RNA was reverse transcribed using the TaqMan RT kit or the Superscript III kit (Life Technologies) with oligo(dT) or random hexamers according the manufacturer’s protocols. Reverse-transcribed RNA was amplified by PCR using GoTaq polymerase (Promega) using primers and conditions detailed in Additional file 1 : Table S2. For PCR across the G4C2 repeats, reactions were supplemented with betaine (Sigma), DMSO and 7-deaza GTP (New England Biosystems) [1 (link)]. No RT controls were used to confirm absence of DNA contamination. RT-PCR products were separated in 1.5 % (w/v) agarose gels and stained with ethidium bromide. The amount of PCR product was estimated by densitometric analysis of the gels using the VisionWorks®LS analysis software (UVP).
Visionworks ls analysis software
Manufactured by Analytik Jena
Sourced in United States
VisionWorks®LS is an analysis software for digital imaging and gel documentation. It provides essential functionalities for image capture, analysis, and quantification of gel-based assays.
Automatically generated - may contain errors
Lab products found in correlation
Goat anti rabbit secondary antibody, by Bio-Rad (2 mentions)
Anti mmp9, by Abcam (2 mentions)
Goat anti mouse secondary antibody, by Bio-Rad (2 mentions)
Superscript 3 kit, by Thermo Fisher Scientific (1 mentions)
Betaine, by Merck Group (1 mentions)
Taqman rt kit, by Thermo Fisher Scientific (1 mentions)
Gotaq polymerase, by Promega (1 mentions)
Pageruler prestained protein ladder, by Thermo Fisher Scientific (1 mentions)
Bradford protein assay, by Thermo Fisher Scientific (1 mentions)
Sds page, by Bio-Rad (1 mentions)
Cdp star reagent, by New England Biolabs (1 mentions)
Gel doc xr imaging system, by Bio-Rad (1 mentions)
Quantity one 1 d analysis software, by Bio-Rad (1 mentions)
Anti claudin 4, by Thermo Fisher Scientific (1 mentions)
Anti claudin 5, by Thermo Fisher Scientific (1 mentions)
Anti occludin, by Thermo Fisher Scientific (1 mentions)
Anti claudin 2, by Thermo Fisher Scientific (1 mentions)
Anti egfr, by Cell Signaling Technology (1 mentions)
Anti stat3, by Cell Signaling Technology (1 mentions)
Phosphoserine 473 akt, by Cell Signaling Technology (1 mentions)
11 protocols using visionworks ls analysis software
1
Quantitative RT-PCR of G4C2 Repeats
2
Western Blot Analysis of Apoptosis Markers
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Protein was extracted from IC25 and IC50 DHAQC (2)-treated and untreated control cells using radioimmunoprecipitation assay buffer, and the protein concentration was determined using Bradford protein assay (Thermo Fisher Scientific). The cell homogenates were electrophoresed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) (Bio-Rad Laboratories, Inc., Hercules, CA, USA), transferred onto nitrocellulose membrane, blocked with Tris-buffered saline–Tween buffer containing 5% skimmed milk powder for 1 hour, and incubated with primary antibodies (anti-p53, anti-BAX, anti-Bcl-2, anti-cytochrome c, and anti-β-actin at dilution 1:1000) (Abcam plc, Cambridge, UK) for another hour. Then, membranes were washed and incubated with the anti-rabbit secondary antibodies-conjugated with alkaline phosphatase (1:5000 dilution) for 1 hours, followed by washing with Tris-buffered saline–Tween buffer, and finally visualized by chemiluminescence with CDP-Star® reagent (NEB [UK], Hitchin, UK) using a BioSpectrum® system (UVP, Upland, CA, USA). The size of the targeted proteins was determined by protein marker (PageRuler Prestained Protein Ladder; Thermo Fisher Scientific), and the density results obtained from this targeted protein were analyzed using VisionWorks®LS analysis software (Version 7.1 RC3.54; UVP) by normalizing against β-actin.
3
Gel and Blot Image Analysis Protocols
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Gels images from Figs 2A , 4D and 5B were acquired and analyzed using the Gel Doc XR Imaging System and Quantity One 1-D analysis software (BioRad).
Blots images from Figs2B and 5A were acquired and analyzed using ChemiDoc-It (UVP) and VisionWorks LS analysis software.
If processing in brightness and contrast of gel and blot images has been made it was applied to the entire image including controls. No high-contrast gels or blots has been displayed. When necessary, cropped gels and juxtaposing images were displayed to improve the clarity and conciseness of the presentation, being explicited in the figure.
SigmaPlot - Scientific Data Analysis and Graphing Software (Systat Software Inc.) was used for statystical analysis an graphing of experimental data in Figs3 , 4A–C , 5C , 6 and 7 .
Blots images from Figs
If processing in brightness and contrast of gel and blot images has been made it was applied to the entire image including controls. No high-contrast gels or blots has been displayed. When necessary, cropped gels and juxtaposing images were displayed to improve the clarity and conciseness of the presentation, being explicited in the figure.
SigmaPlot - Scientific Data Analysis and Graphing Software (Systat Software Inc.) was used for statystical analysis an graphing of experimental data in Figs
4
Evaluating Colonic Tight Junction Integrity
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As described previously [10 (link)], for WB analysis, colonic mucosal stripping was obtained from the TgM9 and WT mice (n=20 per group) with and without CAC. In vivo WB analysis was performed with 30μg/ well of cell lysates. WB analysis for in vitro model was performed using whole cell lysates (30μg/ well) of CaCo2BBE cells with and without MMP9. 106 cells of CaCo2BBE were grown on 6 well plate for 100% confluency. As described by Bazzoni et al [62 (link)], we collected Triton X-100 soluble and insoluble fraction of CaCo2BBE cells with and without MMP9 to assess TJ integrity and 10μg/ well was used for WB analysis. The antibodies used were anti-MMP9 (Abcam, Cambridge, MA), anti-EGFR (Cell Signaling, Beverly, MA), anti-Claudin-2 (Life Technologies, Rockford, IL), anti-Claudin-4 (Invitrogen, Rockford, IL), anti-Claudin-5 (Invitrogen), anti-TFF3 (Cloud-Clone Corp., Katy, TX), anti-Sp1 (Upstate Cell Signaling Solutions, Lake Placid, NY), anti-Occludin (Invitrogen), anti-STAT3 (Cell Signaling). Goat anti-mouse secondary antibody (Bio-Rad, Hercules, CA) or goat anti-rabbit secondary antibody (Bio-Rad) were used. Densitometry graphs were generated by using image acquisition and analysis software by VisionWorksLS Analysis Software (UVP, Upland, CA).
5
Adipose Tissue Glucose Uptake and Lipid Analysis
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Adipose tissue biopsies were digested with collagenase before determination of d ‐[U‐14C]glucose uptake as previously described (Foley et al. 1983 ; Kashiwagi et al. 1983 ; Betts et al. 2011 ). The lipid content of adipocytes was determined by extraction and weighing of total lipids of a 50 μL aliquot of 20% adipocytes in suspension. The cell suspension was mixed with 2.7 mL of isopropanol:heptane:H2SO4 (40:10:1 ratio) followed by the addition of 1.8 mL heptane and 1.0 mL of ddH2O before vortexing and centrifugation. A 1.0 mL aliquot of the organic layer was evaporated and the lipid subsequently weighed.
Proteins were separated by SDS‐PAGE and transferred using semidry electrotransfer to a nitrocellulose membrane and were normalised to glyceraldehyde 3‐phosphate dehydrogenase (GAPDH) and per milligram lipid within each sample. Western blotting analysis was performed with the following antibodies: Akt1 (Millipore, Billerica, MA, USA), Akt2 (Millipore), phosphoserine 473 Akt (Cell Signaling Technology, Danvers, MA, USA), GLUT4 (Holman et al.1990 ), GAPDH (Proteintech, Rosemont, IL, USA). The images were acquired in an EPI Chemi II darkroom (UVP, Upland, CA, USA) and bands quantified using VisionWorks LS analysis software (UVP).
Proteins were separated by SDS‐PAGE and transferred using semidry electrotransfer to a nitrocellulose membrane and were normalised to glyceraldehyde 3‐phosphate dehydrogenase (GAPDH) and per milligram lipid within each sample. Western blotting analysis was performed with the following antibodies: Akt1 (Millipore, Billerica, MA, USA), Akt2 (Millipore), phosphoserine 473 Akt (Cell Signaling Technology, Danvers, MA, USA), GLUT4 (Holman et al.
6
Mucosal Protein Analysis in Colitis-Associated Cancer
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As described previously [10 (link)] mucosal stripping were obtained from the TgM9 and WT mice (n = 20 per group) with and without CAC after the sacrifice for WB analysis. The antibodies used were anti-MMP9 (Abcam, Cambridge, MA), anti-NICD (Abcam), anti-caspase-3 (Cell Signaling, Beverly, MA) anti-p53 (Cell Signaling), anti-p21WAF1/Cip1 (BD Bioscience, San Jose, CA), anti-cyclin D1 (Santa Cruz, Dallas, TX), anti-Cyclin A (Cell Signaling), anti-Cyclin E1 (Santa Cruz), anti-γH2AX (Abcam), anti-p19ARF (Abcam), and anti-p14ARF (Abcam). Goat anti-mouse secondary antibody (1:2000; Bio-Rad, Hercules, CA) or goat anti-rabbit secondary antibody (1:2000, Bio-Rad) were used. Densitometry graphs were generated by using image acquisition and analysis software by VisionWorksLS Analysis Software (UVP, Upland, CA)
7
ISSR Markers For Genetic Diversity
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Six primers of ISSR markers were selected (Gupta et al. 1994; (link)Stepansky et al. 1999 ) (Table 1). The analysis with ISSR markers was based on protocols developed by Żabicka et al. (2020) ; the same reagents and laboratory equipment were used. The PCR products were separated in 1% agarose gel with 1X TBE and SimplySafe (EURx Sp. z o.o. Gdansk, Poland) for about 90 min at 120 V. Band patterns of 38 samples were captured with a MultiDoc-It™ Imaging System with VisionWorks® LS Analysis Software (UVP, Upland, CA, USA). PCR reaction cycle were repeated on selected samples to test reproducibility of obtained results.
Split phylogenetic network (NeighborNet) was constructed in SplitsTree v. 4.6 (Huson and Bryant 2006) (link) based on Dice coefficient to evaluate the relationships between examined individuals, bootstrap was calculated on 1000 replicates.
Split phylogenetic network (NeighborNet) was constructed in SplitsTree v. 4.6 (Huson and Bryant 2006) (link) based on Dice coefficient to evaluate the relationships between examined individuals, bootstrap was calculated on 1000 replicates.
8
Methylation Assays of Bacterial Cultures
9
Quantifying mRNA and Protein Expression
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For RT-qPCR studies, relative mRNA expression was calculated for each gene of interest using the comparative CT method with 18s as the reference. Fold change was calculated as the ratio of the reperfused to the non-ischemic lobe for each animal. For the Signosis transcription factor array results, transcription factors that changed significantly in the 0.5 hr reperfused lobes were identified by one-way ANOVA comparisons to a sham group.
Densitometric analysis of western blots was carried out using the VisionWorks LS Analysis Software (Analytik Jena US LLC, Upland, CA). Raw data was exported to GraphPad Prism (GraphPad Software, La Jolla, CA) for statistical analyses. For each antibody probe, the protein bands were normalized to control band on the blot and expressed as a ratio. For phospho-specific immunoblotting, the normalized phospho-band was divided by the normalized total protein band in order to reflect the stoichiometry of phosphorylation. These data were then used for a paired t-test (non-ischemic to reperfused lobe) in Prism.
Densitometric analysis of western blots was carried out using the VisionWorks LS Analysis Software (Analytik Jena US LLC, Upland, CA). Raw data was exported to GraphPad Prism (GraphPad Software, La Jolla, CA) for statistical analyses. For each antibody probe, the protein bands were normalized to control band on the blot and expressed as a ratio. For phospho-specific immunoblotting, the normalized phospho-band was divided by the normalized total protein band in order to reflect the stoichiometry of phosphorylation. These data were then used for a paired t-test (non-ischemic to reperfused lobe) in Prism.
10
Western Blot Protein Analysis Protocol
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For western blotting, 15–30 μl of the lysates were incubated in 1x Laemmli sample buffer containing DTT at 72 °C for 10 min. For ABCA4, 30 µl of lysates were incubated with Protein Loading Buffer 5x (National Diagnostics) at 37 °C for 15 min. The proteins were separated on an SDS-polyacrylamide gel via gel electrophoresis. Cerulean and Myo7b blots were stained for 1 h at room temperature (RT) or overnight at 4 °C using the mouse anti-GFP (detects cerulean, 1:2000, JL-8, Clontech, Takara) and the mouse anti-β-tubulin antibody (1:500, D3U1W, Cell Signaling) or with the rabbit anti-Myo7b (1:1000, Sigma-Aldrich) and the mouse anti-β-actin antibody (1:3000, AC-15, Sigma-Aldrich). ABCA4 blots were stained overnight at 4 °C using the rabbit anti-ABCA4 (1:1000, abcam, detecting the C-terminus of the protein) and the rabbit anti-hexokinase II (1:1000, C64G5, Cell Signaling) antibody. Secondary antibodies (Peroxidase AffiniPure anti-rabbit, 1:2000, Jackson, or goat anti-mouse IgG-HRP, 1:2000, Santa Cruz Biotechnology) were added for 1 h at RT. All antibodies used for western blotting have been validated by the manufacturer. The western blots were imaged, and the relative band intensities quantified using the ImageLab software (Bio-Rad, v5) or VisionWorks LS Analysis Software (for ABCA4, Analytik Jena).
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