EMBO/SourceData huggingface dataset:
Figure 4A. Immunoblot of extracts of RAW264.7 cells stably overexpressing flag-RKIP infected with VSV (MOI, 0.1) for the indicated times followed by immunoprecipitation (IP). WCL, whole cell lysates.B. Coimmunoprecipitation and immunoblot analysis of 293T cells transfected with vector for myc-RKIP alone or with the deletion mutants of TBK1. Numbers in parentheses indicate amino acids included in construct. FL, full length; KD, kinase domain; ULD, ubiquitin-like domain; SDD, α-helical scaffold domain; NBD, C-terminal domain.* indicates targeted band.C. Immunoblot of 293T cells transfected with vector for myc-TBK1 together with plasmid for HA-GSK3β or HA-RKIP, followed by immunoprecipitation. * indicates targeted band.D. In vitro kinase assay was carried out in the reaction mixture containing 2 mM ATP, 100 μM GST-RKIP or 100 μM GST-GFP and 5 μM flag-TBK1 at 30 °C for 30 min and then followed by immunoblotting with the indicated Antibody.
Figure 5A. Real-time PCR analysis of IL-6, TNF-α and IFN-β mRNA in RAW264.7 cells stably overexpressing flag-RKIP, flag-S109A and flag-S109D, followed by infection with VSV (MOI, 0.1).B. Immunoblot analysis of p- TBK1, -IRF3, -P38 of cells as in (A).C. FACS analysis of RAW264.7 cells stably overexpressing flag-RKIP, flag-S109A and flag-S109D infected with VSV-eGFP at MOI of 1.D. Immunoblot analysis of phosphorylated-TBK1, -IRF3, -P38 in lysates of RKIP+/+ or RKIP-/- bone marrow derived macrophages (BMDM) transfected with flag-RKIP, flag-S109A and flag-S109D, and then stimulated with VSV (MOI, 0.1).E. ELISA of IFN-β and TNF-α of cells in (D).
Figure 6A. BMDM cells were infected with VSV (MOI, 0.1) or HSV (MOI, 5) for the indicated times. Cell lysates were analyzed by immunoblotting with the indicated antibodies.B. BMDM cells were transfected with control or TBK1 siRNA. 72 hours after transfection, cells were analyzed by immunoblotting with antibody for phospho-RKIP or TBK1.C. Immunoblot analysis of RKIP phosphorylation in control or TBK1-/- mouse embryonic fibroblasts after VSV (MOI, 0.1) infection for the indicated times.D. Recombinant flag-tagged TBK1 was immunoprecipitated with anti-flag M2 beads in flag-TBK1 transfected 293T cells. Followed by in vitro kinase assay, which was carried out in the reaction mixture containing 2 mM ATP, 100 μM GST-RKIP or 100 μM GST-GFP together with 5 μM flag-TBK1 at 30 °C for 30 min. Assay mixtures were immunoblotted with the indicated antibody for phospho-RKIP, flag, GST or his.E. Immunoblot analysis of extracts of RAW264.7 cells stably overexpressing flag-RKIP, flag-S109A and flag-S109D, infected with VSV (MOI, 0.1) for the indicated times, followed by immunoprecipitation with anti-flag beads.F. BMDM cells were infected with VSV (MOI, 0.1) for the indicated times. The cell lysates were immunoprecipitated with anti-TBK1 (left) or anti-RKIP (right). The immunoprecipitates were analyzed by immunoblot with anti-RKIP or anti-TBK1 antibody. The levels of the endogenous RKIP, TBK1, and p-RKIP were detected by immunoblot analysis.G. Co-immunoprecipitation and immunoblot analysis of 293T cells transfected with myc-TBK1 alone or with flag-RKIP, flag-S109A or flag-S109D.H. GST-pull down experiment was performed with 1 μg fusion protein GST-GFP, GST-his-RKIP or GST-his-S109D mixed with 20 μl precleared agarose beads in 800 μl reaction medium, followed by adding 1 μg flag-TBK1 and incubating at 4°C for 3 hours with gentle rotation. Pull down (1st-3th lane) and input samples (4th lane) were separated by SDS-PAGE followed by immunoblotting with anti-GST or anti-flag antibody.I. Immunoprecipitation and immunoblot analysis of 293T cells transfected with indicated combinations of vector for myc-TBK1, flag-RKIP, flag-S109A, flag-S109D and HA-GSK3β or flag-GFP.J. In vitro kinase assay was carried out in the reaction mixture containing 2 mM ATP, 50 μM GST-TBK1 together with 100 μM GST-his-RKIP, 100 μM GST-his-S109D, 100 μM GST-his-S109A or 100 μM GST, respectively. React at 30 °C for 30 min. Then followed by immunoblotting with anti-p-TBK1or anti-GST antibody.
Figure 1(D) Immunostaining of PGCLCs d5 cryosections for SOX2 (magenta) and TFAP2C (cyan). Barplot indicates the quantification of SOX2+ cells, TFAP2C+ cells, and SOX2+/TFAP2C+. n=1150 cells, from n=3 separate inductions, using two biological clones. The white squares represent the position of the magnified region at the bottom. Scale bar, 50 µm and 10 µm for the magnified region.
Figure 2(C) MA plot of differential gene expression changes between XGFP- and XGFP+ PGCLCs as determined by RNA-seq. Log2-mean expression (log2-normalised counts from DESeq2) on the x-axis and the log2-fold change on the y-axis are shown. Significantly upregulated and downregulated genes are highlighted in red and green respectively. False Discovery Rate (FDR) < 0.001. Non-significant genes with log2-mean expression between 0 and 0.2 were removed for easier plot visualisation.(D) Expression levels (normalised DEseq2 counts) of selected differentially expressed genes between XGFP- and XGFP+ PGCLCs during the differentiation time course. Genes with FDR < 0.001 were considered significantly differentially expressed. Points indicate expression of individual biological replicates.(E) Heatmap of RNA-seq normalised counts showing the Zscore across PGCLC induction timepoints of 31 manually selected and manually ordered marker genes belonging to the categories reported on the side.(G) FACS analysis of cell cycle using DAPI. Numbers indicate the percentage of cells in G1, S and G2/M respectively.(H) Alkaline phosphatase staining for ESCs, XGFP+ PGCLCs and XGFP- PGCLCs grown for 7 days in 2i/LIF medium on immortalised mouse embryonic fibroblasts. (I) Barplot indicating the absolute numbers of Alkaline Phosphatase (AP) positive colonies in each cell type after 7 days of culture in 2i/LIF medium on immortalised mouse embryonic fibroblasts. Y-axis is in square root scale (sqrt) for better plot visualisation. Each white dot represents one technical replicate.
Figure 4(H) Immunofluorescence images of SYCP3 (red), DAZL (yellow) and DAPI at agg11 of IVDi tissue maturation from XGFP- PGCLCs. IVDi = in vitro differentiation. White squares indicate the positions of the magnified section shown below. Top panel scale bar = 100 μm. Middle panel scale bar = 10 μm. Bottom panel scale bar = 50 μm. (I) Quantification of SYCP3+ cells (oocytes in cyst and primary follicles) in IVDi tissues at agg11. Each dot represents one IVDi tissue performed in 3 biological replicates.
Figure 6(A) Schematic representation of the m220 stromal feeders expansion culture to compare the meiotic capacity of XGFP- and XGFP+ PGCLCs. Meiosis is induced via addition of Retinoic Acid (RA) and Bone Morphogenetic Protein 2 (BMP2). c0 = starting day of culture, c9 = culture day 9 and last day of culture. (B) Representative images for the expression of XGFP (green) and SYCP3 (red) in germ cells at c5, c7 and c9 from XGFP+ and XGFP- PGCLCs. All cells with SYCP3 signal irrespective of localization pattern or intensity were scored as SYCP3+ cells. Cells were counterstained with DAPI (grey). Scale bars = 10 μm. (C) Number of SYCP3+ cells per m220 culture day originating from XGFP+ and XGFP- PGCLCs. Each white dot represents a biological replicate (n = 3). P-values shown are from a two-sided Mann-Whitney U test. n.s. = not significant. (D) Percentage of XGFP+ cells among SYCP3+ cells at the indicated m220 culture day, originating from XGFP+ or XGFP- PGCLCs. Green bars show constitutive GFP expression in XGFP+ PGCLC-derived cells, while striped bars signify XGFP reactivation in XGFP- cells. Each white dot represents a biological replicate (n = 3).(E) Representative images showing stages of meiotic prophase I from culture day 9 (c9) germ cells from XGFP+ and XGFP- PGCLCs. c9 germ cells were spread and immunostained for SYCP3 (red), and γH2AX (grey). Scale bars = 10 µm. (F) Quantification of meiotic progression in culture day (c9) expanded germ cells derived from XGFP+ and XGFP- PGCLCs. The graphs show the percentages of the meiotic stage for SYCP3+ cells. Lepto, leptotene; Zygo, zygotene; Pachy, pachytene; Diplo, diplotene. Numbers indicate detected cells per meiotic stage and original XGFP status.(G) Representative tile scan merged images of XGFP (green) and Xtomato (red) in germ cells at c7 from XGFP+ and XGFP- PGCLCs. Cells were counterstained with DAPI (grey). Scale Bar = 1 mm. The white squares contain a magnified image of the region depicted by the white dotted squares (Scale Bar = 1 µm). (H) Density ridge plot showing size of cellular aggregates in (G) measured using the XTomato signal. X-Axis shows area in µm2 scaled as log2. Dashed line represents the mean. XFGP- mean = 1320 µm2, XGFP+ mean = 5514 µm2. XGFP- n = 297 aggregates, XGFP+ n = 373 aggregates. P-value from two-sample unpaired Wilcoxon-Mann-Whitney test with R defaults.
Figure 1(B) The EC50 values (95% confidence interval) for VHH-B9, VHH-10C4 and VHH-4A2 are 99.2 nM (83.8-117.5 nM), 112.5 nM (101.1-125.2 nM) and 788.7 nM (682.7-911.2 nM), respectively. Values are mean ± S.D., n=3, technical replicates.(C, D) Primary cultured neurons were transduced by Semliki Forest virus (SFV) expressing wild type human APP695 and treated with 3 µM of the indicated VHHs. PBS and anti-GFP VHH were used as controls. (C) Western blot analysis of conditioned media for sAPP⍺ and sAPPβ, as well as cell extracts for full length APP, CTF⍺ and CTFβ. (D) ELISA measurements of Aβ1-40 and Aβ1-42 in conditioned media. Values are mean ± S.E.M., n=3 cultures for each analysis. One-way ANOVA, *p<0.5, **p<0.01, *** p<0.0001.(E, F) Dose-dependent inhibition of BACE1 in primary cultured neurons by VHH-B9. Cultured neurons were transduced by SFV expressing wild type human APP695 and treated with PBS (control: CT) or decreasing concentrations of VHH-B9, ranging from 10 µM to 0.7 nM. (E) Western blot analysis of conditioned media for sAPP⍺ and sAPPβ, as well as cell extracts for full length APP, CTF⍺ and CTFβ. (F) Conditioned media was analyzed by ELISA to assess levels of Aβ1-40. Values are mean ± S.D., n=3 cultures for each analysis. The EC50 value (95 % confidence interval) was estimated as 85.4 nM (52.5-125.6 nM).
Figure 3(A) Co-staining with anti-N-Terminal Aβ 82E1 (green) and Thioflavin-S (magenta) reveals Aβ plaques (arrow heads) in regions of interest. Representative images are shown. Scale bar: 50 μm. (B, C) The number of Aβ plaques in regions of interest was significantly reduced by treatment AAV-VHH-B9 (B), as was total plaque area (C). (
Figure 4(A) (left) Representative images of anti-GFAP immunostaining in Prefrontal Cortex and Amygdala from AppNL-G-F mice receiving either AAV-VHH-B9 or AAV-GFP; (right) quantification of the GFAP+ cell number in the indicated brain regions across experimental cohorts.(B) (left) Representative images of anti-IBA1 immunostaining in dentate gyrus and amygdala from AppNL-G-F mice receiving either AAV-VHH-B9 or AAV-GFP; (right) quantification of the IBA1+ cell number in the indicated brain regions across experimental cohorts.
f1(a) Δ133p53α expression is induced by cycloheximide (CHX). MRC-5 fibroblasts (at population doubling levels (PDLs) 51) were treated with CHX (25 μg ml−1) for indicated time periods and examined in immunoblot for Δ133p53α and full-length p53. β-actin was a loading control for normalization. The relative expression levels of Δ133p53α and full-length p53 are shown below the images.(b) Δ133p53α expression is induced by phosphoinositide 3-kinase inhibitors. MRC-5 fibroblasts (PDLs 51) were treated for 4 h with LY303511 (10 μM), LY294002 (10 μM), Wortmannin (50 nM) or dimethylsulphoxide alone (as control) and examined in immunoblot for Δ133p53α and β-actin expression.(c) Diminished Δ133p53α at replicative senescence is restored by treatment with bafilomycin A1. The immunoblot analyses were performed in early-passage (Y) and replicatively senescent (S) human fibroblast strains MRC-5 and WI-38. The examined PDLs were 30 (Y) and 60 (S) for MRC-5; and 30 (Y) and 54 (S) for WI-38. Bafilomycin A 1 treatment was at 100 nM for 4 h. The relative expression levels of Δ133p53α, full-length p53 and p62/SQSTM1 (normalized with β-actin) are shown. Anti-LC3B antibody detected LC3-I and LC3-II, which was used in quantitative analysis shown in d. (d) Replicative senescence is not associated with enhanced autophagy in general. LC3-II expression levels were normalized with β-actin (data in c) and are shown in the bar graphs as relative values to replicatively senescent cells without bafilomycin A1 (S/−; defined as 1). The LC3-II levels in replicatively senescent cells were higher than those in early-passage cells whether in the presence or absence of bafilomycin A1, which may suggest increased synthesis of autophagosomes at replicative senescence. However, the differences in LC3-II levels between the presence and absence of bafilomycin A1 were similar in early-passage and replicatively senescent cells: in MRC-5, 1.9 for Y (2.1−0.2) and 2.0 for S (3.0−1); in WI-38, 1.6 for Y (2.2−0.6) and 1.6 for S (2.6−1). These data indicate that early-passage and replicatively senescent cells have similar rates of autophagic flux, that is, the number of autophagosomes that are delivered to lysosomes for degradation.
f2(a-c) Knockdown of pro-autophagic proteins restores Δ133p53α expression. MRC-5 fibroblasts at late-passage (PDLs 51) were transfected with three independent siRNA oligonucleotides (lanes 1-3) against ATG5 (a), ATG7 (b) and Beclin-1 (c), along with their control siRNA (lane C), for 4 days. Knockdown of ATG5 was confirmed by the loss of ATG5-ATG12 conjugate (detected by anti-ATG5 antibody). Knockdown of the other two proteins was confirmed using the corresponding antibodies. Δ133p53α, full-length p53 and LC3B (LC3-I and LC3-II) were examined in all samples. The relative expression levels of Δ133p53α and full-length p53 (normalized with β-actin) are shown below the images.(d) Knockdown of pro-autophagic proteins and treatment with bafilomycin A1 show similar effects on Δ133p53α and p62/SQSTM1. MRC-5 fibroblasts at late-passage (PDLs 51) were transfected with control siRNA (c), ATG5 siRNA (A5, no. 1 in a), ATG7 siRNA (A7, no. 1 in b) and Beclin-1 siRNA (B1, no. 2 in c) as in a-c, untreated (−) or treated with bafilomycin A1 (+, 100 nM for 4 h), and examined in immunoblots for indicated proteins. The relative expression levels of Δ133p53α and p62/SQSTM1 (normalized with β-actin) are shown below the images.
f3(a) Endogenous STUB1 protein is downregulated at replicative senescence. Immunoblot was performed using early-passage (Y) and replicatively senescent (S) fibroblasts (the same set of cells without treatment as in Fig. 1c).(b) STUB1 mRNA is downregulated at replicative senescence. The same set of cells as in a were examined by qRT-PCR. Data were from triplicate experiments (mean±s.d.). *P0.01; **P0.001 (Student's t-test).(c) Knockdown of STUB1 represses Δ133p53α. MRC-5 fibroblasts (30 PDLs) were transfected with two siRNAs against STUB1 (no. 1 and no. 2) and control siRNA. At 4 days after transfection, immunoblots were performed using anti-STUB1, anti-Δ133p53α, DO-1 (detecting full-length p53), CM1 (detecting both Δ133p53α and full-length p53), anti-p62/SQSTM1 and anti-β-actin antibodies.(d) STUB1 knockdown inhibits cell proliferation. MRC-5 fibroblasts (30 PDLs) were seeded on six-well plates on day 0 (4 × 104 cells per well in 6-well plate; nine wells for each siRNA). Cells were counted on days 1, 4 and 7 (three wells each; mean±s.d.), while siRNA transfection was performed on days 1 and 4. *P0.01 (Student's t-test).(e) STUB1 knockdown induces cellular senescence. The siRNA transfection was performed as in c and repeated 4 days later, followed by incubation for 3 days and SA-β-Gal staining. Percentages of SA-β-Gal-positive cells were from triplicate experiments (mean±s.d.). **P0.001 (Student's t-test).(f,g) STUB1 knockdown upregulates p21WAF1 and IL-8. The same set of cells as in e were examined in qRT-PCR for p21WAF1 mRNA (f) and IL-8 mRNA (g). Data were from triplicate experiments (mean±s.d.). **P0.001 (Student's t-test).(h) Δ133p53α rescues STUB1 knockdown-induced senescence. MRC-5 fibroblasts were transfected with STUB1 siRNA (no. 1) as in e. At the next day after the second transfection (on day 5), the cells were transduced with a lentiviral vector driving Δ133p53α or its vector control. At 5 days after the lentiviral transduction, the cells were stained for SA-β-Gal activity. Representative pictures (left) and quantitative data (right) are shown. Percentages of SA-β-Gal-positive cells were from triplicate experiments (mean±s.d.). **P0.001 (Student's t-test).
f4(a) STUB1 knockdown-induced repression of Δ133p53α is abrogated by bafilomycin A1 treatment. The siRNA transfection in MRC-5 fibroblasts was performed as in Fig. 3e. At 7 days after the initial transfection, the cells were untreated, treated with bafilomycin A1 (100 nM for 4 h) or treated with MG-132 (15 μM for 4 h), and examined in immunoblots as indicated. The results of Δ133p53α, full-length p53 and p62/SQSTM1 in untreated cells replicate those shown in Fig. 3c. The inhibition of autophagy by bafilomycin A1 was confirmed by increased levels of p62/SQSTM1 and LC3B. The inhibition of proteasomal degradation by MG-132 was confirmed by increased full-length p53.(b) STUB1 knockdown-induced repression of Δ133p53α is abrogated by knockdown of pro-autophagic proteins. MRC-5 fibroblasts (PDLs 30) were transfected for 4 days with control siRNA (−) or STUB1 siRNA (no. 1, +), in combination with control siRNA (c), p62/SQSTM1 siRNA (62), ATG5 siRNA (A5, no. 1 in Fig. 2a), ATG7 siRNA (A7, no. 1 in Fig. 2b) or Beclin-1 siRNA (B1, no. 2 in Fig. 2c), and examined in immunoblots for indicated proteins.(c) Δ133p53α is recruited to autophagosomes upon STUB1 downregulation. MRC-5 fibroblasts at early passage (PDLs 30; Y), those at or close to replicative senescence (PDLs 58; S (Rep)) and those induced to senesce by STUB1 knockdown (S (KD)) were used in immunoprecipitation (IP) with anti-STUB1, anti-Hsp70 and anti-LC3B antibodies, followed by immunoblots (IB) as indicated. Whole-protein lysates without IP (input) were also examined for Δ133p53α and β-actin levels.(d) Bafilomycin A1 stabilizes LC3B-interacting Δ133p53α in senescent cells. The same set of cells as in c were treated with bafilomycin A1 (+, 100 nM for 4 h) or untreated (−). These cells were used in IP with anti-LC3B antibody, followed by IB with anti-Δ133p53 antibody. Whole-protein lysates without IP were also examined in IB with anti-LC3B antibody. While untreated samples replicate the result shown in c, the differences between early passage and senescent cells become more evident with bafilomycin A1 treatment.
f5(a) Δ133p53α protein is polyubiquitinated. FLAG-tagged versions of wild-type Δ133p53α (wt) and mutant Δ133p53α (mut; from c below), along with vector control (−), were retrovirally transduced into MRC-5 fibroblasts. The wild-type Δ133p53α-expressing cells had a pair of STUB1 siRNA-transfected (+, siRNA no. 1 for 4 days) and untransfected (−) counterparts. These cells were maintained for 8 h under amino acid- and serum-starved conditions with bafilomycin A1 (100 nM). Protein lysates were used either in IP with anti-ubiquitin (Ub) antibody, followed by IB with anti-FLAG antibody (top), or directly in IB for FLAG-Δ133p53α, STUB1 and β-actin (lower three panels). Smear signals indicate polyubiquitinated FLAG-Δ133p53α protein (Poly-Ub). Asterisk indicates a nonspecific band in negative control. These experimental conditions resulted in similar amounts of FLAG-Δ133p53α in the presence and absence of STUB1 knockdown, allowing quantitative analysis of ubiquitination. The relative densitometric values of wild-type FLAG-Δ133p53α polyubiquitination in the presence and absence of STUB1 knockdown (normalized with total FLAG-Δ133p53α) are shown. ((b) Δ133p53α is ubiquitinated at the C-terminal lysine residues. MRC-5 fibroblasts expressing wild-type FLAG-Δ133p53α in the absence of STUB1 knockdown were used in IP with anti-FLAG antibody, and the resulting immunoprecipitates were analysed using mass spectrometry. Representative MS/MS spectrum of an [M+3H]3+ peptide from FLAG-Δ133p53α shows sites of ubiquitin modification. The peptide sequence for the spectrum is shown, with the sites of modification marked as asterisks: amino-acid positions 248 and 249, which correspond to positions 381 and 382 in full-length p53. The same peptide was also observed in the unmodified state.(c) Lysine (K)-to-arginine (R) substitutions at amino-acid residues 248 and 249.(d) Mutation of the ubiquitination sites renders Δ133p53α resistant to degradation. The same three cells as in a were cultured under amino-acid- and serum-starved conditions for 8 h (without bafilomycin A1), transfected with STUB1 siRNA (no. 1 for 4 days) or untreated, and examined in IBs for FLAG-Δ133p53α, STUB1, p62/SQSTM1 and β-actin. The effect of starvation was confirmed by decreased p62/SQSTM1 in each cell. The upregulation of p62/SQSTM1 by wild-type and mutant FLAG-Δ133p53α is likely associated with enhanced cell proliferation by Δ133p53α overexpression.
f6(a) Δ133p53α is colocalized with p62/SQSTM1 upon autophagy induction. MRC-5 fibroblasts expressing FLAG-Δ133p53α (wild-type) were cultured under amino-acid- and serum-starved conditions for 4 h, and co-immunostained with anti-FLAG antibody (Δ133p53α, green) and anti-p62/SQSTM1 antibody (red). Two representative sets of images, including nuclear staining with 4′,6-diamidino-2-phenylindole (DAPI) and merged images, are shown. Scale bars, 10 μm.(b) Knockdown of p62/SQSTM1 stabilizes endogenous Δ133p53α. MRC-5 fibroblasts at late passage (PDLs 51) were transfected with p62/SQSTM1 siRNA (p62) or control siRNA (C) for 3 days, and examined in immunoblots for Δ133p53α and p62/SQSTM1 expression. The relative expression levels of Δ133p53α (normalized with β-actin) are shown.(c) Δ133p53α is colocalized with LC3B upon autophagy induction. MRC-5 fibroblasts with FLAG-Δ133p53α under starved conditions (as in a) were co-immunostained with anti-FLAG antibody (Δ133p53α, green) and anti-LC3B antibody (red). Two representative sets of images, including DAPI and merged images, are shown. Scale bars, 10 μm. (d) Quantitative analysis of Δ133p53α-LC3B colocalization. The number of colocalized signals out of total number of Δ133p53α signals or out of total number of LC3B signals was counted in 26 cells. Data (percent colocalization) are mean±s.d. Almost all Δ133p53α signals were colocalized with LC3B, while ~80% of LC3B signals were colocalized with Δ133p53α.