PMC

Relative contribution of Ub genes to total Ub levels in various mouse tissues. Total RNA was isolated from various tissues in 5-month-old wild-type mice (n=7 for testis; n=3 for all other tissues). UbC, UbB, UbA52 and UbA80 mRNA levels were measured by quantitative real-time RT–PCR and contribution of UbC (A) or four different Ub genes (B) to total Ub levels are shown after normalization by the number of Ub moieties that each Ub transcript generates. Data are expressed as mean±s.e.m. from the indicated number of tissues.

Cell-cycle defect in UbC^−/− MEFs and its rescue by ectopically expressed HA-Ub. (A) Increased apoptosis, reduced G1 and increased G2/M boundaries in UbC^−/− (−/−) MEFs (passage=3) that can be rescued by ectopic expression of HA-Ub in UbC^−/−; HA-Ub (−/−, +Ub) MEFs. Representative cell-cycle profile analyzed by flow cytometry is shown. ^#P<0.1 versus wild-type (+/+) MEFs, ^**P<0.01 versus wild-type (+/+) MEFs. (B) GFP fluorescence of UbC^+/+ (+/+), UbC^+/− (+/−) and UbC^−/− (−/−) MEFs as a function of DNA content. GFP fluorescence in cells with 4n DNA content (G2/M) is higher than with 2n DNA content (G1). (C) GFP fluorescence in each phase of cell cycle in MEFs. Mean GFP fluorescence from population of UbC^+/− (+/−), UbC^−/− (−/−) and UbC^−/−; HA-Ub (−/−, +Ub) MEFs is shown after subtracting background fluorescence of wild-type MEFs. Data are expressed as mean±s.e.m. from 5–8 MEFs originated from different embryos per genotype. (D) GFP fluorescence in S and G2/M phase is expressed relative to G1 phase. ^*P<0.05.

Effect of proteasome inhibitor on Ub gene expression. (A) Various Ub transcript levels in MEFs after exposure to DMSO (−ALLN) or 10 μg/ml ALLN for 24 h (+ALLN). Total RNA was isolated from UbC^+/+ (+/+), UbC^+/− (+/−) and UbC^−/− (−/−) MEFs with or without proteasome inhibition and UbC, UbB, UbA52 and UbA80 mRNA levels were measured by quantitative real-time RT–PCR and normalized to 18S rRNA levels. mRNA levels of wild-type (+/+) MEFs without proteasome inhibition (−ALLN) were arbitrarily assigned as 1 for each transcript. Data are expressed as mean±s.e.m. from five MEFs originated from different embryos per genotype. ^#P<0.05 versus wild-type (+/+) MEFs/−ALLN, ^*P<0.05 versus −ALLN, ^***P<0.001 versus −ALLN. (B) Contribution of Ub genes to total Ub levels with or without proteasome inhibition. Ub transcript levels in MEFs shown in (B) are normalized by the number of Ub moieties that each Ub transcript generates.

Disruption of UbC gene results in reduced cellular Ub content. (A) Indirect competitive ELISA for total Ub (left panel) or HA-Ub (right panel) levels in MEFs. Total cell lysates from UbC^+/+ (+/+), UbC^+/− (+/−), UbC^−/− (−/−) and UbC^−/−; HA-Ub (−/−, +Ub) MEFs were digested with Usp2-cc and subjected to indirect competitive ELISA. Data are expressed as mean±s.e.m. from four MEFs originated from different embryos per genotype with triplicate experiments. ^*P<0.05 versus wild-type (+/+) MEFs. (B) Various Ub transcript levels in MEFs. Total RNA was isolated from UbC^+/+ (+/+), UbC^+/− (+/−) and UbC^−/− (−/−) MEFs and UbC, UbB, UbA52 and UbA80 mRNA levels were measured by quantitative real-time RT–PCR and normalized to 18S rRNA levels. Data are expressed as mean±s.e.m. from five MEFs originated from different embryos per genotype. ^*P<0.05 versus the corresponding transcript levels in wild-type (+/+) MEFs. (C) Contribution of Ub genes to total Ub levels. Ub transcript levels in MEFs shown in (B) are normalized by the number of Ub moieties that each Ub transcript generates.

Reduced proliferation and premature senescence of UbC^−/− MEFs. (A) Proliferation curve of UbC^+/+ (+/+), UbC^+/− (+/−) and UbC^−/− (−/−) MEFs with (+Ub) or without ectopic expression of HA-Ub. A total of 2.5 × 10⁴ cells were plated on 12-well plate and counted over a 6-day period. Data are expressed as mean±s.e.m. from 4–10 MEFs originated from different embryos per genotype. (B, C) UbC^+/+ (+/+) and UbC^−/− (−/−) MEFs stained with Ki-67 and SA-β-gal. Representative data from passage=5 are shown. Ki-67 positive cells (%) were counted in three MEFs originated from different embryos per genotype. At least four different fields were counted in each MEFs. Scale bar, 100 μm, ^**P<0.01. (D) GFP fluorescence as an indicator of transcriptional activity of UbC gene. Mean GFP fluorescence from population of UbC^+/− (+/−), UbC^−/− (−/−) and UbC^−/−; HA-Ub (−/−, +Ub) MEFs is shown after subtracting background fluorescence of wild-type MEFs. Data are expressed as mean±s.e.m. from 5–8 MEFs originated from different embryos per genotype. (E) Increase of GFP fluorescence in MEFs with passage number. Mean GFP fluorescence from population of UbC^+/− (+/−), UbC^−/− (−/−), UbC^+/−; HA-Ub (+/−, +Ub) and UbC^−/−; HA-Ub (−/−, +Ub) MEFs is shown after subtracting background fluorescence of wild-type MEFs at each passage. Data are from two MEFs originated from different embryos per genotype at different passage number.

Partial rescue of UbC^−/− phenotypes by ectopically expressed HA-Ub. (A) Targeting strategy. From top to bottom: wild-type mouse Hprt locus (Hprt wt), disrupted Hprt locus from HM-1 cells (HM-1) and targeted Hprt locus (HA-Ub). Sites recognized by the StuI restriction enzyme are indicated. The map is not drawn to scale. (B) Southern blot analysis and genotyping by PCR. Left panel: Southern blot analysis after digestion of genomic ES cell DNA with StuI restriction enzyme is shown. DNA was used from wild-type ES cells (Hprt wt), disrupted Hprt containing HM-1 ES cell (HM-1) and a targeted ES cell clone (HA-Ub) that was used for subsequent injections. Right panel: PCR results for C57BL/6J (B6), hemizygous wild-type (−/^*) or HA-Ub knock-in (+/^*) male mice, and heterozygous (+/−) or homozygous (+/+) HA-Ub knock-in female mice are displayed. (C) Breeding strategy to generate UbC^−/−; HA-Ub mice. Owing to random inactivation of X chromosome in females, rescued phenotype is expected from UbC^−/−; HA-Ub^+/* male or UbC^−/−; HA-Ub^+/+ female mice. Both genotypes will be referred to in this work simply as UbC^−/−; HA-Ub when the gender does not need to be defined. X^UbX^Ub=HA-Ub^+/+ female; X^UbX=HA-Ub^+/− female; X^UbY=HA-Ub^+/* male; XY=HA-Ub^−/* male. (D) Morphology of UbC^+/+; HA-Ub (+/+, +Ub) and UbC^−/−; HA-Ub (−/−, +Ub) embryos at E15.5 and E17.5. Scale bar, 2 mm. (E) Histology of sagittal liver sections from E15.5 and E17.5 embryos. Liver sections were stained with H&E. Note that no developmental defects are found in UbC^−/− livers with ectopic expression of HA-Ub. Upper panel, scale bar, 1 mm; lower panel, scale bar, 200 μm.

Effect of heat shock and proteasome inhibitor on UbC gene expression and cellular Ub content. (A) Change of GFP fluorescence in UbC^−/− (−/−) MEFs upon heat stress. MEFs were exposed to heat stress as indicated and mean GFP fluorescence from population of viable (PI-negative) cells was measured. Change of GFP fluorescence relative to no treatment was calculated in each MEFs and expressed as mean±s.e.m. from six MEFs originated from different embryos. ^*P<0.05, ^**P<0.01, ^***P<0.001 versus MEFs with no treatment. (B) Indirect competitive ELISA for total Ub levels in MEFs before and after exposure to mild heat shock at 43°C for 30 min and recovery at 37°C for 6 h. Total cell lysates from UbC^+/+ (+/+) and UbC^−/− (−/−) MEFs were digested with Usp2-cc and subjected to indirect competitive ELISA. Data are expressed as mean±s.e.m. from 5–6 MEFs originated from different embryos per genotype with triplicate experiments. ^**P<0.01 versus wild-type (+/+) MEFs/no treatment. (C) Immunoblot analysis of ubiquitinated proteins in MEFs exposed to DMSO (−ALLN) or 10 μg/ml ALLN (+ALLN) for 24 h. Total cell lysates from UbC^+/+ (+/+), UbC^+/− (+/−) and UbC^−/− (−/−) MEFs (50 μg) were subjected to SDS–PAGE followed by immunodetection with anti-Ub antibody. β-actin was used as a loading control. Ub_n, ubiquitin conjugates; uH2A, mono-ubiquitinated histone 2A; Ub₁, ubiquitin monomer. (D) Indirect competitive ELISA for total Ub levels in MEFs exposed to DMSO (−ALLN) or 10 μg/ml ALLN (+ALLN) for 24 h. Total cell lysates from UbC^+/+ (+/+), UbC^+/− (+/−), UbC^−/− (−/−) and UbC^−/−; HA-Ub (−/−, +Ub) MEFs were digested with Usp2-cc and subjected to indirect competitive ELISA. Data are expressed as mean±s.e.m. from four MEFs originated from different embryos per genotype with triplicate experiments. ^#P<0.05 versus wild-type (+/+) MEFs/−ALLN, ^*P<0.05 versus −ALLN, ^**P<0.01 versus −ALLN. (E) Transcriptional activation of UbC gene under proteasome inhibition as monitored by the increase of GFP fluorescence. GFP fluorescence of UbC^+/− (+/−), UbC^−/− (−/−) and UbC^−/−; HA-Ub (−/−, +Ub) MEFs is shown after subtracting background fluorescence of wild-type MEFs. Data are expressed as mean±s.e.m. from 5–8 MEFs originated from different embryos per genotype. ^*P<0.05 versus −ALLN.

Targeted disruption of UbC locus results in impaired fetal liver development. (A) Schematic representation of targeting strategy. From top to bottom: partial restriction map of UbC locus, targeting vector, genomic structure of disrupted allele before and after Cre recombination. The position of 5′ probe and DTA probe for Southern blotting and the location of PCR primers used for screening homologous recombinants and genotyping are shown. The map is not drawn to scale. (B) Southern blot analysis of SacI-digested genomic DNA from ES cell clones. Upper left panel: 5′ probe detected 9.7 and 5.9 kb fragments from wild-type (wt) and disrupted alleles, in homologous recombinants (hr), but only 9.7 kb fragment from wt allele in non-recombinant, wt ES cells. It also hybridized to linearized 6.8 kb targeting vector (+vec). Upper right panel: DTA probe detected linearized 6.8 kb targeting vector (+vec) and >3.9 kb fragment from non-homologous recombinants (nhr), but not from homologous recombinants (hr) or wt ES cells. Lower panel; PCR results for wt (+/+), heterozygous (+/−) and homozygous (−/−) knockout embryos are displayed. (C) Morphology of wt (+/+), heterozygous (+/−) and homozygous (−/−) knockout embryos at E13.5 (upper panel) and histology of sagittal embryonic sections stained with H&E (lower panel). Fetal liver is indicated by white arrow. Scale bar, 1 mm. (D) Histology of sagittal embryonic (upper panel) and liver (lower panel) sections from E13.5 embryos. Note the reduced size of UbC^−/− embryonic liver (indicated with asterisk). Sections were stained with H&E. Hepatocytes (white arrow, lower) are stained lightly, whereas hematopoietic precursors (yellow arrow, upper) are stained darkly. Upper panel, scale bar, 1 mm; lower panel, scale bar, 50 μm. (E) Morphology of wt (+/+), heterozygous (+/−) and homozygous (−/−) knockout embryos at E13.5 with floxed GFP-puro cassette removed by Zp3-Cre recombinase. Fetal liver is indicated by white arrow. Scale bar, 1 mm.

Enhanced sensitivity of UbC^−/− MEFs to heat stress and proteasome inhibition. (A) Immunoblot analysis for inducible Hsp70 expression in MEFs before and after exposure to mild heat shock (HS) at 43°C for 30 min and recovery at 37°C for 6 h. Total cell lysates from UbC^+/+ (+/+) and UbC^−/− (−/−) MEFs (20 μg) were subjected to SDS–PAGE followed by immunodetection with anti-Hsp70 antibody. α-tubulin was used as a loading control. (B) Viability of MEFs upon heat stress. UbC^+/+ (+/+) and UbC^−/− (−/−) MEFs were exposed to heat stress as described in Materials and methods. Preconditioned (PC+) or non-preconditioned (PC−) MEFs were exposed to lethal HS at 45°C for the time as indicated and recovered at 37°C for 24 h. After recovery, the percent of viable cell populations (both adherent and floating) was determined by propidium iodide (PI) staining. Data are expressed as mean±s.e.m. from 6–7 MEFs originated from different embryos per genotype. ^#P<0.05, ^##P<0.01,^###P<0.001 versus wild-type (+/+) MEFs with no treatment (PC−, HS 0 min), ^*P<0.05 versus wild-type (+/+) MEFs with the same treatment. (C) MTT cell viability assay for MEFs. UbC^+/− (+/−) or UbC^−/− (−/−) MEFs were treated with indicated concentration of ALLN for 24 h. At the end of incubation, viability was accessed by the percentage of MTT conversion. Data are expressed as mean±s.e.m. from three MEFs originated from different embryos per genotype. ^*P<0.05 versus UbC^+/− (+/−) MEFs. (D) Cell-cycle abnormality in UbC^−/− (−/−) MEFs (passage=3) and failure of its rescue by ectopic expression of HA-Ub in UbC^−/−; HA-Ub (−/−, +Ub) MEFs in the presence of proteasome inhibition. MEFs were exposed to 10 μg/ml ALLN (+ALLN) for 24 h and subjected to cell-cycle analysis by flow cytometry. Representative cell-cycle profile is shown. ^*P<0.05 versus wild-type (+/+) MEFs/+ALLN.