Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Positive selection screen identifies natural product β-catenin inactivators

doi: 10.1073/pnas.2625189123

Figure Lengend Snippet: Compound 2 specifically activates novel PKC family members. ( A ) Heatmap showing cell survival data (GFP+ objects) for 293FT TK1 KO β-cat S37C -DCK* and 293FT TK1 KO β-cat S37C/S715A -DCK* cells treated with positive subfractions from the indicated lineages for 24 h followed by BVdU (50 µM) for 96 h. Subfractions were dosed at a nominal concentration of 2.5 µg/mL. *: L90865_5 series was dosed at 5 µg/mL. n = 2 technical replicates. ( B ) Relative GFP+ objects for 293FT TK1 KO β-cat S37C -DCK* and 293FT TK1 KO β-cat S37C/S715A -DCK* cells treated with L90865 subfractions for 24 h followed by BVdU treatment (50 µM) for 96 h. n = 2 technical replicates. ( C ) Immunoblot analysis of 293FT cells treated, where indicated, with 2 (25 µM), 3 (25 µM), or ING (1 µM) for 20 min. n = 3 biological replicates. ( D ) Immunoblot analysis of 293FT cells treated, where indicated, with CHIR99021 (5 µM), ING (1 µM), Bis-I (1 µM), or Go6976 (0.5 µM) for 24 h. n = 3 biological replicates. ( E ) Immunofluorescence of DLD-1 cells treated with indicated compounds 2 (25 µM), Bis-I (1 µM), or Go6976 (0.5 µM) for 24 h. Blue: DAPI stain, Red: β-Cat. (Scale bar, 10 µm.) ( F ) Immunoblot analysis of 293FT cells treated, where indicated, with PMA (1 µM), ING (1 µM), 2 (25 µM), or 3 (25 µM) for 24 h. n = 2 biological replicates. ( G ) Summary of the K d values measured by microscale thermophoresis for each of the indicated compounds in the presence of full-length protein PKCδ or PKCα. ( H ) Three-dimensional representation of the molecular docking of compound 2 to the C1b domain of PKCδ (PDB: 7LF3). Key hydrogen bonds are shown with red dots. ( I – L ) Space-filled models for the simulated binding of compound 2 ( I ) and the reported crystal structures for AJH-836 ( J , PDB: 7LF3), ING ( K , PDB: 7KO6), and phorbol 12,13-dibutyrate ( L , PDB: 7KNJ). Key hydrogen bonds are shown with red dots, and the coloring is based on the Eisenberg consensus hydrophobicity scale (note: a value less than −0.73 is set to −0.73).

Article Snippet: The following compounds were purchased: DMSO (Sigma Aldrich, D2650), thymidine (dThD, MedChemExpress, HY-N1150), ( E )-5-(2-Bromovinyl)-2’-deoxyuridine (BVdU, Fisher Scientific 50-536-044 from Chem-Impex International Inc. MFCD00058585), CHIR99021 (MedChemExpress, HY-10182), Ingenol-3-angelate (ING, Sigma Aldrich, SML1318), Phorbol-12-myristate (PMA, VWR, 80055-400), Bisindolylmaleimide-I (Bis-I, VWR, 80055-904 and MedChemExpress, HY-13867), Go6976 (Selleck, S7119), Euphorbia factor L1 (Selleck, S9410), Euphorbia factor L2 (MedChemExpress, HY-N5001), Euphorbia factor L3 (MedChemExpress, HY-N0562), Euphorbia factor L7A (MedChemExpress, HY-N8121), Euphorbia factor L7B (MedChemExpress, HY-N8118), 17-AAG (MedChemExpress, HY-10211), Actinomycin D (Thermo Fisher Scientific, 11805017), BIX-02565 (MedChemExpress, HY-16104), WNTinib (MedChemExpress, HY-160765), AZ-628 (MedChemExpress, HY-11004), doxycycline (Takara Bio, 631311), AJH-836 (from the Kazanietz-Cooke Laboratory University of Miami), YSE-028 (a gift from Hirokazu Tamamura, Department of Medicinal Chemistry, Laboratory for Biomaterials and Bioengineering, Institute of Integrated Research, Institute of Science Tokyo, Japan), Zotatifin (gift from Neal Rosen, Emeritus Faculty at Memorial Sloan Kettering Cancer Center, New York, New York), DAPI (Cell Signaling, 4083), Blasticidin (Thermo Fisher Scientific, NC9016621), Puromycin (Thermo Fisher Scientific, NC9138068), hygromycin (Life Technologies, 10687010).

Techniques: Concentration Assay, Western Blot, Analysis, Immunofluorescence, Staining, Microscale Thermophoresis, Docking Assay, Binding Assay

Journal: Neurotherapeutics

Article Title: Regulatory T cells attenuate astrogliosis via IL-10/STAT3/PKC/vimentin signaling and promote neurological recovery after spinal cord injury

doi: 10.1016/j.neurot.2025.e00827

Figure Lengend Snippet: IL-10 supplementation and PKC phosphorylation inhibition recapitulate Treg-mediated downregulation of vimentin in vitro . A Representative immunofluorescence images of Foxp3 (green) and IL-10 (red) of magnetically sorted Tregs from mouse spleen. B ELISA quantification of IL-10 levels in supernatants of isolated Tregs. Cohen's d = 4.7545. C Experimental timeline of in vitro IL-10 intervention in astrocytes (rIL-10: recombinant IL-10; αIL-10, IL-10 neutralizing antibody). D-H Western blot quantification of p-STAT3/STAT3 (η 2 = 0.8939), SOCS3 (η 2 = 0.9199), p -PKC/PKC (η 2 = 0.9414), and vimentin (η 2 = 0.9252) protein levels in astrocytes following IL-10 intervention. I Representative immunofluorescence images of vimentin in astrocytes following IL-10 intervention. J Experimental timeline of in vitro PKC phosphorylation modulation in astrocytes (Go6976: PKC inhibitor; PMA: PKC activator). K, L Western blot quantification of vimentin protein levels in astrocytes following PKC modulation. η 2 = 0.8597. M, N Immunofluorescence quantification of vimentin levels in astrocytes following PKC modulation. η 2 = 0.6114. Data are mean ± SEM (n = 6 independent cultures). Statistical analyses were performed using two-tailed Student's t -test (B) or one-way ANOVA (E-H, L, N). P values are indicated on the graphs. n.s., not significant.

Article Snippet: For in vitro PKC modulation, we established optimal concentrations through gradient testing: astrocytes were treated with the PKC inhibitor Go6976 (100 nM, Selleck) for 24 h or the PKC activator Phorbol 12-myristate 13-acetate (PMA; 10 nM, Selleck) for 30 min.

Techniques: Phospho-proteomics, Inhibition, In Vitro, Immunofluorescence, Enzyme-linked Immunosorbent Assay, Isolation, Recombinant, Western Blot, Two Tailed Test