Fig 1: Schematic diagram for the putative regulatory mechanism with MSC/chondrocyte-derived CM. The activation of PI3K signaling or the administration of TNFa generated the chondroprotective MSC/chondrocyte-derived CM. The CM suppressed tumor progression via the GAPDH-L1CAM axis and downregulated TNFa by inhibiting p38 signaling. It also blocked the differentiation of bone-resorptive osteoclasts by downregulating NFATc1 and cathepsin K. By contrast, the activation of PI3K or the administration of TNFa to CS and chondrocytes stimulated tumorigenic and inflammatory responses.
Fig 2: a-Helical domain (aa 230–281) of PspA is required for host GAPDH selectively(A) Diagram of PspA and the corresponding rPspA fragments (F-) used in pull-down experiments. These truncated fragments were used to test PspA binding to GAPDH (see Figure S3). The region colored red within the aHD of PspA was identified as the subdomain that mediates GAPDH binding.(B) Cell lysates from different sources were used in pull-down experiments using rPspA as bait. Versions of GAPDH that interacted with PspA were detected by immunoblot.(C) F-6 was pulled down with purified human GAPDH and visualized by SDS-PAGE separation and staining with Coomassie blue dye. Results shown are representative with experiments performed three times with identical results.
Fig 3: IKKß activity directly phosphorylates VEEV nsP3. (A) Schematic of Kinase HotSpot Assay performed by Reaction Biology Corp. as described in Section 2. Illustration was generated using Biorender. (B) VEEV TC-83 nsP3 was expressed and purified from a bacterial expression system as described in Section 2. A cell-free, in vitro assay of IKKß enzyme vs. purified VEEV nsP3 measured the amount of 33P-?-ATP transferred onto substrate. GAPDH was included as a negative control substrate and ‘IKKtide’, a small validated peptide, was included as a positive control. Graph is representative of the average 33P counts measured for duplicate reactions of substrate, corrected for purity, and incubated with 200 nM IKKß for 1 h (n = 2). (C) WT or IKKß-/- MEF cells were infected with TC-83 at MOI of 2 for 1 h and media was replaced after removal of virus. At 6 hpi, total protein lysates were obtained and subjected to LC-MS/MS. Mass spectra were fitted against NCBI reference sequence L01443 for VEEV TC-83 nsP3. (D) U-87MG cells were untreated or pre-treated with 1 µM BAY-11-7082 for 2 h, infected with TC-83 at MOI of 1 for 1 h and conditioned media containing 1 µM BAY-11-7082 or standard media was replaced after removal of virus. At 8 hpi, total protein lysates were obtained and subjected to LC-MS/MS. Mass spectra were fitted against NCBI reference sequence NP_740698.1 for putative VEEV nsP3. Phosphorylated amino acid residue sites detected on VEEV nsP3 by LC-MS/MS are listed in panels (B,C). * p < 0.0332 and **** p < 0.0001.
Fig 4: Interaction of GAPDH and L1CAM. The single and double asterisks indicate p < 0.05 and 0.01, respectively. (A) Expression levels of Hsp90ab1 and GAPDH after their silencing or overexpression in C28/I2 chondrocytes. (B) Upregulation of GAPDH in response to recombinant Hsp90ab1proteins, and downregulation of Hsp90ab1 by silencing GAPDH in C28/I2 chondrocytes. (C) Elevation of p-p38 and TNFa by silencing Hsp90ab1 in C28/I2 chondrocytes, while their reduction in response to recombinant Hsp90ab1 protein. Elevation of p-p38 and TNFa by silencing GAPDH in C28/I2 chondrocytes, while their reduction in GAPDH-overexpressing C28/I2 chondrocytes. (D,E) Reduction in MTT-based viability and transwell invasion by silencing GAPDH in SW1353 cells and the opposite effect in GAPDH overexpressed SW1353 cells (n = 6). (F) Downregulation of Snail by silencing GAPDH in SW1353 cells and elevation of Snail in GAPDH overexpressed SW1353 cells. (G) Co-immunoprecipitated L1CAM with GAPDH. (H,I) Suppression of GAPDH-mediated inhibition of MTT-based viability and EdU-based proliferation of SW1353 cells by RNA silencing of L1CAM (n = 6). (J) Downregulation of Runx2 and Snail in response to recombinant GAPDH in SW1353 cells, and its suppression by silencing L1CAM. Full Western blot images are available in Figure S8.
Fig 5: Pneumococcal PspA interacts with human GAPDH(A) Recombinant His-tagged PspA protein composed of the aHD and PRD was used in pulldown experiments with A549 and D562 cell lysates. The PspA bound proteins were separated by SDS-PAGE and visualized by Coomassie blue dye. Interacted proteins were identified by liquid chromatography tandem mass spectrometry (Table S1). Red arrowheads indicate GAPDH.(B) Interaction of PspA with host GAPDH was analyzed by immunoblot after A549 and D562 cell lysates were pulled down using PspA as bait.(C) Interactions of PspA with huGAPDH were analyzed by surface plasmon resonance spectroscopy under different PspA concentrations. (D) Spn WU2, Spn EF3030, or its pspA isogenic mutants, and (E) S. aureus, E. coli, and S.mutans were incubated with FITC-conjugated huGAPDH. The huGAPDH binding to bacterium was measured by flow cytometry. Mock represents Spn bacteria only.Data are representative of (A and B) two or (C–D) three independent experiments.
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