EMBO/soda-nel huggingface dataset:
ENTREP/FAM189A2 encodes a new ITCH ubiquitin ligase activator that is downregulated in breast cancer (2021)
Figure 2

Figure 2. ENTREP associates with ITCH.

A Schema of the expression vectors of ENTREP and ITCH. SS, signal sequence; CD20, CD20-homology extracellular domain; TM, transmembrane domain; C2, C2 domain; WW, tryptophan-tryptophan domain; HECT, the HECT domain; C830A, the catalytically inactive mutation. Red bar, cytoplasmic domain of ENTREP and a bait for the yeast two-hybrid screening.

B, C The immunoprecipitation analysis. Co-precipitation was impaired by either the WW domain deletion of ITCH or the mutation in the PPxY motifs of ENTREP. Data shown are representative of at least two independent experiments. IP, immunoprecipitation; IB, immunoblot.

D The immunofluorescence staining of Cos7 cells transiently transfected with indicated vectors. Merged, merged images of FLAG, Halo and DAPI staining. Scale bar, 20mm. Data shown are representative of at least three independent experiments.

E Representative images of the proximity ligation assay (PLA) using Cos7 cells transfected with indicated vectors. PLA signals, granular dots of green color, were presented with DAPI images. Arrowheads, the plasma membrane. Scale bar, 10mm.

F The box-and-whisker plot presentation of the PLA. A minimum of 10 cells per condition were counted from two independent experiments. P-values obtained by Student's t-tests and P < 0.05 was considered as statistically significant. * P < 0.05. Box-whisker plot represents the interquartile range (25th and 75th percentiles) as a box and the median as a line. The maximn and minimun values within 1.5 x interquartile range are shown as whiskers.
ENTREP/FAM189A2 encodes a new ITCH ubiquitin ligase activator that is downregulated in breast cancer (2021)
Figure 3

Figure 3. ITCH ubiquitinates ENTREP.

A The nickel-pull down assay using myc/HIS-tagged ENTREP along with HA-tagged ubiquitin. Arrows indicate HA-ubiquitin-incorporating ENTREP. Arrowhead indicates ENTREP without ubiquitination. Data shown are representative of three independent experiments.

B The ubiquitination of ENTREP. The immunoprecipitated samples using anti-FLAG antibody under the denaturing condition were separated by the SDS-PAGE. The gel area corresponding to band #A, #B and #C of ENTREP-FLAG were applied to the ubiquitin-AQUA/PRM analysis. The absolute number of each ubiquitins of ENTREP #A, #B and #C were calculated by subtraction of those of the corresponding area of the control vector sample (Appendix Table S2 for raw data of the ubiquitin-AQUA/PRM analysis). Based on the ratios of each ubiquitins to the total ubiquitin of indicated gel area, which are showed as Circular graphs (ENTREP #A, #B and #C), the number of ENTREP-linked ubiquitin (Ub) is summerized as schema: #A is modified with one Ub, and about two thirds of #B molecules and half of #C molecules are modified with two and three of single Ubs (multi-monoubiquitin), respectively. The result was based on three biological replicates.
ENTREP/FAM189A2 encodes a new ITCH ubiquitin ligase activator that is downregulated in breast cancer (2021)
Figure 4

Figure 4. ENTREP associates with EPN1.

A The immunoprecipitation analysis. The cytoplasmic ENTREP (wild or mut1+2 mutant) co-precipitated with EPN1myc in the presence of ITCH wild or C830A. The blot bands were semi-quantified using ImageJ software. ENTREP ratio was caluculated as a ratio of ENTREP in the myc-coprecipitaed sample to that of the total lysate. The ratios shown as mean ± SD from three biological replicates. P-values were obtained by Student's t-tests and P < 0.005 was considered as statistically significant. No significant difference was observed.

B Representative images of the proximity ligation assay (PLA) using Cos7 cells transfected with indicated vectors. PLA signals, granular dots of green color, were presented with DAPI images. Scale bar, 10mm.

C The box-and-whisker plot presentation of the PLA. A minimum of 10 cells per condition were counted from two independent experiments. P-values obtained by Student's t-tests and P < 0.05 was considered as statistically significant. * P < 0.05. Box-whisker plot represents the interquartile range (25th and 75th percentiles) as a box and the median as a line. The maximn and minimun values within 1.5 x interquartile range are shown as whiskers.

D The expression of endogenous ENTREP in HMEC. ENTREP co-localized with Clathrin, RAB5, RAB7 and RAB11. Inserts, the ring-shaped structures of the marked areas. The images are representative of at least two independent experiments. Scale bar 10mm.

E Co-localization of ENTREP with ITCH and EPN1. Cos7 cells transfected with indicated vectors were stained with specific antibodies and observed under a laser confocal microscope. Arrowheads, full length ENTREP (wild or mut1+2 mutant) expression at the plasma membrane. Inserts, the high-magnification images of the marked area indicating the co-localizaion of ENTREP-FLAG, EPN1myc and Halo-ITCHwild. The images are representative of at least two independent experiments. Scale bar 10mm.
ENTREP/FAM189A2 encodes a new ITCH ubiquitin ligase activator that is downregulated in breast cancer (2021)
Figure 5

Figure 5. ENTREP enhances the ubiquitination of CXCR4.

A Schema of the expression vectors of ITCH deletion mutants and the carboxyl-terminal tail of CXCR4. TM7, the seventh transmembrane domain of CXCR4; HA, hemagglutinin tag; DsRed, DsRed-momoner (Clontech/TAKARA).

B The immunoprecipitation analysis. Cytoplasmic ENTREP co-precipitated with either ITCH C830A, delWW12 or delWW34 but not with delWW14. Data shown are representative of at least two independent experiments.

C The immunoprecipitation analysis. HA-CXCR4SS-DsRed hardly co-precipitated with either ITCHC830A or delWW mutants. HA-CXCR4DD-DsRed co-precipitated with ITCH C830A and delWW12 but not with delWW14 and delWW34. Data shown are representative of at least two independent experiments.

D The immunoprecipitation analysis. HA-CXCR4DD-DsRed co-precipitated with ENTREP when co-transfected with either ITCH C830A or delWW12. Data shown are representative of at least two independent experiments.

E The immunoprecipitation analysis. The increased transfection of ENTREP vector enhanced precipitation of HA-CXCR4DD-DsRed with ITCH C830A. Data shown are representative of at least two independent experiments.

F The nickel-pull down assay using indicated vectors along with HISx6-tagged wild type ubiquitin vector. HA-tagged DsRed was used as a control for HA-CXCR4DD-DsRed. Arrowhead, FLAG-tagged ITCH (wild or C830A); arrow, FLAG-ENTREP wild cyt. Data shown are representative of at least two independent experiments.

G Co-localization of ENTREP and CXCR4 in the endosome. Cos7 cells were transfected with ENTREP-FLAG, CXCR4-DsRed and Halo-ITCH wild vectors and treated with CXCL12. ENTREP-FLAG and CXCR4-DsRed co-localized in the RAB7-positive endosome. Inserts, the high-magnification images of the marked area indicating the colocalizaion of ENTREP-FLAG, CXCR4-DsRed and RAB7. Scale bar 10mm. The image is representative of at least two independent experiments.
ENTREP/FAM189A2 encodes a new ITCH ubiquitin ligase activator that is downregulated in breast cancer (2021)
Figure 6

Figure 6. ENTREP participates in CXCL12-induced endocytosis of CXCR4.

A The immunoblot analyses of MCF-7 ENTREP-KO and parent MCF-7 cells. Note that the amount of CXCR4 protein was not changed. The immunoblot analyses were carried out using six independent samples, and their blot bands were semi-quantified using ImageJ software. CXCR4/GAPDH ratio was caluculated as a ratio of GAPDH-adjusted CXCR4 expression. CXCR4/GAPDH ratios shown as mean ± SD from at least four biological replicates. P-values were obtained by Student's t-tests and P < 0.005 was considered as statistically significant. No significant difference was observed.

B Cell proliferation analysis. Cells were cultured in DMEM supplemented with 10% FBS and counted at day1 to day3. Data are shown as mean ± SD from six biological replicates. P-values were obtained by Student's t-tests and P < 0.005 was considered as statistically significant. NS, not significant.

C The expression of endogenous CXCR4 in lentivirally-transduced MCF-7 ENTREP-KO cells. CXCL12 (+) and (-) indicate the presence and absence of CXCL12 treatment. In CXCL12-treated ENTREP-EGFP expressing cells, a non-negligible amount of CXCR4 was observed in the cytoplasm, in which CXCR4 was overlapped with ENTREP-EGFP. CXCR4 expression on the cell surface still remained (arrows). Scale bar 10 mm. The images are representative of at least three independet experiments.
ENTREP/FAM189A2 encodes a new ITCH ubiquitin ligase activator that is downregulated in breast cancer (2021)
Figure 7

Figure 7. ENTREP fine-tunes CXCR4 function.

A Dot plot presentation of the chemotaxis analyses using the Boyden chamber. CXCL12 (+) and (-) indicate the presence and absence of CXCL12 as an attractant in the bottom chambers. ENTREP-knockout enhanced the chemotaxis toward CXCL12, which was blocked by pre-treatment with AMD3100 (upper panel). The doxycyclin (DOX)-induced ENTREP expression suppressed the chemotaxis of mouse 4T1-Luc cells toward CXCL12 (lower panel). Ratio of migrated cells into the bottom chamber against the applied cells on the top chambers was normalized by the results of non-treated cells (n=7, upper panel, and n=9, lower panel, from at least two independent experiments, respectively). P-values were obtained by Student's t-tests and P < 0.005 was considered as statistically significant. Insert, the immunoblot analysis of the DOX-induced ENTREP expression in 4T1-Luc cells. NS, not significant.

B Dot plot presentation of mammosphere assay (left panel) and the images of mammosphere (right panel). Each mark in dot plot presentation indicates the number of mammosphere from 3,000 cells per well in an ultra-low attachment plate from two independent experiments. To estimate the effect of CXCL12/CXCR4 pathway, 100 ng/ml of CXCL12 and 12 μM of AMD3100 were applied as indicated. For statistical analysis, variance was assessed using two-way ANOVA and significance was calculated using Tukey post hoc test correcting for multiple comparison. NS, not significant. Scale bar in the images of mammosphere 100 mm.
Hexokinase 2 displacement from mitochondria-associated membranes prompts Ca2+-dependent death of cancer cells (2020)
Figure 1

Figure 1. HK2 locates in MAM of cancer cells and is displaced by HK2pep

A Immunofluorescence staining of HK2 with an AlexaFluor488-conjugated antibody in HeLa cells expressing mitochondria-targeted RFP. Yellow signals in the merge analysis indicate mitochondrial localization of HK2. Scale bar: 15 µm.

B Immunofluorescence staining of HK2 with a secondary AlexaFluor555-conjugated antibody in HeLa cells expressing both mitochondria-targeted YFP and ER-targeted CFP. The merged white signal indicates MAM localization of HK2 and is quantified in the bar graph on the right (n=24). Image magnifications are shown in the lower part of the panel; arrows indicate HK2 dots in mito-ER contact sites. Scale bar: 15 µm.

C Fluorescence co-staining of HK2 and split-GFP-based probe for ER-mitochondria contacts (SPLICSL) on HeLa cells; HK2 is revealed with a secondary AlexaFluor555-conjugated antibody, the merged signal is white. Scale bar: 15 µm.

D Quantification of panel B experiment showing the percentage of HK2 dots that merge with MAMs in HeLa cells (n=24 cells analyzed from three independent experiments; mean±SD).

E Quantification of panel C experiment showing the percentage of SPLICSL dots positive for HK2 in HeLa cells (n=10 cells analyzed from three independent experiments; mean±SD).

F Percentage of HK2 dots positive for SPLICSL in HeLa, COLO 741, MDA-MB-231, PN 04.4 and S462 cells (n≥6 cells for each cell line; mean±SD).

G Functional unit composition of HK2pep (left); the HK2-targeting sequence is in red; the polycation and polyanion stretches are in light blue and light green, respectively; the MMP2/9 target sequence is in yellow. On the right, mass spectrometry profile of HK2pep before and after incubation (cl-HK2pep) with human MMP9.

H HK2 displacement from HeLa MAMs after a 2 min treatment with cl-HK2pep is shown by loss of merging signal analyzed as in (C). cl-SCRpep is used as a negative control and Pearson's co-localization coefficient is indicated in figure (cl-SCRpep n=26 cells; cl-HK2pep n=24 cells; p<0.001 with a Student's t test). Scale bar: 10 µm.

I, J Effect of cl-HK2pep on glucose phosphorylation by human recombinant HK2 (I) or in 4T1 cell extracts (J), where both total hexokinase activity and HK1/HK2 specific activities are measured.
Hexokinase 2 displacement from mitochondria-associated membranes prompts Ca2+-dependent death of cancer cells (2020)
Figure 2

Figure 2. HK2 detachment from MAMs prompts Ca2+ influx and depolarization in mitochondria following IP3R opening

A-F Effect of cl-HK2pep on cellular Ca2+ dynamics and IP3 levels. ER Ca2+ levels are measured by the FRET-based, D4ER fluorescent probe expressed in the lumen of ER (A); IP3 levels are assessed with the GFP-PHD probe; histamine (100μM) is used as a positive control for IP3 generation; data are reported as mean of fluorescent signals±SEM (n=3 independent experiments; B); changes in mitochondrial Ca2+ levels are recorded (C; scale bar: 20µm) and quantified using the GCAMP6f sensor (in D, as mean of 475/410 nm ratio; signal±SEM of at least 5 independent experiments and more than 20 cells analized), in (E) as percentage of cells with increased Ca2+ in mitochondria, with a threshold 475/410 ratio for positivity >3; baseline mean ratio=1.84±0.54) or with mitochondria-targeted aequorin (F, where data are reported as mean of [Ca2+]±SEM of 3 independent experimental days).

G-J Effect of cl-HK2pep treatment on mitochondrial membrane potential assessed with the TMRM probe. Kinetic experiments (G, single cell analysis, n=216 cells analyzed in at least 10 independent experiments; H, representative field; scale bar: 20µm) are quantified (I and J; TMRM fluorescence is normalized to initial value and expressed in percentage for each time point, with a depolarization threshold placed at 40% of initial value).

Data information: Experiments throughout the Figure are carried out on HeLa cells; cl-SCRpep, negative control of cl-HK2pep (2 μM each). Where indicated, cells are kept in Ca2+ free medium plus 500 µM EGTA with or without 10 µM BAPTA-AM; Xe-C is Xestospongin C, which selectively inhibits IP3R at the 5 μM concentration used here [46].
Hexokinase 2 displacement from mitochondria-associated membranes prompts Ca2+-dependent death of cancer cells (2020)
Figure 3

Figure 3. HK2pep induces PTP- and calpain-dependent cell death

A, B Effects of the PTP desensitizers CsA or C63 (5 µM each, 1 h preincubation) on mitochondrial Ca2+ levels recorded with GCAMP6f (A) and on mitochondrial membrane potential assessed with TMRM (B) in cells treated with cl-HK2pep.

C-F cell death induction by cl-HK2pep; in the cytofluorimetric analyses reported in (C, E and F), viable cells are double negative for Annexin V-FITC and 7-AAD staining and measured 15 min after peptide treatment; in the kinetic experiment shown in (D; scale bar: 50µm) viable cells are double negative for Annexin V-FITC and 7-AAD and TMRM positive. In C and F data are presented as mean±SD and they were obtained from 3 different experiments or more; 3 technical replicates in all analyzed experiment.

G, H Effect of calpain inhibition on mitochondrial membrane potential assessed with TMRM (G, normalized fluorescence signal mean±SD; 3 different experiments including 3 technical replicates each) and on mitochondrial Ca2+ levels recorded with GCAMP6f (H) in cells treated with cl-HK2pep .

Data information: Experiments throughout the Figure are carried out on HeLa cells; cl-SCRpep, negative control of cl-HK2pep (2 μM each); where indicated, the caspase inhibitor Z-VAD-fmk or the calpain inhibitor PD150606 (50 µM each) are pre-incubated 1 h before peptide treatment. Experiments using TMRM or GCAMP6f probes are analyzed as in Figure 2. In C, F and G data are presented as mean±SD of at least 3 independent experiments. In (C) *** p<0.001 with a Student's t test; in (F, G), p<0.0001 with a two-way ANOVA (cl-HK2pep treatment vs cl-HK2pep+PD150606 or cl-SCRpep treatments); Bonferroni post-test in graph ***p<0.001 (cl-HK2pep vs cl-SCRpep and cl-HK2pep vs PD150606+cl-HK2pep).
Hexokinase 2 displacement from mitochondria-associated membranes prompts Ca2+-dependent death of cancer cells (2020)
Figure 4

Figure 4. HK2pep kills primary B-CLL cells

A Representative IHCs in human reactive lymphoid tissue, where HK2 expression is mainly restricted to proliferating blasts within germinal centers (center), and in patient B-CLL cells. Scale bar 100 µm.

B-G Effect of cl-HK2pep on human B-CLL cells freshly isolated from patients. HK2-expressing B-CLL cells (B; GAPDH and respiratory complex III subunit UQCRC2 are cytosol and mitochondrial loading controls, respectively) undergo mitochondrial depolarization assessed by TMRM staining (C) and cell death, measured by cytofluorimetric analysis of Annexin V-FITC and 7-AAD staining (D-G). Cells are treated with 5 µM cl-HK2pep; PD150606 (50 μM) is pre-incubated for 1 h. Experiments are carried out on samples from at least 10 patients and on CD19+, primary B lymphocytes from 5 healthy controls. In C, D, F and G data are presented as mean±SEM; all data were obtained from 3 technical replicates in all analyzed patient samples. In (C, D) two-way ANOVA: p<0.0001 (cl-HK2pep treatment vs cl-HK2pep+PD150606 or cl-SCRpep treatments); Bonferroni post-test in graph ***p<0.001(cl-HK2pep vs cl-SCRpep and cl-HK2pep vs PD150606+cl-HK2pep), in (F, G), Student's t test; ***p<0.01, *p<0.05.
Hexokinase 2 displacement from mitochondria-associated membranes prompts Ca2+-dependent death of cancer cells (2020)
Figure 5

Figure 5. The HK2-targeting peptide inhibits in vitro and in vivo tumor growth

A-D Effect of HK2 targeting on in vitro tumorigenicity. HK2-silencing with two different shRNAs (shHK2-1 and shHK2-2) in CT26 colon cancer cells (A) inhibits growth in soft agar (B; colony area±SEM; n=6 replicates obtained from 3 independent experiments; Student's t test ***p<0.001; A.U., arbitrary units). Treatment with cl-HK2pep (50 µM, 6 h; C, scale bar: 100 µm) or with the uncleaved HK2pep (2 µM, 48 h; D, scale bar: 200 µm) reverts formation of foci; dead cells are highlighted by Trypan Blue staining.

E-H Effect of HK2-targeting on in vivo neoplastic growth. HK2- and MMP-2-expressing CT26 cells (E, scale bar: 100 µm) are allografted in Balb/c male mice. Intratumor administration (5 injections of 2 nmol peptide every 12 h) of either cl-HK2pep or HK2pep (F; CNT n=4; cl-HK2pep n=5; HK2pep n=5 mice), or intraperitoneal administration of HK2pep (G; 5 injections of 60 nmol peptide every 12 h; SCRpep n=6; HK2pep n=7 mice) reduce neoplastic growth; the same effect is observed on allografts of breast carcinoma 4T1 cells (H; 5 injections of 60 nmol peptide every 12 h; SCRpep n=5; HK2pep n=7 mice) in Balb/c female mice.

Data information: Throughout the Figure, cl-SCRpep or SCRpep are used as peptide negative control. In G, representative ultrasound inspections and 3D reconstructions of tumor masses are shown (light grey: SCRpep treated animal; light blue: HK2pep treated animal). In F, mean tumor volume±SD is reported; Student's t test ***p<0.001; in G, H, normalized volume±SEM; a two-way ANOVA is performed (p<0.001 - HK2pep vs SCRpep); Bonferroni post-test in the graphs *p<0.05; ***p<0.001.

TABLE

Table 1. Biological and clinical characteristics of the 22 B-CLL patients analyzed. *Mutated was deﬁned as having a frequency of mutations greater than 2% from the germline IGHV sequence. §N = normal karyotype. WBC, white blood cell.

Expanded view figure legends
p38γ and p38δ reprogram liver metabolism by modulating neutrophil infiltration (2015)
Figure 1

Figure 1. p38γ and p38δ are up-regulated in NAFDL.

(A) Left: qRT-PCR analysis of mRNA expression of MAPK12 (p38gamma) and MAPK13 (p38delta) in liver extracts prepared from obese patients with alcoholic fatty liver disease (NAFLD) and control individuals without NAFLD. mRNA expression was normalized to the amount of Gapdh mRNA. Right: Representative H&E-stained liver sections. Scale Bar: 50μm. (n= 11-74) (B) Left: qRT-PCR analysis of mRNA expression of MAPK12 (p38gamma) and MAPK13 (p38delta) in liver extracts prepared from control patients with NAFLD/non-alcoholic steatohepatitis (NASH) and control individuals without NAFLD/NASH. mRNA expression was normalized to the amount of Gapdh mRNA. Right: Representative H&E-stained liver sections. Scale Bar: 50μm. (n= 11-9) (C) Quantification of immunoblot analysis of p38δ expression in liver extracts prepared from obese patients with NAFLD and individuals without NAFLD. Representative blots are shown. (n= 7-40) (D) qRT-PCR analysis of Mapk12 (p38gamma) and Mapk13 (p38delta) mRNA expression in liver extracts prepared from wild-type mice (WT) fed a diet deficient in methionine and choline (MCD) or control diet (ND) for 3 weeks; mRNA expression was normalized to the amount of Gapdh mRNA. (n=5-10) (E) Immunoprecipitation analysis of activation and protein levels of p38γ and δ isoforms in liver extracts prepared from WT fed a MCD or ND diet for the times indicated. (F) Immunoprecipitation analysis of activation and protein levels of p38γ and δ isoforms in liver extracts prepared from WT fed a MCD or ND for 3 weeks (n=5). Western blot against vinculin was used to assay the protein amount in the total lysate (TL) used for each IP. Protein expression was normalized to vinculin. Data are means ± SEM. *P<0.05; **P<0.01. (Statistical significance by two-tailed Student’s t test. Characteristics of patients and controls were compared by means of χ2 or Mann-Whitney U tests.)
p38γ and p38δ reprogram liver metabolism by modulating neutrophil infiltration (2015)
Figure 2

Figure 2. p38γ/δ-/- mice are protected against steatohepatitis and fibrosis.

(A) Representative H&E and oil red-stained liver sections prepared from WT and p38γ/δ-/- mice fed a ND or the MCD diet for 3 weeks. Scale Bar: 50μm. (B) Liver triglycerides and (C) plasma transaminase activity (ALT) measured in WT and p38γ/δ-/- mice after 3 weeks of MCD diet. (D) TBARS and hydrogen peroxide detected in liver samples from mice fasted overnight after the 3-week MCD diet. (E) qRT-PCR analysis of Col1a1, Acta2 and Timp1 mRNA expression. mRNA expression was normalized to the amount of Gapdh mRNA.(F) Representative Masson's trichrome-stained liver sections prepared from WT and p38γ/δ-/- mice fed a ND or the MCD diet for 3 weeks. Scale Bar: 50μm. Data are means ± SEM (n=5-10). *P<0.05; **P<0.01; ***P<0.001 (2-way ANOVA coupled to Bonferroni’s post tests).
p38γ and p38δ reprogram liver metabolism by modulating neutrophil infiltration (2015)
Figure 3

Figure 3. p38γ/δLyzs-KO mice are protected against steatohepatitis induced by MCD diet

Lyzs-Cre and p38γ/δLyzs-KO mice were fed a ND or a MCD diet for 3 weeks. (A) Representative H&E and oil red stained liver sections. Scale Bar: 50μm. (n=5-10). (B) Liver triglycerides (C) and plasma ALT at the end of the diet period. (D) Measurement of plasma TNF-α and IL-6. (E) qRT-PCR analysis of myeloid cell markers and cytokine mRNA expression from liver tissue; mRNA expression was normalized to the amount of Gapdh mRNA. (F) qRT-PCR analysis of M1 and M2 polarization cell markers from liver infiltrated macrophages. mRNA expression was normalized to the amount of Gapdh mRNA. Data are means ± SEM (n=5-10). *P<0.05; **P<0.01; ***P<0.001 (2-way ANOVA coupled to Bonferroni’s post tests).