Table 1.
Sequences of gene-specific primer pairs used in RT-qPCR.
Fig 1.
Effects of doxycycline on expression of TNF-α protein by Western blotting analysis and ELISA.
(A) HaCaT-TNF-α (TNF-α) and HaCaT-GFP (GFP) cells were treated with increasing doxycycline (Dox) concentrations (0, 0.5, 1.0, 2.0, and 3.0 μg/mL) for 48 hrs. Cells were lyzed and analyzed using Western blot with TNF-α-specific antibody rabbit monoclonal antibody. Fifteen micrograms of proteins were loaded on each lane. GAPDH was used as loading control. Bar graphs represent relative TNF-α expression normalized with GAPDH. (B) Same as (A) except HaCaT-TNF-α and HaCaT-GFP cells were treated with 1.0 μg/ml Dox and incubated for 24, 48 and 72 hrs. (C) HaCaT-TNF-α were treated with increasing doses of Dox (1.0, 2.0, and 3.0 μg/mL) and incubated for 24, 48 and 72 hrs to induce secretion of TNF-α proteins. The supernatants were prepared and detected by ELISA. Line graphs represent concentration of TNF-α secreted into the supernatant after treated with doxycycline. All data represent mean ± SEM from three independent experiments. (D) HaCaT-TNF-α were treated with and without 1.0 μg/ml Dox and incubated for 48. Cells were harvested and subjected to subcellular fractionation into cytoplasmic (Cyt) and membrane (Mem) fractions. Fifteen micrograms of proteins were loaded on each lane. GAPDH was used as cytoplasmic markers and loading control. Bar graphs represent relative TNF-α expression normalized with GAPDH. ** (p< 0.01)
Fig 2.
Effects of TNF-α expression on HaCaT cell viabilities.
Line graphs represent percent cell viability of HaCaT-GFP (A) HaCaT-TNF-α (B) after treated with doxycycline (1.0, 2.0 and 3.0 μg/mL) and 10 ng/mL TNF-α was used as a positive control. Values are represented as mean ± SEM (n = 3). (Percentage cell viability is shown as a percent of doxycycline-treated cells normalized with control untreated cells.)
Fig 3.
Effects of doxycycline induces pro-inflamatory cytokines (IL-1β and IL-8) expression in HaCaT cells.
HaCaT cells were treated with 1.0 μg/mL doxycycline (Dox) or 10 ng/mL TNF-α for 24 hrs. and then treated with or without 10 ng/mL IFN-γ for a further 24 hrs. Total RNA was extracted and reverse-transcribed. cDNA was amplified by qRT-PCR using IL-1β and IL-8 primers (Table 1). GAPDH was used as loading control. Bar graphs represent relative gene expression (A) IL-1β (B) IL-8 expression where values represent relative gene expression normalized with GAPDH. Mean ± SEM from three independent experiments. *(p<0.05), **(p<0.01) compared with control group.
Fig 4.
Effects of doxycycline induces pro-inflamatory cytokines (IL-1β, IL-8, IL-6, NF-κB1, KRT16, FOSL1 and MMP9) expression in HaCaT-TNF-α cells.
HaCaT-TNF-α cells treated with 1.0 μg/mL Dox or 10 ng/mL TNF-α for 24 hrs were then treated with or without 10 ng/mL IFN-γ for a further 24 hrs. Total RNA was extracted and reverse-transcribed. cDNA was amplified by qRT-PCR using IL-1β, IL-8, IL-6, NF-κB1, KRT16, FOSL1 and MMP9 primers (Table 1). GAPDH was used as loading control. Relative expression of IL-1β (A) IL-8 (B) IL-6 (C) NF-κB1 (D) KRT16 (E) FOSL1 (F) and MMP9 expression (G) are shown. Values represent relative gene expression normalized with GAPDH. Mean ± SEM from three independent experiments. *(p<0.05), **(p<0.01) compared with control group.
Fig 5.
Effects of secreted TNF-α in supernatant stimulated pro-inflammatory cytokines (IL-1β and IL-8) expression and effects of membrane TNF-α on IL-8 expression in HaCaT cells.
HaCaT-TNF-α cells were treated with 1.0 μg/mL doxycycline (Dox) for 48 hrs. Supernatant collected was serially diluted with fresh medium in the ratio (supernatant fresh medium) 1:1.5, 1:2, 1:3 and 1:4. HaCaT cells were incubated with diluted supernatant for 24 hrs and subsequently treated with 10 ng/mL IFN-γ for a further 24 hrs. Expression levels of IL-1β and IL-8 were quantitated by qRT-PCR. Relative expression of IL-1β (A) and IL-8 (B) are shown. (C) HaCaT-TNF-α cells were cultured at different seeding densities and treated with 1 μg/ml Dox for 48 hr. Cells harvested at approximately 50% or 90% confluence. IL-8 gene expression from cells at 50% and 90% confluency were analyzed by RT qPCR. Relative expression of IL-8 are shown. Results are represented as the Mean ± SEM from three independent experiments compared with control untreated *(p<0.05), **(p<0.01).
Fig 6.
Quercetin inhibits pro-inflammatory cytokine (IL-1β and IL-8) expression.
HaCaT-TNF-α cells pre-treated with 1.0 μg/mL Dox or 10 ng/mL TNF-α for 24 hrs. Cells were then treated with 10 μM and 20 μM Quercetin for an additional 24 hrs. Total RNA was extracted and reverse-transcribed. cDNA was amplified by qRT-PCR. Relative expression of IL-1β (A-B) and IL-8 expression (C-D) are shown. Results are represented as Mean ± SEM from three independent experiments compared with control # (p<0.01) and compared with Dox or TNF-α treated *(p<0.05),**(p<0.01).