Journal: bioRxiv

Article Title: Activation-induced shift in nutrient preference and function-specific nutrient dependence in human neutrophils

doi: 10.1101/2023.09.25.559385

Figure Lengend Snippet: a. Schematic of glucose uptake, glycogen synthesis, and glycogenolysis. Important enzymes are listed. b. Glucose consumed from the media within 1 hour by unstimulated neutrophils or neutrophils that are treated with indicated stimuli (300μg/mL zymosan A, 100ng/mL TNFα, or 100nM PMA) or inhibitor (20μM GPi). Bars and error bars show mean ± SD from n=3-4 independent donors, as represented by individual dots. Statistical analysis was done by unpaired t-test, * indicates p<0.05. c. (Left) Representative flow cytometry result measuring the distribution of GLUT1 level on cell surface within a population of neutrophils that are untreated or treated with indicated stimuli or inhibitor for 30min. (Right) The experiment was repeated with neutrophils isolated from n=4 independent donors (indicated by different markers). Bars and error bars show mean ± SD of quantified mean GLUT1 level from each donor. Statistical analysis was performed by paired t-test. d-e. Immunoblot analysis of (d) total GLUT1 level and phosphorylated GLUT1 (Ser226), and (e) total PYGL and phosphorylated PYGL (Ser15) in neutrophils treated with indicated stimuli or inhibitor for 30min. Representative blots were shown on the left. The experiment was repeated multiple times with neutrophils isolated for different donors (indicated by individual dots), and quantified results were compiled and shown on the right. Bars and error bars show mean ± SD. Statistical analysis performed by unpaired t-test. f. Level of glycogen store in human peripheral blood neutrophils that are untreated, treated with GPi (20μM) for 2h, or stimulated with zymosan A (300μg/mL), or LPS (1μg/mL) for 2h. Bars and error bars show mean ± SD from n=4-5 independent donors, as represented by individual dots. p-value determined by paired t-test. g. Fraction of labeled glycogen after 2hr incubation of neutrophils that are untreated or treated with indicated stimuli or inhibitor in media containing U- 13 C-glucose. Bars and error bars show mean ± SD from n=3-5 independent donors, as represented by individual dots. p-values were determined by paired t-test.

Article Snippet: Primary antibodies: PYGL (abcam, ab198268), phospho(S15)-PYGL (abcam, ab227043), GLUT1 (Alomone Labs, AGT-041), P-GLUT1 (Millipore Sigma, ABN991), B-actin (Cell Signaling, no. 4967); and secondary antibodies: Goat-anti-rabbit 800 (LI-COR, no. 925-32211), Goat-anti-mouse 680 (LI-COR, no. 925-68070).

Techniques: Flow Cytometry, Isolation, Western Blot, Labeling, Incubation

Journal: International Journal of Environmental Research and Public Health

Article Title: The Salutary Influence of Forest Bathing on Elderly Patients with Chronic Heart Failure

doi: 10.3390/ijerph14040368

Figure Lengend Snippet: Effect of forest bathing on change for the endothelin-1 (ET-1) production and components of the renin-angiotensin system (RAS). Renin, angiotensinogen (AGT), angiotensin II (Ang II), angiotensin II type 1 receptor (AT1), and angiotensin II type 2 receptor (AT2) of subjects were evaluated before and after the experiment (forest group ( n = 23) and control group ( n = 10)). ** p < 0.01, # p < 0.05, analyzed by the Kruskal-Wallis test followed by the Dunn-Bonferroni test.

Article Snippet: The bio-markers for heart failure BNP and NT-ProBNP, cardiovascular disease-related factors, including ET-1, and constituents of the renin-angiotensin system (RAS), such as renin, angiotensinogen (AGT), angiotensin II (Ang II), angiotensin II type 1 receptor (AT1), angiotensin II type 2 receptor (AT2), as well as pro-inflammatory cytokines, including interleukin-6 (IL-6), tumor necrosis factor α (TNF-α), were analyzed using commercially available enzyme-linked immunoassay (ELISA) kits (CUSABIO, Wuhan, China) according to the manufacturer’s protocols.

Techniques: Control

Journal: Cellular and Molecular Neurobiology

Article Title: Sevoflurane Induces Learning and Memory Impairment in Young Mice Through a Reduction in Neuronal Glucose Transporter 3

doi: 10.1007/s10571-019-00779-0

Figure Lengend Snippet: Sevoflurane decreases GLUT3 protein expression in the hippocampus and temporal lobe. a WB analysis of GLUT1, GLUT4, and GLUT3 protein expression in the hippocampus and temporal lobe of young mice 24 h after the last exposure to sevoflurane. n = 4 for each group. b, c, d Histograms showing the quantification of GLUT1, GLUT4, and GLUT3 blots in the hippocampus and temporal lobe. In the hippocampus, * P < 0.05 versus the Con group. In the temporal lobe, # P < 0.05 versus the Con group. e Fluorescent images showing GLUT3 expression in neurons of the hippocampal CA1 area and temporal lobe (left panel). Quantification of GLUT3 intensity in the CA1 region of the hippocampus and temporal lobe 24 h after the last exposure to sevoflurane (right panel). In the CA1 region of the hippocampus, * P < 0.05 versus the Con group. In the temporal lobe, # P < 0.05 versus the Con group. n = 4 for each group. The data are presented as the mean ± s.e.m

Article Snippet: After incubation with WB-specific blocking solution (5% skimmed milk powder (AS1033, Aspen, China) diluted in TBST), the PVDF membranes were separately incubated with anti-GLUT1 (1:1000, ab652, Abcam), anti-GLUT4 (1:2000, ab654, Abcam), anti-(extracellular) GLUT3 (1:1000, AGT-023, Alomone), anti-Bax (1:800, sc-7480, Santa Cruz), anti-Bcl-2 (1:800, sc-7382, Santa Cruz), anti-caspase-3 (cleaved) (1:100, AB3623, Millipore), anti-poly (ADP-ribose) polymerase (PARP) (cleaved) (1:1000, 5625, Cell Signaling Technology), and anti-β-actin (1:10,000, Aspen) antibodies at 4 °C overnight.

Techniques: Expressing

Journal: Cellular and Molecular Neurobiology

Article Title: Sevoflurane Induces Learning and Memory Impairment in Young Mice Through a Reduction in Neuronal Glucose Transporter 3

doi: 10.1007/s10571-019-00779-0

Figure Lengend Snippet: Sevoflurane reduces GLUT3 protein expression in primary hippocampal neurons. a Primary hippocampal neurons were transfected with LV5-GLUT3. The transfection efficiency was examined by GFP expression (green) 36 h post infection, which was analysed by phase contrast (left panel) and fluorescence microscopy (right panel). b WB analysis of GLUT3 protein expression in primary hippocampal neurons 24 h after the last exposure to sevoflurane. Neurons were normally cultured (the Con group), only transfected with LV5-GLUT3 (the LV5-GLUT3 group), transfected with LV5-GLUT3 and then exposed to sevoflurane (the LV5-GLUT3 + Sev group), and only exposed to sevoflurane (the Sev group). c Immunofluorescence of GLUT3 expression in the somata and dendrites of primary hippocampal neurons 24 h after the last exposure to sevoflurane. The four groups: the LV5 group (neurons transfected with LV5 empty vectors), the LV5 + Sev group (neurons transfected with LV5 empty vectors and then exposed to sevoflurane), the LV5-GLUT3 and the LV5-GLUT3 + Sev group. d Histograms showing the results of GLUT3 blots in neurons among the four groups. n = 5 for each group. * P < 0.05 versus the Con group. # P < 0.05 versus the LV5-GLUT3 group. e Histograms showing the intensity of GLUT3 in the four treatment groups. n = 5 for each group. * P < 0.05 versus the LV5 group. # P < 0.05 versus the LV5-GLUT3 group. The data are presented as the mean ± s.e.m

Article Snippet: After incubation with WB-specific blocking solution (5% skimmed milk powder (AS1033, Aspen, China) diluted in TBST), the PVDF membranes were separately incubated with anti-GLUT1 (1:1000, ab652, Abcam), anti-GLUT4 (1:2000, ab654, Abcam), anti-(extracellular) GLUT3 (1:1000, AGT-023, Alomone), anti-Bax (1:800, sc-7480, Santa Cruz), anti-Bcl-2 (1:800, sc-7382, Santa Cruz), anti-caspase-3 (cleaved) (1:100, AB3623, Millipore), anti-poly (ADP-ribose) polymerase (PARP) (cleaved) (1:1000, 5625, Cell Signaling Technology), and anti-β-actin (1:10,000, Aspen) antibodies at 4 °C overnight.

Techniques: Expressing, Transfection, Infection, Fluorescence, Microscopy, Cell Culture, Immunofluorescence

Journal: Cellular and Molecular Neurobiology

Article Title: Sevoflurane Induces Learning and Memory Impairment in Young Mice Through a Reduction in Neuronal Glucose Transporter 3

doi: 10.1007/s10571-019-00779-0

Figure Lengend Snippet: Sevoflurane inhibits GLUT3 surface expression and GLUT3 mRNA expression in neurons. a PC12 cells were transfected with LV5-GLUT3. The transfection efficiency was examined by GFP expression (green) 36 h post infection, which was analysed by phase contrast (left panel) and fluorescence microscopy (right panel). b The expression levels of GLUT3 protein in PC12 cells infected with LV5-GLUT3 were determined by WB analysis. PC12 cells were normally cultured (the Con group), transfected with LV5 empty vectors (the LV5 group), or transfected with LV5-GLUT3 (the LV5-GLUT3 group). c Flow cytometry analysis of GLUT3 surface expression in PC12 cells 24 h after the last exposure to sevoflurane. Four groups: the Con group, the Sev group (PC12 cells only exposed to sevoflurane), the LV5-GLUT3 group and the LV5-GLUT3 + Sev group (PC12 cells transfected with LV5-GLUT3 and then exposed to sevoflurane). n = 5 for each group. * P < 0.05 versus the Con group. # P < 0.05 versus the LV5-GLUT3 group. d Representative G6P concentrations showing glucose uptake in PC12 cells 24 h after the last exposure to sevoflurane. n = 5 for each group. * P < 0.05 versus the Con group. # P < 0.05 versus the LV5-GLUT3 group. e Representative G6P concentration showing glucose uptake in primary hippocampal neurons 24 h after the last exposure to sevoflurane. n = 5 for each group. * P < 0.05 versus the Con group. # P < 0.05 versus the LV5-GLUT3 group. f Representative quantification of GLUT3 mRNA expression in the hippocampus and temporal lobe 24 h after the last exposure to sevoflurane. n = 4 for each group. In the hippocampus, * P < 0.05 versus the Con group. In the temporal lobe, # P < 0.05 versus the Con group. g Representative quantification of GLUT3 mRNA expression in primary hippocampal neurons 24 h after the last exposure to sevoflurane. n = 5 for each group. * P < 0.05 versus the Con group. # P < 0.05 versus the LV5-GLUT3 group. The data are presented as the mean ± s.e.m

Article Snippet: After incubation with WB-specific blocking solution (5% skimmed milk powder (AS1033, Aspen, China) diluted in TBST), the PVDF membranes were separately incubated with anti-GLUT1 (1:1000, ab652, Abcam), anti-GLUT4 (1:2000, ab654, Abcam), anti-(extracellular) GLUT3 (1:1000, AGT-023, Alomone), anti-Bax (1:800, sc-7480, Santa Cruz), anti-Bcl-2 (1:800, sc-7382, Santa Cruz), anti-caspase-3 (cleaved) (1:100, AB3623, Millipore), anti-poly (ADP-ribose) polymerase (PARP) (cleaved) (1:1000, 5625, Cell Signaling Technology), and anti-β-actin (1:10,000, Aspen) antibodies at 4 °C overnight.

Techniques: Expressing, Transfection, Infection, Fluorescence, Microscopy, Cell Culture, Flow Cytometry, Concentration Assay

Journal: Cellular and Molecular Neurobiology

Article Title: Sevoflurane Induces Learning and Memory Impairment in Young Mice Through a Reduction in Neuronal Glucose Transporter 3

doi: 10.1007/s10571-019-00779-0

Figure Lengend Snippet: Sevoflurane induces neural apoptosis in the hippocampus and temporal lobe. a Representative immunoblots showing the protein expression of Bax, Bcl-2, cleaved caspase-3 and cleaved PARP in the hippocampus and temporal lobe of young mice 24 h after the last exposure to sevoflurane. n = 4 for each group. b, c, d, e Histograms showing the results of Bax, Bcl-2, cleaved caspase-3, and cleaved PARP blots in the hippocampus and temporal lobe. * P < 0.05 versus the Con group. In the temporal lobe, # P < 0.05 versus the Con group. f Representative images showing the flow cytometry analysis of the apoptosis ratio in PC12 cells 24 h after the last exposure to sevoflurane. Early apoptotic populations (PE + 7-AAD − cells) are in the lower-right quadrant; late apoptotic cells (PE + 7-AAD + ) are in the upper-right quadrant in each dot plot. g Representative quantification of the flow cytometry analysis of the apoptosis ratio of PC12 cells 24 h after the last exposure to sevoflurane. h Representative CCK-8 assay showing cell viability in primary hippocampal neurons 24 h after the last exposure to sevoflurane. n = 5 for each group. * P < 0.05 versus the Con group. # P < 0.05 versus the LV5-GLUT3 group. The data are presented as the mean ± s.e.m

Article Snippet: After incubation with WB-specific blocking solution (5% skimmed milk powder (AS1033, Aspen, China) diluted in TBST), the PVDF membranes were separately incubated with anti-GLUT1 (1:1000, ab652, Abcam), anti-GLUT4 (1:2000, ab654, Abcam), anti-(extracellular) GLUT3 (1:1000, AGT-023, Alomone), anti-Bax (1:800, sc-7480, Santa Cruz), anti-Bcl-2 (1:800, sc-7382, Santa Cruz), anti-caspase-3 (cleaved) (1:100, AB3623, Millipore), anti-poly (ADP-ribose) polymerase (PARP) (cleaved) (1:1000, 5625, Cell Signaling Technology), and anti-β-actin (1:10,000, Aspen) antibodies at 4 °C overnight.

Techniques: Western Blot, Expressing, Flow Cytometry, CCK-8 Assay

Journal: Cell reports

Article Title: Dysregulated oxalate metabolism is a driver and therapeutic target in atherosclerosis.

doi: 10.1016/j.celrep.2021.109420

Figure Lengend Snippet: Figure 1. Dysregulated glycine and oxalate metabolism in patients and mice with atherosclerosis (A) Schematic representation of glycine metabolic pathways. (B–E) Targeted metabolomics assessing the ratios of glycine to (B) serine, (C) threonine, (D) alanine, and (E) oxalate in serum from age- and sex- matched patients with or without sCAD (n = 24). (F) En face analysis of atherosclerotic lesions in male Apoe/ mice fed a standard diet (SD) or Western diet (WD) for 12 weeks (n = 5). (G–J) Targeted metabolomics assessing the ratios of glycine to (G) serine, (H) threonine, (I) alanine, and (J) oxalate in plasma from male Apoe/ mice fed a SD or WD for 12 weeks (n = 5). (K) Western blot analysis of AGXT protein abun- dance in livers from Apoe/ mice fed a SD or WD for 12 weeks (n = 5). Mann-Whitney U test for (B)-(E), (G), (H), and (K). Unpaired t test for (F), (I), and (J). Data are pre- sented as mean ± SEM. All points and p values are shown.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Primer sequences for Agxt genotyping:, forward: 50-ACACCTCCACTGTCCTG TCC-30 Integrated DNA Technologies N/A Primer sequences for Agxt genotyping:, reverse: 50-GGTCAGATCTGCCTGCTA CC-30 Integrated DNA Technologies N/A Primer for Sanger sequencing:, 50-GCAGAGCTAGCTGGGAAATG-30 Integrated DNA Technologies N/A Primer sequences for Apoe genotyping:, forward: 50-GCCTAGCCGAGGGAGAG CCG-30 Integrated DNA Technologies N/A Primer sequences for Apoe genotyping:, reverse 1: 50-TGTGACTTGGGAGCTC TGCAGC-30 Integrated DNA Technologies N/A Primer sequences for Apoe genotyping:, reverse 2: 50-GCCGCCCCGACTGC ATCT-30 Integrated DNA Technologies N/A Primer sequences for qPCR, see Table S2 Integrated DNA Technologies N/A Recombinant DNA pAdDeltaF6 Jiandie Lin’s lab, University of Michigan; Guo et al., 2017 N/A pAAV2/8 Jiandie Lin’s lab, University of Michigan; Guo et al., 2017 N/A pAAV-TBG-GFP Jiandie Lin’s lab, University of Michigan; Guo et al., 2017 N/A pAAV-TBG-MCS Jiandie Lin’s lab, University of Michigan; Guo et al., 2017 N/A AGXT Human Tagged ORF Clone Origene CAT# RG212899 Software and algorithms ImageJ National Institutes of Health RRID:SCR_003070 https://imagej.nih.gov/ ij/download.html GraphPad Prism GraphPad Software Version 8.0 RRID:SCR_002798 https:// www.graphpad.com/scientific-software/ prism/ R statistics Hosted by Vienna, University of Economics and Business http://www.R-project.org/ RStudio RStudio Team (2020). https://www.rstudio.com/ RRID:SCR_000432 IBM SPSS software IBM Corporation Version 23.0; RRID:SCR_019096 https:// www.ibm.com/analytics/ spss-statistics-software FastQC Babraham Bioinformatics Version 0.11.8 RRID:SCR_014583 https:// www.bioinformatics.babraham.ac.uk/ projects/fastqc/ Trimmomatic Bolger et al., 2014 Version 0.35 RRID:SCR_011848 http:// www.usadellab.org/cms/? page=trimmomatic HISAT2 Kim et al., 2015 Version 2.1.0.13; RRID:SCR_015530 http:// daehwankimlab.github.io/hisat2/ download/ HTSeq Anders et al., 2015 Version 0.6.0; RRID:SCR_005514 https:// htseq.readthedocs.io/en/master/install. html (Continued on next page) Cell Reports 36, 109420, July 27, 2021 e3 Article ll OPEN ACCESS

Techniques: Western Blot, Clinical Proteomics, MANN-WHITNEY

Journal: Cell reports

Article Title: Dysregulated oxalate metabolism is a driver and therapeutic target in atherosclerosis.

doi: 10.1016/j.celrep.2021.109420

Figure Lengend Snippet: Figure 2. AGXT deficiency exacerbates atherosclerosis in male Apoe/ mice (A) Western blot analysis confirming the loss of AGXT in livers from male Agxt//Apoe/ mice (n = 6). (B) Glycine/oxalate ratio in plasma from male Agxt//Apoe/ mice and their Agxt+/+/Apoe/

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Primer sequences for Agxt genotyping:, forward: 50-ACACCTCCACTGTCCTG TCC-30 Integrated DNA Technologies N/A Primer sequences for Agxt genotyping:, reverse: 50-GGTCAGATCTGCCTGCTA CC-30 Integrated DNA Technologies N/A Primer for Sanger sequencing:, 50-GCAGAGCTAGCTGGGAAATG-30 Integrated DNA Technologies N/A Primer sequences for Apoe genotyping:, forward: 50-GCCTAGCCGAGGGAGAG CCG-30 Integrated DNA Technologies N/A Primer sequences for Apoe genotyping:, reverse 1: 50-TGTGACTTGGGAGCTC TGCAGC-30 Integrated DNA Technologies N/A Primer sequences for Apoe genotyping:, reverse 2: 50-GCCGCCCCGACTGC ATCT-30 Integrated DNA Technologies N/A Primer sequences for qPCR, see Table S2 Integrated DNA Technologies N/A Recombinant DNA pAdDeltaF6 Jiandie Lin’s lab, University of Michigan; Guo et al., 2017 N/A pAAV2/8 Jiandie Lin’s lab, University of Michigan; Guo et al., 2017 N/A pAAV-TBG-GFP Jiandie Lin’s lab, University of Michigan; Guo et al., 2017 N/A pAAV-TBG-MCS Jiandie Lin’s lab, University of Michigan; Guo et al., 2017 N/A AGXT Human Tagged ORF Clone Origene CAT# RG212899 Software and algorithms ImageJ National Institutes of Health RRID:SCR_003070 https://imagej.nih.gov/ ij/download.html GraphPad Prism GraphPad Software Version 8.0 RRID:SCR_002798 https:// www.graphpad.com/scientific-software/ prism/ R statistics Hosted by Vienna, University of Economics and Business http://www.R-project.org/ RStudio RStudio Team (2020). https://www.rstudio.com/ RRID:SCR_000432 IBM SPSS software IBM Corporation Version 23.0; RRID:SCR_019096 https:// www.ibm.com/analytics/ spss-statistics-software FastQC Babraham Bioinformatics Version 0.11.8 RRID:SCR_014583 https:// www.bioinformatics.babraham.ac.uk/ projects/fastqc/ Trimmomatic Bolger et al., 2014 Version 0.35 RRID:SCR_011848 http:// www.usadellab.org/cms/? page=trimmomatic HISAT2 Kim et al., 2015 Version 2.1.0.13; RRID:SCR_015530 http:// daehwankimlab.github.io/hisat2/ download/ HTSeq Anders et al., 2015 Version 0.6.0; RRID:SCR_005514 https:// htseq.readthedocs.io/en/master/install. html (Continued on next page) Cell Reports 36, 109420, July 27, 2021 e3 Article ll OPEN ACCESS

Techniques: Western Blot, Clinical Proteomics

Journal: Cell reports

Article Title: Dysregulated oxalate metabolism is a driver and therapeutic target in atherosclerosis.

doi: 10.1016/j.celrep.2021.109420

Figure Lengend Snippet: Figure 3. Oxalate homeostasis is main- tained and atherosclerosis is unaltered in fe- male Agxt//Apoe/ mice (A) Western blot analysis confirming the loss of AGXT in livers from female Agxt//Apoe/ mice (n = 6). (B) GlGlycine/oxalate ratio in plasma from female Agxt//Apoe/ and Agxt+/+/Apoe/ mice (n = 10). (C–G) Female Agxt//Apoe/ and Agxt+/+/ Apoe/ mice were fed a WD for 12 weeks (n = 10): (C) plasma TC, (D) atherosclerosis in the aortic tree, (E) H&E staining, (F) ORO staining, and (G) Mac-2 immunohistochemistry of aortic sinus. Scale bars, 200 mm. Unpaired t test for (B), (D), (E), and (G). Mann- Whitney U test for (C) and (F). Data are presented as mean ± SEM. All points and p values are shown.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Primer sequences for Agxt genotyping:, forward: 50-ACACCTCCACTGTCCTG TCC-30 Integrated DNA Technologies N/A Primer sequences for Agxt genotyping:, reverse: 50-GGTCAGATCTGCCTGCTA CC-30 Integrated DNA Technologies N/A Primer for Sanger sequencing:, 50-GCAGAGCTAGCTGGGAAATG-30 Integrated DNA Technologies N/A Primer sequences for Apoe genotyping:, forward: 50-GCCTAGCCGAGGGAGAG CCG-30 Integrated DNA Technologies N/A Primer sequences for Apoe genotyping:, reverse 1: 50-TGTGACTTGGGAGCTC TGCAGC-30 Integrated DNA Technologies N/A Primer sequences for Apoe genotyping:, reverse 2: 50-GCCGCCCCGACTGC ATCT-30 Integrated DNA Technologies N/A Primer sequences for qPCR, see Table S2 Integrated DNA Technologies N/A Recombinant DNA pAdDeltaF6 Jiandie Lin’s lab, University of Michigan; Guo et al., 2017 N/A pAAV2/8 Jiandie Lin’s lab, University of Michigan; Guo et al., 2017 N/A pAAV-TBG-GFP Jiandie Lin’s lab, University of Michigan; Guo et al., 2017 N/A pAAV-TBG-MCS Jiandie Lin’s lab, University of Michigan; Guo et al., 2017 N/A AGXT Human Tagged ORF Clone Origene CAT# RG212899 Software and algorithms ImageJ National Institutes of Health RRID:SCR_003070 https://imagej.nih.gov/ ij/download.html GraphPad Prism GraphPad Software Version 8.0 RRID:SCR_002798 https:// www.graphpad.com/scientific-software/ prism/ R statistics Hosted by Vienna, University of Economics and Business http://www.R-project.org/ RStudio RStudio Team (2020). https://www.rstudio.com/ RRID:SCR_000432 IBM SPSS software IBM Corporation Version 23.0; RRID:SCR_019096 https:// www.ibm.com/analytics/ spss-statistics-software FastQC Babraham Bioinformatics Version 0.11.8 RRID:SCR_014583 https:// www.bioinformatics.babraham.ac.uk/ projects/fastqc/ Trimmomatic Bolger et al., 2014 Version 0.35 RRID:SCR_011848 http:// www.usadellab.org/cms/? page=trimmomatic HISAT2 Kim et al., 2015 Version 2.1.0.13; RRID:SCR_015530 http:// daehwankimlab.github.io/hisat2/ download/ HTSeq Anders et al., 2015 Version 0.6.0; RRID:SCR_005514 https:// htseq.readthedocs.io/en/master/install. html (Continued on next page) Cell Reports 36, 109420, July 27, 2021 e3 Article ll OPEN ACCESS

Techniques: Western Blot, Clinical Proteomics, Staining, Immunohistochemistry, MANN-WHITNEY

Journal: Cell reports

Article Title: Dysregulated oxalate metabolism is a driver and therapeutic target in atherosclerosis.

doi: 10.1016/j.celrep.2021.109420

Figure Lengend Snippet: Figure 4. Dysregulated oxalate metabolism induces a pro-inflammatory response and CCL5 release (A–C) RNA sequencing of livers collected from male Agxt//Apoe/ and Agxt+/+/Apoe/ mice fed a WD for 12 weeks (n = 5): (A) volcano plot of DEGs (padj < 0.05, log2 fold change > 1) in male Agxt//Apoe/ versus Agxt+/+/Apoe/ mice (blue indicates downregulated; red indicates upregulated). (B) Kyoto Encyclopedia of Genes and Genomes (KEGG)-based pathway analysis. The significance of the enrichment was determined by a right-tailed Fisher’s exact test followed by a Benjamini-Hochberg multiple testing adjustment. (C) Heatmap-based representation of 50 DEGs linking inflammation, fibrogenesis, and the metabolism of lipids and sterols/steroids with atherosclerosis.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Primer sequences for Agxt genotyping:, forward: 50-ACACCTCCACTGTCCTG TCC-30 Integrated DNA Technologies N/A Primer sequences for Agxt genotyping:, reverse: 50-GGTCAGATCTGCCTGCTA CC-30 Integrated DNA Technologies N/A Primer for Sanger sequencing:, 50-GCAGAGCTAGCTGGGAAATG-30 Integrated DNA Technologies N/A Primer sequences for Apoe genotyping:, forward: 50-GCCTAGCCGAGGGAGAG CCG-30 Integrated DNA Technologies N/A Primer sequences for Apoe genotyping:, reverse 1: 50-TGTGACTTGGGAGCTC TGCAGC-30 Integrated DNA Technologies N/A Primer sequences for Apoe genotyping:, reverse 2: 50-GCCGCCCCGACTGC ATCT-30 Integrated DNA Technologies N/A Primer sequences for qPCR, see Table S2 Integrated DNA Technologies N/A Recombinant DNA pAdDeltaF6 Jiandie Lin’s lab, University of Michigan; Guo et al., 2017 N/A pAAV2/8 Jiandie Lin’s lab, University of Michigan; Guo et al., 2017 N/A pAAV-TBG-GFP Jiandie Lin’s lab, University of Michigan; Guo et al., 2017 N/A pAAV-TBG-MCS Jiandie Lin’s lab, University of Michigan; Guo et al., 2017 N/A AGXT Human Tagged ORF Clone Origene CAT# RG212899 Software and algorithms ImageJ National Institutes of Health RRID:SCR_003070 https://imagej.nih.gov/ ij/download.html GraphPad Prism GraphPad Software Version 8.0 RRID:SCR_002798 https:// www.graphpad.com/scientific-software/ prism/ R statistics Hosted by Vienna, University of Economics and Business http://www.R-project.org/ RStudio RStudio Team (2020). https://www.rstudio.com/ RRID:SCR_000432 IBM SPSS software IBM Corporation Version 23.0; RRID:SCR_019096 https:// www.ibm.com/analytics/ spss-statistics-software FastQC Babraham Bioinformatics Version 0.11.8 RRID:SCR_014583 https:// www.bioinformatics.babraham.ac.uk/ projects/fastqc/ Trimmomatic Bolger et al., 2014 Version 0.35 RRID:SCR_011848 http:// www.usadellab.org/cms/? page=trimmomatic HISAT2 Kim et al., 2015 Version 2.1.0.13; RRID:SCR_015530 http:// daehwankimlab.github.io/hisat2/ download/ HTSeq Anders et al., 2015 Version 0.6.0; RRID:SCR_005514 https:// htseq.readthedocs.io/en/master/install. html (Continued on next page) Cell Reports 36, 109420, July 27, 2021 e3 Article ll OPEN ACCESS

Techniques: RNA Sequencing

Journal: Cell reports

Article Title: Dysregulated oxalate metabolism is a driver and therapeutic target in atherosclerosis.

doi: 10.1016/j.celrep.2021.109420

Figure Lengend Snippet: Figure 5. Oxalate overload induces mito- chondrial dysfunction and overproduction of superoxide, leading to CCL5 release in macrophages (A) qPCR analyses of genes regulating redox ho- meostasis in livers from male Agxt//Apoe/

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Primer sequences for Agxt genotyping:, forward: 50-ACACCTCCACTGTCCTG TCC-30 Integrated DNA Technologies N/A Primer sequences for Agxt genotyping:, reverse: 50-GGTCAGATCTGCCTGCTA CC-30 Integrated DNA Technologies N/A Primer for Sanger sequencing:, 50-GCAGAGCTAGCTGGGAAATG-30 Integrated DNA Technologies N/A Primer sequences for Apoe genotyping:, forward: 50-GCCTAGCCGAGGGAGAG CCG-30 Integrated DNA Technologies N/A Primer sequences for Apoe genotyping:, reverse 1: 50-TGTGACTTGGGAGCTC TGCAGC-30 Integrated DNA Technologies N/A Primer sequences for Apoe genotyping:, reverse 2: 50-GCCGCCCCGACTGC ATCT-30 Integrated DNA Technologies N/A Primer sequences for qPCR, see Table S2 Integrated DNA Technologies N/A Recombinant DNA pAdDeltaF6 Jiandie Lin’s lab, University of Michigan; Guo et al., 2017 N/A pAAV2/8 Jiandie Lin’s lab, University of Michigan; Guo et al., 2017 N/A pAAV-TBG-GFP Jiandie Lin’s lab, University of Michigan; Guo et al., 2017 N/A pAAV-TBG-MCS Jiandie Lin’s lab, University of Michigan; Guo et al., 2017 N/A AGXT Human Tagged ORF Clone Origene CAT# RG212899 Software and algorithms ImageJ National Institutes of Health RRID:SCR_003070 https://imagej.nih.gov/ ij/download.html GraphPad Prism GraphPad Software Version 8.0 RRID:SCR_002798 https:// www.graphpad.com/scientific-software/ prism/ R statistics Hosted by Vienna, University of Economics and Business http://www.R-project.org/ RStudio RStudio Team (2020). https://www.rstudio.com/ RRID:SCR_000432 IBM SPSS software IBM Corporation Version 23.0; RRID:SCR_019096 https:// www.ibm.com/analytics/ spss-statistics-software FastQC Babraham Bioinformatics Version 0.11.8 RRID:SCR_014583 https:// www.bioinformatics.babraham.ac.uk/ projects/fastqc/ Trimmomatic Bolger et al., 2014 Version 0.35 RRID:SCR_011848 http:// www.usadellab.org/cms/? page=trimmomatic HISAT2 Kim et al., 2015 Version 2.1.0.13; RRID:SCR_015530 http:// daehwankimlab.github.io/hisat2/ download/ HTSeq Anders et al., 2015 Version 0.6.0; RRID:SCR_005514 https:// htseq.readthedocs.io/en/master/install. html (Continued on next page) Cell Reports 36, 109420, July 27, 2021 e3 Article ll OPEN ACCESS

Techniques:

Journal: Cell reports

Article Title: Dysregulated oxalate metabolism is a driver and therapeutic target in atherosclerosis.

doi: 10.1016/j.celrep.2021.109420

Figure Lengend Snippet: Figure 6. AAV-mediated overexpression of AGXT reduces oxidative stress and inflammation AAV-AGXT or AAV-GFP was injected into male Apoe/ mice and the mice were fed a WD for 12 weeks (n = 8). (A) Western blot analysis confirming the over- expression of AGXT in livers from mice treated with AAV-AGXT. (B) Plasma glycine/oxalate ratio. (C and D) qPCR analyses of genes regulating (C) redox homeostasis and (D) inflammatory re- sponses. Gene expression levels were normalized to 18S. (E and F) Plasma concentrations of (E) CCL2 and (F) CCL5. (G) DHE fluorescence and Mac-2 immunofluores- cence in the aortic sinuses. Scale bars, 50 mm. Unpaired t test for (B), (E), and (G), and Mann- Whitney U test for (F). Statistical differences in gene expression in (C) and (D) were tested using unpaired t test or Mann-Whitney U test, depending on normality tests. *P < 0.05, **P <0.01, ***P <0.001 versus mice injected with AAV-GFP. Data are presented as mean ± SEM. All points and p values are shown.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Primer sequences for Agxt genotyping:, forward: 50-ACACCTCCACTGTCCTG TCC-30 Integrated DNA Technologies N/A Primer sequences for Agxt genotyping:, reverse: 50-GGTCAGATCTGCCTGCTA CC-30 Integrated DNA Technologies N/A Primer for Sanger sequencing:, 50-GCAGAGCTAGCTGGGAAATG-30 Integrated DNA Technologies N/A Primer sequences for Apoe genotyping:, forward: 50-GCCTAGCCGAGGGAGAG CCG-30 Integrated DNA Technologies N/A Primer sequences for Apoe genotyping:, reverse 1: 50-TGTGACTTGGGAGCTC TGCAGC-30 Integrated DNA Technologies N/A Primer sequences for Apoe genotyping:, reverse 2: 50-GCCGCCCCGACTGC ATCT-30 Integrated DNA Technologies N/A Primer sequences for qPCR, see Table S2 Integrated DNA Technologies N/A Recombinant DNA pAdDeltaF6 Jiandie Lin’s lab, University of Michigan; Guo et al., 2017 N/A pAAV2/8 Jiandie Lin’s lab, University of Michigan; Guo et al., 2017 N/A pAAV-TBG-GFP Jiandie Lin’s lab, University of Michigan; Guo et al., 2017 N/A pAAV-TBG-MCS Jiandie Lin’s lab, University of Michigan; Guo et al., 2017 N/A AGXT Human Tagged ORF Clone Origene CAT# RG212899 Software and algorithms ImageJ National Institutes of Health RRID:SCR_003070 https://imagej.nih.gov/ ij/download.html GraphPad Prism GraphPad Software Version 8.0 RRID:SCR_002798 https:// www.graphpad.com/scientific-software/ prism/ R statistics Hosted by Vienna, University of Economics and Business http://www.R-project.org/ RStudio RStudio Team (2020). https://www.rstudio.com/ RRID:SCR_000432 IBM SPSS software IBM Corporation Version 23.0; RRID:SCR_019096 https:// www.ibm.com/analytics/ spss-statistics-software FastQC Babraham Bioinformatics Version 0.11.8 RRID:SCR_014583 https:// www.bioinformatics.babraham.ac.uk/ projects/fastqc/ Trimmomatic Bolger et al., 2014 Version 0.35 RRID:SCR_011848 http:// www.usadellab.org/cms/? page=trimmomatic HISAT2 Kim et al., 2015 Version 2.1.0.13; RRID:SCR_015530 http:// daehwankimlab.github.io/hisat2/ download/ HTSeq Anders et al., 2015 Version 0.6.0; RRID:SCR_005514 https:// htseq.readthedocs.io/en/master/install. html (Continued on next page) Cell Reports 36, 109420, July 27, 2021 e3 Article ll OPEN ACCESS

Techniques: Over Expression, Injection, Western Blot, Clinical Proteomics, Gene Expression, MANN-WHITNEY

Journal: Cell reports

Article Title: Dysregulated oxalate metabolism is a driver and therapeutic target in atherosclerosis.

doi: 10.1016/j.celrep.2021.109420

Figure Lengend Snippet: Figure 7. AAV-mediated overexpression of AGXT reduces atherosclerosis independent of circulating cholesterol AAV-AGXT or AAV-GFP was injected into male Apoe/ mice and the mice were fed a WD for 12 weeks (n = 8). (A) Plasma TC. (B) Atherosclerosis in the aortic tree. (C–E) H&E staining (D), ORO staining (D), and Mac- 2 immunohistochemistry (E) of aortic sinus. Scale bars, 200 mm. (F) Proposed model of dysregulated oxalate metabolism as a driver and therapeutic target in atherosclerosis. Unpaired t test for (A)–(E). Data are presented as mean ± SEM. All points and p values are shown.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Primer sequences for Agxt genotyping:, forward: 50-ACACCTCCACTGTCCTG TCC-30 Integrated DNA Technologies N/A Primer sequences for Agxt genotyping:, reverse: 50-GGTCAGATCTGCCTGCTA CC-30 Integrated DNA Technologies N/A Primer for Sanger sequencing:, 50-GCAGAGCTAGCTGGGAAATG-30 Integrated DNA Technologies N/A Primer sequences for Apoe genotyping:, forward: 50-GCCTAGCCGAGGGAGAG CCG-30 Integrated DNA Technologies N/A Primer sequences for Apoe genotyping:, reverse 1: 50-TGTGACTTGGGAGCTC TGCAGC-30 Integrated DNA Technologies N/A Primer sequences for Apoe genotyping:, reverse 2: 50-GCCGCCCCGACTGC ATCT-30 Integrated DNA Technologies N/A Primer sequences for qPCR, see Table S2 Integrated DNA Technologies N/A Recombinant DNA pAdDeltaF6 Jiandie Lin’s lab, University of Michigan; Guo et al., 2017 N/A pAAV2/8 Jiandie Lin’s lab, University of Michigan; Guo et al., 2017 N/A pAAV-TBG-GFP Jiandie Lin’s lab, University of Michigan; Guo et al., 2017 N/A pAAV-TBG-MCS Jiandie Lin’s lab, University of Michigan; Guo et al., 2017 N/A AGXT Human Tagged ORF Clone Origene CAT# RG212899 Software and algorithms ImageJ National Institutes of Health RRID:SCR_003070 https://imagej.nih.gov/ ij/download.html GraphPad Prism GraphPad Software Version 8.0 RRID:SCR_002798 https:// www.graphpad.com/scientific-software/ prism/ R statistics Hosted by Vienna, University of Economics and Business http://www.R-project.org/ RStudio RStudio Team (2020). https://www.rstudio.com/ RRID:SCR_000432 IBM SPSS software IBM Corporation Version 23.0; RRID:SCR_019096 https:// www.ibm.com/analytics/ spss-statistics-software FastQC Babraham Bioinformatics Version 0.11.8 RRID:SCR_014583 https:// www.bioinformatics.babraham.ac.uk/ projects/fastqc/ Trimmomatic Bolger et al., 2014 Version 0.35 RRID:SCR_011848 http:// www.usadellab.org/cms/? page=trimmomatic HISAT2 Kim et al., 2015 Version 2.1.0.13; RRID:SCR_015530 http:// daehwankimlab.github.io/hisat2/ download/ HTSeq Anders et al., 2015 Version 0.6.0; RRID:SCR_005514 https:// htseq.readthedocs.io/en/master/install. html (Continued on next page) Cell Reports 36, 109420, July 27, 2021 e3 Article ll OPEN ACCESS

Techniques: Over Expression, Injection, Clinical Proteomics, Staining, Immunohistochemistry

Journal: Journal of Hepatocellular Carcinoma

Article Title: AGXT2 Suppresses the Proliferation and Dissemination of Hepatocellular Carcinoma Cells by Modulating Intracellular Lipid Metabolism

doi: 10.2147/JHC.S470250

Figure Lengend Snippet: The Primers Used for qPCR Analysis

Article Snippet: The membranes were then incubated with primary antibodies targeting AGXT2 (1:7000; Cat. no. 66602-1-Ig; Proteintech), LDLR (1:800; Cat. no. R380860; Zenbio), PCSK9 (1:800; Cat. no. R25297; Zenbio), angiopoietin like 4 (ANGPTL4) (1:800; Cat. no. R23468; Zenbio), and β-catenin (1:6000; Cat. no. CPA9066; cohesion).

Techniques: Sequencing

Journal: Journal of Hepatocellular Carcinoma

Article Title: AGXT2 Suppresses the Proliferation and Dissemination of Hepatocellular Carcinoma Cells by Modulating Intracellular Lipid Metabolism

doi: 10.2147/JHC.S470250

Figure Lengend Snippet: Expression profile of AGXT2 at the mRNA level. The expression of the AGXT2 gene was analyzed across various cancer types and specific subtypes utilizing datasets from TCGA. ( A ) The BioGPS database indicates that AGXT2 is predominantly expressed in liver and kidney tissues. ( B ) Analysis of TCGA and GTEx datasets revealed a significant decrease in AGXT2 mRNA expression in HCC tissues compared to normal liver tissues. ( C ) AGXT2 expression was significantly lower in HCC tissues compared to normal liver tissues in the GSE63898 dataset. ( D ) Among a cohort of 32 patients with HCC, there was a notable decrease in AGXT2 gene mRNA expression.

Article Snippet: The membranes were then incubated with primary antibodies targeting AGXT2 (1:7000; Cat. no. 66602-1-Ig; Proteintech), LDLR (1:800; Cat. no. R380860; Zenbio), PCSK9 (1:800; Cat. no. R25297; Zenbio), angiopoietin like 4 (ANGPTL4) (1:800; Cat. no. R23468; Zenbio), and β-catenin (1:6000; Cat. no. CPA9066; cohesion).

Techniques: Expressing

Journal: Journal of Hepatocellular Carcinoma

Article Title: AGXT2 Suppresses the Proliferation and Dissemination of Hepatocellular Carcinoma Cells by Modulating Intracellular Lipid Metabolism

doi: 10.2147/JHC.S470250

Figure Lengend Snippet: Expression of AGXT2 protein in HCC tissues and serum.( A ) The AGXT2 gene expression status in tumor tissue ( T ) and adjacent normal tissue ( N ) of HCC was analyzed using Western blot assay. ( B ) Statistical analysis of AGXT2 protein levels in 12 pairs of human liver tumors and adjacent normal liver tissues was conducted through Western blot. AGXT2 protein levels were quantified using Images J software. ( C ) AGXT2 protein expression in the sera of 40 patients with liver cancer was assessed by ELISA. ( D ) Tumor tissue ( T ) and adjacent normal tissue ( N ) were visualized using IHC staining for observation. ( E ) The immunohistochemical staining technique was utilized to detect the expression level of AGXT2 protein in 40 pairs of human liver tumors and their corresponding adjacent normal liver tissues. A subset of four pairs of specimens were randomly selected for inclusion in the figure depicting AGXT2 immunohistochemical staining. ( F ) The immunohistochemical results of AGXT2 in a biopsy specimen from a liver cancer patient were examined.

Article Snippet: The membranes were then incubated with primary antibodies targeting AGXT2 (1:7000; Cat. no. 66602-1-Ig; Proteintech), LDLR (1:800; Cat. no. R380860; Zenbio), PCSK9 (1:800; Cat. no. R25297; Zenbio), angiopoietin like 4 (ANGPTL4) (1:800; Cat. no. R23468; Zenbio), and β-catenin (1:6000; Cat. no. CPA9066; cohesion).

Techniques: Expressing, Gene Expression, Western Blot, Software, Enzyme-linked Immunosorbent Assay, Immunohistochemistry, Immunohistochemical staining, Staining

Journal: Journal of Hepatocellular Carcinoma

Article Title: AGXT2 Suppresses the Proliferation and Dissemination of Hepatocellular Carcinoma Cells by Modulating Intracellular Lipid Metabolism

doi: 10.2147/JHC.S470250

Figure Lengend Snippet: RNA-Seq data and LC-MS data analysis from the AGXT2-overexpression model.( A ) Utilizing a heat map as a visual representation to illustrate the DEGs across multiple datasets. The heat map employs a color gradient to indicate the level of transcript enrichment, with red denoting upregulated genes and green denoting downregulated genes. ( B ) Identifying the top 60 genes exhibiting variance in expression levels attributed to AGXT2, with red representing upregulation and blue representing downregulation. ( C ) Volcano plot displaying genes that are expressed differentially in DEGs. The log2 fold change difference associating with the Control and Ad-AGXT2 samples is showed on the x - axis, and negative log of p - values is showed on the y - axis. Every point represents a gene that has measurable expression in both samples. ( D ) Volcano plot of DETs in Huh7 cell with the AGXT2-overexpression. ( E ) Visualization of DETs using a heat map across various sample data sets. Red indicates the transcripts that are upregulated in the heatmaps, while blue indicates the downregulated transcripts. F. Statistics of 17 upregulated and 25 downregulated DETs. ( F ) Cholesterol was most significantly downregulated in metabolites (Log FC = −7.01, p = 0.0129).

Article Snippet: The membranes were then incubated with primary antibodies targeting AGXT2 (1:7000; Cat. no. 66602-1-Ig; Proteintech), LDLR (1:800; Cat. no. R380860; Zenbio), PCSK9 (1:800; Cat. no. R25297; Zenbio), angiopoietin like 4 (ANGPTL4) (1:800; Cat. no. R23468; Zenbio), and β-catenin (1:6000; Cat. no. CPA9066; cohesion).

Techniques: RNA Sequencing, Liquid Chromatography with Mass Spectroscopy, Over Expression, Expressing, Control

Journal: Journal of Hepatocellular Carcinoma

Article Title: AGXT2 Suppresses the Proliferation and Dissemination of Hepatocellular Carcinoma Cells by Modulating Intracellular Lipid Metabolism

doi: 10.2147/JHC.S470250

Figure Lengend Snippet: AGXT2 regulated the cholesterol level of liver cancer cells by down-regulating LDLR.( A ) KEGG pathway enrichment joint analysis by DEGs and DETs. ( B ) Intersected 51 genes of the cholesterol metabolic pathway with the top 500 DEGs genes of overexpressing AGXT2. ( C ) LDLR and PCSK9 mRNA were increased after AGXT2 overexpression. ( D ) AGXT2 reduces intracellular cholesterol levels in liver cancer by an oil red O staining. ( E ) Protein-protein molecular docking technique predicted that AGXT2 and LDLR had 6 bonding hydrogen bonds and 1 salt bridge. ( F ) Ectopic expression of AGXT2 induced LDLR protein level and increase PCSK9 protein level which were detected by Western blotting. ( G ) AGXT2 protein is bound to LDLR protein by co-immunoprecipitation.

Article Snippet: The membranes were then incubated with primary antibodies targeting AGXT2 (1:7000; Cat. no. 66602-1-Ig; Proteintech), LDLR (1:800; Cat. no. R380860; Zenbio), PCSK9 (1:800; Cat. no. R25297; Zenbio), angiopoietin like 4 (ANGPTL4) (1:800; Cat. no. R23468; Zenbio), and β-catenin (1:6000; Cat. no. CPA9066; cohesion).

Techniques: Over Expression, Staining, Expressing, Western Blot, Immunoprecipitation

Journal: Journal of Hepatocellular Carcinoma

Article Title: AGXT2 Suppresses the Proliferation and Dissemination of Hepatocellular Carcinoma Cells by Modulating Intracellular Lipid Metabolism

doi: 10.2147/JHC.S470250

Figure Lengend Snippet: AGXT2 overexpression inhibited the growth and metastasis of hepatocellular carcinoma cells.( A ).Experimental wound healing area of Huh7 cells after AGXT2 activation or inhibition. ( B ) ImageJ computer software analyzed the scratch test results. Scratch assay of Huh7 cells after activation or inhibition. Healing ratio = (Initial area - 48h area) / Initial area. ( C ) The representative images of wound healing assays in Huh7 cell lines. The invasion ability of Huh7 were measured by count of cells. ( D ) Flow cytometry was used to observe the cyclical impacts of AGXT2 overexpression and knockdown on HepG2 and Huh7 cells. ( E ) The experiment of subcutaneous tumor formation in NTG mice. ( F ) Quantitative statistical analysis of subcutaneous tumor formation in NTG mice. ( G ) HE staining of the tumor. ( H ) Low expression of AGXT2 protein indicated poor OS by GEPIA2.

Article Snippet: The membranes were then incubated with primary antibodies targeting AGXT2 (1:7000; Cat. no. 66602-1-Ig; Proteintech), LDLR (1:800; Cat. no. R380860; Zenbio), PCSK9 (1:800; Cat. no. R25297; Zenbio), angiopoietin like 4 (ANGPTL4) (1:800; Cat. no. R23468; Zenbio), and β-catenin (1:6000; Cat. no. CPA9066; cohesion).

Techniques: Over Expression, Activation Assay, Inhibition, Software, Wound Healing Assay, Flow Cytometry, Knockdown, Staining, Expressing