EMBO/soda-nel huggingface dataset:
SMCHD1 Promotes ATM-dependent DNA Damage Signaling and Repair of Uncapped Telomeres (2020)
Figure 6

Figure 6. ATR signalling induction by TPP1 removal rescues the telomere fusion defect in SMCHD1 knockout cells.

(A) Western Blot detection of SMCHD1, TRF2, TPP1 and hnRNPA1 in wild-type or SMCHD1 knockout HeLa cells transfected with the indicated shRNAs (shTRF2, shTPP1, shTRF2/shTPP1) or EV control.

(B) Representative metaphase spreads (Scale bar: 5µm) from HeLa cells transfected with indicated shRNAs or EV control and quantification of telomere fusions. Bars represent average number of fused chromosome ends. SDs were obtained from 3 independent experiments (>2,500 telomeres counted/condition/experiment). (***) P < 0.001; (**) P < 0.01 unpaired two-tailed Student's t-test.

(C) Representative metaphase spreads (Scale bar: 5µm) from HeLa cells transfected with indicated shRNAs or EV control treated for 4 days with ATRi (VE-821) and quantification of telomere fusions. Bars represent average number of fused chromosome ends. SDs were obtained from 3 independent experiments (>2,000 telomeres counted/condition/experiment). (***) P < 0.001; (**) P < 0.01; (*) P < 0.05 unpaired two-tailed Student's t-test.
GRWD1 negatively regulates p53 via the RPL11-MDM2 pathway and promotes tumorigenesis (2016)
Figure 1

Figure 1. GRWD1 silencing increases, whereas GRWD1 overexpression reduces, p53 induction and stability upon nucleolar stress

(A) Wild type (left and middle panels) and p53-/- (right panel) HCT116 cells were transfected with control (mixture of control DS scrambledNeg, siLuci, and siGFP) or GRWD1-targeting (mixture of siGRWD1-3 and 4) siRNAs for 36 h, treated with 5 nM actinomycin D, and harvested at the indicated times. Whole cell extracts were analyzed by immunoblotting with the indicated antibodies. Coomassie Brilliant Blue (CBB) staining serves as a loading control.

(B) Control U2OS cells infected with control retroviruses with the empty vector and U2OS cells overexpressing the siRNA-resistant HA-GRWD1 were treated and analyzed as above.

(C) HCT116 cells were transfected with control (mixture of control DS scrambledNeg, siLuci, and siGFP) or GRWD1-targeting (mixture of siGRWD1-3 and 4) siRNAs for 24 h, treated with 50 µg/ml cycloheximide, and harvested at the indicated times. Whole cell extracts were analyzed by immunoblotting with the indicated antibodies.

(D) HFF2/T cells stably overexpressing HA-GRWD1 were established by retroviral infection. Cells were cultured in the presence or absence of actinomycin D (5 nM) for 12h and then treated with or without MG132 (20uM) for 6 h as indicated. Whole cell extracts were analyzed as above.

(E) Control and HA-GRWD1-overexpressing HFF2/T cells were first treated with actinomycin D (5nM) for 12 h, then further treated with 50 ug/ml cycloheximide, and harvested at the indicated times. Whole cell extracts were analyzed by immunoblotting with the indicated antibodies. The means and SDs from two independent experiments are shown.
GRWD1 negatively regulates p53 via the RPL11-MDM2 pathway and promotes tumorigenesis (2016)
Figure 2

Figure 2. GRWD1 depletion by itself impairs nucleolar integrity and induces nucleolar stress response

(A) U2OS cells were transfected with control (mixture of control DS scrambledNeg, siLuci, and siGFP) or GRWD1-targeting (mixture of siGRWD1-3 and 4 or siGRWD1-1) siRNAs for 24 h. Whole cell extracts were then analyzed by immunoblotting with the indicated antibodies.

(B) U2OS cells treated as above were stained with propidium iodide and subjected to flow cytometry.

(C) HCT116 cells treated with actinomycinD (5nM) or vehicle (DMSO) for 12h were first extracted with Triton X-100 to remove nucleoplasmic proteins, double-immunostained with anti-GRWD1 (green) and anti-fibrillarin (red) antibodies as a marker for nucleoli, and counterstained with DAPI. Scale bar, 20 um.

(D) HCT116 cells were transfected with control (mixture of control siLuci and siGFP) or GRWD1-targeting (mixture of siGRWD1-3 and 4 or siGRWD1-1) siRNAs for 24 h and then immunostained as above. Scale bar, 20 um.
GRWD1 negatively regulates p53 via the RPL11-MDM2 pathway and promotes tumorigenesis (2016)
Figure 3

Figure 3. GRWD1 interacts with RPL11 via the N-terminal domain including the acidic domain

(A) 293T cells were co-transfected with HA-GRWD1 (1.6 ug) and RPL5-FLAG (2.4 ug) or RPL11-FLAG (2.4 ug) as indicated for 42h and subjected to immunoprecipitation with anti-FLAG antibody. Immunoprecipitates (IPs) and 3.125% of inputs were immunoblotted with the indicated antibodies. *, immunoglobulin light chains.

(B) Lysates from untransfected 293T cells were immunoprecipitated with anti-GRWD1 antibody or control IgG. Immunoprecipitates (IPs) and 2.7% of inputs were immunoblotted with the indicated antibodies.

(C) HCT116 cells were treated with 5nM actinomycin D for 12 h or left untreated and subjected to immunoprecipitation with anti-GRWD1 antibody or control IgG. Immunoprecipitates (IPs) and 3.5% of inputs were immunoblotted with the indicated antibodies.

(D) GST-RPL11 or GST alone was incubated with purified GRWD1 and bound proteins analyzed by immunoblotting with the indicated antibodies. I, input. P, pull-down.

(E) Cell lysates were prepared from 293T cells transfected with wild-type (WT) HA-GRWD1-FLAG (12 µg) or various N-terminal truncated mutants of HA-GRWD1-FLAG (12 µg) for 42h, and then subjected to GST pull-down assay with purified GST-RPL11 or GST. Bound proteins were analyzed by CBB staining (GST and GSR-RPL11) and immunoblotting with the indicated antibodies. *, non-specific bands.

(F) Schematic representation of WT GRWD1 and N-terminal truncated mutants used in (D) and following studies.

(G) HCT116 cells were transfected with RPL11-FLAG expression vector (0.56 ug) or empty vector (0.56 ug) for 24 h and then treated with 5 nM actinomycinD or vehicle (PBS) for 12 h. The cells were first extracted with Triton X-100 to remove nucleoplasmic proteins, double-immunostained with anti-GRWD1 antibody (green) and anti-FLAG M2 antibody (red), and counterstained with DAPI. Scale bar, 20 um.
GRWD1 negatively regulates p53 via the RPL11-MDM2 pathway and promotes tumorigenesis (2016)
Figure 4

Figure 4. GRWD1 inhibits the RPL11-MDM2 interaction and alleviates RPL11-mediated suppression of MDM2 ubiquitin ligase activity

(A) H1299 cells were co-transfected with the indicated expression vectors (p53, 7.5 ng; HA-Ub, 0.5 ug; His-Xpress-MDM2, 2 ug; RPL11-FLAG, 1 ug; HA-GRWD1-FLAG, 1.5 ug; GFP, 0.04 ug) for 48 h and analyzed by immunoblotting with the indicated antibodies. GFP serves as a control protein to show equal transfection efficiencies.

(B) Lysates were prepared from 293T cells co-transfected with His-Xpress-MDM2 (1.2 ug), FLAG-RPL11 (1.8 ug), and HA-GRWD1 (1 ug) as indicated for 42 h and then immunoprecipitated with anti-FLAG antibody. Immunoprecipitates (IPs) and 1.5% of inputs were immunoblotted with the indicated antibodies.

(C) In vivo ubiquitination assay to detect MDM2 autoubiquitination in H1299 cells. Lysates were prepared from H1299 cells transfected with the indicated expression vectors (His-Xpress-MDM2, 2 ug; HA-Ub, 0.5 ug; RPL11-FLAG, 1 ug; HA-GRWD1-FLAG, 1.5 ug) for 48 h, treated with proteasome inhibitors for 6 h before harvest, and then immunoprecipitated with anti-MDM2 antibody. Immunoprecipitates (IPs) and inputs were immunoblotted with the indicated antibodies.

(D) In vitro ubiquitination of p53 by immunopurified MDM2. His-Xpress-MDM2 was immunopurified from transfected 293T cells with anti-Omni probe antibody. Recombinant p53 was incubated with E1, E2 (UbcH5a), His-ubiquitin, ATP, GST-RPL11, GRWD1-His, and immunopurified His-Xpress-MDM2 or control immunoprecipitates at 30℃ for 120 min as indicated. The samples were resolved by SDS-PAGE followed by immunoblotting with the indicated antibodies.
GRWD1 negatively regulates p53 via the RPL11-MDM2 pathway and promotes tumorigenesis (2016)
Figure 5

Figure 5. GRWD1 overexpression promotes oncogenic transformation in combination with E7 and activated KRAS

(A) Expression of the introduced proteins and the effect on p53. HFF2/T cells stably expressing HA-GRWD1, E7, and KRAS G12V were established with retroviral vectors and analyzed by immunoblotting with the indicated antibodies. *, endogenous RAS proteins.

(B and C) Anchorage-independent growth of HFF2/T cells overexpressing E7 with or without HA-GRWD1. Three weeks after seeding, images were acquired and colonies >100 µm in diameter were counted. Representative images are shown in (B). Scale bars, 500 um. Average numbers of colonies per field, with SDs, are shown in (C). Six randomly selected areas were counted per sample.

(D and E) Anchorage-independent growth of HFF2/T cells overexpressing E7 and activated KRAS G12V with or without HA-GRWD1. At 9 days after seeding, the colonies were analyzed as above. Representative images are shown in (D). Scale bars, 500 um. The average numbers of colonies (>200 um in diameter) per field, with SDs, are shown in (E). Twenty randomly selected areas were counted.

(F) Tumorigenesis of HFF2/T cells expressing E7 and KRAS G12V with or without HA-GRWD1. Cells were subcutaneously injected into nude mice (1 x 106 cells/mouse), and tumor size was measured. Average tumor volumes, with SDs, are shown (n=5).

(G) High expression of GRWD1 is associated with poor prognosis of brain lower grade glioma patients with wild type p53 but not with mutated p53. The data were obtained from the TCGA databases (the cBioPortal for Cancer Genomics; http://www.cbioportal.org). For detail, see Materials and Methods.
GRWD1 negatively regulates p53 via the RPL11-MDM2 pathway and promotes tumorigenesis (2016)
Figure 6

Figure 6. The abilities of GRWD1 to inhibit the nucleolar stress response and promote tumorigenesis require interaction with RPL11 via the N-terminal domain

(A) 293T cells were co-transfected with HA-GRWD1 wild-type (WT) (0.5 ug) or HA-GRWD1 Δ1-136 (3.5 ug) and RPL11-FLAG (1.5 ug) as indicated for 42h, and then immunoprecipitated with an anti-FLAG antibody. Immunoprecipitates (IPs) and 1.6% of inputs were immunoblotted with the indicated antibodies.

(B) 293T cells were co-transfected with His-Xpress-MDM2 (0.6 ug), RPL11-FLAG (0.9 ug), and HA-GRWD1 WT (0.5 ug) or HA-GRWD1 Δ1-136 (4.5 ug) as indicated for 42h and subjected to immunoprecipitation with an anti-FLAG antibody. Immunoprecipitates (IPs) and 1.6% of inputs were immunoblotted with the indicated antibodies.

(C) H1299 cells were co-transfected with the indicated expression vectors (p53, 7.5 ng; HA-Ub, 0.5 ug; His-Xpress-MDM2, 2 ug; RPL11-FLAG, 1 ug; HA-GRWD1-FLAG WT, 1.5 ug; HA-GRWD1-FLAG Δ1-136, 1.5 ug; GFP, 0.04 ug) for 48 h, and then analyzed by immunoblotting with the indicated antibodies. GFP serves as a control protein to show equal transfection efficiencies.

(D) HFF2/T cells stably expressing HA-GRWD1 wild-type (WT) or Δ1-136, E7, and KRAS G12V were established using retroviral vectors and analyzed by immunoblotting with the indicated antibodies. *, endogenous RAS proteins.

(E and F) Anchorage-independent growth of HFF2/T cells overexpressing E7 and activated KRAS G12V with HA-GRWD1 WT or Δ1-136 (or without HA-GRWD1). At 9 days after seeding, colonies were analyzed as above. Representative images are shown in (E). Scale bars, 500 um. Average numbers of colonies (>200 um in diameter) per photographed area with SDs are shown in (F). Seventy randomly selected areas were counted.
Transcriptional repression of NFKBIA triggers constitutive IKK- and proteasome-independent p65/RelA activation in senescence (2020)
Figure 1

Figure 1. NF-κB is activated in two distinct phases in response to DNA damage corresponding to anti-apoptotic and pro-inflammatory gene programs

(A) SA-β-gal staining of U2-OS cells, harvested at indicated times post irradiation (IR); h, hours; D, days; UT, untreated. n = 3. Scale bar: 50 μm. Bar Graph: Significance was determined by Brown Forsythe ANOVA with Dunnett's 3T test, using n = 3 biological replicates. SD shown. *** p < 0.001, n.s. = not significant.

(B) Nuclear fractions from U2-OS cells were harvested at indicated time points post IR (20 Gy) as in (A). Top panel: NF-κB DNA binding was analyzed by EMSA. Lower panels: Western blot of indicated NF-κB subunits and PARP1 as loading control. The two phases of nuclear translocation and DNA binding are indicated. Representative gel shown from n = 5 biological replicates.

(C) Gene expression analysis of two NF-κB phases following damage. RNA-Seq analysis (n = 5 biological replicates per group) of U2-OS cells, either untreated or analyzed 1.5 hours or 7 days after irradiation (20 Gy), as indicated. The heatmap shows significantly regulated genes (log2 value > 0.5 and p value < 0.05). Black arrow points to NFKBIA. Significance determined by ANOVA with Bonferroni correction for multiple testing. See also Dataset EV1.

(D) Female mice 12-16 weeks old (n = 6 mice per condition (with three technical repeats per mouse), were irradiated (5 Gy) and sacrificed after 1 hour (first phase), 7 days (second phase) or left untreated. Skin or renal tissue RNA was analyzed by RT-qPCR, shown as a mean +/- SD. Significance was confirmed by ANOVA with Tukey multiple comparisons test. SD shown: * = p < 0.05, ** = p < 0.01.

(E) In situ hybridization using an anti-sense IκBα/Nfkbia riboprobe on longitudinal skin sections from male and female mice (5-8 weeks of age) treated as in (D) for the time indicated. Representative sections shown from n = 6 mice per condition. Bu, bulge region (dashed lines), HS, hair shaft, IFE, interfollicular epidermis. Scale bar: 50 μm.

(F) Quantitation of (E) from n = 6 mice per condition and 3-4 sections per mouse. Bulge regions positive for Nfkbia were counted and presented as percentage of total. One way ANOVA analysis with Tukey multiple comparisons test was performed. SD shown. ** = p < 0.01.

(G) Skin sections as in (E) analyzed by immunofluorescence with IκBα (red) or IL-6 antibody (green) and nuclear DAPI staining (blue). Dotted lines delineate hair follicles, Bu, bulge region, HS, hair shaft, IFE, interfollicular epidermis, SG, sebaceous gland. Scale bar: 50 μm.

(H) Quantitation of (G) from n = 6 mice per condition and 3-4 sections per mouse. Bulge regions positive for IκBα or IL-6 were counted and presented as percentage of total. One way ANOVA analysis with Tukey multiple comparisons test was performed. SD shown. * = p < 0.05, *** = p < 0.001.
Transcriptional repression of NFKBIA triggers constitutive IKK- and proteasome-independent p65/RelA activation in senescence (2020)
Figure 2

Figure 2. Knockdown of NFKBIA in cell lines and knockout in vivo mimics second phase NF-κB activity and triggers SASP

(A) Left panel: IκBα western blot of U2-OS cells. Lane 1, untreated (ut); Lanes 2-4, single IR (20 Gy) at indicated time points prior to harvest; Lane 5, IR at 5 days prior to harvest, plus 1.5 hours prior to harvest. Right panel: Samples treated as in the left panel, analyzed by RT-qPCR. Significance from n = 3 replicates per condition determined by ANOVA analysis with Tukey multiple comparisons test. SD shown. *** = p < 0.001.

(B) NFKBIA knockdown by dox-inducible shRNA in U2-OS cells. Cells were pre-treated with Dox to induce knockdown. For knockdown efficiency see Fig. S2F. Cells were irradiated (20 Gy) or not, as indicated and described above. NF-κB activity was analyzed by EMSA. ns, non-specific band. A representative gel from n = 3 biological replicates is shown.

(C) Venn diagram of RNA-Seq analysis of genes activated following irradiation in the first and second NF-κB activity phases or following shRNA-mediated NFKBIA knockdown in non-irradiated cells (as in B). Numbers of overlapping genes are shown in red. Hypergeometric probability calculation showed significance between overlaps (adjusted with Bonferroni correction for multiple testing). SD shown. *** = p < 0.001.

(D) Top GO terms obtained using DAVID Functional Annotation Bioinformatics Microarray 6.7 for first and second phase genes and genes activated upon NFKBIA knockdown, as in (C), as indicated.

(E) GSEA was performed comparing the IκBα IEC‐KO gene list (n = 4 villin-Cre x floxed IκBα mice and n = 4 littermate controls (GSE 139251) Mikuda et al, 2020(), with a gene signature based on a list of SASP factors Coppe et al, 2010(). NES: normalized enrichment score. FDR: false discovery rate.

(F) RT-qPCR analysis of indicated genes was performed using RNA extracted from the duodenum of 5 and 8 weeks old control villin-Cre (n = 6) or villin-Cre x floxed IκBα mice (n = 6) both male and female. Expression is shown as fold change between littermates, paired t-test. SD shown. * p < 0.05 ** = P < 0.01.
Transcriptional repression of NFKBIA triggers constitutive IKK- and proteasome-independent p65/RelA activation in senescence (2020)
Figure 3

Figure 3. RelA regulates SASP but not cell cycle arrest

(A) Nuclear fractions from either untreated or irradiated (20 Gy) U2-OS cells, 1.5 hours or 7 days prior to harvest, were analyzed by EMSA with supershift analysis. For supershifting, lysates were incubated with antibodies or water, as indicated. Arrows point to shift location of a given subunit, due to antibody binding. ns, non-specific band. All antibodies were previously tested for supershift compatibility. Representative gel shown from n = 3 replicates.

(B) Expression of SASP targets of NF-κB obtained from RNA-seq analysis (Dataset EV1B), was quantitated using RT-qPCR from the shRNA (RELA) stably expressing U2-OS cells. For knockdown efficiency see Appendix Fig S4A. Cells were treated with doxycycline (Dox) to induce knockdown of p65. Heatmap represents targets normalized to the untreated Scrambled control. Expression is shown as log2 change with p values < 0.05. Statistical analysis performed using ANOVA with Bonferroni correction for multiple testing. First-phase and second-phase samples were irradiated (20 Gy) 1.5h and 7 Days prior to harvest, respectively. Analysis based on n = 3 biological replicates.

(C) Cell duplication was measured at time points indicated in U2-OS cells bearing either scrambled control or Dox-inducible shRNA against RELA (n = 3 biological replicates). Treatment with Dox was initiated at two days prior to IR (20 Gy) or after IR. Statistical significance in total duplication number between groups at day 6 was determined by ANOVA with Tukey multiple comparisons test. SD shown. * = P < 0.05, *** = p < 0.001.
Transcriptional repression of NFKBIA triggers constitutive IKK- and proteasome-independent p65/RelA activation in senescence (2020)
Figure 4

Figure 4. p65 Ser 468 phosphorylation in senescence inhibits NFKBIA expression

(A) Nuclear (Nuc.) and cytoplasmic (Cyto.) fractions of U2-OS cells were analyzed at times indicated after IR exposure by SDS-PAGE western blotting. PARP1 and LDHA are fractionation controls and phospho-specific p65 signals are indicated. Representative gels from n = 3 biological replicates are shown. Right panel: quantitation of nuclear p65 and phosphorylated p65 species (n = 3). Fold changes compared to untreated samples (ut) and time points post IR are indicated on Y and X axes, respectively.

(B) U2-OS cells treated with dox to deplete endogenous p65, were irradiated (20 Gy) and transfected with plasmids encoding p65, p65-S276A or p65-S468A, as indicated. Nuclear (N) and cytoplasmic (C) lysates were analyzed by SDS-PAGE at day seven post IR. Representative gel from n = 3 biological replicates is shown. PARP1 and αTubulin serve as fractionation and loading controls.

(C) ChIP performed with U2-OS cells irradiated (20 Gy) 1.5h or 7 days prior to assay, or left untreated (UT). Normalized relative enrichment of NFKBIA is shown (relative to input and two non-recruiting reference regions). Left panel: p65 antibody. n = 5 biological replicates with 3 technical repeats per biological replicate. Right: Antibody against p65 pSer468. n = 2 biological replicates with three technical repeats per biological replicate. Statistical significance was determined by ANOVA with Tukey multiple comparisons test. SD shown. * = p < 0.05, ** = p < 0.01, *** = p < 0.001

(D) Kinase proteome array was performed with nuclear fractions of U2-OS cells that were either left untreated (UT) or irradiated (20 Gy) at time points indicated prior to harvest. Kinase binding to membrane was quantitated (n = 2) and only significant results (p < 0.05) are shown. Significance was determined by ANOVA with Tukey multiple comparisons test.

(E) U2-OS cells left untreated (ut) or irradiated (20 Gy) 7 days before harvesting, as indicated. Two days prior to harvest cells were transfected with siRNA against GSK3β or scrambled control (Scr). Whole cell lysates were analyzed by SDS-PAGE and western blotting. A representative example from n = 3 experiments is shown.

(F) U2-OS cells were left untreated or exposed overnight to 10 mM LiCl, with or without prior irradiation, as indicated, and analyzed as in (E). Representative data from n = 3 experiments are shown.

(G) RT-qPCR of U2-OS cells irradiated (20 Gy) either 1.5h or 7 days prior to harvest. CHIR denotes treatment with GSK3 inhibitor CHIR-99021 (10nM overnight). DMSO overnight treatment was used as control. Quantitation was performed and statistical significance obtained from n = 3 replicates, using ANOVA with Tukey multiple comparisons test. SD shown. * = p < 0.05, ** = p < 0.01, *** = p < 0.001.
Transcriptional repression of NFKBIA triggers constitutive IKK- and proteasome-independent p65/RelA activation in senescence (2020)
Figure 5

Figure 5. The second phase is IKK- and proteasome-independent

(A) Diagram showing DNA double strand break (DSB) induced first phase NF-κB activation through an ATM/TRAF6 and PARP1/PIASy dependent IKK activation mechanism Hinz et al, 2010Stilmann et al, 2009Wu et al, 2006(; ; ).

(B) SDS-PAGE western blot of whole cell lysates of U2-OS cells irradiated (20 Gy) at time points indicated prior to harvest. ut, untreated. LDHA. Representative gel shown from n = 4 biological replicates.

(C) U2-OS cells transfected with TRAF6 siRNA or scrambled control siRNA and TRAF6 and IKKβ levels analyzed by western blotting (top panels). NF-κB activity was determined by EMSA in untreated cells (ut) and after IR at indicated time points (lower panel). ns, unspecific band. Representative gels are shown from n = 2 biological replicates.

(D) U2-OS cells were exposed to IR at indicated time points prior to harvest and analyzed by western blotting for levels of IκBα, NIK, IL-1α and PARP1. Treatment with proteasomal inhibitor MG132 (10 μM) started 4 hours prior to harvest. NIK serves as positive control for efficient proteasome inhibition. Quantitation from n = 4 biological replicates and SD is shown for IκBα (right panel). Brown-Forsynthe ANOVA test: p = 0.0005.

(E) U2-OS CRISPR IKBKB knockout and control cell lines were irradiated (20 Gy) and harvested at indicated time points. NF-κB activity was analyzed by EMSA (lower panel). ns indicates an non-specific band. Upper panel, western blot analysis of actin and IKKβ. Representative gels shown from n = 3 replicates.

(F) Irradiated U2-O2 control and IKBKB knockout cells (as above) were analyzed by RT-qPCR at the time points indicated for expression of NF-κB target genes identified by RNA-Seq (Dataset EV1). Significantly regulated targets, cutoff set at p = 0.05 (as determined by ANOVA with Tukey multiple comparison), are shown.