Journal: bioRxiv

Article Title: Metformin inhibits PDGF signaling to suppress hyaluronan and IL-6 production in Thyroid Eye Disease

doi: 10.1101/2025.05.19.654298

Figure Lengend Snippet: A) non-TED (n=3) and TED (n=3) OFs were treated with or without PDGFβ for 72 hours. Secreted HA levels in media supernatant collected after treatment were analyzed on agarose gel electrophoresis and stained using Stains-All. 15 MDa and HiLadder were used as a size standard. B) Quantitative HA levels in both non-TED and TED after treatment were measured as described in the Methods section. C) and D) The relative production of IL6 and IL8 by OFs after PDGFβ treatment for 72 hours was measured with ELISA assay. The experiments were conducted in triplicate. The data represent mean ± SEM and were analyzed statistically using One-way ANOVA with Tukey’s multiple comparisons. Vehicle versus PDGFβ: * P ≤ 0.05, ** P = 0.001, *** P < 0.0008, **** P < 0.0001, and non-TED versus TED: #### P ≤ 0.0001. HA, hyaluronan.

Article Snippet: The Hyaluronan DuoSet ELISA kit was used for HA quantification (R&D Systems, Minneapolis, MN).

Techniques: Agarose Gel Electrophoresis, Staining, Enzyme-linked Immunosorbent Assay

Journal: bioRxiv

Article Title: Metformin inhibits PDGF signaling to suppress hyaluronan and IL-6 production in Thyroid Eye Disease

doi: 10.1101/2025.05.19.654298

Figure Lengend Snippet: A) TED OFs were treated with different doses of metformin as indicated for 72 hours; thereafter, cells were analyzed by Western blot for pAMPKɑ Thr172 , total AMPKɑ, and β-tubulin (loading control). B) Relative levels of pAMPKɑ Thr172 normalized to total AMPKɑ in three strains with representative results. Band intensities were quantified using Image Lab software. C) and D) The relative production of IL6 and IL8 by OFs after metformin treatment for 72 hours was measured by ELISA. The experiments were conducted in triplicate on three different strains. The data represent mean ± SEM. One-way ANOVA with Dunnett’s multiple comparisons was used to compare vehicle versus metformin treatment. *p≤ 0.05, **p≤ 0.001.

Article Snippet: The Hyaluronan DuoSet ELISA kit was used for HA quantification (R&D Systems, Minneapolis, MN).

Techniques: Western Blot, Control, Software, Enzyme-linked Immunosorbent Assay

Journal: bioRxiv

Article Title: Metformin inhibits PDGF signaling to suppress hyaluronan and IL-6 production in Thyroid Eye Disease

doi: 10.1101/2025.05.19.654298

Figure Lengend Snippet: A) TED OFs were pretreated with AMPK activators metformin (1000 μM) or AICAR (400 μM) for 1 hour and then added PDGFβ (25 ng/mL) for the next 72 hours; thereafter, cells were analyzed by western blots for pAMPKɑ Thr172 , total AMPKɑ and β-tubulin as a loading control. B) Relative levels of pAMPKɑ Thr172 normalized to total AMPKɑ in three strains with representative results. Band intensities were quantified using Image Lab software. C) Quantitative HA levels in TED OFs after treatment with PDGFβ and AMPK activators, metformin/AICAR, were measured with the HA ELISA assay. D) and E) The relative production of IL6 and IL8 by OFs after treatment with PDGFβ and AMPK activators, metformin/AICAR, was measured by ELISA. The experiments were conducted in triplicate on three different strains. The data represent mean ± SEM. Student’s T-test and One-way ANOVA with Tukey’s multiple comparisons were used to compare PDGFβ and PDGFβ + metformin/AICAR. *P ≤ 0.05, **P ≤ 0.001, ***P < 0.0009, ****P < 0.0001, and vehicle versus metformin/AICAR treatment. # P ≤ 0.05, ### P ≤ 0.0005.

Article Snippet: The Hyaluronan DuoSet ELISA kit was used for HA quantification (R&D Systems, Minneapolis, MN).

Techniques: Western Blot, Control, Software, Enzyme-linked Immunosorbent Assay

Journal: Arthritis and rheumatism

Article Title: Diminished cartilage-lubricating ability of human osteoarthritic synovial fluid deficient in proteoglycan 4: Restoration through proteoglycan 4 supplementation.

doi: 10.1002/art.34674

Figure Lengend Snippet: Figure 1. Characterization of the proteoglycan 4 (PRG4) enzyme- linked immunosorbent assay control by protein staining (A) and characterization of high molecular weight PRG4 immunoreactivity in PRG4 control, normal (NL) human synovial fluid (hSF), and osteo- arthritic (OA) SF samples by Western blotting using antipeptide antibody LPN (capture) (B) and horseradish peroxidase (HRP)– conjugated peanut agglutinin (PNA) (detection) (C). Samples were subjected to 3–8% sodium dodecyl sulfate–polyacrylamide gel electro- phoresis, followed by protein staining or Western blotting as described in Materials and Methods. PRG4 controls treated with neuraminidase and SF treated with hyaluronidase and neuraminidase were probed with LPN and with HRP–conjugated PNA.

Article Snippet: A hyaluronan DuoSet ELISA development kit was obtained from R&D Systems, proteinase K was from Roche Applied Science, and MegaLadder and HiLadder HA molecular weight markers were from Hyalose.

Techniques: Enzyme-linked Immunosorbent Assay, Control, Staining, High Molecular Weight, Western Blot

Journal: Arthritis and rheumatism

Article Title: Diminished cartilage-lubricating ability of human osteoarthritic synovial fluid deficient in proteoglycan 4: Restoration through proteoglycan 4 supplementation.

doi: 10.1002/art.34674

Figure Lengend Snippet: Figure 3. Characterization of hyaluronan (HA) in normal and PRG4- deficient OA SF samples. A, Concentrations of HA in normal and PRG4-deficient OA SF samples. B, Molecular weight distribution of HA in normal SF samples (n 8) and in PRG4-deficient OA (OA-LO) SF samples (n 5). Values are the mean SEM. P 0.05. See Figure 1 for other definitions.

Article Snippet: A hyaluronan DuoSet ELISA development kit was obtained from R&D Systems, proteinase K was from Roche Applied Science, and MegaLadder and HiLadder HA molecular weight markers were from Hyalose.

Techniques: Molecular Weight

Journal: Arthritis and rheumatism

Article Title: Diminished cartilage-lubricating ability of human osteoarthritic synovial fluid deficient in proteoglycan 4: Restoration through proteoglycan 4 supplementation.

doi: 10.1002/art.34674

Figure Lengend Snippet: Figure 4. Effect of hyaluronan (HA) and proteoglycan 4 (PRG4) supplementation on the cartilage boundary–lubricating ability of PRG4-deficient osteoarthritic (OA) synovial fluid (SF) samples, as determined by cartilage-on-cartilage friction testing. Two friction coefficients (), static (static, Neq) (A) and kinetic (kinetic, Neq ; at a presliding duration of 1.2 seconds) (B), in phosphate buffered saline (PBS; negative control lubricant), PRG4-deficient OA (OA-LO) SF alone, PRG4-deficient OA SF plus PRG4, PRG4-deficient OA SF plus PRG4 and HA, and normal SF (NL; positive control lubricant) were calculated. Values are the mean SEM. P 0.05. Neq represents equilibrium axial load; angle brackets indicate that the value is an average.

Article Snippet: A hyaluronan DuoSet ELISA development kit was obtained from R&D Systems, proteinase K was from Roche Applied Science, and MegaLadder and HiLadder HA molecular weight markers were from Hyalose.

Techniques: Saline, Negative Control, Positive Control