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(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 <t>BJ-5ta</t> 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.
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(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 <t>BJ-5ta</t> 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.
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(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.

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