EMBO/soda-nel huggingface dataset:
HMCES safeguards replication from oxidative stress and ensures error-free repair (2020)
Figure 2

Figure 2. HMCES participates in cell proliferation and DNA damage/replication stress response.

(A) Western blotting analysis confirmed HMCES knockout in 293A cells (293A HMCES KO). Blots also included knockout cells reconstituted with WT SFB- HMCES.

(B, C) Colony-forming capacity of HMCES KO cells was compared to that of control wild-type 293A cells.

(C) Box plot showing colony forming efficiencies of 293A wild-type control and HMCES KO cells. Box limits represent 25th and 75th percentile, centerline shows the median and whiskers extend minimum to maximum. Data include more than three independent repeats performed in duplicates. Student's t-test were performed for statistical analysis (****, p<0.0001).

(D) Colonogenic survival assay of 293A wild-type control and HMCES KO cells treated with various concentrations of hydroxyurea (HU) was shown. Seeding density with respect to control was indicated below each image. The images shown are representative of three biological repeats, each performed in duplicate.

(E) Quantification showing survival fraction of wild-type control (WT) and HMCES KO cells upon HU treatment. Data are presented as mean±s.e.m (n=3, each performed in duplicates) and student's t-test were performed for statistical analysis (ns, not significant; *, p<0.05).

(F) Quantification of colonogenic survival assay upon exposure of WT and HMCES KO cells to ionizing radiation (IR). Data are presented as mean±s.e.m. Quantifications are from three biological repeats performed in duplicates. Student's t-test was performed for statistical analysis (*, p<0.05; **, p<0.01).

(G, H) Sensitivity assay and quantification of surviving WT control and HMCES KO cells determined by colony-forming assay upon exposure to H2O2. Experiments were repeated three independent times, each time performed in duplicates and data are presented as mean±s.e.m. Statistical significance for the corresponding experiment was analyzed by student's t-test (*, p<0.05).

(I) Bar graph showing sensitivity of WT, HMCES KO and stably reconstituted WT cells towards H2O2 treatment. Experiment was repeated two independent times with technical repeats.
HMCES safeguards replication from oxidative stress and ensures error-free repair (2020)
Figure 3

Figure 3. HMCES-mediated tolerance is imparted by the bypass of oxidative damage.

(A, B) Colonogenic assays were performed by adding increasing concentrations of pemetrexed to control wild-type (WT) 293A cells and HMCES knockout (HMCES KO) cells (A) and quantification of results were shown (B). Increasing numbers of cells were seeded with increasing concentrations of the compound to obtain optimal drug-dose response curve. Seeding density with respect to control is indicated below each image. The images shown are representative of three biological repeats, each performed in duplicate. (B) Data are presented as mean±s.e.m. and student's t-test was performed for statistical analysis (****, p<0.0001).

(C) Western blot showing APEX2 levels upon 293A cells infected with viruses encoding scrambled or two independent APEX2 shRNAs.

(D) Colony forming assay was performed to indicate colony forming efficiency in 293A and HMCES KO cells upon infection with viruses encoding scrambled or APEX2 shRNAs.

(E) Sensitivity assay was conducted with WT and HMCES KO cells upon treatment with AP Endonuclease inhibitor III (APEi). Results are presented from two independent experiments and data are presented as mean±s.e.m.

(F) Colony forming assay was performed with WT and HMCES KO cells upon either pemetrexed treatment alone or in combination with APEi. Data are presented as mean±s.e.m (n=2 each with technical repeats).

(G, H) Western blotting revealed levels of POLH (G) and REV1 (H) in two independent clones upon selection following transfection with indicated shRNA.

(I) Colony formation assay was conducted to reveal proliferation of WT, POLH KD, REV1 KD, HMCES KO, HMCES KO + POLH KD and HMCES KO +REV1 KD cells.

(J) Representative images showing proliferation of respective shRNA resistant reconstitution of POLH in 293A and HMCES KO cells stably expressing POLH shRNA. Experiment was repeated two independent times in duplicates.

(K) Mutagenesis frequency analysis was conducted in 293T scrambled and 293T HMCES shRNA cells using sup F shuttle vector-based assay system. Experiments were repeated three independent times and data are presented as mean±s.e.m. Student's t-test was used to calculate statistical significance (**, p<0.01).
HMCES safeguards replication from oxidative stress and ensures error-free repair (2020)
Figure 4

Figure 4. CRISPR-CAS9 based synthetic lethalilty screen conducted in HMCES KO cells reveal genetic interaction between HMCES and homologous recombination pathway.

(A) Schematic representation of DDR CRISPR-CAS9 screen performed in WT and HMCES KO cells.

(B) Volcano plot showing DDR CRISPR-CAS9 screen for HMCES KO cells. -log10(p-value) is plotted against log2(HMCES KO/ WT) fold change. Genes showing significant positive and negative enrichment are colored in red and top enriched candidates are also labeled.

(C) Plot showing number of sgRNAs that are negatively or positively enriched for each gene in DDR library.

(D, E) Western blots showing levels of CtIP (D) and BRCA2 (E) upon shRNA mediated knockdown in 293A cells.

(F, G) Cell proliferation measured using CellTiter-Glo assay in 293A and HMCES KO cells without or with transfection with viruses encoding CtIP (F) and BRCA2 (G) shRNAs. Each experiment was repeated three independent times done in triplicates each time. Data was represented as mean±s.e.m. Student's t-test was used to calculate statistical significance (***, p<0.001; ****, p<0.0001).

(H) Our current working model depicting HMCES mechanism of action on replication fork. Upon encountering oxidative base damage on replication fork, HMCES along with BER machinery can successfully remove modified base to resume replication. This mechanism would result in error-free replication and maintain genomic integrity between parental and nascent replication forks. In the absence of HMCES, however, the replication fork can proceed only by replicating through the damage, often by misrecognition and misincorporation of bases by replicative or translesion polymerases, leading to replication errors. HRR factors such as CtIP, BRCA2, BRCA1 and PALB2 are indispensable for processing the damage, possible recruitment of TLS polymerase, and replication fork restart.
Ubiquilin 1 Promotes IFN-γ-Induced Xenophagy of Mycobacterium tuberculosis. (2015)
Fig 1

MUPs bind ubiquilins.

(A) Domain structure of human UBQLN1 (Interpro: http://www.ebi.ac.uk/interpro/). (B) Gal4 DNA-binding domain fusions of MUPs were tested with Gal4 activation domain fusions of ubiquilins (murine) in Y2H. (C) HEK293 cells transfected with indicated MUP-V5, myc-UBQLN1 (human), or empty vector were immunoprecipitated (IP) with anti-myc or anti-UBQLN1 antibodies. Western blot (WB) of IP and lysate were probed as indicated. (D-E) WB of input lysate and IP from HEK293 cells expressing myc-UBQLN1 and Rv1926-V5 (D) or Rv1566-V5 (E). IP was performed using antibodies directed against myc, V5, or isotype control (IgG).
Ubiquilin 1 Promotes IFN-γ-Induced Xenophagy of Mycobacterium tuberculosis. (2015)
Fig 2

UBQLN1 binds Mtb <italic>in vitro</italic>.

(A) Mtb-UBQLN1 binding assay. Mtb were incubated with HEK293 cell lysate (B, C) or recombinant protein (D) (input) and washed 5 times to remove unbound proteins. The pellet contains Mtb and associated host proteins (elution). (B) Mtb were incubated with lysate from HEK293 cells transfected with vector control, myc-UBQLN1, GFP, or V5-parkin. WB analysis of the input (I), 5th wash (W5), and elution (E), probed with indicated antibodies. Actin is a loading control for the cell lysate and Ag85b for the bacterial pellet. (C) Mtb were incubated with lysate from HEK293 cells transfected with myc-UBQLN1-ΔUBA, myc-UBQLN1-ΔUBL, and myc-UBQLN1-UBA. WB containing the input, 5th wash, and elution were incubated with anti-myc antibody. The predicted sizes of myc-UBQLN1-ΔUBL, myc-UBQLN1-ΔUBA, and myc-UBQLN1-UBA are 55 kD, 50 kD, and 6 kD, respectively. (D) Mtb were incubated with recombinant GST-UBQLN1, GST-UBQLN1-ΔUBL, or GST-UBQLN1-ΔUBA. WB containing the input and elution was probed with an anti-GST antibody.
Ubiquilin 1 Promotes IFN-γ-Induced Xenophagy of Mycobacterium tuberculosis. (2015)
Fig 3

UBQLN1 localizes to Mtb, restricts its growth, and promotes the ability of macrophages to active CD4+ T cell.

(A) BMDMs were infected with GFP-expressing wt Mtb or ΔesxA for 24h and immunostained with UBQLN1 antibody. IFN-γ was added 1d prior to infection as indicated. (B) Quantification of results from A from 3 independent experiments with at least 100 bacteria analyzed per experiment; ***P< 0.001, Fisher’s exact test, comparing the proportion of UBQLN1 positive versus negative Mtb in indicated samples. (C) Cellular lysate from BMDMs that were treated with IFN-γ as indicated were analyzed by WB with an antibody that recognizes UBQLN1 and UBQLN2. (D) BMDMs were transfected with siRNAs targeting UBQLN1 or a non-targeting control (siCON) and lysates were analyzed by WB using an antibody that recognizes UBQLN1 and UBQLN2. (C-D) Actin served as a loading control. (E) BMDMs were infected with Mtb live/dead strain and treated with AnTc 24h prior to fixation. (F) BMDMs were transfected with siRNAs targeting UBQLN1 or siCON and infected with live/dead Mtb strain. IFN-γ was added 1d prior to infection if indicated. %Viable bacteria were quantified 24 and 48 hpi. Data are from 2 independent experiments. *P<0.05; ***P<0.001, Fisher’s exact test, comparing the proportion of viable and non-viable bacteria in indicated samples. (G) BMDMs were treated with the indicated siRNAs three days prior to infection and then activated with IFN-γ one day prior to infection with H37Rv or ΔesxA. CFU were enumerated 4 and 96 hpi. *P< 0.05, unpaired Student’s t-test. Data are representative of three independent experiments. (H) BMDMs transfected with siRNA targeting UBQLN1 or siCON were infected with wt Mtb (H37Rv) (I) UBQLN1-silenced BMDMs and controls were infected with H37Rv or ΔesxA. (H-I) 24 hpi infected BMDMs were co-cultured with P25TCR-Tg CD4+ T cells, and IFN-γ was measured by ELISA. Data is representative of 3 independent experiments. *P<0.05, **P<0.005, unpaired Student’s t-test. (B, F, Γ, H) Results are mean +/- SEM; ns, not significant.
Ubiquilin 1 Promotes IFN-γ-Induced Xenophagy of Mycobacterium tuberculosis. (2015)
Fig 4

UBQLN1 co-localizes with Mtb that are cleared through autophagy.

(A) BMDMs treated with IFN-γ and infected for 24h with GFP-Mtb were immunostained for UBQLN1 and either ubiquitin (using the FK2 antibody) or p62. GFP-LC3 BMDMs infected with DsRed-Mtb were immunostained for UBQLN1. (B) Quantification of those Mtb that co-localize with at least one of the cellular markers shown in A. More than 375 total bacteria were counted in two independent experiments, and the number co-localizing with at least one marker is indicated as “total” under each bar. (C) Wt macrophages (Atg16L1flox/flox; Cre-) and autophagy-deficient macrophages (Atg16L1flox/flox Lyz-cre; Cre+) were activated with IFN-γ or not, followed by infection with the live/dead Mtb reporter strain. % viable was quantified after addition of AnTc. **P<0.005, ***P<0.0001, Fisher’s exact test (in which the proportions of viable to non-viable Mtb was compared). (D) Atg16L1flox/flox Cre+ and Cre- BMDMs treated with IFN-γ and infected for 24h with GFP-Mtb were immunostained for UBQLN1 and p62. The number of Mtb co-localizating with the cellular markers is shown. (C-D) At least 100 bacteria were analyzed in three independent experiments. (E) Images from IFN-γ treated BMDMs of indicated genotype infected with GFP-Mtb and immunostained with UBQLN1 and p62. Arrows indicates p62+ UBQLN1+ Mtb.
Ubiquilin 1 Promotes IFN-γ-Induced Xenophagy of Mycobacterium tuberculosis. (2015)
Fig 5

UBQLN1 promotes ubiquitin, p62, and LC3 recruitment to Mtb.

(A-C) BMDMs transfected with siRNA targeting UBQLN1 or control siRNA. Images show IFN-γ treated macrophages infected with GFP-Mtb (A-B) or DsRed-Mtb (C) for 24h. Cells were immunostained for ubiquitin (Ub) with the FK2 antibody (A) or p62 (B), which are shown in red. (C) GFP-LC3 BMDMs were infected with DsRed-Mtb. LC3 is shown in green, whereas Mtb are red. (A-C) Quantification shows the percentage of Mtb that colocalized with the indicated cellular marker in macrophages that were treated with IFN-γ as indicated. Results are mean +/- SEM from two independent experiments, with at least 100 bacteria analyzed per experiment. ***P<0.001, Fisher’s exact test (in which the proportion of Mtb associated with the cellular marker in a given condition was compared to the proportion found in the sample treated with siCON and IFN-γ).
Ubiquilin 1 Promotes IFN-γ-Induced Xenophagy of Mycobacterium tuberculosis. (2015)
Fig 6

UBQLN1 acts in parallel to parkin.

(A) WT and Park2-/- BMDMs were pretreated with IFN-γ, infected with GFP-Mtb for 24h, and immunostained with the FK2 antibody. Bacterial colocalization with FK2 immunoreactivity was quantified from 2 independent experiments. (B) Park2-/- BMDMs pretreated or not treated with IFN-γ were infected with GFP-Mtb for 24h and immunostained with UBQLN1 specific antibody; Arrows indicate Mtb co-localized with UBQLN1. ns- not significant. (C) Park2-/- BMDMs were transfected with siRNA targeting UBQLN1 or control siRNA 3d prior to infection, treated with IFN-γ, and infected with GFP-Mtb for 24h prior to immunostaining for ubiquitin (FK2 antibody). Ubiquitin (FK2+) bacteria were quantified from two independent experiments. (A, C) **P<0.01, Fisher’s exact test (comparing the proportion of FK2 positive bacteria in indicated samples). Results are mean +/- SEM. A minimum of 100 Mtb were counted in each experiment.
Inhibition of IRGM Establishes a Robust Antiviral Immune State to Restrict Pathogenic Viruses (2021)
Figure 1

Figure 1. Viruses and viral PAMP-induced IRGM expression suppresses IFN response.

(A-C) Western blot analysis of THP-1 cells infected with (A) HSV-1 at MOI 5 for 3 h, 6 h, 12 h, 18 h, and 24 h (B) CHIKV at MOI 5 for 3 h, 6 h, 12 h, 18 h, and 24 h (C) JEV at MOI 5 for 3 h, 6 h, 12 h, and 24 h.

(D-H) Western blot analysis of THP-1 cells untreated or treated with (D) 5'pppRNA ( 1 μg/ml) for 3 h, 6 h, 12 h, 18 h and 24 h (E) Poly I:C (1 μg/ml) for 3 h, 6 h, 9 h, 12 h and 24 h (F) Poly dA:dT (1 μg/ml) for 3 h, 6 h, 9 h,12 h and 24 h (G) cGAMP (1 μg/ml) for 6 h, 12 h, 18 h, and 24 h (H) dsDNA (1 μg/ml) for 3 h, 6 h, and 12 h and probed with antibodies as indicated.

(I-L) Control and IRGM knockdown THP-1 IFN reporter cells were treated with (I) Poly I:C (1 μg/ml) for 8 h, 12 h and 24 h or (J) Poly dA:dT (1 μg/ml) for 12 h and 24 h or (K) IFN-β (500 ng/ml) for 8 h and 12 h or (L) cGAMP (1 μg/ml) for 12 h and 24 h and the supernatant was subjected to luciferase reporter assay using QuantiLuc reagent (n=3, mean ± SD ,*P < 0.05, **P < 0.005, ***P < 0.0005, Student's unpaired t-test).

(M-P) Control and IRGM knockdown THP-1 cells were untreated and treated with Poly I:C (1 μg/ml) for 8 h and the total RNA was subjected to qRT PCR with primers of (M) MX2 (N) ISG15 (O) OAS1 and (P) IFN-β. (n=3, mean ± SD, *P ≤ 0.05, **P ≤ 0.005, ***P < 0.0005, Student's unpaired t-test).

(Q-T) Control and IRGM knockdown THP-1 cells were untreated and treated with Poly dA:dT (1 μg/ml) for 8 h and the total RNA was subjected to qRT PCR with primers of (Q) MX2 (R) ISG15 (S) OAS1 and (T) IFN-β. (n=3, mean ± SD, **P ≤ 0.005, ***P < 0.0005, Student's unpaired t-test).
Inhibition of IRGM Establishes a Robust Antiviral Immune State to Restrict Pathogenic Viruses (2021)
Figure 4

Figure 4. Irgm1-/- mice are resistant to CHIKV Infection.

(A) The stages of paralysis in C57BL/6 mice post CHIKV infection (MOI 1x107 PFU/mouse, 6 dpi) with scoring according to the severity of the disease.

(B) The graph depicts percentage change in body weight of CHIKV infected irgm1+/+ and irgm1-/- neonates for duration as indicated (n=12, mean ± SD, *p < 0.05, **p < 0.005, ***p < 0.0005, Student's unpaired t-test).

(C) The graph depicts total paralysis scores of CHIKV infected irgm1+/+and irgm1-/-neonates (n=12) until 5 days post-infection.

(D) Kaplan-Meier survival graph depicts percentage survival of mock and CHIKV infected irgm1+/+ and irgm1-/- neonates during the course of infection (n=12, ****p < 0.0001, Log-rank (Mantel-Cox) test).

(E, F) Total RNA isolated from muscles of mock and CHIKV infected (MOI 1x107 PFU/mouse, 6 and 11dpi) irgm1+/+and irgm1-/-mice and was subjected to qRT-PCR to quantitate the viral load (n=3, mean ± SD, ***p < 0.0005, Student's unpaired t-test).

(G) Total RNA isolated from the brain of mock and CHIKV infected (MOI 1x107 PFU/mouse, 6 dpi) irgm1+/+ and irgm1-/-mice and was subjected to qRT PCR for quantitation of viral load (n=3, mean ± SD, ***p < 0.0005, Student's unpaired t-test). The total RNA used for qRT PCR with the brain is four times more than muscles.

(H, I) Total RNA isolated from muscles and brains of mock and CHIKV infected (MOI 1x107 PFU/mouse) (6 dpi) irgm1+/+ and irgm1-/-mice and was subjected to qRT PCR with MX2 (n=3, mean ± SD, *p < 0.05, ***p < 0.0005, Student's unpaired t-test). The total RNA used for qRT PCR with the brain is four times more than muscles.
Inhibition of IRGM Establishes a Robust Antiviral Immune State to Restrict Pathogenic Viruses (2021)
Figure 5

Figure 5. Type I IFN response is blunted in virus-infected IRGM knockdown cells.

(A-D) Control and si-IRGM transfected THP-1 IFN reporter cells were kept uninfected (Mock) or infected with (A) JEV (MOI 5) or (B) CHIKV (MOI 5) or (C) HSV-1 (MOI 2.5) or (D) VSV-eGFP (MOI 1) and the supernatant collected 8 hpi were subjected to luciferase assay. The graphs depict fold change in interferon response. (n = 3, mean ± SD, *p < 0.05, **p < 0.005, ***p < 0.0005, Student's unpaired t-test).

(E-J) Control and si-IRGM transfected HT-29 cells uninfected or infected with CHIKV and qRT-PCR analysis were performed with several ISG's (E) SAMHD1 (F) HERC5 (G) ISG15 (H) Viperin/RSAD2 (I) MX1 (J) APOBEC3G. (n = 3, mean ± SE, **p < 0.005, ***p < 0.0005, ****p < 0.00005, Student's unpaired t-test).

(K) Western blot analysis with cell lysates of mock and CHIKV (MOI 5, 24 h) infected control and si-IRGM transfected THP-1 cells and probed with the indicated antibodies. S.E, short exposure; L.E, long exposure.

(L) Western blot analysis with cell lysates of mock and CHIKV (MOI 5, 24 h) infected control and IRGM knockdown HT-29 cells and probed with the indicated antibodies.

(M) Western blot analysis with cell lysates of mock and JEV (MOI 5, 24 h) infected control and si-IRGM transfected HT-29 cells and probed with the indicated antibodies.

(N) Western blot analysis with cell lysates of THP-1 control or IRGM knockdown cells, untransfected or transfected with, heat-killed whole CHIKV or CHIKV viral RNA and probed with the indicated antibodies.

(O) qRT-PCR analysis to determine the knockdown efficiencies of PRR's and adaptor proteins as indicated.(n = 3, mean ± SE, ***p < 0.0005, Student's unpaired t-test)

(P) qRT PCR analysis with total RNA isolated from control and IRGM knockdown HT-29 cells transfected with siRNA combinations as indicated that were infected with CHIKV (MOI 1, 24 h). (n = 3, mean ± SE, ***p < 0.0005, ****p < 0.00005, Student's unpaired t-test).

(Q) Western blot analysis with cell lysates of mock and CHIKV (MOI 1, 24 h) infected control and IRGM knockdown HT-29 cells transfected with indicated siRNA and probed with the indicated antibodies.

Expanded view Figure Legends
H2B monoubiquitination is a key epigenetic regulator of ferroptosis and is regulated by p53 (2019)
Figure 1

Figure 1. Loss of H2Bub1 promotes ferroptosis.

A, Western blot analysis of 293T cells treated with 20 μM erastin for the indicated times.

B, H1299 cells transfected with a control siRNA (siCont.) or an RNF20-specific siRNA (siRNF20) and a wild-type H2B (H2BWT) or a K120R-mutated H2B (H2BK120R) were treated with 12 μM erastin (+) or untreated (-) for 24 h. Representative phase-contrast images were recorded (magnification, ×20), and the surviving cells were counted.

C, Lipid ROS levels were assessed by flow cytometry after C11-BODIPY staining in cells treated as in (B).

D, H1299 cells treated as in (B) were subjected to transmission electron microscopy. Representative images are shown.

E, Control H1299 cells (transfected with siCont. or H2BWT) and H2Bub1-depleted H1299 cells (transfected with siRNF20 or H2BK120R) were treated with erastin either with or without a ferroptosis-specific inhibitor, ferrostatin-1 (2 μM), for 24 h. Representative phase-contrast images were recorded (magnification, ×20), and the surviving cells were counted.

Data information: Bars and error bars are mean±s.d., n=3 independent repeats. Two-tailed unpaired Student's t-test was performed. *: p<0.05, **: p<0.01.
H2B monoubiquitination is a key epigenetic regulator of ferroptosis and is regulated by p53 (2019)
Figure 2

Figure 2. Identification of SLC7A11 as a target of H2Bub1.

A, qRT-PCR (left) and Western blot (right) analyses of H1299 cells transfected with a control siRNA (siCont.) or an RNF20-specific siRNA (siRNF20) and a wild-type H2B (H2BWT) or a K120R-mutated H2B (H2BK120R) for 24 h.

B, Chromatin immunoprecipitation (ChIP) assay was carried out with anti-H2Bub1 antibodies in H1299 cells (left) or 293T cells either untreated or treated with 20 μM erastin for 24 h (right). The intergenic region was used as a negative control for the occupancy of H2Bub1.

C, Intracellular GSH levels were examined in H1299 cells treated as indicated, and bar graphs are shown.

D, H1299 cells transfected as indicated were treated with 12 μM erastin (+) or untreated (-) for 24 h. Representative phase-contrast images were recorded (magnification, ×20).

E, Surviving cells from the assay shown in (D) were counted.

F, GO analysis with the genes downregulated in H2BK120R (black) or RNF20-sepecific siRNA (siRNF20) (red) transfected 293T cells by employing a previously reported microarray data (44).

G, Affected metal ion-binding genes in (F) were selected and subjected to cluster analysis.

H, Labile iron levels were assessed by flow cytometry with a standard method in H1299 cells.

I, Labile iron levels examined in (H) were quantified.

Data information: Bars and error bars are mean±s.d., n=3 independent repeats. Two-tailed unpaired Student's t-test was performed. *: p<0.05, **: p<0.01.
H2B monoubiquitination is a key epigenetic regulator of ferroptosis and is regulated by p53 (2019)
Figure 3

Figure 3. p53 negatively regulates H2Bub1.

A and B, Western blot analysis was performed with H1299 or Hep3B cells transfected with a Flag-tagged p53 or an empty plasmid (Cont.) for 48 h.

C, Immunofluorescence (IF) assay for H1299 cells transfected with a Flag-tagged p53 (Flag-p53) or an empty plasmid (Cont.) for 48 h. Representative images are shown (magnification, ×40). Arrowhead indicates a Flag-p53 overexpressing cell.

D, Western blot analysis was performed with H1299 transfected with a Flag-tagged p53 or an empty vector (Cont.) for 48 h.

E, 293T cells transfected with a control siRNA (siCont.) and three p53-specific siRNAs (sip53) were subjected to Western blot analysis.

F, SMMC cells were treated with 100 μM etoposide (+) or untreated (-) for 24 h, and Western blot analysis was performed.

G, Western blot analysis of A549 cells and H1299 cells.

H, H1299 cells transfected with a Flag-tagged p53 or an empty plasmid (Cont.) were analyzed by micrococcal nuclease (MNase) digestion assay.

I, Western blot analysis of H1299 cells transfected with a wild-type p53 (p53-WT) or two "gain-of-function" mutants (p53-R273H and p53-R175H) for 48 h.