody (ABclonal, A8555), rat anti-L1CAM antibody (R&D Systems, MAB5674), rabbit anti-Myh10 (N-terminus) antibody (GeneTex, GTX133378), abbit anti-Myh9 (N-terminus) antibody (GeneTex, GTX101751), rabbit anti-Myh10 (C-terminus) antibody (Biolegend, 909901), rabbit anti-GFP antibody (Thermo Fisher Scientific, A11122). rabbit anti-β-actin antibody (Proteintech, 20536-1-AP).

Techniques: Fluorescence, Transfection, Expressing, Control, shRNA, Knockdown, Cell Culture, Knock-Out

Journal: bioRxiv

Article Title: Proteomic and functional analyses of the periodic membrane skeleton in neurons

doi: 10.1101/2020.12.23.424206

Figure Lengend Snippet: In neurites, actin filaments form ring-like structures that are connected by spectrin tetramers, and actin filaments in these rings are likely capped by adducin and tropomodulin at their fast- and slow-growing ends, respectively. The length and structure of the actin filaments within the actin rings remains an open question. Dematin and coronin bind to the actin filaments, and ankyrin B and ankyrin G bind to the site near the center of each spectrin tetramer. The NMII bipolar filaments bind preferentially to actin filaments within the actin rings and can regulate the diameter of neurites by exerting radial contractile forces. It is possible that a smaller fraction of NMII bipolar filaments may connect the two adjacent actin rings. Membrane proteins, such as ion channels, cell adhesion molecules, receptors and signaling molecules, are associated either with the actin filaments through adaptor proteins dematin, or associated with the center positions of spectrin tetramers through the adaptor protein ankyrin, to form actin and ankyrin junctional complexes at the plasma membrane of neurites. Recruitment of cell adhesion molecules to periodic sites on the MPS and interactions of cell adhesion molecules between two abutting neurites bring their respective MPS structures in phase, which could in turn bring additional MPS-bound cell adhesion molecules from the two neurites into proximity, enhancing neurite-neurite interactions. Moreover, the MPS could serve as a structural platform that recruits and organizes transmembrane proteins and membrane-associated signaling molecules, potentially facilitating a variety of signaling pathways in neurons.

Article Snippet: The following primary antibodies were used in this study: guinea pig anti-MAP2 antibody (Synaptic Systems, 188004), rabbit anti-MAP2 antibody (Synaptic Systems, 188002), mouse anti-αII spectrin antibody (Biolegend, 803201), mouse anti-βII spectrin antibody (BD Biosciences, 612563), mouse anti-dematin antibody (Santa Cruz Biotechnology, sc-135881), rabbit anti-coronin 2B antibody (Novus Biologicals, NBP 1-85567), mouse anti-tubulin antibody (Santa Cruz Biotechnology, sc-5286), rabbit anti-Tau antibody (Synaptic Systems, 314002), mouse anti-K v 1.2 channel antibody (Neuromab, 75-008), rabbit anti-neurofascin antibody (Neuromab, 75-172), rabbit anti-NrCAM (Abcam, ab24344), goat anti-CHL1 antibody (R&D systems, AF2147), rabbit anti-NCAM1 antibody (EMD Millipore, AB5032), mouse anti-ankyrin G antibody (Santa Cruz Biotechnology, sc-12719), mouse anti-bassoon antibody (Enzo, ADI-VAM-PS003-F), rabbit anti-homer antibody (Synaptic Systems, 160003), rabbit anti-protein 4.1B antibody (LifeSpan BioSciences, LS-C83790-100), rabbit anti-L1CAM antibody (ABclonal, A8555), rat anti-L1CAM antibody (R&D Systems, MAB5674), rabbit anti-Myh10 (N-terminus) antibody (GeneTex, GTX133378), abbit anti-Myh9 (N-terminus) antibody (GeneTex, GTX101751), rabbit anti-Myh10 (C-terminus) antibody (Biolegend, 909901), rabbit anti-GFP antibody (Thermo Fisher Scientific, A11122). rabbit anti-β-actin antibody (Proteintech, 20536-1-AP).

Techniques: Membrane, Clinical Proteomics, Protein-Protein interactions

Journal: Nature Communications

Article Title: Tumor-penetrating therapy for β5 integrin-rich pancreas cancer

doi: 10.1038/s41467-021-21858-1

Figure Lengend Snippet: a Dot plots representing uptake of Alexa 647-labeled iRGD-AgNPs or control AgNPs by mCherry-labeled CAF spheroids. The bar diagram shows the proportion of CAFs that internalized the AgNPs. n = 4 independent experiments. Two-tailed unpaired t test; p = 0.0008. b Dot plots representing iRGD-AgNP uptake by mCherry-labeled CAF spheroids in the presence of anti-NRP-1, αvβ3, or αvβ5 antibodies or control IgG. The bar diagram shows the proportion of CAFs that internalized the AgNPs normalized against IgG. n = 4 (Rabbit IgG and NRP-1), n = 5 (Mouse IgG, αvβ3, and αvβ5) independent experiments. One-way ANOVA; p = 0.4784 (NRP-1 vs. αvβ3), p < 0.0001 (NRP-1 vs. αvβ5 and αvβ3 vs. αvβ5). c Representative confocal images from three independent experiments of GFP-positive hPCF1424 CAFs (green) treated with Alexa 647-labeled iRGD-AgNPs (red) in the presence of mouse IgG (left panels) or an anti-αvβ5 blocking antibody (right panels). The cells were etched to remove the AgNPs bound to the surface and highlight the internalized particles. Pre-etch and post-etch images are shown. Scale bars 100 µm. d Bar diagram showing the % expression of αvβ5 or NRP-1 in hPCF1424 CAFs transiently transfected with non-specific siRNA (NS), two different pools of siRNAs against integrin β5 (ITGb5-1 and -2), or NRP-1 siRNA (siNRP-1), as measured by median fluorescence intensity (left panel). Representative data of three biological replicates. e Bar diagram showing flow cytometric analysis of iRGD-AgNP uptake by the siRNA-treated CAFs in ( d ). The results are shown as the proportion of CAFs that internalized the AgNPs. n = 3 (NRP-1), n = 5 (ITGb5-1 and -2) independent experiments. One-way ANOVA; p < 0.0001 (NS vs. siITGb5-1 and NS vs. siITGb5-2), p = 0.9997 (NS vs. siNRP-1). All error bars, SEM. *** p < 0.001; **** p < 0.0001. Source data provided in Source Data file.

Article Snippet: Other antibodies used for immunofluorescence were anti-FAP (1:100) (Cat. no. ABT-11, Millipore Sigma, Temecula, CA), Cy3-anti-αSMA (1:100) (Clone 1A4, Cat. no. C6198, Millipore Sigma), anti-ER-TR7 (1:100) (Cat. no. SC-73355, Santa Cruz Biotechnology, Dallas, TX), rabbit anti-mouse β5 integrin (1:200) (Cat. no. 15459, GeneTex, Irvine, CA), rabbit monoclonal anti-Ki67 (1:200) (Clone SP6, Cat. no. ab16667, Abcam), mouse monoclonal anti-αSMA (0.034 μg/ml) (Clone 1A4, Cat. no. ab7817, Abcam), and rat anti-mouse CD31(1:100) (Clone MEC 13.3, Cat. no. 550274, BD Biosciences) antibodies.

Techniques: Labeling, Control, Two Tailed Test, Blocking Assay, Expressing, Transfection, Fluorescence

Journal: Nature Communications

Article Title: Tumor-penetrating therapy for β5 integrin-rich pancreas cancer

doi: 10.1038/s41467-021-21858-1

Figure Lengend Snippet: a Expression of αvβ5 integrin in PC3 cells after 2 days of incubation in normal media (NM) or CM prepared from cultured hPCF1424 CAFs ( n = 9), mPCFAA0779 CAFs ( n = 7), or MIA PaCa-2 human PDAC cells ( n = 4) performed in independent experiments. Fold over NM is shown. One-way ANOVA; p < 0.0001 (NM vs. hPCF1424 CM), p = 0.5085 (NM vs. mPCFAA0779 CM), p = 0.9886 (NM vs. MIA PaCa-2). b Dot plots representing iRGD-AgNP or control AgNP uptake in PC3 cells cultured in NM or CM from hPCF1424 CAFs. c The bar diagrams show the proportion of PC3 (left) or LM-PmC (right) cells that internalized iRGD-AgNPs. The cells were incubated in NM or CM from hPCF1424 CAFs for 2 days prior to study. n = 6 (PC3), n = 3 (LM-P) independent experiments. Two-tailed unpaired Student’s t test; p = 0.0001 (PC3 NM vs. CAF CM), p = 0.0127 (LM-P NM vs. CAF CM). d Expression of actin, αv, and β5 integrin mRNAs normalized against cyclophilin A analyzed by qPCR in PC3 cells incubated with NM or CM from hPCF1424 CAFs. n = 3 independent experiments. Two-tailed unpaired Student’s t test; p = 0.00016 (αv), p < 0.0001 (β5), p = 0.923 (actin). e , f Expression of αvβ5 integrin ( e ) or NRP-1 ( f ) in PC3 cells cultured in normal media (NM) or CM from hPCF1424 CAFs in the presence or absence of exogenous TGF-β, a TGF-β-specific inhibitor LY2157299, or vehicle alone. Mean fluorescence intensity (MFI) assessed by flow cytometry is shown. n = 4 independent experiments. Two-tailed unpaired Student’s t test; p = 0.0008 ( e: NM vs. TGF-β), p = 0.0009 ( e: TGF-β vs. TGF-β + LY2157299), p = 0.7082 ( e: CAF CM vs. CAF CM + DMSO), p = 0.0177 ( e: CAF CM + DMSO vs. CAF CM + LY2157299). No significant differences in ( f ). All error bars, SEM; * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001. Source data provided in Source Data file.

Article Snippet: Other antibodies used for immunofluorescence were anti-FAP (1:100) (Cat. no. ABT-11, Millipore Sigma, Temecula, CA), Cy3-anti-αSMA (1:100) (Clone 1A4, Cat. no. C6198, Millipore Sigma), anti-ER-TR7 (1:100) (Cat. no. SC-73355, Santa Cruz Biotechnology, Dallas, TX), rabbit anti-mouse β5 integrin (1:200) (Cat. no. 15459, GeneTex, Irvine, CA), rabbit monoclonal anti-Ki67 (1:200) (Clone SP6, Cat. no. ab16667, Abcam), mouse monoclonal anti-αSMA (0.034 μg/ml) (Clone 1A4, Cat. no. ab7817, Abcam), and rat anti-mouse CD31(1:100) (Clone MEC 13.3, Cat. no. 550274, BD Biosciences) antibodies.

Techniques: Expressing, Incubation, Cell Culture, Control, Two Tailed Test, Fluorescence, Flow Cytometry

Journal: Nature Communications

Article Title: Tumor-penetrating therapy for β5 integrin-rich pancreas cancer

doi: 10.1038/s41467-021-21858-1

Figure Lengend Snippet: a Representative confocal image of a tumor section showing in vivo homing of IV FAM-iRGD (green) to β5 integrin-positive areas (red) in a subcutaneous LM-P mouse tumor. Blue, DAPI; scale bar 1 mm. ( n = 4 mice). b Mice bearing orthotopic PDAC created with LM-PmC cells (WT, n = 4) or the β5 integrin-deficient D3 clone (KO, n = 3). mCherry signals were detected with an IVIS Spectrum 19 days after tumor implantation. Representative mouse images are shown. Left bar diagram, total tumor burden (radiant efficiency) p = 0.0186; right bar diagram, degree of tumor spreading (mCherry-positive area) p = 0.0061, error bars, SEM, statistical analyses, two-tailed unpaired Student’s t test. c Confocal micrographs showing spreading of IV FAM-iRGD (green) in WT and KO tumors. Red, tumor cells; cyan, CD31; blue, DAPI; scale bars, 50 μm. The mice 19 days after tumor implantation in ( b ) were used. The mean FAM intensity was measured to quantify the amount of iRGD that homed to the tumor (left bar diagram, p = 0.0473), and % area positive for FAM was used to determine the degree of iRGD spreading (right bar diagram, p = 0.0071). WT ( n = 4), β5 KO ( n = 3). Two-tailed unpaired Student’s t test. d Mice bearing orthotopic PDAC created with WT LM-PmC cells or the β5-deficient D3 clone were subjected to IVIS imaging. mCherry was detected at the indicated time points. e Kaplan–Meier survival curves of the mice in ( d ). WT ( n = 7), D3 β5 KO ( n = 3). Log-rank (Mantel–Cox) test; p = 0.0074, HR = 3.57. f Survival curves of mice bearing orthotopic PDAC created with WT ( n = 9) or two different β5 integrin-deficient clones, G5 β5 KO ( n = 4) and D3 β5 KO ( n = 6). Log-rank (Mantel–Cox) test; p = 0.0026, HR = 3.5 (WT vs. G5 β5 KO); p = 0.0026, HR = 3.1 (WT vs. D3 β5 KO). All error bars, SEM; * p < 0.05; ** p < 0.01. Source data provided in Source Data file.

Article Snippet: Other antibodies used for immunofluorescence were anti-FAP (1:100) (Cat. no. ABT-11, Millipore Sigma, Temecula, CA), Cy3-anti-αSMA (1:100) (Clone 1A4, Cat. no. C6198, Millipore Sigma), anti-ER-TR7 (1:100) (Cat. no. SC-73355, Santa Cruz Biotechnology, Dallas, TX), rabbit anti-mouse β5 integrin (1:200) (Cat. no. 15459, GeneTex, Irvine, CA), rabbit monoclonal anti-Ki67 (1:200) (Clone SP6, Cat. no. ab16667, Abcam), mouse monoclonal anti-αSMA (0.034 μg/ml) (Clone 1A4, Cat. no. ab7817, Abcam), and rat anti-mouse CD31(1:100) (Clone MEC 13.3, Cat. no. 550274, BD Biosciences) antibodies.

Techniques: In Vivo, Tumor Implantation, Two Tailed Test, Imaging, Clone Assay

Journal: Nature Communications

Article Title: Tumor-penetrating therapy for β5 integrin-rich pancreas cancer

doi: 10.1038/s41467-021-21858-1

Figure Lengend Snippet: a IV delivery of fluorescent dextran by iRGD co-injection to orthotopic PDAC created with LM-PmC cells (WT; n = 5) or the β5 integrin-deficient D3 clone (KO; n = 3) in mice. Two-tailed unpaired Student’s t test; p = 0.002. b Kaplan–Meier survival curve of KPC mice treated with gemcitabine alone ( n = 18) or in combination with iRGD ( n = 16). Log-rank (Mantel–Cox) test; p = 0.0385, HR = 0.53. c Numbers of metastatic lung (left) and liver (right) nodules identified in the mice from ( b ). iRGD/Gem ( n = 7); Gem ( n = 8). Two-tailed unpaired Student’s t test; p = 0.0263 (lung metastasis), p = 0.4 (liver metastasis). d The weight of tumors harvested from KPC mice treated with iRGD + gemcitabine + nab-paclitaxel ( n = 14) or gemcitabine + nab-paclitaxel alone ( n = 13) for 6 weeks. Two-tailed unpaired Student’s t test; p = 0.0193. All error bars, SEM; * p < 0.05; ** p < 0.01. Source data provided in Source Data file.

Article Snippet: Other antibodies used for immunofluorescence were anti-FAP (1:100) (Cat. no. ABT-11, Millipore Sigma, Temecula, CA), Cy3-anti-αSMA (1:100) (Clone 1A4, Cat. no. C6198, Millipore Sigma), anti-ER-TR7 (1:100) (Cat. no. SC-73355, Santa Cruz Biotechnology, Dallas, TX), rabbit anti-mouse β5 integrin (1:200) (Cat. no. 15459, GeneTex, Irvine, CA), rabbit monoclonal anti-Ki67 (1:200) (Clone SP6, Cat. no. ab16667, Abcam), mouse monoclonal anti-αSMA (0.034 μg/ml) (Clone 1A4, Cat. no. ab7817, Abcam), and rat anti-mouse CD31(1:100) (Clone MEC 13.3, Cat. no. 550274, BD Biosciences) antibodies.

Techniques: Injection, Two Tailed Test