Journal: American Journal of Translational Research

Article Title: Distinct effects of ANGPT2 on gene expression of glomerular podocytes and mesangial cells

doi:

Figure Lengend Snippet: ANGPT2 mRNA expression in glomerular cells of human and mouse. A. The single-cell RNA-seq database search with KIT (http://humphreyslab.com/SingleCell/displaycharts.php) identified ANGPT2 mRNA expression in various kidney cell types, including glomerular cells (podocytes, mesangial cells and endothelial cells) and various tubular cell types. EC, endothelial cells; PT (S1), proximal tubular cells (segment 1); LH (DL), Loop of Henle (descending limb); LH (AL), Loop of Henle (ascending limb); DCT, distal convoluted tubule; PC, principal cells; IC, intercalated cells. B. GEO database search (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE123179) revealed ANGPT2 mRNA in podocytes (Pod), mesangial cells (Mes) and endothelial cells (Endo) from mice by bulk sequencing. N=3 for each group; *P=0.00046, mesangial cells vs. podocytes; P=0.00037, mesangial cells vs. endothelial cells.

Article Snippet: The information of the reagents used in the present study is as follows: Antibodies against ANGPT2 (Affinity), SYNPO (Santa Cruz), GAPDH (Proteintech), total ERK1/2, phospho-ERK1/2, total AKT and phospho-AKT (Cell Signaling Technologies); Reverse transcription kit (DRR037A, Takara); Quantitative PCR kit (Thermo Fisher Scientific); RIPA cell lysis buffer, BCA protein quantification kit (Beyotime, Shanghai); RNA extraction kit (Takara); ANGPT2 (HY-P7510, MCE); Trizol (Invitrogen).

Techniques: Expressing, RNA Sequencing, Sequencing

Journal: American Journal of Translational Research

Article Title: Distinct effects of ANGPT2 on gene expression of glomerular podocytes and mesangial cells

doi:

Figure Lengend Snippet: ANGPT2 protein expression in glomerular cells of human and mouse. A. Immunofluorescence staining of ANGPT2 in normal human glomeruli. Podocyte marker, SYNPO, was co-stained, thereby localizing ANGPT2 to podocytes and non-podocytes. B. Similar co-staining of ANGPT2 and SYNPO in mouse glomeruli showing ANGPT2 is present in both podocytes and non-podocytes in mice.

Article Snippet: The information of the reagents used in the present study is as follows: Antibodies against ANGPT2 (Affinity), SYNPO (Santa Cruz), GAPDH (Proteintech), total ERK1/2, phospho-ERK1/2, total AKT and phospho-AKT (Cell Signaling Technologies); Reverse transcription kit (DRR037A, Takara); Quantitative PCR kit (Thermo Fisher Scientific); RIPA cell lysis buffer, BCA protein quantification kit (Beyotime, Shanghai); RNA extraction kit (Takara); ANGPT2 (HY-P7510, MCE); Trizol (Invitrogen).

Techniques: Expressing, Immunofluorescence, Staining, Marker

Journal: American Journal of Translational Research

Article Title: Distinct effects of ANGPT2 on gene expression of glomerular podocytes and mesangial cells

doi:

Figure Lengend Snippet: ANGPT2 expression in glomeruli was upregulated in patients with diabetic nephropathy. From the gene expression dataset of glomeruli from diabetic patients (GSE96804), the glomerular ANGPT2 levels in DN patients at different stages of the diseases were obtained. Early: urinary albumin <0.5 g/24 h with normal serum creatinine; middle: urinary albumin >0.5 g/24 h with normal serum creatinine; late: serum creatinine >1.24 mg/dL. *P<0.05, **P<0.01, significant difference vs. normal subjects (Ctrl).

Article Snippet: The information of the reagents used in the present study is as follows: Antibodies against ANGPT2 (Affinity), SYNPO (Santa Cruz), GAPDH (Proteintech), total ERK1/2, phospho-ERK1/2, total AKT and phospho-AKT (Cell Signaling Technologies); Reverse transcription kit (DRR037A, Takara); Quantitative PCR kit (Thermo Fisher Scientific); RIPA cell lysis buffer, BCA protein quantification kit (Beyotime, Shanghai); RNA extraction kit (Takara); ANGPT2 (HY-P7510, MCE); Trizol (Invitrogen).

Techniques: Expressing, Gene Expression

Journal: American Journal of Translational Research

Article Title: Distinct effects of ANGPT2 on gene expression of glomerular podocytes and mesangial cells

doi:

Figure Lengend Snippet: ANGPT2 expression in glomeruli was upregulated in patients with multiple glomerular diseases. A. Nephroseq searches identified ANGPT2 upregulation in glomeruli of patients with CKD (Normal n=3; CKD n=5; fold change 6.98, P=0.001). B. ANGPT2 upregulation in hypertension (Normal n=4; hypertension n=14; fold change 1.22, P=0.023). C. ANGPT2 upregulation in FSGS (Normal n=9; FSGS n=8; fold change 1.36, P=0.033). D. ANGPT2 in LN (Normal n=14; LN n=32; fold change 1.41, P=0.005). *P<0.05, significant vs. normal subjects.

Article Snippet: The information of the reagents used in the present study is as follows: Antibodies against ANGPT2 (Affinity), SYNPO (Santa Cruz), GAPDH (Proteintech), total ERK1/2, phospho-ERK1/2, total AKT and phospho-AKT (Cell Signaling Technologies); Reverse transcription kit (DRR037A, Takara); Quantitative PCR kit (Thermo Fisher Scientific); RIPA cell lysis buffer, BCA protein quantification kit (Beyotime, Shanghai); RNA extraction kit (Takara); ANGPT2 (HY-P7510, MCE); Trizol (Invitrogen).

Techniques: Expressing

Journal: American Journal of Translational Research

Article Title: Distinct effects of ANGPT2 on gene expression of glomerular podocytes and mesangial cells

doi:

Figure Lengend Snippet: Gene expression regulation by ANGPT2 in podocytes and mesangial cells in culture. A. Volcano plot visualization of statistically significant gene expression changes in ANGPT2-treated podocytes (left) and mesangial cells (right) versus untreated cells, respectively. X-axis, log fold change; Y-axis, -log10 P value. B. Venn diagrams showing the huge difference in the list of genes regulated by ANGPT2 in podocytes vs. mesangial cells.

Article Snippet: The information of the reagents used in the present study is as follows: Antibodies against ANGPT2 (Affinity), SYNPO (Santa Cruz), GAPDH (Proteintech), total ERK1/2, phospho-ERK1/2, total AKT and phospho-AKT (Cell Signaling Technologies); Reverse transcription kit (DRR037A, Takara); Quantitative PCR kit (Thermo Fisher Scientific); RIPA cell lysis buffer, BCA protein quantification kit (Beyotime, Shanghai); RNA extraction kit (Takara); ANGPT2 (HY-P7510, MCE); Trizol (Invitrogen).

Techniques: Gene Expression

Journal: American Journal of Translational Research

Article Title: Distinct effects of ANGPT2 on gene expression of glomerular podocytes and mesangial cells

doi:

Figure Lengend Snippet: GO analysis shows fundamental differences in enrichments between the regulated genes in podocytes (A) and those in mesangial cells (B) treated with ANGPT2.

Article Snippet: The information of the reagents used in the present study is as follows: Antibodies against ANGPT2 (Affinity), SYNPO (Santa Cruz), GAPDH (Proteintech), total ERK1/2, phospho-ERK1/2, total AKT and phospho-AKT (Cell Signaling Technologies); Reverse transcription kit (DRR037A, Takara); Quantitative PCR kit (Thermo Fisher Scientific); RIPA cell lysis buffer, BCA protein quantification kit (Beyotime, Shanghai); RNA extraction kit (Takara); ANGPT2 (HY-P7510, MCE); Trizol (Invitrogen).

Techniques:

Journal: American Journal of Translational Research

Article Title: Distinct effects of ANGPT2 on gene expression of glomerular podocytes and mesangial cells

doi:

Figure Lengend Snippet: KEGG pathways analysis shows the difference in KEGG pathways between the group of genes regulated in podocytes (A) and that in mesangial cells (B) after treatment with ANGPT2.

Article Snippet: The information of the reagents used in the present study is as follows: Antibodies against ANGPT2 (Affinity), SYNPO (Santa Cruz), GAPDH (Proteintech), total ERK1/2, phospho-ERK1/2, total AKT and phospho-AKT (Cell Signaling Technologies); Reverse transcription kit (DRR037A, Takara); Quantitative PCR kit (Thermo Fisher Scientific); RIPA cell lysis buffer, BCA protein quantification kit (Beyotime, Shanghai); RNA extraction kit (Takara); ANGPT2 (HY-P7510, MCE); Trizol (Invitrogen).

Techniques:

Journal: American Journal of Translational Research

Article Title: Distinct effects of ANGPT2 on gene expression of glomerular podocytes and mesangial cells

doi:

Figure Lengend Snippet: pERK was increased in ANGPT2-treated podocytes but decreased in mesangial cells in culture. ANGPT2 (500 ng/ml) was used to treat podocytes and mesangial cells for 24 h. The results represent the data from three independent experiments and are expressed as mean ± SD. *P<0.05; **P<0.01 vs. control.

Article Snippet: The information of the reagents used in the present study is as follows: Antibodies against ANGPT2 (Affinity), SYNPO (Santa Cruz), GAPDH (Proteintech), total ERK1/2, phospho-ERK1/2, total AKT and phospho-AKT (Cell Signaling Technologies); Reverse transcription kit (DRR037A, Takara); Quantitative PCR kit (Thermo Fisher Scientific); RIPA cell lysis buffer, BCA protein quantification kit (Beyotime, Shanghai); RNA extraction kit (Takara); ANGPT2 (HY-P7510, MCE); Trizol (Invitrogen).

Techniques: Control

Journal: American Journal of Translational Research

Article Title: Distinct effects of ANGPT2 on gene expression of glomerular podocytes and mesangial cells

doi:

Figure Lengend Snippet: pAKT was increased in ANGPT2-treated podocytes but downregulated in mesangial cells in culture. Podocytes and mesangial cells were treated with 500 ng/ml ANGPT2 for 24 h and subject to immnunoblotting. The results represent the data from three independent experiments and are expressed as mean ± SD. **P<0.01 vs. control.

Article Snippet: The information of the reagents used in the present study is as follows: Antibodies against ANGPT2 (Affinity), SYNPO (Santa Cruz), GAPDH (Proteintech), total ERK1/2, phospho-ERK1/2, total AKT and phospho-AKT (Cell Signaling Technologies); Reverse transcription kit (DRR037A, Takara); Quantitative PCR kit (Thermo Fisher Scientific); RIPA cell lysis buffer, BCA protein quantification kit (Beyotime, Shanghai); RNA extraction kit (Takara); ANGPT2 (HY-P7510, MCE); Trizol (Invitrogen).

Techniques: Control

Journal: American Journal of Translational Research

Article Title: Distinct effects of ANGPT2 on gene expression of glomerular podocytes and mesangial cells

doi:

Figure Lengend Snippet: Expression of ANGPT2 interacting proteins in podocytes and mesangial cells

Article Snippet: The information of the reagents used in the present study is as follows: Antibodies against ANGPT2 (Affinity), SYNPO (Santa Cruz), GAPDH (Proteintech), total ERK1/2, phospho-ERK1/2, total AKT and phospho-AKT (Cell Signaling Technologies); Reverse transcription kit (DRR037A, Takara); Quantitative PCR kit (Thermo Fisher Scientific); RIPA cell lysis buffer, BCA protein quantification kit (Beyotime, Shanghai); RNA extraction kit (Takara); ANGPT2 (HY-P7510, MCE); Trizol (Invitrogen).

Techniques: Expressing

Journal: bioRxiv

Article Title: Meta Learning Improves Robustness and Performance in Machine Learning-Guided Protein Engineering

doi: 10.1101/2023.01.30.526201

Figure Lengend Snippet: Design and screening of yeast display antibody (scFv) libraries. (A) Workflow overview: Degenerate oligonucleotides are computationally designed based on previously published deep mutational scanning data (DMS) ( Mason et al ., 2021 ; Koenig et al ., 2015 ) and crystal structures (PDB: 1N8Z, 4ZFF, 4ZFG), transformed into yeast, and screened by FACS for binding to antigen. The sorted populations are then used for targeted deep sequencing of the antibody variable regions. (B) The crystal structure of 4D5 scFv (grey) in complex with HER2 (blue) (PDB: 1N8Z). The amino acid positions of 4D5 targeted for combinatorial mutagenesis are highlighted in pink. (C) The 4D5 scFv library is screened for binding to HER2 antigen by FACS; dot plots show the library is fractionated into HER2-binding populations (High-, Low-, or Non-binding) based on fluorescence intensity. Multiple rounds of FACS are performed (Fig. S1). (D) Protein sequence logo plots represent the mutagenesis regions of the 4D5 scFV and are derived from deep sequencing of the various HER2-binding populations. (E) The crystal structure of 5A12 scFv (grey) in complex with VEGF antigen (orange) (PDB: 4ZFF). The amino acid positions of 5A12 targeted for combinatorial mutagenesis are highlighted in pink (F) The 5A12 scFv library is screened for binding to VEGF antigen by FACS; dot plots show the library is fractionated into VEGF-binding populations (High- or Low/Non-binding) based on fluorescence intensity. Multiple rounds of FACS are performed (Fig. S2). (G) Protein sequence logo plots represent the mutagenesis regions of the 5A12 scFV and are derived from deep sequencing of the various VEGF-binding populations. (H) The crystal structure of 5A12 scFv (grey) in complex with Ang2 antigen (orange) (PDB: 4ZFG). The amino acid positions of 5A12 targeted for combinatorial mutagenesis are highlighted in pink (I) The 5A12 scFv library is screened for binding to VEGF antigen by FACS; dot plots show the library is fractionated into Ang2-binding populations (High- or Low/Non-binding) based on fluorescence intensity. Multiple rounds of FACS are performed (Fig. S3). (J) Protein sequence logo plots represent the mutagenesis regions of the 5A12 scFV and are derived from deep sequencing of the various Ang2-binding populations.

Article Snippet: The 5A12 library was first incubated with either 0.5 nM VEGF (Acro, VE5-H82Q0-200ug) or 12 nM Ang2 (Sino Biological, 10691-H08S-B) plus Streptavidin-AlexaFluor647 (Bioloegend, 405237) for 30 minutes at 4 C. Following the fist stain, cells were centrifuged at 8000 G for 30 seconds and washed once before incubation with 1ng/μL anti-FLAG-PE for 30 minutes at 4 C. Following staining, cells were washed twice and kept on ice and away from light until sorting. scFv expressing (FLAG+) cells in the 4D5 library were sorted by FACS (BD Aria Fusion) first into HER2 binding and non-binding fractions, then into three populations based on AlexaFluor647-conjugated HER2 Mean Fluorescent Intensity: High, Low, and Negative. scFv expressing (FLAG+) cells in the 5A12 library were sorted by FACS (BD Aria Fusion) into High binding (VEGF+ or Ang2+) or Low/Non-binding (VEGF- or Ang2-) populations.

Techniques: Transformation Assay, Binding Assay, Sequencing, Mutagenesis, Fluorescence, Derivative Assay

Journal: bioRxiv

Article Title: Meta Learning Improves Robustness and Performance in Machine Learning-Guided Protein Engineering

doi: 10.1101/2023.01.30.526201

Figure Lengend Snippet: Meta learning applied to supervised machine learning models trained to predict multi-antigen binding classification using yeast antibody (scFv) sequence data with largely single-antigen binding classification labels. (A) Schematic representation of machine learning task. The training set is constructed from deep sequencing of 5A12 libraries following the final round of the VEGF FACS screen (Fig. S2). Positive (High-binding) and negative (Low/Non-binding) VEGF labels are retained and sequences are arbitrarily assigned an Ang2 binding classification. Test and meta sets consist of 5A12 variants with binding labels for both targets and are batched by combining deep sequencing from both VEGF and Ang2 final round FACS screens (Fig. S2, S3). (B,C, and D) Meta learning and baseline prediction performance (Matthew’s Correlation Coefficient) as a function of the number of training samples. FT refers to Fine-Tune Baseline. Points correspond to mean performance and shaded regions to 95 % confidence intervals across 3 random seeds. Performance curves plotted for meta sets consisting of 32 (bright yellow), 96 (dark yellow), 288 (red), and 864 (purple) sequences.

Article Snippet: The 5A12 library was first incubated with either 0.5 nM VEGF (Acro, VE5-H82Q0-200ug) or 12 nM Ang2 (Sino Biological, 10691-H08S-B) plus Streptavidin-AlexaFluor647 (Bioloegend, 405237) for 30 minutes at 4 C. Following the fist stain, cells were centrifuged at 8000 G for 30 seconds and washed once before incubation with 1ng/μL anti-FLAG-PE for 30 minutes at 4 C. Following staining, cells were washed twice and kept on ice and away from light until sorting. scFv expressing (FLAG+) cells in the 4D5 library were sorted by FACS (BD Aria Fusion) first into HER2 binding and non-binding fractions, then into three populations based on AlexaFluor647-conjugated HER2 Mean Fluorescent Intensity: High, Low, and Negative. scFv expressing (FLAG+) cells in the 5A12 library were sorted by FACS (BD Aria Fusion) into High binding (VEGF+ or Ang2+) or Low/Non-binding (VEGF- or Ang2-) populations.

Techniques: Binding Assay, Sequencing, Construct