UBQLN1 depletion hinders HR repair during S phase.

(A) RAD51 recruitment was hampered after UBQLN1 knockdown. HeLa cells were treated as indicated in Fig 5A. IF was performed with RAD51 antibody. Images were taken by ZEISS Axio Observer 7. (B) Quantification of A. Cells contain more than one RAD51 foci were calculated (n ≥ 50 cells). (C) γH2AX foci were accumulated after UBQLN1 knockdown. HeLa cells were treated as indicated in Fig 5A. IF was performed with γH2AX antibody. (D) Quantification of C. Cells contain more than one γH2AX foci were calculated (n ≥ 50 cells). All values are means ± SEM of more than three independent experiments.</p

UBQLN1 deficiency leads to DNA replication stress.

(A) RPA1 foci increased in UBQLN1 depleted VA13 cells. Cells were transfected with indicated siRNAs for 72 h and IF was performed. Scale bars, 5μm. (B-C) Quantification of (A). Cells contain more than one RPA1 foci were calculated. Total (B) or telomere localized (C) RPA1 foci were counted respectively. All values are means ± SEM of more than three independent experiments (* P (TIF)</p

Depletion of UBQLN1 leads to cell cycle arrest.

(A) UBQLN1 depletion increases EdU positive cells. HeLa cells were transfected with siRNAs for 72 h, and treated with DMSO or CPT (100 nM) during the last 12 h. Cells were treated with EdU for 15 min before detection. Scale bars, 5 μm. (B) Quantification of A. The proportion of EdU positive cells was calculated. (C) PCNA foci increased in UBQLN1 depleted HeLa cells. Cells were transfected with siRNAs and treated with CPT same as A before IF was performed. Scale bars, 5 μm. (D) Quantification of C. PCNA foci in each cell was counted, and mean number was calculated. (E) Cell cycle arrested in UBQLN1 depleted HeLa cells. Cells were transfected with siRNAs and treated with CPT same as A before flow cytometry was performed (n = 3). All values are means ± SEM of more than three independent experiments (* P<0.05, ** P<0.01, *** P<0.001, ****P<0.0001).</p

UBQLN1 regulates the departure of RPA1 from replication forks.

(A) Experimental schematic diagram of B. (B) RPA1 retained at replication fork after UBQLN1 knockdown. 293T cells were transfected with siRNAs and plasmids expressing Flag-RPA1, BASU-PCNA and HA-Ubiquitin for 72h, and then labeled with biotin for 6h. After crosslinking and sonication, Co-IP and western were performed with indicated antibodies. (C) RPA1 ChIP. Half of the precipitate was detected by Western blot using ubiquitin antibody, and the other half was detected by Southern blot using telomeric probe. (D) HeLa cells were transfected with GFP-RPA1 and mCherry-UBQLN1 and synchronizated at S phase as indicated in Fig 5A. The live cell was photographed using live cell imaging technology for 6 hours (n = 1). (TIF)</p

Deficiency of UBQLN1 aggravates idiopathic pulmonary fibrosis.

(A) UBQLN1 transcription level decreased in human lungs of IPF patients. FPKM value of UBQLN1 gene was downloaded from RNA-seq dataset GSE124685 and GSE134692 in the GEO database. n ≥ 26 in each group. (B) Schematic diagram of mouse IPF model construction and UBQLN1 knockdown. (C) UBQLN1 knockdown efficiency in mouse model. RT-PCR analysis of UBQLN1 mRNA level in mouse lungs infected AAV carrying indicate shDNA. (D) Telomere length shortened after UBQLN1 knockdown in mouse lung. The DNA from mouse lungs in B were plotted on nylon membrane and detected by telomere C-probe. Total DNA was stained with gel-red as loading control. (E) RT-PCR analysis of Telomere length. (F) Senescent cells increased in mouse IPF lungs after UBQLN1 knockdown. Lungs from IPF mice were stained by SA-β-gal. (G) p21 protein increased in mouse IPF lungs after UBQLN1 knockdown. p21 protein in lungs from IPF mice were detected by IHC. (H) Quantification of (G). (I) Pathology of IPF after UBQLN1 knockdown. Lung tissues from mice in B were detected by Masson staining. (J) Quantification of (I). The percentage of blue area (collagenous fiber) was quantified. (K) Survival analysis of IPF patients. Data of IPF patients (GSE93606) was downloaded from GEO database. n ≥ 21 in each group. All values are means ± SEM of 4 to 6 mice. (* P<0.05, ** P<0.01, *** P<0.001, ****P<0.0001).</p

UBQLN1 regulates the departure of RPA1 from replication forks.

(A) Schematic diagram of the “double thymidine” synchronization, CPT treatment and release. (B) RPA1 foci retained after UBQLN1 or RFWD3 knockdown. HeLa cells were treated as indicated in A. IF was performed with RPA1 antibody. Images were taken by Nikon Eclipse Ni-E. (C) Quantification of B. Cells contain more than one RPA1 foci were calculated (n ≥ 150 cells). All values are means ± SEM of more than three independent experiments. (D) RPA1 binds DNA persistently in UBQLN1 knockdown cells. GFP-RPA1 were stably expressed in HeLa cells, and cells were treated as indicated in A with HU treatment instead of CPT. (E) UBQLN1 foci dynamics after releasing from replication stress. HeLa cells were treated as indicated in A. IF was performed with UBQLN1 antibody at indicated time points. (F) Quantification of E. Cells contain more than one UBQLN1 foci were calculated (n ≥ 50 cells). All values are means ± SEM of more than three independent experiments.</p

UBQLN1 depletion increase telomere instability.

(A) 53BP1 foci increased in UBQLN1 depleted HeLa cells. Cells were transfected with indicated siRNAs for 72 h and IF was performed. Scale bars, 5μm. (B-C) Quantification of (A). Cells contain more than one 53BP1 foci were calculated. Total (B) or telomere localized (C) 53BP1 foci were counted respectively. All values are means ± SEM of more than three independent experiments (* P (TIF)</p

Depletion of UBQLN1 leads to genome instability.

(A&B) Telomeres shortened after UBQLN1 depletion. HeLa cells were transfected with siRNAs for three days, and telomere length was detected by Q-FISH analysis (A). The telomere signal intensity was quantified (B). The numbers of quantified telomeres in Q-FISH were annotated as n. (C) Telomeres shortened after UBQLN1 depletion. HeLa cells were transfected with siRNAs twice in a week, and telomere length was detected by TRF analysis (n = 3). (D) UBQLN1 knockdown efficiency. HeLa cells were transfected as in C, total RNA were extracted and UBQLN1 mRNA was detected by RT-q PCR. (E) C-circles increased after UBQLN1 depletion in U2OS cells. Cells were transfected with siRNAs for 72h. Telomere c-circles were amplified by Φ29 DNA polymerase and detected by slot blot (DNA was plotted on nylon membrane and hybridized with telomere C-probe) (n = 3). (F) C-circles decreased after expressing synonymous UBQLN1 in 293T cells. Cells were transfected with siRNAs for 24 h and then transfected with plasmid expressing synonymous UBQLN1 for 48 h. Telomere c-circles were amplified by Φ29 DNA polymerase and detected by slot blot (n = 3). (G) Micronuclei increased after UBQLN1 depletion. HeLa cells were transfected with siRNAs for 72h, and nuclei were stained by DAPI. The micronuclei were counted. Scale bars, 5μm. All values are means ± SEM of more than three independent experiments (* P<0.05, ** P<0.01, *** P<0.001, ****P<0.0001).</p

UBQLN1 is involved in DNA replication.

(A) RPA1 foci increased in UBQLN1 depleted HeLa cells. Cells were transfected with indicated siRNAs for 72 h and IF was performed. Scale bars, 5μm. (B-C) Quantification of A. Cells contain more than one RPA1 foci were calculated. Total (B) or telomere localized (C) RPA1 foci were counted respectively. (D-F) Experiments in A-C were repeated in U2OS cells. (G) Protein levels in UBQLN1 knockdown cells. Immunoblot analysis of indicated proteins in HeLa cells 72 h after siRNA transfection (n = 3). (H) DNA fiber assay in UBQLN1 knockdown cells. HeLa cells were transfected with indicated siRNAs for 72 h. Cells were labeled with CldU for 30 min and subsequently with IdU for 20 min during the last 50 minutes, then the DNA fiber assay was performed. (I) Quantification of H. The length of IdU labeled DNA (green) was calculated (n ≥ 500 DNA). All values are means ± SEM of more than three independent experiments (* P<0.05, ** P<0.01, *** P<0.001, ****P<0.0001).</p

RPA1 is ubiquitinated in response to replication stress.

(A) 293T cells were transfected with plasmids expressing HA-Ubiquitin and mCherry-RPA1 or vector for 72 h, and treated with HU (1 mM), CPT (100 nM) or DMSO during the last 12 h. IP-western was performed with indicated antibodies (n = 3). (B) 293T cells were transfected with plasmids expressing HA-Ubiquitin, Flag-RPA1, GFP-UBQLN1 or vector for 72 h, and treated with DMSO, HU (1 mM) or CPT (100 nM) during the last 12 h. Co-IP and western were performed with indicated antibodies (n = 3). (TIF)</p