Journal: Oncotarget

Article Title: P73 tumor suppressor and its targets, p21 and PUMA, are required for madin-darby canine kidney cell morphogenesis by maintaining an appropriate level of epithelial to mesenchymal transition

doi:

Figure Lengend Snippet: A. Knockdown of TAp73 decreases, whereas knockdown of ΔNp73 increases, the expression of p21 and PUMA. The levels of p21, PUMA, and actin were measured in parental MDCK, MDCK-TAp73-KD, and MDCK-ΔNp73-KD cells by Western blotting. B. Generation of MDCK cell lines in which p21 was stably knocked down. Parental MDCK and MDCK-p21-KD cells were mock-treated or treated with camptothecin for 18h and the level of p21 and actin protein was measured by Western blotting. C. Representative microscopic images of parental MDCK and MDCK-p21-KD cells in 2-D cultures. D. Top panel: colony formation assay was performed with parental MDCK or MDCK-p21-KD cells. Bottom panel: the number of colonies was counted and presented as Mean ± S.D. from three separate experiments. E. Wound healing assay was performed with parental MDCK and MDCK-p21-KD cells. Top panel: cell migration was determined by visual assessment of cells migrating into the wound for 24 h using a phase-contrast microscopy. The dashed lines indicate the wound edge. Bottom panel: the time required for would closure was measured and presented as Mean ± s.d. from three separate experiments. F. Representative images of parental MDCK and MDCK-p21-KD cells in 3-D culture for 6 d or 12 d. Scale bar: 50 μM.

Article Snippet: Antibodies used were purchased from Bethyl (anti-p73), ProSci (anti-PUMA), Santa Cruz Biotechnology (anti-p21 (H164), anti-β-catenin (E-5), anti-Snail-1, anti-Twist), BD Transduction Laboratories (anti-E-cadherin, San Jose, CA), Sigma (anti-actin, St. Louis, MO), and BioRad (secondary antibodies against rabbit or mouse IgG conjugated with HRP, Life Science Research, Hercules, CA).

Techniques: Expressing, Western Blot, Stable Transfection, Colony Assay, Wound Healing Assay, Migration, Microscopy

Journal: Oncotarget

Article Title: P73 tumor suppressor and its targets, p21 and PUMA, are required for madin-darby canine kidney cell morphogenesis by maintaining an appropriate level of epithelial to mesenchymal transition

doi:

Figure Lengend Snippet: A. - D. The levels of β-catenin, E-cadherin, Snail, Twist, and actin were determined by Western blotting with extracts from parental MDCK (A-D), MDCK-TAp73-KD A. , MDCK-p21-KD B. , MDCK-PUMA-KD C. , and MDCK-ΔNp73-KD D. cells. E. The levels of E-cadherin, Snail and Twist transcripts were measured by qRT-PCR in parental MDCK cells and MDCK cells with knockdown of TAp73, ΔNp73, p21 or PUMA. The level of Actin was measured as an internal control. F. A model for the role of p73, p21 and PUMA in MDCK cell morphogenesis.

Article Snippet: Antibodies used were purchased from Bethyl (anti-p73), ProSci (anti-PUMA), Santa Cruz Biotechnology (anti-p21 (H164), anti-β-catenin (E-5), anti-Snail-1, anti-Twist), BD Transduction Laboratories (anti-E-cadherin, San Jose, CA), Sigma (anti-actin, St. Louis, MO), and BioRad (secondary antibodies against rabbit or mouse IgG conjugated with HRP, Life Science Research, Hercules, CA).

Techniques: Western Blot, Quantitative RT-PCR

Journal: Cell reports

Article Title: Complement C4A Regulates Autoreactive B Cells in Murine Lupus

doi: 10.1016/j.celrep.2020.108330

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Goat IgG fraction to mouse complement C3 , MP Biomedicals , Cat# ICN55463, RRID:AB_2334481.

Techniques: Purification, Recombinant, Software

Journal: The Journal of Immunology Author Choice

Article Title: The Ox40/Ox40 Ligand Pathway Promotes Pathogenic Th Cell Responses, Plasmablast Accumulation, and Lupus Nephritis in NZB/W F1 Mice

doi: 10.4049/jimmunol.1700608

Figure Lengend Snippet: Anti-Ox40 agonist mAb treatment exacerbates renal disease and IgM deposition in NZB/W F1 mice. Renal disease monitoring following anti-Ox40 agonist mAb (red) and a control Ig (blue) treatment (10 mg/kg, three times per week for 3 wk) in NZB/W F1 mice. Black arrows (Rx) refer to treatment days. The age and proteinuria status of each cohort (at the time of first treatment) is indicated at the top of each plot. (A) Thirteen-week-old proteinuria-free NZB/W F1 mice at treatment start. (B) Three independent cohorts (21-, 26-, and 27-wk-old) of NZB/W F1 mice with proteinuria 30–100 mg/dl at treatment start. (C) Representative H&E- and PAS-stained kidney sections (left and middle panels) highlighting glomeruli (top panels), periarterial regions (middle panels), and tubulointerstitium (bottom panels). Scale bars, 100 μm. Corresponding disease severity scores (right panels). Group means are plotted in black (n = 36 per group combined). (D) Representative images from frozen kidney sections (left panels) stained for glomerular deposits of IgM (top panel), IgG (middle panel), and C3 (bottom panel). Scale bars, 50 μm. Corresponding signal intensity in the cortex regions is presented as pixel intensity/μM2 of cortex with group mean ± SD from two independent cohorts (n = 25–28 per group, combined) (right panels). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Article Snippet: Glomerular IC deposits were visualized on 5-μm acetone-fixed OCT-embedded kidney sections by direct immunofluorescence staining using Alexa Fluor 488–conjugated donkey anti-mouse IgG (cat. no. A-21202; Invitrogen), goat anti-mouse IgM (cat. no. A-21042; Invitrogen), or fluorescein-conjugated goat anti-complement C3 (cat. no. 55510; MP Biomedicals).

Techniques: Staining

Journal: Frontiers in Endocrinology

Article Title: Integrated multi-omics analysis reveals complement component 3 as a central driver of immune dysregulation in polycystic ovary syndrome

doi: 10.3389/fendo.2025.1523488

Figure Lengend Snippet: Functional effects of elevated C3 levels in KGN and H295R cells. (A) Western blot analysis of C3 and IL1B protein expression levels in KGN cells transfected with Myc-C3 or negative control (NC). (B) qPCR analysis of mRNA levels for CYP17A1 , CRP , and IL6 in Myc-C3-transfected KGN cells, indicating significant increases in these markers compared to NC. (C) ELISA assay measuring IL6 secretion levels, showing an increase in IL6 secretion in the Myc-C3 group relative to NC group in KGN cells. (D) Proliferation assay demonstrating increased proliferation rates in Myc-C3-transfected KGN cells compared to NC over 5 days. (E) Colony formation assay illustrating a decrease in colony formation rate in Myc-C3 KGN cells versus NC. (F) Schematic of the C3 treatment setup in H295R cells, comparing NC and C3-treated groups. (G) Proliferation assay in H295R cells treated with C3 recombinant protein. (H) qPCR analysis of mRNA levels for CRP , IL6 , and IL1B in C3-treated H295R cells, with significant upregulation of inflammatory markers in the C3 group. (I) Schematic representation of the upregulation of complement C3 contributing to the PCOS. P values < 0.05 was considered statistically significant (*), ** indicated p < 0.01, *** indicated p < 0.001 and **** indicated p < 0.001.

Article Snippet: After blocking in 5% skim milk, the membranes were treated 4 hours with the following antibodies: mouse monoclonal antibody against GAPDH (1:2,000 dilution; 0411; sc47724); Rabbit recombinant multiclonal to IL-1 beta (1:1,000 dilution; abcam; ab283818); Rabbit monoclonal antibody against Complement C3 (1: 1,000 dilutions; CST; #38977).

Techniques: Functional Assay, Western Blot, Expressing, Transfection, Negative Control, Enzyme-linked Immunosorbent Assay, Proliferation Assay, Colony Assay, Recombinant