EMBO/soda-nel huggingface dataset:
Non-canonical phosphoglycerate dehydrogenase activity promotes liver cancer growth via mitochondrial translation and respiratory metabolism (2022)
Figure 5

Figure 5 PHGDH promotes the proliferation of liver cancer cells by facilitating mitochondrial activity.

A PLC cells overexpressing EV, PHGDHWT, or PHGDHR236E were further infected with viruses expressing NTC or shmtEFG2. The OCR was measured after successive injections of oligomycin (oligo), FCCP, and antimycin A/rotenone (AA/Rot). Basal respiration, ATP-linked respiration, and maximal respiration were analysed with the OCR curve.

B PLC cells overexpressing EV, PHGDHWT, or PHGDHR236E were further infected with viruses expressing NTC or shmtEFG2 prior to western blot analysis. β-Actin was used as the loading control (left). Cell growth curves were constructed by cell counting with trypan blue exclusion (right).

C PLC cells overexpressing EV, PHGDHWT, or PHGDHR236E were further treated with H2O or tigecycline prior to western blot analysis. β-Actin was used as the loading control (left). Cell growth curves were determined by cell counting with trypan blue exclusion (right).

D-F PLC cells overexpressing EV or PHGDH were further infected with viruses expressing NTC or shmtEFG2. Then, these cells were subcutaneously injected into male nude mice (n = 5 in each group). Tumour sizes were measured beginning 14 days after inoculation (D). At the end of the experiment, the tumours were excised (E), and PHGDH and mtEFG2 expression in tumour lysates was analysed by western blotting (F).

G-I PLC cells overexpressing EV or PHGDH were subcutaneously injected into male nude mice (n = 5 in each group). Mice were intraperitoneally injected with 100 mg/kg tigecycline daily beginning 14 days after inoculation. Tumour sizes were measured beginning 14 days after inoculation (G). At the end of the experiment, the tumours were excised (H), and PHGDH expression in tumour lysates was analysed by western blotting (I).

Data information: n = 3 independent experiments in (A-C), and data are presented as the mean ± SD values in (A-C, D, G). Statistical significance was determined by one-way ANOVA. *P < 0.05 between the indicated groups. NS: not significant.

Source data are available online for this figure.

Expanded View Figure Legends
p53 status determines the role of autophagy in pancreatic tumour development (2013)
f1

a, Pancreatic sections stained with Atg7, LC3 and p62 showing Atg7−/− next to Atg7+/+ regions after Pdx-Cre-mediated recombination. Right-side images are cropped and magnified regions (boxed) from left-side images. The magnification is × 2.5. b, Haematoxylin and eosin (HE), p53 and caspase-3 staining of exocrine tissue in Atg7+/+ and Atg7−/− pancreases. c, Quantification (median, s.e.m.) of p53 and caspase-3 activation in Atg7-deficient acinar tissue. d, Kaplan–Meier analysis comparing overall survival of mice either wild type (blue) or Atg7−/− (red) in the pancreas. Mantel–Cox test was used for statistics. ‘n/a’, 14/16 mice were killed >500 days when completely healthy. e, Biochemical analysis of endocrine function in moribund Atg7−/− or age-matched healthy control mice. Detailed information about the mice is tabulated. A Mann–Whitney U-test was used for statistics. Scale bars: 50 &mgr;m. **P  0.01 and ***P  0.001.
p53 status determines the role of autophagy in pancreatic tumour development (2013)
f2

a, Alcian blue/PAS (AB/PAS) staining in pancreases from 150-day-old Atg7-proficient or Atg7-deficient KrasG12D/+ mice (wild-type mice with expression of KrasG12D in their pancreases). b, Quantification of PanIN from mice of the indicated genotype and age. Fold increase and male/female ratios are shown. Box plots express the first (Q1) and third (Q3) quartiles set by the upper and lower limits of the box, whereas the median is shown by the horizontal line inside the box. The whiskers extend upward and downward to the highest or lowest observation within the upper (Q3 + 1.5 × the interquartile range) and lower (Q1 − 1.5 × interquartile range) limits. Values outside the upper and lower limits are ‘outliers’ and are shown by individual symbols. c, d, Kaplan–Meier analysis comparing overall survival (c) or PDAC-free survival (d) of Atg7+/+ (blue) or Atg7−/− (red) KrasG12D/+ mice. e, PanIN grading of pancreases from 150-day-old Atg7+/+ or Atg7−/− Pdx-Cre KrasG12D/+ mice. Values are mean and error bars are s.d. b, e, A Mann–Whitney U-test was used for statistics. f, Representative p53 staining from 150-day-old mice of both genotypes. g, PDAC-free survival of autophagy-proficient (blue) and Atg5-deficient (red) KrasG12D/+ mice. d, g, Median survival ± s.d. and male/female (m/f) ratio are provided. ‘n/a’, no animals succumbed to PDAC. A log-rank test (Mantel–Cox) was used for statistics. a, f, Scale bars: 500 &mgr;m (a), 50 &mgr;m (f). *P  0.05, **P  0.01; NS, not significant.
p53 status determines the role of autophagy in pancreatic tumour development (2013)
f3

a, Kaplan–Meier analysis comparing PDAC-free survival in Atg7+/+ (blue) or Atg7−/− (red) KrasG12D/+ p53−/− mice. b, Pancreatic tumours of indicated genotypes stained for Atg7, LC3 and p62. Insets are magnified crops from the same images to show specifics of LC3 and p62 staining. The magnification is × 4. c, Tumour incidence and tumour weight (median, s.e.m.) in mice from indicated genotypes, killed at 21, 29 and 38 days of age. Numbers in the incidence diagram represent tumour-bearing mice versus all mice. Significance was assessed with Fisher’s exact test (incidence) and Mann–Whitney U-test (weight). d, Representative haematoxylin and eosin stainings of pancreases from indicated time points and genotypes. e, Kaplan–Meier analysis comparing PDAC-free survival of Atg5+/+ (blue) or Atg5−/− (green) KrasG12D/+ p53−/− mice. f, Kaplan–Meier analysis comparing PDAC-free survival of KrasG12D/+ p53−/− mice that were chloroquine-treated (CQ, red), vehicle-treated (PBS, black) or untreated (blue). a, e, f, The blue curve in all Kaplan–Meier plots represents the same colony of mice (KrasG12D/+, p53−/−, Atg7+/+, Atg5+/+). A Mantel–Cox test was used for statistics. b, d, Scale bars: 100 &mgr;m (b), 200 &mgr;m (d). *P  0.05, **P  0.01.
p53 status determines the role of autophagy in pancreatic tumour development (2013)
f4

a, LC3 staining of representative pancreatic tumours from mice of indicated genotypes. Scale bars: 50 &mgr;m. b, Western blotting for LC3, p53 and Atg7 in cell lines derived from individual tumours which developed in the absence or presence of p53. c, d, Average oxygen consumption rate (OCR, c) and extracellular acidification rate (ECAR, d) of eight Atg7+/+ and seven Atg7−/− cell lines revealed increased ECAR in autophagy-deficient cells. Error bars are s.e.m. e, Treatment with 2-deocyglucose (2-DG) reduces ECAR and abrogates the difference between cell lines from Atg7+/+ and Atg7−/− tumours. Values are the average of three biological replicates. Error bars are s.e.m. f, g, LC-MS analysis revealed increased glucose consumption from medium (f) and extracellular lactate accumulation (g) in Atg7-null cell lines. Values are the average of eight (Atg7+/+) and seven (Atg7−/−) biological replicates. h, LC-MS analysis shows increased accumulation of 2-DG and its metabolite 2-DG-6-phosphate in Atg7-deficient cell lines. Values are averages of five biological replicates for each genotype. i, Background levels of 18F-FDG uptake in brain and heart can be seen in sagittal and coronal views (middle and right panels, respectively) of wild-type Atg7 mice (top row). In contrast, intense 18F-FDG uptake is seen in pancreatic tumours of Atg7 deficient mice (bottom row). j, LC-MS of intracellular glycolytic and pentose phosphate pathway intermediates shows enhanced accumulation in Atg7−/− tumour cell lines. Values are average of eight (Atg7+/+) and six (Atg7−/−) biological replicates. f–h, j, Error bars are s.e.m. a, f–h, j, A Mann–Whitney U-test was used to ascertain significance. *P  0.05, **P  0.01.
Endolysosomal degradation of Tau and its role in glucocorticoid-driven hippocampal malfunction (2018)
Figure 2

Figure 2. Tau degradation occurs through the Rab35/ESCRT pathway.

(A) Schematic diagram of the Rab35/ESCRT pathway, indicating the manipulation of targets (Rab35 and TSG101) used in our studies, and the final readout of Tau degradation; green and red arrows represent overexpression and knockdown of the target proteins, respectively.

(B, C) Representative immunoblots (B) and quantification of Tau degradation (C) from 14 DIV primary neurons transduced with EGFP or shTSG101, treated for 24 h with either DMSO (CON) or cycloheximide (CHX), and probed for Tau and tubulin. shTSG101-expressing neurons exhibit markedly decreased Tau degradation compared to EGFP-expressing controls (n=18-19 per condition, unpaired student's t-test, *p=0.0108).

(D, E) Representative immunoblots (D) and quantification of Tau degradation (E) from 14 DIV neurons transduced with mCh or mCh-TSG101, treated for 24 h with either DMSO (CON) or cycloheximide (CHX), and probed for Tau and tubulin. Overexpression of TSG101 increases Tau degradation (n=17/condition, unpaired student's t-test, *p=0.0115).

(F, G) Representative immunoblots (F) and quantification of Tau degradation (G) from 14 DIV neurons transduced with mCh or shRab35, treated for 24 h with either DMSO (CON) or cycloheximide (CHX), and probed for Tau and tubulin. shRab35-expressing neurons exhibit markedly decreased Tau degradation compared to mCh-expressing controls (n=23-26 per condition, unpaired student's t-test, ***p=0.0008).

(H, I) Representative immunoblots (H) and quantification of Tau degradation (I) from 14 DIV neurons transduced with mCh or mCh-Rab35, treated for 24 h with either DMSO (CON) or cycloheximide (CHX), and probed for Tau or tubulin. Rab35 overexpression increases Tau degradation (n=18 per condition, unpaired student's t-test, *p=0.0126).

(J) Flow cytometry distribution of N2a cells cotransfected with either HA vector (black) or HA-Rab35 (green) and medium fluoresence timer-tagged wild-type Tau (FT-Tau); blue fluorescence (y axis) indicates "younger" Tau protein while red fluorescence (x axis) indicates "older" Tau.

(K) The ratio of cells expressing red to blue (older:younger) FT-Tau is reduced in cells overexpressing Rab35, indicating faster Tau turnover (n=9/condition, unpaired student's t-test , ****p<0.0001).

(L) The ratio of cells expressing red to blue (older:younger) FT-tagged phospho-mimetic E14 Tau (FT-TauE14) is also reduced in cells overexpressing Rab35, indicating that Rab35 also triggers degradation of phosphorylated Tau (n=10/condition, unpaired student's t-test, *p=0.0032).

Data information: All numeric data represent mean ± SEM.
Endolysosomal degradation of Tau and its role in glucocorticoid-driven hippocampal malfunction (2018)
Figure 3

Figure 3. Phospho-dependent selectivity of Tau sorting into the Rab35/ESCRT pathway.

(A, B) Representative immunoblots (A) and quantification of Tau degradation (B) from 14 DIV primary neurons transduced with mCh or shRab35, treated for 24 h with either DMSO (CON) or cycloheximide (CHX), and probed for pSer396/404-Tau (PHF1), pSer262-Tau, or pSer202-Tau (CP13) and tubulin. shRab35-expressing neurons exhibit markedly decreased pSer262- and p396/404-Tau degradation compared to mCh-expressing controls, while pSer202-Tau degradation is unaffected (n=4 per condition for pSer396/404-Tau and pSer202-Tau, n=3 for pSer262-Tau, unpaired student's t-test, **p=0.0027, *p=0.0120).

(C) Images of PLA signal (green) for Hrs/Tau interaction in N2a cells co-transfected with FLAG-Hrs and either mCh or mCh-Rab35 (red), and probed with antibodies against FLAG and total (DA9) or phospho-Tau species (pSer396/404-Tau, pSer262-Tau, or pSer202-Tau); scale bar: 5 μm.

(D) Rab35 overexpression significantly increases PLA puncta for total, pSer396/404-Tau, pSer262-Tau but not pSer202-Tau (n=121-132 cells for total Tau, 178-195 cells for pSer396/404-Tau, 71-98 cells for pSer262-Tau, 25-29 cells for pSer202-Tau; Mann-Whitney test, ****p<0.0001).

Data information: All numeric data represent mean ± SEM.
Endolysosomal degradation of Tau and its role in glucocorticoid-driven hippocampal malfunction (2018)
Figure 5

Figure 5. Glucocorticoids decrease Rab35 levels in vitro and in vivo.

(A, B) Representative immunoblots (A) and quantification of Hrs, TSG101, CHMP2b, and Rab35 protein levels (B) from 14 DIV neurons treated with either DMSO (CON) or glucocorticoids (GC). GC treatment selectively decreases Rab35 protein levels without affecting levels of Hrs, TSG101, or CHMP2b (n=9-10/condition, unpaired student's t-test, *p=0.0221).

(C) Rab35 mRNA levels are decreased by GC in 14 DIV hippocampal neurons and N2a cells (for neurons, n=9/condition, unpaired student's t-test, **p=0.0036; for N2a cells, n=6/condition, unpaired student's t-test, *p=0.0138).

(D) Schematic diagram of GC and vehicle control (CON) treatment in rats for 15 days.

(E, F) Representative immunoblots (E) and quantification of levels of different Rab proteins (F) in hippocampus of GC-treated and CON animals. Protein levels of Rab35, but not other Rabs, are decreased in GC-treated animals compared to CON ones (n=5 animals/condition, unpaired student's t-test, *p=0.0257).

(G, H) Immunofluorescence staining of Rab35 (green) and DAPI (blue) (G) showing that Rab35 fluorescence intensity is reduced in hippocampal area (CA1) of GC-treated aniamls (H) (n=15 slices/condition; unpaired student's t-test, ***p=0.0003).

Data information: All numeric data represent mean ± SEM.
Endolysosomal degradation of Tau and its role in glucocorticoid-driven hippocampal malfunction (2018)
Figure 6

Figure 6. AAV-mediated Rab35 expression rescues glucocorticoid-induced Tau accumulation and associated neuronal atrophy in rat hippocampus.

(A, B) Representative immunoblots (A) and quantification of Tau degradation (B) in 14 DIV neurons expressing mCh-Rab35 or mCh, treated for 24 h with cycloheximide (CHX) or DMSO (CON) under GC conditions. GC significantly decreases Tau degradation, whereas Rab35 overexpression blocks this effect (n=12-14/condition; 1-way ANOVA, Dunnet post-hoc analysis, *pmCh vs mCh GC=0,0319, *pmCh vs Rab35 GC=0,047).

(C) Injection of AAV to express EGFP or EGFP-Rab35, driven by the CaMKIIα promoter, in rat hippocampus prior to GC or vehicle (CON) treatment.

(D, E) Representative immunoblots (D) and quantification of Tau levels (E) in hippocampal synaptosomes reveal that GC increases total Tau levels in animals expressing EGFP, but not Rab35 (n=5-6 animals/group, 2 replicates, 2-way ANOVA, GC x Rab35 interaction F1, 39=10,51 p=0.002; Sidak posthoc analysis *p=0.0168).

(F) Golgi-based 3D neuronal reconstruction of hippocampal pyramidal neurons (CA1 area).

(G) GC treatment reduces the length of apical dendrites in animals expressing EGFP, but not EGFP-Rab35 (n=6-7 animals/group; 6-8 neurons/animal, 2-way ANOVA , GC x Rab35 interaction F1, 155=3.969 p=0.0481, overall GC effect F1, 155=8.998 p=0.0031, Sidak posthoc analysis **p=0.0021).

(H) GC treatment reduces mature spine density in animals expressing EGFP, but not EGFP-Rab35 (2-way ANOVA, GC x Rab35 interaction F1, 499=12.33 p=0.0005, overall GC effect F1, 499=4.373 p=0.0370, Sidak posthoc analysis *** p=0.0003; n=6-7 animals/group; 6-8 neurons per animal).

(I, J) Sholl analysis of apical dendrites in rat hippocampus shows reduced dendritic intersections after GC treatment in EGFP-expressing animals; however, this effect is not seen in EGFP-Rab35-expressing animals (3-way ANOVA, GC x Rab35 interaction F1, 4212= 14,926 p<0.0001, Simple effect analysis, Sidak test for multiple comparisons *p120=0.012, **p140=0.001, **p220=0.002, ***p240<0.001, ***p260<0.001, **p280=0.001, **p300=0.001, *p320=0.021, *p340=0.047, n=6-7 animals/group, 6-8 neurons per animal).

Data information: All numeric data represent mean ± SEM.
CBX4 contributes to HIV-1 latency by forming phase-separated nuclear bodies and SUMOylating EZH2 (2022)
Figure 1

Figure 1. CBX4 contributes to HIV-1 latency.

A. An siRNA library targeting major PcG proteins was transfected into TZM-bl cells, respectively. Three different siRNAs targeting each gene were transfected as a mixture. Forty-eight hours post transfection, cells were harvested and lysed, followed by measuring the luciferase activity of each group. Fold changes of luciferase expression were calculated for each gene by normalizing to the negative control siNC. The red dashed line represented the base line.

B. Different siRNAs targeting each gene were transfected as in (A). Twenty-four hours post siRNA-transfection, cells in each group were transfected with equal amounts of Tat-expressing plasmids. Another 24 hours later, cells were proceeded to luciferase assay.

C. siRNAs targeting different genes were transfected into TZM-bl cells as in (A). About 24 hours later, cells were treated with TNFα. Another 24 hours later, cells were proceeded to luciferase assay.

D. The endogenous CBX4 in TZM-bl cells was knocked down by three different siRNAs targeting 3'UTR of CBX4 mRNA, or treated with siNC. Different gradients of CBX4 construct were co-transfected. The expression of luciferase from each group was measured and normalized to the siNC group which was not co-transfected with CBX4 construct.

E. Total RNAs from naïve CD4+ T cells, PHA-stimulated CD4+ T cells and resting memory CD4+ T cells were extracted and proceeded to RT-qPCR to quantitate the relative expression of CBX4. The expression of CBX4 in PHA-stimulated and resting memory CD4+ T cells was normalized to naïve CD4+ T cells.

F, G. The percentages of GFP-positive J-Lat 10.6 cells in shluc and shCBX4 groups. LRAs SAHA and JQ-1 were used as supplements. The representative flow cytometry figure of each group was shown in (F). The overall statistical results were shown in (G).

H, I. One heterogeneous latency model J-Lat Mix and one monoclonal model J-Lat A2 were treated as in (F). The reactivation efficiencies of each group were indicated by the percentages of GFP-positive cells.

Data information: Data represented mean ± SEM in biological triplicate. p-Values in (A-C) were calculated by one-way ANOVA with Dunnett's multiple comparisons test. p-Values in (D) were calculated by two-way ANOVA with Sidak's multiple comparisons test. p-Values in (E-I) were calculated by one-way ANOVA with Tukey's multiple comparisons test. *p < 0.05, **p < 0.01, ***p < 0.001.
CBX4 contributes to HIV-1 latency by forming phase-separated nuclear bodies and SUMOylating EZH2 (2022)
Figure 3

Figure 3. CBX4 recruits PcG proteins and forms nuclear bodies.

A. TZM-bl cells were transfected with empty vectors and CBX4-expressing plasmids respectively, followed by the enrichment of CBX4-bound proteins. The heatmap represented CBX4-enriched proteins. Representative genes were shown in table aside the heatmap. The heatmap scale represented fold changes of gene expression (min: -8 fold; max: 8 fold).

B. Super-resolution SIM image of CBX4 in HEK293T cells. DAPI was used to dye DNA which was colored into blue. FITC-tagged antibodies were used to label endogenous CBX4 which was colored into green.

C. The distribution of CBX4 bodies diameters. The black line indicated the median value of diameters which was 512 nm. Data were collected from three cells of three independent samples.

D. Rendered 3D shapes of CBX4 bodies. Three panels represented XY, YZ and XZ planes of one CBX4 body. The right scatter plot represented the distribution of CBX4 bodies sphericities. The black line indicated the median value of sphericities which was 0.9. Data were collected from three cells of three independent samples.

E. ImmunoFISH images of the pseudotyped HIV-1 genomic DNA and CBX4 bodies in J-Lat 10.6. Naïve cells were treated with DMSO. Activated cells were treated with TNFα.

F-L. RFP-tagged EZH2, RING1B, SUMO4, SUZ12, CHAF1A, DNMT1 and HP1α were co-overexpressed with GFP-tagged CBX4 in HEK293T cells, respectively. SIM imaging was performed for each combination. The line scan profiles of SIM images which showed co-localization and non-co-localization between CBX4 and its partners were shown on the right of each panel. The red arrows in merged images indicated the positions where the line scans profiled.

M-O. GFP-tagged CBX4 was overexpressed in HEK293T cells, followed by treating with AF568-tagged antibodies against H3K4me3, H3K9me3 and H3K27me3, respectively. CBX4 and each histone modification were imaged with SIM. Line scan profiles were shown on the right of each panel. Red arrows in merged images indicated the positions where line scans profiled.

Data information: Scale bars in (B) and (E-O) represented 5 μm. Scale bars in (D) represented 100 nm. Scale bars in amplified images of (E) represented 500 nm. All the samples were imaged to obtain at least three sets of images.
CBX4 contributes to HIV-1 latency by forming phase-separated nuclear bodies and SUMOylating EZH2 (2022)
Figure 4

Figure 4. CBX4 bodies are phase-separated condensates.

A. FRAP images of CBX4 bodies to indicate the internal diffusion and diffusion across boundary properties. GFP-tagged CBX4 was overexpressed in HEK293T cells. These live cells were proceeded to time series imaging 24 hours post transfection. Six GFP-CBX4 bodies were circled and marked as region of interest 1 (ROI 1), ROI 2, ROI 3, ROI 4, ROI 5 and ROI 6. Three CBX4 bodies (ROI 4, ROI 5 and ROI 6) were bleached with strong 488 nm laser pulse. Another three unbleached CBX4 bodies (ROI 1, ROI 2 and ROI 3) were set as control. Images were captured every 4 s. The right histogram showed relative fluorescence intensities of unbleached and bleached CBX4 bodies in each time point.

B, C. Representative images of two CBX4 bodies fusing into one and one CBX4 body splitting into two. Images were captured every 5 s.

D. 1.5% 1,6-hexanediol (1,6-Hex) compound was used to treat GFP-tagged CBX4-expressing live cells. The DNA was stained with Hoechst and colored into blue. Images were captured every 5 s.

E. The schematic of CBX4 backbone. P1 represented Chromodomain (CD). P2 and P3 represented two sub-regions of IDR, which harbored two SUMO-interacting motifs named SIM1 and SIM2. P4 represented CBox domain.

F-I. GFP-tagged CBX4 truncation mutants including CBX4-dCD, CBX4-dP2, CBX4-dP3 and CBX4-dCBox were overexpressed in HEK293T cells and imaged with SIM.

J. The schematic of five motifs within CBox domain, including Specific peptide 1 (KPF), β-sheet 1 (IIITD), β-sheet 2 (LTVT), Specific peptide 2 (FKEY) and C-terminal disordered motif (VTV).

K-O. GFP-tagged CBX4 mutants including CBox-KPFmut, CBox-IIITDmut, CBox-LTVTmut, CBox-FKEYmut and CBox-VTVmut were overexpressed in HEK293T cells and imaged with SIM. The motif mutants were constructed by mutating these motifs into corresponding numbers of alanine residues.

P. RFP-tagged EZH2 was co-overexpressed with GFP-tagged CBX4 mutant named CBX4mut which was CBX4-LTVTmut. SIM images were captured. The line scan profile was shown on the right. The red arrow within merged image indicated the line scan position.

Data information: Data represented mean ± SEM in biological triplicate in (A). Scale bars in (A), (B-D), (F-I) and (K-P) represented 5 μm. All the samples were imaged to obtain at least three sets of images.