Journal: Frontiers in Medicine

Article Title: Human epidermis models demonstrate mediator role of TLR2 and TLR3 for psoriatic inflammation

doi: 10.3389/fmed.2025.1663279

Figure Lengend Snippet: Treatmentof 3D epidermis models with TLR agonists. Epidermis models were set up from primary immortalized keratinocytes and stimulated with TLR2 agonist Pam 2 CSK 4 (500 ng/mL), TLR3 agonist poly(I:C) (5 μg/mL) or TLR4 agonist LPS (0.8 ng/mL) for 6 days. The epidermis models were analyzed for the expression of S100A7 either immunohistochemically (A) or by western blot (B) . The protein molecular weight on western blot is indicated in kDa.

Article Snippet: For staining of TLR2, 1×10 6 cells were resuspended in 100 μL DermaLife K keratinocyte growth medium with 1% FCS, 2 μL of fluorescence labeled antibody [anti-TLR2-PE (Miltenyi, ref. no. 130–127-922)] or the corresponding isotype control antibody [REA control antibody IgG (Miltenyi, ref. no. 130–104-613)] was added and samples were incubated for 30 min at 37°C, 5% CO 2 in the dark.

Techniques: Expressing, Western Blot, Molecular Weight

Journal: Frontiers in Medicine

Article Title: Human epidermis models demonstrate mediator role of TLR2 and TLR3 for psoriatic inflammation

doi: 10.3389/fmed.2025.1663279

Figure Lengend Snippet: Stimulation of TLR2 KO epidermis models with Pam 2 CSK 4 . 3D in vitro epidermis models were set up from wild type or TLR2 KO keratinocytes and treated with 500 ng/mL Pam 2 CSK 4 for 6 days during airlift culture. (A) FACS analysis of TLR2 expression (plasma membrane) in wild type and TLR2 KO keratinocytes. (B) Immunohistochemical staining of S100A7 in TLR2 KO and wild type (wt) epidermis models with and without Pam 2 CSK 4 treatment. Representative images are shown for the visualization of the staining pattern. (C) Western blot analysis of S100A7 in wild type and TLR2 KO epidermis models ( N = 2, n = 3). Full length blots can be found in the supplement . Protein levels were normalized to actin and are shown as fold changes relative to the untreated wild type models in a box plot. The boxes show the interquartile range (IQR) with the median as horizontal line and the mean as unfilled square. Whiskers extend to the most extreme values within 1.5 × IQR from the quartiles, points beyond are plotted as outliers. * p < 0.05 (Mann–Whitney test).

Article Snippet: For staining of TLR2, 1×10 6 cells were resuspended in 100 μL DermaLife K keratinocyte growth medium with 1% FCS, 2 μL of fluorescence labeled antibody [anti-TLR2-PE (Miltenyi, ref. no. 130–127-922)] or the corresponding isotype control antibody [REA control antibody IgG (Miltenyi, ref. no. 130–104-613)] was added and samples were incubated for 30 min at 37°C, 5% CO 2 in the dark.

Techniques: In Vitro, Expressing, Clinical Proteomics, Membrane, Immunohistochemical staining, Staining, Western Blot, MANN-WHITNEY

Journal: Frontiers in Medicine

Article Title: Human epidermis models demonstrate mediator role of TLR2 and TLR3 for psoriatic inflammation

doi: 10.3389/fmed.2025.1663279

Figure Lengend Snippet: Multiplexanalysis of secreted immune mediators after TLR stimulation. 3D in vitro epidermis models were set up from wild type or TLR2 or TLR3 KO keratinocytes and treated with 5 μg/mL poly(I:C) or 500 ng/mL Pam 2 CSK 4 for 6 days during airlift culture ( N = 2, n = 4). Data is shown as box plots. The boxes indicate the interquartile range (IQR) with the median as horizontal line and the mean as black square. Whiskers extend to the most extreme values within 1.5 × IQR from the quartiles, points beyond are plotted as outliers. p -value (* p < 0.05, ** p < 0.01) was determined with Mann–Whitney test. (A) Comparison of wild type (gray boxes) and TLR2 KO epidermis models (white boxes) with and without Pam 2 CSK 4 treatment (indicated as - / P 2 CSK 4 ). (B) Comparison of wild type (gray boxes) and TLR3 KO epidermis models (white boxes) with and without poly(I:C) treatment (indicated as - / p(I:C)).

Article Snippet: For staining of TLR2, 1×10 6 cells were resuspended in 100 μL DermaLife K keratinocyte growth medium with 1% FCS, 2 μL of fluorescence labeled antibody [anti-TLR2-PE (Miltenyi, ref. no. 130–127-922)] or the corresponding isotype control antibody [REA control antibody IgG (Miltenyi, ref. no. 130–104-613)] was added and samples were incubated for 30 min at 37°C, 5% CO 2 in the dark.

Techniques: In Vitro, MANN-WHITNEY, Comparison