sCLU expression is increased during senescence.

<p>Young (Y), middle-age (M), premature-senescent (PS), senescent (S), and late senescent (LS) IMR-90 cells were generated by continuous culture. (A) The “age” of the IMR-90 cells was determined by population doublings and %SA-β-gal+ cell measurements. Experiments were repeated three or more times in triplicate each, and representative results are shown. (PD, population doubling). (B) Senescence markers, phospho-ser15-p53, total p53, p21 and p16 levels, were markedly increased during senescence. (C) Both the precursor (psCLU) and the mature secretory (sCLU) forms of clusterin were induced in total cell lysates during senescence. sCLU levels increased in the media of senescent IMR-90 cells. (D) The promoter activity of sCLU was increased during senescence. *, p-value≤0.05, **, p-value≤0.01.</p

sCLU induction during senescence is mediated through IGF-1R/MAPK/Erk/Egr-1 signaling pathway.

<p>(A) IGF-1 expression was increased in senescent IMR-90 cells. IGF-1 levels in media from young or senescent IMR-90 cells were measured using ELISAs as described above, and normalized to media volume and adjusted cell number (4×10⁴ cells). (B) Total IGF-1 receptor (IGF-1R), phosphorylated IGF-1 receptor (p-IGF-1R), Src, phosphorylated Src, Erk-1/2, phosphorylated-Erk-1/2, and Egr-1 levels in young (Y), middle-age (M), premature-senescent (PS), and senescent (S) IMR-90 cells were determined by western blot analyses. β-Actin levels were measured to monitor loading. (C) Exposure to the indicated doses of the IGF-1R tyrosine kinase inhibitor (AG1024) repressed sCLU levels in senescent IMR-90 cells. psCLU and sCLU were detected by western blot analysis. β-Actin levels were monitored to show equal loading. Senescent IMR-90 cells exposed to varying doses of AG1024 (µM as indicated for 24 h) showed dose-dependent increases in apoptosis (monitored by TUNEL + staining). Only senescent, and not young or middle-aged IMR-90, cells were hypersensitive to AG1024. In the experiment outlined below the western blot analyses cells were exposed to AG1024 (5 µM) for 48 h and therefore higher levels of apoptotic cells were noted in senescent IMR-90 cells compared to cells exposed for 24 h above. Note that only senescent cells were hypersensitive to these inhibitor treatments. All experiments were repeated three times in triplicate.</p

sCLU protects cells from senescence.

<p>(A) sCLU was knocked down in an IMR-90 pooled population containing a lentiviral-mediated inducible shRNA-sCLU construct cultured in presence of doxycycline (dox), while expression of sCLU was not affect in the same pooled population in the absence of doxycycline (dox). The population doubling of IMR-90 cells with inducible shRNA-sCLU decreased faster when cultured in the presence of dox compared to in cells cultured without dox. **, p-value of two curves was ≤0.01. Experiments were repeated three times, starting from infection of young IMR-90 cells with selection of a pTRIPZ-shCLU-containing pooled population. Similar results were observed in all three experiments and representative results from one experiment are shown. (B) IMR-90 cells with sCLU knockdown had significantly higher %SA-β-gal+ cells after being cultured for 33, 39, and 53 days with or without dox. SA-β-gal+ staining was performed to identify senescent cells. *, p-value ≤0.05. (C) Controls included a pooled IMR-90 cell population contain an inducible shRNA-non-targeted, scrambled shRNA construct were cultured in presence or absence of dox. Growth rates and sCLU levels were not affected in the shRNA-Scr (non-targeted) IMR-90 cell populations. Population doubling of IMR-90 cells with inducible non-targeting shRNA was not different when cultured with/without doxycycline. The experiments were repeated three times similarly as inducible shCLU experiments. Representative results of three experiments were shown here. (D) After culturing for 38, 45, and 65 days with or without dox, SA-β-gal+ staining was performed to identify senescent cells in IMR-90 pooled populations with inducible shRNA-Scr (non-targeting) vector. (E) Specificity of shCLU knockdown in conditional IMR-90 senescent cells. IMR-90 pooled populations containing the pTRIPZ-shCLU vector was cultured with or without doxcycline (100 nM for 3 days) and sCLU and nCLU levels were monitored using Western blot analyses. α-Tubulin levels were shown as a loading control. Note that sCLU and not nCLU levels were specifically knocked down (>90%) in cells exposed to dox. In these experiments, IMR-90 cells were cultured in 95% air-5% CO₂, estimated at ∼20% O₂.</p

sCLU expression is differently regulated in SIPS, RS and oncogene-induced senescence.

<p>(A) sCLU levels were increased during senescence in BJ, HE45 and HBEC cells. BJ, HE45 and HBEC cells were aged by continuous cell culture and senescence status determined by %SA-β-gal+ cells. (B) Forced expression of Ras12V represses sCLU expression. Young IMR-90 cells were transduced with lentiviral-mediated Ras12V expression. After cells exhibited typical senescence morphology, stained SA-β-gal+, and were growth arrested, they were harvested and sCLU levels monitored by Western blotting. Phospho-Erk-1/2 was probed to monitor the effects of Ras12V over-expression. α-Tubulin was used for a loading control. (C) IMR-90 cells cultured in 20% oxygen (O₂) underwent senescence faster than cells cultured in 2% O₂. The population doubling of IMR-90 cells cultured in 20% O₂ decreased faster than cells cultured in 2% O₂. (D) BJ cells were not affected by different O₂ tensions. In contrast to IMR-90 cells, the population doublings of BJ cells were identical whether cultured in 20% or 2% O₂. (E) sCLU levels increased in IMR-90 cells cultured in 20% O₂ compared to levels found in IMR-90 cells cultured in 2% O₂ in both early and late passage cells. (F) Young (Y), middle-aged (M), and senescent (S) IMR-90 cells were treated with IR (10 Gy). psCLU, sCLU, total Erk-1/2 (Erk) and phosphorylated Erk-1/2 (pErk) were detected by western blot analyses. For (C) and (D), representative results are presented from at least three repeat experiments with similar results.</p

DDR regulators accumulate at the 3’ end of genes after XRN2 loss.

<p>(<b>A-E</b>) Accumulation of the DNA damage regulators, ATM, BRCA1, γ-H2AX, 53BP1, and CtIP, was monitored at the 3’ pause sites of the (<b>A</b>) <i>ENSA</i>, (<b>B</b>) <i>β-actin</i>, (<b>C</b>) <i>Akirin</i>,<i>1</i> (<b>D</b>) <i>Gemin7</i> genes and (<b>E</b>) an intronic region of <i>Gemin7</i> by chromatin immunoprecipitation. n = 3, S.E. is indicated. (<b>F</b>) Model for XRN2 functions in DNA repair pathway choice. In normal conditions, XRN2 binds to the NHEJ factor 53BP1 promoting DSB repair via the NHEJ pathway. In the absence of XRN2, NHEJ is inhibited downstream of 53BP1, allowing DSB repair via the HR pathway.</p

Loss of XRN2 leads to increased DSB formation and genomic instability.

<p>(<b>A</b>) Steady state levels of XRN2 protein in shScr (+) compared to shXRN2 (-) fibroblast cells were monitored by western blotting and immunofluorescence. (<b>B, C</b>) Basal levels of 53BP1, pATM^ser1981, γ-H2AX, and BRCA1 foci/nuclei were quantitated in shScr, shXRN2, and MCF-7 cells treated with control and XRN2 specific siRNA. (<b>D, E</b>) Genomic aberrations were quantified in shScr, shXRN2, and shk-h cells using derived metaphase spreads. (**p<0.01).</p

XRN2-deficient cells are hypersensitive to various chemotherapeutic agents.

<p>(<b>A-D</b>) shScr and shXRN2 fibroblasts and MDA-MB-231 cells transfected with a siRNA control or targeting XRN2 were either mock-treated or exposed to: (<b>A, B</b>) ionizing radiation (IR); (<b>C, D</b>) ultraviolet light (UV). (<b>E-F</b>) shScr and shXRN2 fibroblasts were either mock-treated or exposed to: (<b>E</b>) H₂O₂ or (<b>F</b>) Aphidicolin (APH). Cells were then monitored for survival using colony forming assays. Colonies of >50 normal-appearing cells were quantified for mock- versus agent-exposed cells. (**p<0.01).</p

R-loop formation and transcription contribute to delayed DSB repair kinetics in XRN2-deficient cells.

<p><b>(A)</b> Levels of R loops were monitored in mock- or IR (1 Gy)-exposed shXRN2 compared to shScr fibroblast cells by immunofluorescence. <b>(B-C)</b> Regression of 53BP1 foci/nucleus was monitored by IF in IR (1 Gy)-treated shXRN2 and shScr cells that were exposed to α-amanitin (α-aman) or transfected with GFP-RNaseH. (*p<0.05).</p

XRN2 is a DNA damage-responsive factor.

<p>(<b>A</b>) To interrogate potential <i>in vivo</i> XRN2-interacting partners, exponentially growing HeLa cells were collected and lysed for FPLC analyses. Individual fractions were separated by SDS-PAGE and indicated proteins visualized by immunoblot (IB). (<b>B</b>) Interactions between XRN2 and the DNA damage regulators Ku80, 53BP1, and BRCA1 were interrogated by immunoprecipitation (IP). (<b>C</b>) XRN2 foci were quantitated in human fibroblasts stably expressing an shRNA control (shScr) or a Kub5-Hera shRNA (shk-h) in mock (unt)-, IR (1 Gy)-, and UV (20 J/m²)-treated cells. (<b>D</b>) Sub-cellular XRN2 localization and S9.6 foci were visualized in mock-, UV (20 J/m²)-, or UV (20 J/m²)- + α amanitin (α-aman)-treated shScr fibroblast cells by immunofluorescence. (*p<0.05).</p

Loss of XRN2 leads to increase amounts of replication stress.

<p>(<b>A</b>) Basal levels of 53BP1 were monitored in PCNA positive cells, an S-phase indicator, in shXRN2 and shScr cells by immunofluorescence (IF). (<b>B, C</b>) Basal levels of phosphorylated RPA, were monitored by IF in shXRN2, shScr, shk-h fibroblasts and MCF-7 cells transfected with control or XRN2 specific siRNAs. (<b>D</b>) Basal levels of phosphorylated ATR were monitored by IF in MCF-7 cells transfected with a siRNA control and a siXRN2. (<b>E</b>) Basal levels of phosphorylated Chk1 were monitored in.shScr, shXRN2, and shk-h fibroblasts by IF. (<b>F)</b> Basal levels of phospho-Chk1-pS-317 and RPA32-pS-(4/8), replication stress indicators, in shXRN2 (-) versus shScr (+) cells were monitored by western blotting. (<b>G</b>) DNA replication elongation was monitored in log-phase shScr, shXRN2, or shk-h fibroblasts by DNA fiber analyses. (**p<0.01).</p