EMBO/SourceData huggingface dataset:
Figure 3A. Jurkat cells were pre-labeled with light or medium L-Arg isotopes. A label swap was performed of cells growing in medium Arg, whereas light cells were cultured in the same medium. Cells were harvested at different time points and equal proteome amounts were mixed, followed by N-terminal COFRADIC fractionation and LC-MS/MS analysis.C. The distribution of turnover times (N=1,894) in our experiment was not unimodal, with a sizable number of quickly turned over proteins, with the median protein turnover rate being 21.6 h. The doubling time of Jurkat cells (24 h) is indicated in red.
Figure 6A. Degradation profiles of AIMP2, MARE2 and AN32E dbTIS and aTIS proteoforms as representative examples of N-terminal proteoforms that differ in stability.B. MS spectra of the N-termini of two proteoforms originating from the aminoacyl tRNA synthase complex-interacting multifunctional protein 2 (AIMP2): a dbTIS indicative N-terminus with processed iMet and starting at the second amino acid Pro, an aTIS indicative peptide starting at position 3 and retaining its iMet. As evident from the MS spectra shown, these N-terminal proteoforms displayed largely different turnover rates.
Figure 8A. Jurkat cells were treated with 100 µg/ml CHX for 0, 0.5, 1.5, 4, 8, 12 or 24 hours. Protein degradation was monitored by Western blotting and stabilities of several short-lived endogenous proteins were confirmed using antibodies (including lamin B, securin, β-catenin, GCIP interacting protein p29) and compared to stable proteins (such as GAPDH and actin). Proteoform-specific bands were used to calculate protein half-lives which were in a good agreement with turnover times obtained from pSILACB-C. Validation of the differential turnover time of the dbTIS and aTIS derived proteoforms of MARE2 and AN32E. Selective C-terminal V5-tagged proteoforms were overexpressed in HCT116 cells for 24 hours and CHXpulse-chase experiments were performed as described for Jurkat cells. Degradation of overexpressed proteoforms was monitored by an anti-V5 antibody and compared to the turnover of stable proteins such as actin and tubulin.
Figure 5H, I WT and lincRNA-EPS-/- iBMMs were infected with VSV (MOI 0.1) for 6 h or 10 h (H) or transfected with polyI:C (1 μg/mL) for 4 h or 8 h (I), phosphorylation and total protein expression were analyzed by Western blot with α-tubulin and β-Actin as loading controls.
Figure 6B Western blot for verification of RNA pulldown-MS results with β-Actin as a negative loading control.D 20 nM siRNA targeting negative control (NC) and PKR were transfected into iBMMs for 48 h, the protein and mRNA expression level of PKR were tested by RT-qPCR and Western blot, respectively.G VSV-N and VSV-G mRNA were transcribed from virus particles for RNA-pulldown, the interactions between VSV RNA with PKR protein was checked by Western blot, β-Actin was shown as a negative control.H LincRNA-EPS was transcribed in vitro and pulled down proteins from iBMMs infected with VSV for 4 h, PKR protein was detected by Western blot and GAPDH was shown as a loading control.I RNA-pulldown using biotinylated lincRNA-EPS from cell lysates of polyI:C (1 and 2.5 μg/mL) transfected iBMMs for 4 h, PKR protein was detected by Western blot and β-Actin was shown as a loading control.
Figure 7A 20 nM siRNA for negative control (NC) and PKR were transfected into iBMMs following infected with VSV (MOI 0.1) for 4 h or 8 h, phosphorylation and total levels of STAT1, eIF2α and PKR were detected by Western blot and β-Actin was shown as a loading control.B PKR was transfected into A549 cells for 24 h following stimulated with IFN-β (50 ng/mL) for 1 h to test STAT1 phosphorylation by Western blot and β-Actin was shown as a loading control.
Figure 1(A) HT29 cells were infected with Shigella WT, S325, or ospD deletion mutants, and incubated for 8 h. Aliquots of cellular supernatants were subjected to cytotoxicity assays. *P<0.05 (one-way ANOVA).(B) HT29 cells were infected with Shigella WT or ∆ospD3 and incubated for 8 h. Infected cells were fixed and subjected to TUNEL and PI staining. Percentages of positive cells (TUNEL, green; PI, red) are shown in graph at right. The nuclei were stained with DAPI (blue). Scale bar: 100 μm. n.s., not significant; *P<0.05 (unpaired two-tailed Student's t-test).(C) HT29 cells were infected with Shigella WT, S325, or ∆ospD3 and incubated for 8 h. Infected cells were subjected to Giemsa staining. Arrows indicate cells in which the cytoplasm disappeared. Scale bar: 20 μm.
Figure 2(A) HT29 cells were infected with Shigella WT, S325, or ospD deletion mutants, and incubated for 8 h. Cell lysates were subjected to immunoblotting.(B HT29 cells were infected with the indicated Shigella strains in the presence or absence of RIPK1 inhibitor, RIPK3 inhibitor, or caspase inhibitor (z-VAD), and then incubated for 8 h. Cell lysates and aliquots of cellular supernatants were subjected to immunoblottingC) HT29 cells were infected with the indicated Shigella strains in the presence or absence of RIPK1 inhibitor, RIPK3 inhibitor, or caspase inhibitor (z-VAD), and then incubated for 8 h. Cell lysates and aliquots of cellular supernatants were subjected to cytotoxicity assay(D HT29 cells treated with the indicated siRNAs were infected with Shigella WT or ∆ospD3. Cell lysates and aliquots of cellular supernatants were subjected to immunoblotting (top) and cytotoxicity assays (bottom), respectively. The knockdown efficiency of the indicated siRNAs was assessed by immunoblotting (inset). n.s., not significant; *P<0.05 (unpaired two-tailed Student's t-test).E) HT29 cells treated with the indicated siRNAs were infected with Shigella WT or ∆ospD3. Cell lysates and aliquots of cellular supernatants were subjected to immunoblotting (top) and cytotoxicity assays (bottom), respectively. The knockdown efficiency of the indicated siRNAs was assessed by immunoblotting (inset). n.s., not significant; *P<0.05 (unpaired two-tailed Student's t-test).
Figure 3(A) HT29 cells were infected with the indicated Shigella strains and incubated for 8 h, and then cell lysates were subjected to immunoblotting.(B) 293T cells were transfected with Myc-tagged ospD expression plasmids. After 24 h, cells were harvested and subjected to immunoblotting. Arrows indicate cleaved RIPK1.D) 293T cells were transfected with a series of plasmids expressing ospD3 point mutants. After 24 h, cells were harvested and subjected to immunoblotting. Arrows indicate cleaved RIPK1.(E HT29 cells were infected with Shigella WT, S325, ∆ospD3, ∆ospD3/D3 (∆ospD3 complemented with wild-type ospD3), or ∆ospD3/D3CS (∆ospD3 complemented with a protease activity-deficient mutant, in which the cysteine residue at position 64 was replaced by serine) strains and incubated for 8 h. Cell lysates and aliquots of cellular supernatants were subjected to immunoblottingF) HT29 cells were infected with Shigella WT, S325, ∆ospD3, ∆ospD3/D3 (∆ospD3 complemented with wild-type ospD3), or ∆ospD3/D3CS (∆ospD3 complemented with a protease activity-deficient mutant, in which the cysteine residue at position 64 was replaced by serine) strains and incubated for 8 h. Cell lysates and aliquots of cellular supernatants were subjected to cytotoxicity assays
Figure 4(A) HT29 cells were infected with the indicated Shigella strains and incubated for 8 h. Cells were harvested and subjected to measurement of caspase-8 activation. Caspase-8 activity is reported as relative light units (RLU) of infected samples, minus the value in uninfected samples. *P<0.05 (one-way ANOVA).(B) HT29 cells were infected with the indicated Shigella strains and incubated for 8 h. Cell lysates were subjected to immunoblotting. Arrows indicate cleaved forms of caspase-8, PARP, GSDMD, and IL-18, respectively.(C) HT29 cells were infected with the indicated Shigella strains and incubated for 8 h. Aliquots of cellular supernatants were subjected to cytotoxicity assay. *P<0.05; n.s., not significant (one-way ANOVA).(D) HT29 cells were infected with Shigella WT or ∆ospC1 mutant and incubated for 8 h. Infected cells were then fixed and stained with cleaved caspase-8 (green), rhodamine-phalloidin (red), and DAPI (blue). Percentages of positive cells are shown in the graph at right (*P<0.05; unpaired two-tailed Student's t-test). Scale bar: 100 μm.(E) HT29 cells were infected with the indicated Shigella strains and incubated for 8 h. Cells were harvested and subjected to measurement of caspase-3/7 activation. Caspase-3/7 activity is reported as relative light units (RLU) of infected samples minus uninfected samples. *P<0.05 (one-way ANOVA).(F) HT29 cells were infected with Shigella WT, S325, or ∆ospC1 strains, and incubated for up to 8 h. After incubation for the indicated times, cells were subjected to real-time Annexin V apoptosis assay. Annexin V binding is reported as relative light units (RLU) in infected samples minus the value in uninfected samples. *P<0.05 (one-way ANOVA).
Figure 5(A HT29 cells were infected with Shigella WT, ∆ospC1, ∆ospD3, ∆ospC1∆ospD3, or ∆ospC1∆ospD3/ospC1 (∆ospC1∆ospD3 complemented with ospC1) strains and incubated for 8 h. Cell lysates and aliquots of cellular supernatants were subjected to immunoblottingB) HT29 cells were infected with Shigella WT, ∆ospC1, ∆ospD3, ∆ospC1∆ospD3, or ∆ospC1∆ospD3/ospC1 (∆ospC1∆ospD3 complemented with ospC1) strains and incubated for 8 h. Cell lysates and aliquots of cellular supernatants were subjected to cytotoxicity assay(C) HT29 cells were infected with Shigella WT, ∆ospD3, or ∆ospC1. Cell lysates obtained at the indicated time points were subjected to immunoblotting.(D HT29 cells treated with DMSO or caspase-8 inhibitor were infected with Shigella WT, ∆ospC1, ∆ospD3, or ∆ospC1∆ospD3 strains and incubated for 8 h. Cell lysates and aliquots of cellular supernatants were subjected to immunoblottingE) HT29 cells treated with DMSO or caspase-8 inhibitor were infected with Shigella WT, ∆ospC1, ∆ospD3, or ∆ospC1∆ospD3 strains and incubated for 8 h. Cell lysates and aliquots of cellular supernatants were subjected to cytotoxicity assay(F) HT29 cells treated with the control or caspase-8 siRNAs were infected with the indicated Shigella strains and incubated for 8 h. Cell lysates were subjected to immunoblotting. The knockdown efficiency of the indicated siRNAs was assessed by immunoblotting.
Figure 3 A, B Core histones were prepared from NSPCs treated with various concentrations of crotonate (A) and 1 μM MS-275 (B) for 24 h and subjected to western blotting analysis using anti-Kcr antibody, and the experiments were repeated three times.  C NSPCs were treated with 20 μM C646, 10 mM acetate (Ac), 20 μM C646 and 10 mM acetate (Ac+C646), 10 mM crotonate (Cr), and 20 μM C646 and 10 mM crotonate (Cr+C646) for 24 h, then core histones were extracted and subjected to western blotting analysis using anti-Kcr antibody, the experiments were repeated three times.
Figure 4 E Quantification analysis of changes in histone Kcr levels in NSPCs treated with DMSO (Control), 5 μM triptolide (FP), 5 μM flavopiridol and 10 mM crotonate (FP+CR), and 10 mM crotonate (CR) for 1 h. Data are presented as mean ± SEM of three independent biological replicates, n = 3. One-way ANOVA with post hoc Tukey's test was used to analyze statistical significance; NS: no significance, P < 0.01, P < 0.001.  F Co-IP analysis indicating RNAP2 binding in H3K9cr enriched regions in NSPCs. H3 was used as a loading control for the input nuclear proteins.  G Quantification analysis of changes in chromatin openness at Tgfb1 and Notum promoter regions by FAIRE-qPCR among different groups of NSPCs. Data are presented as mean ± SEM of three independent biological replicates, n = 3. Two-tailed unpaired Student's t-test was used to analyze statistical significance; P < 0.05, P < 0.01.
Figure 5 A, B Representative images (A) and quantification analysis (B) of changes in cell proliferation ability measured by BrdU incorporation rate among different groups of NSPCs. Scale bar, 50 μm. Data are presented as mean ± SEM of three independent biological replicates, n = 3. Two-tailed unpaired Student's t-test was used to analyze statistical significance; P < 0.01.  C, D Representative images (C) and quantification analysis (D) of changes in neuronal differentiation potential defined by TUJ1 positivity among different groups of NSPCs. Scale bar, 20 μm. Data are presented as mean ± SEM of three independent biological replicates, n = 4. Two-tailed unpaired Student's t-test was used to analyze statistical significance; P < 0.001.  I, J Quantification analysis of expression changes of Mir-203 (I) and Bmi1 (J) by qRT-PCR among different groups of NSPCs. Data are presented as mean ± SEM of three independent biological replicates, n = 3. Two-tailed unpaired Student's t-test was used to analyze statistical significance; P < 0.05, P < 0.01.  K Representative blots (top panel) and quantification analysis (bottom panel) of changes in BMI1 protein levels among different groups of NSPCs. Data are presented as mean ± SEM of three independent biological replicates, n = 3. Two-tailed unpaired Student's t-test was used to analyze statistical significance; P < 0.05.
Figure 6 A, B Core histones were prepared from E13.5 forebrain explants treated with various concentrations of crotonate for 3 h and subjected to western blotting analysis using anti-H3K9cr (A) and anti-Kcr (B) antibodies. Naïve: isolated forebrains without subsequent culture and treatment. C, D Quantification analysis of changes in H3K9cr (C) and H3-Kcr (D) levels by western blotting among different groups of explants. Data are presented as mean ± SEM of three independent biological replicates, n = 3. Two-tailed unpaired Student's t-test was used to analyze statistical significance; NS: no significance, P < 0.01, P < 0.001.  G, H Quantitative analysis of changes in H3K9cr (G, left panel of H) and H3K14la (right panel of H) levels by western blotting among different groups of the embryonic forebrain. Data are presented as mean ± SEM of three independent biological replicates, n = 3. Two-tailed unpaired Student's t-test was used to analyze statistical significance; NS: no significance, P < 0.05, P <0.001.
Figure 2Two top-ranked SplashRNA-designed shRNAmirs targeting human Nurd complex components and a control shRNAmir targeting human CD4 were cloned into both miR-AB and miR-E lentiviral vectors with Venus as reporter. The lentiviruses packaged by these constructs were used for transduction of multiple human cell lines. RNAi efficiency was determined by western blot (WB) analysis of target protein levels in the lysates of the transduced cells. -actin served as loading control.The RNAi efficiency of Pten.1524 and Pten.932 shRNAmir mediated by miR-AB vs. miR-E was tested in NIH-3T3 cells in the context of <30% transduction efficiency (GFP as reporter). Representative western blots of Pten protein expression of the FACS-sorted GFP+ cells from two biological replicates were shown. β-actin served as loading control.
Figure 4293T cells were co-transduced with four miR-AB lentiviruses at 1:1 ratio, which carries a neutral shRNAmir targeting CD4 or CD19, with Azurite, GFP, Ametrine or mOrange as reporter respectively. Cells were cultured for 22 days and fluorescence positive cells were quantified at the time points as indicated. Representative contour FACS plots cells on day 4 and day 22 shown (top panels). Cell numbers of untransduced, single fluorescence positive cells and quadruple positive cells were quantified and normalized to untransduced cells on day 4, and plotted over time (bottom panel). Data are shown as mean ± s.d. and n=4. NS indicates no significant difference (P>0.05, two-tailed unpaired Student's t-test).
Figure 1(C) qRT-PCR expression of endogenous repeats following shRNA-mediated KAP1 depletion in NTERA-2 cells. Results were normalized to β2 microglobulin (B2M). KAP1 expression levels were verified by qRT-PCR and Western blot. A representative experiment of 2 experiments is shown. Two-tailed unpaired t tests were done (HERVK14C_2 p-value = 0.002300).(D qRT-PCR expression of endogenous repeats following KAP1 knockout in HeLa Results were normalized to B2M. KAP1 expression levels were verified by qRT-PCR and Western blot. Two-tailed unpaired t tests were done and p-values are: HERVK14C_1: 0.0017, HERVK14C_2: 0.0044, SVA D VNTR: 0.0243 Clones 8, 12 and 15 (from D) were selected for mRNA-sequencing.E) qRT-PCR expression of endogenous repeats following KAP1 knockout in 293T cells Results were normalized to B2M. KAP1 expression levels were verified by qRT-PCR and Western blot. For (E), we found that clone I (also treated with KAP1 sgRNA) retained KAP1 protein expression (E, right panel) so we only used clones II, III and IV to explore phenotype (E, left panel), which represented validated KAP1 knockouts. Two-tailed unpaired t tests were done and p-values are: (E) HERVK14C_2: <0.0001.(F) qRT-PCR expression of endogenous repeats following shRNA-mediated KAP1 depletion in PBMCs (day 6 post-transduction). Results were normalized to B2M. All error bars show standard deviation (SD). All numbers above bars depict fold changes compared to control cells (to one decimal place).
Figure 2(A) Boxplots showing repeats significantly upregulated (>2 fold where p=<0.05 using DESeq2) in knockout (N=3) compared to wildtype (N=3) HeLa cells based on mRNA-sequencing data. P values = 0.0174 (HERV-S), 0.0047 (HERV-K14C), 0.00013 (HERV-T LTR6B) and 1.90E-06 (HERV-T HERVS71). HERV-T and HERV-S but not HERVK14C also reached significance when only adjusted p-values were considered, where differences are compared to the whole of repBase. KAP1 binding (ENCODE data) is shown in Fig S1A. All numbers above bars depict fold changes compared to control cells (to one decimal place).(F) Genomic coordinates of the common KAP1 sites identified in Fig EV3D were subjected to ChIP-seq correlation analyses using ChIP-Cor software (see methods). Each plot shows duplicate ChIP-seq experiments from ENCODE. See Fig EV3B for complete data.
Figure 3(B) NTERA-2 cells were transduced with either an empty vector (WT) or the same vector containing an shRNA against KAP1 (KD) prior to puromycin selection and transduction with increasing doses of GFP reporter vectors. GFP expression was analysed 72 hours post reporter transduction. A representative experiment of 2 experiments is shown. Two-tailed unpaired t test p-values: PBS-LTR-GFP: 0.0148; PBSChr15-LTR-GFP: 0.0377(C) The same as (B) except that here KAP1 wildtype (WT) and knockout (KO) 293T cells were used. P-values: PBS-LTR-GFP: 0.0044; PBSChr15-LTR-GFP: 0.0077.(D) PBMCs were transduced with increasing doses of GFP reporter vectors (vectors were normalized to the same number of infectious units after titering them on permissive cells), left plot. Vectors were integrated at similar levels as measured by GFP Taqman qPCR, right plot. A 293T cell line with a single vector copy integrant (PGK-GFP) was used as a control to estimate the absolute copy numbers. A representative experiment of 3 experiments is shown here. Two-tailed unpaired t test p-value 0.0288 (dose 3). Error bars show SD.
Figure 4(A) qRT-PCR expression of endogenous repeats (left) and ISGs (right) following shRNA-mediated KAP1 depletion in HeLa cells (day 6 post transduction), normalized to B2M and GAPDH respectively. n=2 and two-tailed unpaired t test p-values are: HERVK14C: 0.025, 0.003; SVA VNTR: 0.00027, 0.01; ISG56: 0.002; CCL5: 0.007, 0.00003; CXCL10: 0.0008; Mxa: 0.011; IKba: 0.008, 0.01. See also Fig S2AB.(B) qRT-PCR expression of endogenous repeats (left) and ISGs (right) following 5-AZA treatment of HeLa cells and PBMCs (day 6 post transduction). A representative experiment of two is shown in each case. Two-tailed unpaired t tests were performed. HeLa cell p-values: ISG56: 0.005; CCL5: 0.0006; CXCL10: 0.0007; Mxa: 0.0001; IFNb: 0.02. For repeat expression (left panel), p values not available as N=2. PBMC p-values: HERVK14C: 0.1; SVA D VNTR: 0.003; ISG56: 0.001; CCL5: 0.012; CXCL10: 0.012; Mxa: 0.02; IKba: 0.08. Results were normalized to GAPDH and B2M (GAPDH results shown). See also Fig S2C for results in 293T cells. All error bars show SD.(C) DNA methylation at endogenous SVAs was measured over 18 CpGs in the stated treatment groups (day 5 post transductions, or day 2 post 5-AZA treatment). Each point represents the average methylation state of one sequence with at least 10 sequences analyzed per group. Mann-Whitney tests were performed to compare shControl to shKAP1 (p=0.6593) and DMSO to 5-AZA (0.0027). 5-AZA was added to all experiments at 7uM. All numbers above bars depict fold changes compared to control cells (to one decimal place).
Figure 5(A) H3K9me3 ChIP-PCR where error bars show SD of 3 independent IPs. Results show the IP enrichment relative to the total input normalized to GAPDH as a negative control region. IgG control IPs gave only background enrichments, shown.(B) 293T cells were transduced with the stated shRNAs and cells harvested 4 days after puro selection for Western Blot to verify KAP1 and SETDB1-depletion (left) and ChIP (right). Cell numbers were normalized per treatment group before sonication and IPs were performed in triplicate. Results show IP relative to total input and GAPDH normalized. IgG control IPs gave only background enrichments shown. One representative experiment of two is shown. Unpaired t tests were performed. P values: ZNF239: KAP1, 0.0292; SETDB1, 0.0137; HERVK14C_1: KAP1, 0.0227; SETDB1, 0.0082; HERVK14C_2: SETDB1, 0.0462; SVA D VNTR, SETDB1, 0.0064. The inlay shows qRTPCR expression analysis of HERVK14C in the same experiment, normalized to B2M or GAPDH as stated. All error bars show SD.(C) Venn diagram showing H3K9me3 enriched sites (18271) as those identified in both ChIP replicates in the human cell line K562 (left). Right: % of ISGs or random genes intersecting a H3K9me3 or KAP1 ChIP-seq peak. 5kb upstream and downstream of each gene was also included. N=437 genes per group.(D) An IFIT1 promoter-based Luciferase reporter THP-1 cell line was used or 293T cells or HeLa cells transduced with an IFN-β promoter-based Luciferase reporter. These cell lines were transduced with an shKAP1 or shControl vector or a vFLIP-expressing vector and luciferase measured 6 days later. Error bars show SD. Unpaired t tests were used to compare shControl to shKAP1 samples: p values = 0.122 (THP-1), 0.0001 (HeLa), 0.0001 (293T cells).(E) WT, STING KO and MAVS KO THP-1 cells were verified by Licor blot.(F) Cells from (E) were untreated or transduced with shControl or shKAP1 vectors and Luciferase read over time (left). Cells were also treated with control stimulants (right). Results show the average Luciferase secreted over two time points (days 5 and 6 for the left plot and days 1 and 2 for the right plot) with all error bars showing SEM. HT-DNA: herring testis DNA.