Journal: Toxicology and applied pharmacology

Article Title: Monoubiquitinated γ-H2AX: abundant product and specific biomarker for non-apoptotic DNA double-strand breaks

doi: 10.1016/j.taap.2018.07.007

Figure Lengend Snippet: Panels A-C used antibodies #1. (A) Impact of gel polyacrylamide percentage and transfer conditions to PVDF membranes. Treatments of H460 cells: 6 h with 3 mM hydroxyurea (HU), 6 or 16 h with 5 μM aphidicolin (Aphi) or 1 h with 5 μM bleomycin (Bleo). All γ-H2AX images had 5 s film exposures. (B) Ethanol effect in semi-dry transfer. IMR90 cells were treated with 40 μM etoposide (Eto) for 1 h. (C) Comparison of wet (pH 8.2 and pH 8.8) and semi-dry transfers using 12% ethanol-containing buffers (10 s film exposures for all γ-H2AX images). IMR90 cells were treated with 40 μM etoposide for 1 h. (D) Effectiveness of different antibodies using semi-dry transfer with 12% ethanol. Normal human keratinocytes were treated for 1 h with 30 μM etoposide or 0.5 μM bleomycin. Antibodies #1–3: 1:1000 dilutions and 5 s exposures, antibody #4: 1:1000 dilution and 60 s exposure. (E) Comparison of antibodies #1 and #5 (both at 1:1000 dilutions) in H460 cells treated with bleomycin for 1 h. (F) γ-H2AX blot (Ab#1) in control and H2AX-depleted IMR90 cells treated with 100 μM etoposide for 1h.

Article Snippet: IMR90, WI38, normal human neonatal keratinocytes (PCS-200–010), H460, A549, U2OS, mouse embryonic fibroblasts (MEFs, SCRC-1040), telomerase-immortalized human bronchial epithelial HBEC3 (CRL-4051), Daudi, HCT116, Ramos, Raji and CA46 were obtained from ATCC.

Techniques: Comparison, Control

Journal: Frontiers in Immunology

Article Title: Staphylococcus epidermidis prevents UV-induced skin aging by suppressing TLR3-mediated senescence

doi: 10.3389/fimmu.2026.1796085

Figure Lengend Snippet: Staphylococcus epidermidis inhibits UVB-induced senescence in keratinocytes. (A) Neonatal human epidermal keratinocytes (NHEKs) at 90% confluence were irradiated with 10 mJ/cm 2 UVB, followed by treatment with 75 μg/mL ≤10kDa S. epi . β-gal staining was performed 24 hours post-treatment. The proportion of senescent cells was quantified using Image (J, B) After UVB irradiation, different concentrations of ≤10kDa S. epi were added to NHEKs. RNA was isolated 24 hours later, and P16 expression was measured by quantitative RT-PCR with β-actin as the internal control. (C) NHEKs at 90% confluence were irradiated with 10mJ/cm 2 UVB, followed by treatment with 75μg/mL ≤10kDa S. epi . Six hours later, intracellular ROS were labeled with DCFH-DA and visualized using fluorescence microscope (excitation: 488 nm; emission: 525 nm), and fluorescence intensity was quantified using Image (J, D) NHEKs were treated with different concentrations of ≤10kDa S. epi . Six hours later, proteins were extracted for γH2Ax analysis, and densitometric analysis of protein bands were quantified by Image (J) Immunoblotting results are representative of three independent experiments performed with independent cultured keratinocytes. (E) NHEKs were irradiated with 10mJ/cm 2 UVB and treated with 75μg/mL ≤10kDa S. epi . Six hours post-treatment, γH2Ax expression was visualized by immunofluorescence, and fluorescence intensity was quantified using Image (J, F) After UVB exposure (10mJ/cm 2 ), NHEKs were treated with different concentrations of ≤10kDa S. epi . RNA was isolated 24 hours later, and the expressions of IL-6 , TNFα and IL-1β was analyzed by quantitative RT-PCR using β-actin as internal control. (G) Following UVB irradiation (10mJ/cm 2 ), NHEKs were treated with different concentrations of ≤10kDa S. epi . 24 hours later, cell culture supernatants were collected, and IL-6 and IL-1β secretion was measured by ELISA. Data represent mean ± SEM from independent biological replicates ( n = 3). All the experiments have been repeated three times. Statistical significances were evaluated by One-way ANOVA. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, n.s., no significance.

Article Snippet: Neonatal human epidermal keratinocytes (NHEKs) and neonatal human fibroblasts were purchased from Lifeline Cell Technology.

Techniques: Irradiation, Staining, Isolation, Expressing, Quantitative RT-PCR, Control, Labeling, Fluorescence, Microscopy, Western Blot, Cell Culture, Immunofluorescence, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Immunology

Article Title: Staphylococcus epidermidis prevents UV-induced skin aging by suppressing TLR3-mediated senescence

doi: 10.3389/fimmu.2026.1796085

Figure Lengend Snippet: Staphylococcus epidermidis inhibits fibroblast senescence by inhibiting SASP secretion from UVB-irradiated keratinocytes. (A) Schematic representation of the experimental design. NHEKs were cultured to 90% confluence and then exposed to 10 mJ/cm 2 UVB for induction. Subsequently, 75μg/mL of ≤10kDa S. epi was added to coculture for 48 hours. The conditioned medium containing SASP factors was collected by centrifugation at 2, 000 rpm for 20 minutes, mixed with fresh DMEM at a 1:2 ratio, and the final serum concentration was adjusted to 10%. The mixed medium was used to culture primary human fibroblasts for 48 hours. (B) Fibroblasts were stained with senescence-associated β-gal, and the percentage of senescent cells was quantified by Image (J, C) Protein levels of P16 and P21 in fibroblasts were analyzed by western blotting. Densitometric analysis of protein bands were quantified by Image (J, D) RT-PCR analysis of RNA isolated from fibroblasts was performed to assess the expression of P16, P21 , P53 , TNFα , IL-6 , IL-1β , and MMP1 , with β-actin as the internal control. Con-SASP, SASP collected from control NHEKs; S.epi -SASP, SASP collected from NHEKs treated with 75μg/mL of ≤10kDa S.epi ; UVB-SASP, SASP collected from NHEKs treated with UVB; UVB+ S.epi -SASP, SASP collected from NHEKs treated with UVB and 75μg/mL of ≤10kDa S.epi . Data represent mean ± SEM with n = 3. All the experiments have been repeated three times. Statistical significances were analyzed by One-way ANOVA. ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: Neonatal human epidermal keratinocytes (NHEKs) and neonatal human fibroblasts were purchased from Lifeline Cell Technology.

Techniques: Irradiation, Cell Culture, Centrifugation, Concentration Assay, Staining, Western Blot, Reverse Transcription Polymerase Chain Reaction, Isolation, Expressing, Control

Journal: Frontiers in Immunology

Article Title: Staphylococcus epidermidis prevents UV-induced skin aging by suppressing TLR3-mediated senescence

doi: 10.3389/fimmu.2026.1796085

Figure Lengend Snippet: LP78 inhibits UVB-induced keratinocyte senescence. (A) NHEK at 90% confluency were irradiated with 10mJ/cm 2 UVB, followed by treatment with different concentrations of LP78. RNA was extracted 24 hours later, and P16 expression was detected by RT-PCR, with β-actin as the internal control. (B) NHEKs at 90% confluency were irradiated with 10mJ/cm 2 UVB and treated with different concentrations of LP78. Cell lysates were collected after 24 hours, and P16 and γH2Ax levels were analyzed by western blotting. Densitometric analysis of protein bands were quantified by Image (J, C) NHEKs at 90% confluency were irradiated with 10mJ/cm 2 UVB and treated with 10μg/mL LP78. Six hours post-treatment, intracellular ROS were visualized using fluorescence microscope and the intensity was quantified using Image (J, D) NHEK at 90% confluency were irradiated with 10mJ/cm 2 UVB and treated with different concentrations of LP78. RNA was extracted 24 hours later, and the expression of IL-6 , TNFα and IL-1β was measured by RT-PCR, with β-actin as the internal control. (E) NHEKs at 90% confluency were irradiated with 10mJ/cm 2 UVB and treated with different concentrations of LP78. After 24 hours, culture supernatants were collected, and the secretion of IL-6 and IL-1β was quantified by ELISA. Data represent mean ± SEM with n = 3. All the experiments have been repeated three times. Statistical significances were analyzed by One-way ANOVA. *** p < 0.001, **** p < 0.0001, n.s., no significance.

Article Snippet: Neonatal human epidermal keratinocytes (NHEKs) and neonatal human fibroblasts were purchased from Lifeline Cell Technology.

Techniques: Irradiation, Expressing, Reverse Transcription Polymerase Chain Reaction, Control, Western Blot, Fluorescence, Microscopy, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Immunology

Article Title: Staphylococcus epidermidis prevents UV-induced skin aging by suppressing TLR3-mediated senescence

doi: 10.3389/fimmu.2026.1796085

Figure Lengend Snippet: UV-induced skin photoaging is dependent on TLR3 activation, and Staphylococcus epidermidis and LP78 inhibit TLR3-mediated SASP factors. (A) A long-term photoaging model was established using WT and Tlr3 −/− C57BL/6 mice ( n = 5-7). The dorsal skin of each mouse was divided into upper (shield with tin foil) and lower (exposed) regions. The lower region was irradiated with UV as shown in Figure 1A . Representative photographs of dorsal skin were taken at the end of the experiment for HE staining. (B) Epidermal thickness of dorsal skin from mice treated as (A) was quantified using Image J software. (C) Masson’s trichrome staining of dorsal skin from mice treated as (A) was performed to evaluate dermal collagen content. (D) Western blot analysis of P16, P21 and γH2Ax protein expression in dorsal skin tissues from mice treated as (A) . Densitometric analysis of protein bands were quantified by Image (J, E) ELISA measurement of Il-6 and Il-1β in dorsal skin tissues from mice treated as (A) . (F) RT-PCR analysis of Il-6 , Tnfα , Il-1β and Mmp1 , with β-actin as an internal control. (G) NHEKs with or without TLR3 silencing were irradiated with 10mJ/cm 2 UVB at 90% confluence. Following UVB exposure, cells were treated with 75μg/mL of ≤10kDa S. epi or 10μg/mL of LP78. RNA was extracted 12h hours post-treatment, and IL-6 , TNFα and IL-1β mRNA expression was assessed by RT-PCR, with β-Actin as a reference gene. (I) Supernatants were collected from the same cultures in (G) , and the secretion of IL-6 and IL-1β was measured by ELISA. Data represent mean ± SEM with n = 3-7. All the experiments have been repeated twice or three times. Statistical significances were analyzed by Two-way ANOVA. ** p < 0.01, **** p < 0.0001.

Article Snippet: Neonatal human epidermal keratinocytes (NHEKs) and neonatal human fibroblasts were purchased from Lifeline Cell Technology.

Techniques: Activation Assay, Irradiation, Staining, Software, Western Blot, Expressing, Enzyme-linked Immunosorbent Assay, Reverse Transcription Polymerase Chain Reaction, Control

Journal: Frontiers in Immunology

Article Title: Staphylococcus epidermidis prevents UV-induced skin aging by suppressing TLR3-mediated senescence

doi: 10.3389/fimmu.2026.1796085

Figure Lengend Snippet: Staphylococcus epidermidis and LP78 inhibit SASP factor expression through TRAF1 induction. (A) NHEKs were treated with 75μg/mL of ≤10kDa S. epi or 10μg/mL of LP78. RNA was extracted at different time points, and TRAF1 expression was assessed by RT-PCR, with β-actin as the internal control. (B) Western blot analysis of TRAF1 in NHEKs treated as in (A) . Densitometric analysis of protein bands were quantified by Image (J, C) NHEKs were transfected with siTRAF1 using jetPRIME at 60% confluency. After 24 hours, the cells were exposed to 10mJ/cm 2 UVB and treated with 75μg/mL of ≤10kDa S. epi or 10μg/mL of LP78. RNA was extracted after 12 hours post-treatment, and the expression of IL-6 , TNFα and IL-1β was analyzed by RT-PCR, with β-actin as the internal reference. (C) The supernatant from the same culture in (B) was analyzed by ELISA to measure IL-6 and IL-1β secretion. Data represent mean ± SEM with n = 3. All the experiments have been repeated three times. Statistical significances were analyzed by Two-way ANOVA. * p < 0.05, **** p < 0.0001, n.s., no significance.

Article Snippet: Neonatal human epidermal keratinocytes (NHEKs) and neonatal human fibroblasts were purchased from Lifeline Cell Technology.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Control, Western Blot, Transfection, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Immunology

Article Title: Staphylococcus epidermidis prevents UV-induced skin aging by suppressing TLR3-mediated senescence

doi: 10.3389/fimmu.2026.1796085

Figure Lengend Snippet: Staphylococcus epidermidis and LP78 induce TRAF1 dependent on TLR2. (A) NHEKs were transfected with siTLR2 using jetPRIME at 60% confluency. After 24 hours, the cells were treated with 75μg/mL of ≤10kDa S. epi or 10μg/mL of LP78. Cell lysates was extracted after 12 hours post-treatment, and the expression of TRAF1 was analyzed Western blotting. Densitometric analysis of protein bands were quantified by Image (J, B) NHEKs were transfected with siTLR2 using jetPRIME at 60% confluency. After 24 hours, the cells were exposed to 10mJ/cm 2 UVB and treated with 75μg/mL of ≤10kDa S. epi or 10μg/mL of LP78. RNA was extracted after 12 hours post-treatment, and the expression of IL-6 , TNFα and IL-1β was analyzed by RT-PCR, with β-actin as the internal reference. (C) The supernatant from the same culture in (B) was analyzed by ELISA to measure IL-6 and IL-1β secretion. Data represent mean ± SEM with n = 3. All the experiments have been repeated three times. Statistical significances were analyzed by Two-way ANOVA. **** p < 0.0001, n.s., no significance.

Article Snippet: Neonatal human epidermal keratinocytes (NHEKs) and neonatal human fibroblasts were purchased from Lifeline Cell Technology.

Techniques: Transfection, Expressing, Western Blot, Reverse Transcription Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay