EMBO/soda-nel huggingface dataset:
HOPS/TMUB1 retains p53 in the cytoplasm and sustains p53-dependent mitochondrial apoptosis (2019)
Figure 7

Figure 7 - HOPS role in importin α mediated p53 nuclear import and PCAF mediated p53 acetylation.

A HOPS expression and p53 nuclear accumulation. HOPS was overexpressed in RKO and cells were fixed and stained with antibody against p53 (green). Nuclei were stained with DAPI (blue). Samples were analysed with fluorescent microscope and merged images are shown (right panel). Scale bars, 10µm. Cells were scored for p53 localization. The results are shown as percentage of nuclear versus cytoplasmic p53 localization in untransfected and HOPS transfected cells (left panel).

B Biochemical analysis of HOPS intracellular localization. RKO cells were transfected or not with HOPS and fractionated into nuclear and cytoplasmic component. Aliquots relative to the same cell number per fraction were immunoblotted using anti-p53 and anti-HOPS antibodies. GAPDH and Lamin B1 are purity controls of cytoplasmic and nuclear fraction respectively.

C HOPS and p53 binding to importin α. Untransfected or HOPS transfected RKO cells were treated with etoposide and each sample was evaluated by immunoprecipitation with anti-p53 and analysed by immunoblotting using anti-importin α antibody. Arrowhead indicates the importin α specific signal.

D PCAF-mediated p53 acetylation. p53 and PCAF Flag-tagged were co-expressed in H1299 cells with or without HOPS and treated with trichostatin A and nicotinamide. p53 acetylation levels were evaluated by immunoprecipitation with anti-p53 antibody followed by immunoblotting with anti-acetylated-Lysine (AcK), anti-p53, anti-Flag, anti-HOPS and anti-α-tubulin antibodies.

Data information: all the experiments detailed above were performed three times, and representative panels are shown. In A data are presented as mean ±SD.
Proteasomal degradation within endocytic organelles mediates antigen cross-presentation (2019)
Figure 1

Figure 1. Expression of hβ2m partially rescues surface Kb expression and antigen cross-presentation in TAP1-/- BMDC, but does not rescue endogenous antigen presentation.

A. Cell surface H2-Kb levels of wild type and TAP1-/- BMDC transduced with control vector or a vector expressing hβ2m were analyzed.

B Cross-presentation of OVA by wild type and TAP1-/- BMDC expressing hβ2m were compared to control BMDC, by measuring IL-2 production by a Kb-SIINFEKL specific T-cell hybridoma (B3Z) cultured with paraformaldehyde fixed BMDC that were incubated with varying numbers of OVA-coated latex beads for 6hr, prior to fixation.

C The effect of hβ2m on the presentation of peptides derived from endogenous antigen was assessed by measuring IL-2 levels in the culture supernatant of the B3Z hybridoma incubated with fixed VV-OVA infected WT and TAP1-/- BMDC transduced with control vector or hβ2m.

Data Information: In (B and C), a representative experiment of three independent experiments is shown. The mean± SD of assay triplicates are plotted. p<0.05 represented by "*", p<.01 represented by "**" and p<0.001 is represented by "****"(Students t-test).
Proteasomal degradation within endocytic organelles mediates antigen cross-presentation (2019)
Figure 2

Figure 2. Expression of Rab5ACA, Rab22ACA and Rab7DN, respectively, increases hβ2m-enhanced TAP-independent cross-presentation.

A-C Surface H2-Kb levels of wild-type and TAP1-/- BMDC co-expressing Rab mutants and hβ2m were analyzed by flow cytometry.

D-F Cross-presentation of OVA by wild-type and TAP1-/- BMDC co-expressing Rab mutants and hβ2m were analyzed and IL-2 release is shown (D, E, F).

Data information- In (D-F), representative means (±SD) of at the least three independent experiments per Rab mutants setup in triplicated are plotted. p<0.05 represented by "*", p<.01 represented by "**", p<.005 represented by "***", p<0.001 is represented by "****" and "ns" is not significant (Students t-test).
Proteasomal degradation within endocytic organelles mediates antigen cross-presentation (2019)
Figure 3

Figure 3 Proteasomal activity is required for stable surface Kb expression in TAP1-/- dendritic cells and antigen cross-presentation.

A-D TAP1-/- BMDC transduced with vector control (A) or hβ2m (C) were incubated with varying concentrations of Epoxomicin for 6hr in absence or presence of exogenously added Kb-binding SIINFEKL peptide and analyzed for surface expression of Kb by flow cytometry. Representative plots are shown (A, C). The impact of Epoxomicin on cell surface Kb was analyzed by plotting Kb surface expression on cells incubated with Epoxomicin, normalized to that on cell incubated without Epoxomicin (n=3) (B, D).

E The effect of Epoxomicin on cross-presentation of OVA-coated beads by TAP1-/- BMDC expressing hβ2m was determined by incubating the BMDC with OVA coated latex beads in the presence of varying doses of Epoxomicin and 10μM gB peptide. After 6hrs the cells were fixed, incubated with B3Z cells and IL-2 production was measured

Data information: In (B, D), the means (±SEM) of three independent experiments are plotted. Data was analyzed by performing a nonlinear regression analysis. In (E), a representative experiment of three independent experiments is shown. The mean (±SD) of assay triplicates are plotted. Data was analyzed by performing a linear regression analysis.
Proteasomal degradation within endocytic organelles mediates antigen cross-presentation (2019)
Figure 4

Figure 4. Reconstitution of TAP-independent cross-presentation in 293T-FcR-Kb cells by expressing Rab5ACA, Rab22ACA and Rab7ADN.

A, B 293T-FcR-Kb cells co-expressing empty vector or Rab mutants (Rab5ACA, Rab22ACA, Rab7ADN) individually, with either LacZ as a control or US6 (A) or ICP47 (B), were analyzed for cross-presentation of OVA using B3Z cells. The effects of US6 and ICP47 on cross-presentation were analyzed by plotting the mean percentage of IL-2 release by cells co-expressing US6 (A) or ICP47 (B) with control vector or Rab mutants compared to cells co-expressing LacZ with control vector or Rab mutants, respectively.

C-E 293T-FcR-Kb cells expressing Rab5ACA (C), Rab22ACA (D) and Rab7ADN (E) were incubated with opsonized OVA coated latex beads in the presence of varying doses of Epoxomicin. After 6hrs the cells were fixed, incubated with B3Z cells and IL-2 production was measured.

Data information- In (A and B), means (±SEM) of three independent experiments for each Rab mutants are plotted. p<.01 represented by "**", (Students t-test). Representative experiments of three independent experiments are shown for each Rab mutants (C, D, E). The means (±SD) of assay triplicates are plotted. Data was analyzed by performing a linear regression analysis
LncRNA GUARDIN suppresses cellular senescence through a LRP130-PGC1ɑ-FOXO4-p21-dependent signaling axis (2020)
Figure 1

Figure 1. GUARDIN inhibits cellular senescence through suppression of p21.

A. Senescence-associated β-galactosidase (SA-β-gal) staining in A549, HAFF, HepG2 and H1299 cells after 48h transduction with either negative control lentiviruses (shCtrl) or those targeting GUARDIN (shGUARDIN). The graph shows percentage of SA-β-gal-positive cells.

B. Senescence-associated heterochromatin foci formation (SAHF) in A549 cells with either shCtrl or shGUARDIN. Representative IF staining with H3K9me3 antibodies (green) and nuclear Hoechst counterstaining (blue).

C. Secretion of SASP factors IL-6 and IL-8 in A549 cells with either shCtrl or shGUARDIN.

D. Western blotting for p21, p16 and p15 in HepG2 cells after 48h transduction with shCtrl or shGUARDIN-1, -2, respectively (left). Knockdown efficiencies analyzed by qPCR (right).

E. SA-β-gal staining in A549 cells after 24h transduction with either shCtrl or shGUARDIN in combination with p21 shRNA (shp21). The graph shows percentage of SA-β-gal-positive cells.

F. qPCR measurement of p21 mRNA in A549, HepG2, HAFF and H1299 cells with either GUARDIN shRNA knockdown (top) or GUARDIN overexpression (bottom).

G. Western blotting for p53 and p21 in A549, HepG2, HAFF and H1299 cells with either GUARDIN shRNA knockdown (top) or GUARDIN overexpression (bottom).

H. qPCR assays of NOXA, TIGAR, BAX and p21 mRNA levels in A549 cells with either shCtrl or shGUARDIN.

Data information: (A, C, D-F, H) values are mean ± s.e.m (n = 3 biological replicates). (A, C, D, F, H) two-tailed paired Student's t test; (E) two-way ANOVA with Bonferroni's multiple comparison post-test (ns, not significant, *** P < 0.001).
LncRNA GUARDIN suppresses cellular senescence through a LRP130-PGC1ɑ-FOXO4-p21-dependent signaling axis (2020)
Figure 2

Figure 2. GUARDIN facilitates LRP130-PGC1α interaction that mediates transcriptional repression of p21.

A. SDS-PAGE of RNA pulldown assays using biotin-labelled sense/antisense probes against GUARDIN from whole-cell lysates of A549 cells indicating putative GUARDIN-binding proteins (left); protein identities with high probabilities were determined by mass spectrometry (right).

B. RNA pulldown assays interrogating putative GUARDIN-associated proteins identified in (A) from whole-cell lysates of A549 and HAFF cells. BRCA1, BARD1 served as positive controls and β-actin served as negative controls.

C. RNA immunoprecipitation (RIP) assays against IgG/LRP130 antibodies in whole-cell lysates of A549 cells.

D. Subcellular localization of GUARDIN and its co-localization with LRP130. RNA FISH for GUARDIN (red) and IF for LRP130 (green) in A549 cells with either shCtrl or shGUARDIN. Nucleus were counterstained with Hoechst (blue).

E. RNA pulldown assays using biotin-labelled sense/antisense probes against GUARDIN from whole-cell lysates of A549 cells. GUARDIN levels were measured by RT-PCR and co-precipitated LRP130 and PGC1α detected by Western blotting. BRCA1 and β-actin served as positive and negative controls, respectively.

F. RIP assay using IgG/PGC1α antibodies from whole-cell lysates of A549 cells. GUARDIN, LRP130 and PGC1α levels were measured as per (E).

G. Two-step IP assays in whole-cell lysates of A549 cells transfected with FLAG-tagged PGC1α. First phase IPs were conducted with FLAG antibodies (left) and following elution with FLAG peptides, eluates were further subjected to second phase IPs with LRP130 antibodies (right). Samples were subjected to Western blotting and qPCR analysis for LRP130, PGC1α and GUARDIN, respectively.

H. Co-immunoprecipitation (co-IP) between LRP130 and PGC1α in A549 cells after 48h transduction with shCtrl or shGUARDIN. LRP130 was precipitated and samples subjected to Western blotting analysis for LRP130, PGC1α and β-actin as loading control.

I. Mammalian two-hybrid assays between pACT-LRP130 and pBIND-PGC1α in A549 cells after 48h transduction with shCtrl or shGUARDIN. Samples were subjected to the luciferase activity assays.

J. LRP130/PGC1α and p21 protein expression was measured by Western blot in A549 and HAFF cells after 48h transduction with shCtrl or shLRP130 (top left) or shPGC1α (bottom left) as indicated. qPCR assays for p21 mRNA levels were performed in parallel (right panels).

K. Western blotting analysis of LRP130, PGC1α and p21 protein levels in HAFF and A549 cells after 48h transduction with shCtrl or shGUARDIN.

Data information: (I, J) values are mean ± s.e.m (n = 3 biological replicates). (I, J) two-tailed paired Student's t test (** P< 0.01, *** P < 0.001).
LncRNA GUARDIN suppresses cellular senescence through a LRP130-PGC1ɑ-FOXO4-p21-dependent signaling axis (2020)
Figure 3

Figure 3. LRP130/PGC1α negatively regulates FOXO4 transcription.

A. ChIP assays detecting binding of LRP130/PGC1α to the p21 promoter using qPCR and RT-PCR (top left and right, respectively). IgG and p53 served as a negative and positive controls, respectively. Schematic illustrations of the putative LRP130/PGC1α binding sites within DNase I hypersensitive regions of the p21 promoter (bottom).

B. qPCR assays for GUARDIN, FOXO1, FOXO3a and FOXO4 in A549 cells after 24h transduction with shCtrl or shGUARDIN.

C. Western blotting assays for FOXO4 and p21 protein expression in A549 cells after 48h transduction with shCtrl or shGUARDIN alone or in combination with shFOXO4. β-actin served as loading control.

D. Half-life times of FOXO4 mRNA in A549 cells with shCtrl or shGUARDIN measured by qPCR after treating cells with 5 μg/ml of actinomycin (ActD) for the indicated times.

E. ChIP assays detecting binding of LRP130/PGC1α to putative binding sites in the FOXO4 promoter using qPCR (top). Data were normalized to the IgG negative control. Schematic illustration of the LRP130/PGC1α binding sites within DNase I hypersensitive regions of the FOXO4 promoter (bottom).

F. Luciferase assays conducted in A549 cells transfected with LRP130 or PGC1α using the pGL3 (negative control) or pGL3-FOXO4 promoter reporter plasmids.

G. qPCR assays for LRP130 and FOXO4 mRNA levels (upper) and Western blotting analysis for LRP130 protein level (lower) in A549 cells after 48h transduction with LRP130 overexpression or knockdown.

H. qPCR assays for PGC1α and FOXO4 mRNA level (upper) and Western blotting analysis for PGC1α protein level (lower) in A549 cells after 48h transduction with PGC1α overexpression or knockdown.

I. Luciferase activity assays in A549 cells transduced with shCtrl or shGUARDIN using the pGL3 (negative control) or pGL3-FOXO4 promoter reporter plasmids.

Data information: (A, B, D-I) values are mean ± s.e.m (n = 3 biological replicates). (A, B, E-I) two-tailed paired Student's t test (* P< 0.05, ** P< 0.01, *** P < 0.001).
LncRNA GUARDIN suppresses cellular senescence through a LRP130-PGC1ɑ-FOXO4-p21-dependent signaling axis (2020)
Figure 4

Figure 4. Rapamycin regulates GUARDIN expression via FOSL2.

A,B. qPCR assays for GUARDIN expression in A549 cells treated with increasing doses rapamycin for 24h (A) or 100nM of rapamycin for the indicated times (B).

C. Western blotting assays for p53, LRP130, FOXO4 and p21 expression in A459 and H1299 cells treated with 100nM of rapamycin or vehicle (DMSO) for 48h.

D. A549 cells with shCtrl or shGUARDIN were treated with 100nM rapamycin or DMSO vehicle in the indicated combinations for 48h. Western blotting was used to measure p21 levels (left) while conducting SA-β-gal staining in parallel (middle) with the percentage of senescent cells calculated from the SA-β-gal staining (right).

E. Mammalian two-hybrid assays between pACT-LRP130 and pBIND-PGC1α in A549 cells treated with DMSO or 100nM rapamycin. Samples were subjected to the luciferase activity assays.

F. SIX4 and FOSL2 mRNA (left) and protein levels (right) measured by qPCR and Western blotting, respectively in A549 cells treated with DMSO or 100nM rapamycin. TBP served as a control.

G. qPCR (upper) and Western blotting (lower) assays for GUARDIN, SIX4 and FOSL2 expression in A549 cells comparing shCtrl with shSIX4 (left), shFOSL2 (middle) or FOSL2 (right) after transduction for 48h.

H. Luciferase assays in A549 cells transduced with shCtrl, shSIX4 or shFOSL2 using pGL3 (negative control) or pGL3-FOXO4 promoter reporter plasmids.

I. ChIP assays comparing control IgG versus FOSL2 antibodies demonstrate specific recovery of the GUARDIN promoter by RT-PCR assay. UPe and UPk served as a positive and negative controls, respectively [54].

Data information: (A, B, D-I) values are mean ± s.e.m (n = 3 biological replicates). (A, B, E-I) two-tailed paired Student's t test; (D) two-way ANOVA with Bonferroni's multiple comparison post-test (* P< 0.05, ** P< 0.01, *** P < 0.001).
The chromatin remodeler SRCAP promotes self-renewal of intestinal stem cells (2020)
Figure 1

Figure 1. SRCAP is highly expressed in intestinal epithelium and ISCs.

(A) Srcap expression in murine different tissues was examined by Northern blot. A 277 nt probe of Srcap (81-358nt) was labeled for Northern blot analysis. 18S RNA was used as a loading control.

(B) Two-week-old SRCAP-C-HA tag mice were sacrificed for longitudinal sections followed by immunofluorescence staining. A global look of the section was shown. Scale bar: 500 μm. Green: EpCAM, Red: HA tag, Nuclei were counterstained by DAPI.

(C) Murine intestine tissues were fixed and stained according to iDISCO staining protocol. SRCAP was highly expressed in vast majority of crypts in mouse intestine. Scale bar: 50 μm. Green: EpCAM, Red: SRCAP, Nuclei were counterstained by DAPI.

(D) Duodenum region of intestinal tissues were obtained from Lgr5GFP-CreERT2 (Lgr5GFP) mice for immunofluorescence staining by indicated antibodies with OpalTM 7-color fIHC kit. Nuclei were counterstained with DAPI. Scale bar: 50 μm.

(E) ISCs from Lgr5GFP mice were collected for organoid formation. Organoids were fixed for immunofluorescence staining with indicated antibodies. Scale bar: 50 μm.

Data information: Green: EpCAM, Red: SRCAP, Purple: PCNA, Nuclei were counterstained by DAPI.
The chromatin remodeler SRCAP promotes self-renewal of intestinal stem cells (2020)
Figure 2

Figure 2. Srcap deficiency impairs the self-renewal of ISCs and intestinal epithelial regeneration.

(A) Representative images of intestines from 8-week-old Srcap+/+ and Srcap-/- mice (n=6 per group). Length of 120 villi and 120 crypts (20 villi and 20 crypts per mouse) was measured and shown in right panel as means± S.D. ** P < 0.01 by two-tailed Student's t-test. Scale bar, 100 μm. Dotted lines indicate the boundary between crypts and villi. Arrows indicate the length of crypts or villi.

(B) Intestinal tissues from 8-week-old Srcap+/+ and Srcap-/- mice (n=6 per group) were collected for immunofluorescence staining with indicated antibodies. Typical images were shown in left panel and statistical ISC numbers from 120 crypts (20 crypts per mouse) were shown in right panel as means± S.D. ** P < 0.01 by two-tailed Student's t-test. Scale bar, 50 μm.

(C) 1×104 ISCs from indicated mice were collected for organoid formation. Scale bar, 200 μm. n=3 for each group. Organoid formation ratios was shown as means ± S.D. ** p<0.01 by two-tailed Student's t-test.

(D) Srcap+/+ and Srcap-/- mice were treated with 10 Gy's radiation and sacrificed at different time points. Intestinal tissues were collected for H&E staining. Numbers of intact crypts were shown in lower panel as means± S.D. ** P < 0.01 by two-tailed Student's t-test. 50 fields (10 fields per mouse) were taken for each group (n=5). Scale bars, 100 μm.

(E) Lgr5GFP-CreERT2;Rosa26lsl-YFP (LRYFP) were crossed with Srcapflox/flox mice, followed by administration of tamoxifen (TAM) for lineage tracing analysis. Mice were sacrificed 7 days after TAM induction and typical jejunum sections were stained. Numbers of traced crypt-villus units were shown in right panel as means± S.D. ** P < 0.01 by two-tailed Student's t-test. 50 fields (10 fields per mouse) were taken for each group (n=5). Scale bars, 50 μm.

(F) Proliferation of Srcap+/+ and Srcap-/- ISCs were examined by Ki67 staining followed by flow cytometry analysis. Percentages of proliferated cells were counted as means ± S.D (lower panel). ** P < 0.01 by two-tailed Student's t-test. n=5 per group.

(G) Multiple color staining was performed in Srcap+/+ and Srcap-/- intestine sections. AP-Red, DAB and AP-Blue were used for immunohistochemistray coloration. Numbers of each kind of epithelial cells from 50 crypts or 50 villi (10 crypts and 10 villi per mouse) were shown in right panel as means± S.D. ** P < 0.01 by two-tailed Student's t-test. Scale bars, 50 μm.

Data information: All data are representative of five independent experiments.
The chromatin remodeler SRCAP promotes self-renewal of intestinal stem cells (2020)
Figure 3

Figure 3. SRCAP associates with REST.

(A) Nuclear extracts of murine intestinal crypts were immunoprecipitated with anti-SRCAP antibody or rabbit IgG followed by ADASDS-PAGE, sliver staining and mass spectrometry.

(B) SRCAP and REST were stained in murine intestinal crypts. Scale bar, 10 μm.

(C) A schematic diagram of full-length and truncated fragments of REST protein was shown.

(D) Flag-tagged REST fragments were expressed in 293T and purified by immunoprecipitation with anti-Flag antibody. REST proteins were incubated with crypt lysates for pulldown followed by immunoblotting.

(E) C-terminal domain of REST was sufficient and essential for SRCAP association by pulldown assay.

(F) Intestinal crypt lysates were incubated with anti-REST antibody, followed by immunoblotting with indicated antibodies.

(G) Intestinal crypt lysates were incubated with anti-REST antibody and protein A/G beads in 4℃ overnight. Recombinant SRCAP protein was added with different concentrations, followed by immunoblotting.

Data information: All data are representative of four independent experiments.
The chromatin remodeler SRCAP promotes self-renewal of intestinal stem cells (2020)
Figure 4

Figure 4. REST recruits SRCAP onto Prdm16 promoter for its transcription.

(A) Analysis for genome-wide SRCAP binding regions in ISCs from ChIP-seq was shown. Coordinate information for sequence alignment was from UCSC (intron and exon), Ensemble GRCM38 (mm10, release-90) (upstream 2000bp, downstream 2000bp, distal intergenic region, TSS (transcription start site), TES and promoter), GSE57919 (Dnase-seq peaks, liftover to mm10), and GSE34568 (H3K4me2, liftover to mm10). Distribution of SRCAP peaks in ChIP-seq and its overlap with each kind of region was analyzed by bedtools.

(B) Transcriptome microarray analysis between Srcap-/- ISCs and WT ISCs. Top ten downregulated TFs in Srcap-/- ISCs were listed.

(C) Prdm16 expression was tested in Srcap+/+ and Srcap-/- ISCs by Western blotting.

(D) Prdm16 expression was visualized in indicated intestinal tissues by immunofluorescence staining. Green: GFP; Red: SRCAP; Blue: Prdm16. Scale bar, 10 μm.

(E) Prdm16 promoter (-875~-854) region was used as probe to perform DNA-pulldown assay with murine intestinal crypt lysates followed by immunoblotting.

(F, G) Indicated promoter activation was analyzed with luciferase assay. DKK1 was used as a control. Results of relative fold changes are shown as means ± S.D. Data represent five independent replicates. . ** p<0.01 by two-tailed Student's t-test. .

(H) 1×104 ISCs from indicated mice were collected for organoid formation. For gene depletion or restoration, ISCs were infected with lentivirus. Scale bar, 200 μm. Data information: All data represent five independent experiments.
The chromatin remodeler SRCAP promotes self-renewal of intestinal stem cells (2020)
Figure 5

Figure 5. Prdm16 initiates PPARδ expression to promote ISC stemness.

(A) WT and Srcap-/- ISCs were conducted for transcriptome microarray, followed by Gene Set Enrichment Analysis (GSEA) analysis for PPARδ target genes.

(B) Expression levels of PPARs and PPAR target genes were assessed in sorted ISCs by quantitative RT-PCR. Primers were listed in Table EV1. Relative gene expression folds were normalized to endogenous β-actin and shown as means ± S.D. Data represent five independent replicates. ** p<0.01 and *** p<0.001 by two-tailed Student's t-test.

(C) PPARδ expression was visualized in intestinal tissues by immunofluorescence staining. Scale bar, 50 μm.

(D) ISCs were collected for organoid formation followed by quantitative RT-PCR. For Prdm16 overexpression, ISCs were infected with lentivirus carrying Prdm16 overexpressing plasmids. Primers were listed in Table EV1. Relative gene expression folds were normalized to endogenous β-actin and shown as means ± S.D. Data represent five independent replicates. ** p<0.01 by two-tailed Student's t-test.

(E) mRNA levels of ISC signature genes were examined by quantitative RT-PCR. Primers were listed in Table EV1. Relative gene expression folds were normalized to endogenous β-actin and shown as means ± S.D. Data represent five independent replicates.

(F) Representative images of organoid formation from indicated ISCs were shown (upper panel). Scale bar, 200 μm. Organoid formation ratios was shown as means ± S.D. ** p<0.01 by two-tailed Student's t-test. (lower panel). n=6 for each group. GW501516 (10 μM) were added directly into organoid culture medium.