Journal: bioRxiv

Article Title: HTRA3 protease-chaperone stabilizes cathepsin B for mitochondrial POLG1 depletion in human cell ageing

doi: 10.1101/2025.07.21.647696

Figure Lengend Snippet: (A) WB of CSB, HTRA3 (left panel), and CTSB, and p21 (right panel) in BJ fibroblasts untreated and 10 days post-irradiation using ß-tubulin and GAPDH, respectively, as loading control. (B) Scheme showing the two hypotheses for the mechanism leading to senescence-related HTRA3 and CTSB overexpression. (C) Immunoblots of p21, CSB, HTRA3, CTSB, and POLG1 in BJ-5ta hTERT WT fibroblasts and 2 CRISPR-edited CSB knocked-out clones (BJ-5ta csb-/- hTERT #1 and BJ-5ta csb-/- hTERT #2). Frames show samples on the same blot; each displaying the respective GAPDH used as loading control. (D) Scheme indicating normally blocked senescence in BJ-5ta hTERT cells and activation of senescence upon irradiation at 10 Gy. WB analysis of (E) CSB, CTSB, p21, and (F) HTRA3 three days post-irradiation in BJ-5ta hTERT WT fibroblasts and BJ-5ta hTERT CSB knocked-out fibroblasts (β-tubulin was used as loading control). RT-qPCR of ( G ) p21 Waf1 and ( H ) HTRA3 and ( I ) CTSB in the same experiment and cells schematized in panel D. Samples on the same blot are framed; each frame displays the respective GAPDH or β-tubulin used as loading control. RT-qPCR: n=3 independent experiments; mean ± SD. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001; based on unpaired t test comparisons to respective non-irradiated control.

Article Snippet: Normal female human foetal lung IMR-90 fibroblasts (ATCC; CCL-186) and BJ skin fibroblasts (ATCC; CRL-2522) were cultured in minimum essential medium (MEM, Gibco) supplemented with 2 mM L-glutamin (GlutMAX), 10% FBS, 1% Penicillin–streptomycin, 1% nonessential amino acids (Gibco) and 1% sodium pyruvate (Gibco) BJ-5ta hTERT immortalized human fibroblasts (ATCC; CCL-186) cultured in a 4:1 mixture of DMEM and Medium 199 (Gibco) supplemented with 10% FBS and1% Penicillin–streptomycin.

Techniques: Irradiation, Control, Over Expression, Western Blot, CRISPR, Clone Assay, Activation Assay, Quantitative RT-PCR

Journal: The FASEB Journal

Article Title: Staphylococcus aureus impairs dermal fibroblast functions with deleterious effects on wound healing

doi: 10.1096/fj.201902836r

Figure Lengend Snippet: FIGURE 1 S aureus (SA) affects key functions of dermal fibroblasts. A, The concentration of IL-6 and IL-8 in the culture supernatants was quantified at 72 h by ELISA. B, The gene expression of IL-6, IL-8, and IL-1B was assessed at 72 h by qPCR analysis. C, MMP1 and MMP3 mRNA levels were evaluated at 24 h by qPCR analysis. D, Migration assay was performed at 16 h (BJ-5ta cells) or 28 h (primary cells). E, Pro- collagen 1α concentration in the culture supernatants was quantified at 72 h by ELISA. F, Cell contractility was evaluated at 48 h by collagen gel contraction assay. G, Cell proliferation was evaluated at 72 h using CCK-8. H, TLR2 mRNA levels were measured by qPCR at 24 h. Bars show medians, n = 3 per group. *P < .05, **P < .01 (Kruskal-Wallis test followed by Dunn's multiple comparisons test)

Article Snippet: Human neonatal fibroblasts immortalized with hTERT (BJ- 5ta, ATCC CRL- 4001) were cultured in DMEM and Medium 199 (mix ratio 4:1; Sigma- Aldrich, St Louis, MO) containing 10% FCS and 0.01 mg/mL of hygromycin (Sigma- Aldrich) at 37°C in 5% carbon dioxide.

Techniques: Concentration Assay, Enzyme-linked Immunosorbent Assay, Gene Expression, Migration, Collagen Gel Contraction Assay, CCK-8 Assay

Journal: The FASEB Journal

Article Title: Staphylococcus aureus impairs dermal fibroblast functions with deleterious effects on wound healing

doi: 10.1096/fj.201902836r

Figure Lengend Snippet: FIGURE 3 SA invades dermal fibroblasts by endocytosis, but the invasion does not promote cell death. A, Representative pictures of intra-endosomal SA (arrow heads) in BJ-5ta cells are shown. SA was labeled with CFSE. Scale bar = 5 µm. B, Effect of cytochalasin D on SA internalization, effect of lysostaphin treatment on removal of SA, and internalization of HKSA were assessed respectively in BJ-5ta cells. Intracellular (arrow heads) and extracellular (arrows) are shown. SA and HKSA were labeled with CFSE. Scale bar = 10 µm. C and D, BJ-5ta cells were treated with an endocytosis inhibitor, cytochalasin D (10 µg/mL) for 1 h before SA exposure. Apoptosis (C) and necrosis (D) were evaluated at 7 h. Bars show means, n = 4 per group. **P < .01, ****P < .0001 (Two-way ANOVA followed by Sidak's multiple comparisons test)

Article Snippet: Human neonatal fibroblasts immortalized with hTERT (BJ- 5ta, ATCC CRL- 4001) were cultured in DMEM and Medium 199 (mix ratio 4:1; Sigma- Aldrich, St Louis, MO) containing 10% FCS and 0.01 mg/mL of hygromycin (Sigma- Aldrich) at 37°C in 5% carbon dioxide.

Techniques: Labeling

Journal: The FASEB Journal

Article Title: Staphylococcus aureus impairs dermal fibroblast functions with deleterious effects on wound healing

doi: 10.1096/fj.201902836r

Figure Lengend Snippet: FIGURE 5 STING and MyD88 are crucial for the induction of IFN-β and pro-inflammatory mediators upon SA exposure. A, To present the efficacy of gene knockdown by siRNA, the gene expression data of TMEM173 and MYD88 in the samples are shown. B, BJ-5ta cells were treated with the indicated siRNA or negative control siRNA and then were co-cultured with SA. The gene expression of IFN-β, IL-6, IL-8, and IL-1β was evaluated at 24 h by qPCR analysis. C, BJ-5ta cells were treated with an endocytosis inhibitor, cytochalasin D (10 µg/mL) for 1 h before SA exposure and then were co-cultured with SA. The gene expression of IFN-β, IL-6, and IL-8, and IL-1β was evaluated at 24 h by qPCR analysis. Bars show means, n = 3 per group. *P < .05, **P < .01, ***P < .001, ****P < .0001 (Two-way ANOVA followed by Sidak's multiple comparisons test)

Article Snippet: Human neonatal fibroblasts immortalized with hTERT (BJ- 5ta, ATCC CRL- 4001) were cultured in DMEM and Medium 199 (mix ratio 4:1; Sigma- Aldrich, St Louis, MO) containing 10% FCS and 0.01 mg/mL of hygromycin (Sigma- Aldrich) at 37°C in 5% carbon dioxide.

Techniques: Knockdown, Gene Expression, Negative Control, Cell Culture