EMBO/soda-nel huggingface dataset:
Micropeptide CIP2A-BP encoded by LINC00665 inhibits triple negative breast cancer progression (2019)
Figure 5

Figure 5. Micropeptide CIP2A-BP interacts with the region of CIP2A which is required for interaction with PP2A subunit B56γ

(A) Mass spectrometric analysis of co-immunoprecipitation experiments.

(B) LINC00665 ORF-His plasmid was transfected into MDA-MB-231 cells. Whole-cell lysates were subjected to co-immunoprecipitation using anti-His antibody and immunoblot analysis with CIP2A antibody to verify that CIP2A bound to CIP2A-BP.

(C) LINC00665 ORF-His plasmid was transfected into MDA-MB-231 cells. Whole-cell lysates were subjected to co-immunoprecipitation by CIP2A antibody and immunoblot analysis with CIP2A-BP antibodies to verify that CIP2A-BP bound to CIP2A.

(D) Endogenous CIP2A-BP and CIP2A were co-immunoprecipitated in MCF-10A cell line.

(E) LINC00665 ORF-His plasmid was transfected into MDA-MB-231 cells. Whole-cell lysates were treated with 30 mg/mL RNase A for 30 min, the indicated complexes were co-immunoprecipitated by anti-His or anti-CIP2A antibody, and CIP2A or micropeptide CIP2A-BP were detected.

(F) Upper: A diagram of the domain structure of full length CIP2A protein. Red: RIP=Required for Interaction with PPP2R5C; Orange: RH=Required for Homodimerization; Blue, CC=Coiled coil. Lower: Co-immunoprecipitation coupled western blot analysis assays revealed the interaction of CIP2A-BP with CIP2A via the N-terminus domain of CIP2A.

(G) Upper: A diagram of the domain structure of CIP2A protein fragment (1-245). Red: RIP=Required for Interaction with PPP2R5C. Lower: Co-immunoprecipitation coupled western blot analysis assays revealed the interaction of CIP2A-BP with CIP2A via the N-terminus domain of CIP2A (amino acid 159-245).

(H-K) Co-immunoprecipitation assays showed that the interaction between CIP2A and B56γ in TNBC cells was affected by CIP2A-BP expression.
Micropeptide CIP2A-BP encoded by LINC00665 inhibits triple negative breast cancer progression (2019)
Figure 6

Figure 6. Micropeptide CIP2A-BP inhibits the activation of the PI3K/AKT/NFκB pathway by increasing PP2A activity

(A) The expression levels of the indicated proteins were detected by immunoblotting analysis in CIP2A-BP KO, LINC00665 ORF overexpressed (OE) and respective controls of TNBC cells.

(B) PP2A activity was measured using a protein phosphatase assay kit. PP2A activity assay was performed to evaluate PP2A activity in CIP2A-BP KO, LINC00665 ORF overexpressed (OE) and respective control TNBC cells.

(C) Immunohistochemistry (IHC) staining of p-AKT from TNBC primary tumor (n = 20) with high or low micropeptide CIP2A-BP expression. Lower panels show higher-magnification images of insets in upper panels. Right panel is the quantification of IHC staining p-AKT.

(D) MDA-MB-231 cells were transfected with anti-CIP2A siRNAs. Whole-cell lysates of these cells were subjected to immunoblot analysis with CIP2A, CIP2A-BP, PP2Ac, p-AKT, AKT and β-actin antibodies.

(E) CIP2A-BP KO, LINC00665 ORF overexpressed (OE) and respective control MDA-MB-231 cells were transfected with anti-CIP2A siRNAs. PP2A activity assay was performed to evaluate PP2A activity in the indicated cells.

(F) The indicated cells were pretreated with specific antagonist against AKT (MK-2206，3μM) for 1h. Then whole lysates of these cells were subjected to immunoblot analysis.

Data information: Data are representative of 3 independent experiments (B and E). Data were assessed by paired Student's t test (B, C, and E) and are represented as mean ± SD. *P < 0.05; **P < 0.01. Scale bars: 50μm (C).
Micropeptide CIP2A-BP encoded by LINC00665 inhibits triple negative breast cancer progression (2019)
Figure 7

Figure 7. Micropeptide CIP2A-BP inhibits metastasis and invasion of breast cancer in the MMTV-PyMT mouse model and acts as a potential anti-cancer peptide in vivo

(A) Macroscopic and histological analysis of the lungs of 140 days old MMTV-PyMT mice; lung metastatic nodules were visualized 8 weeks post-transplantation (5 mice per group). Right panel is the quantification of pulmonary metastases.

(B) Immunohistochemistry (IHC) staining of p-AKT of mammary tumors from MMTV-PyMT;CIP2A-BP+/+ or MMTV-PyMT;CIP2A-BP-/- mice. Lower panels show higher-magnification images of insets in upper panels. Right panel is the quantification of IHC staining p-AKT .

(C) MMTV-PyMT mice were injected with CIP2A-BP or svCIP2A-BP via mammary fat pad. Macroscopic and histological analysis of the lungs of 140 days old MMTV-PyMT mice; lung metastatic nodules were visualized 8 weeks post-transplantation (5 mice per group). Right panel is the quantification of pulmonary metastases.

(D) Representative IHC images of p-AKT of mammary tumors from MMTV-PyMT mice that were injected with CIP2A-BP or svCIP2A-BP via mammary fat pad; Lower panels show higher-magnification images of insets in upper panels. Right panel is the quantification of IHC staining p-AKT.

(E) Kaplan-Meier survival curves of nude mice transplanted with MDA-MB-231 cells and injected with CIP2A-BP or svCIP2A-BP (10 mice per group). The horizontal line indicates the time after the mice were injected with the cells via the tail vein.

(F) Macroscopic and histological analysis of the lungs of nude mice injected with MDA-MB-231 cells and CIP2A-BP or svCIP2A-BP via tail vein; lung metastatic nodules were visualized 8 weeks post-transplantation (5 mice per group). Right panel is the quantification of pulmonary metastases.

Data information: Data were assessed by paired Student's t test (A-D, and F) and are represented as mean ± SD. Survival differences were analyzed using the log-rank test (E). *P < 0.05; **P < 0.01. Scale bars: 50μm (B and D), 1000μm (A, C, and F).
Dynamic stroma reorganization drives blood vessel dysmorphia during glioma growth. (2017)
Figure 1

Figure 1 : Blood vessel abnormalities arise during progressive glioma growth. A. Glucose transporter1 (Glut1) immunohistochemistry on sections of 5 weeks growth glioma in ROSAmT/mG::Pdgfb-iCre mouse (50 µm depth stack). Hypoxic tumor cells upregulate Glut1. B. Still image of two-photon live imaging on 2 weeks growth glioma implanted in ROSAmT/mG::Pdgfb-iCre mouse demonstrating tip cell filopodia extension indicative of sprouting. See Supplementary movie 1. C. Representative images of two-photon live imaging of the same glioma area of the same mouse on 2 and 5 weeks growth glioma (BFP positive) implanted in ROSAmT/mG::Pdgfb-iCre mouse (350µm depth stack). Note differences in network complexity and vessel diameter. D. Blood vessel diameter quantification: sprouting blood vessels at 2 weeks growth present caliber comparable to vessels in the healthy brain (hb). At 5 weeks growth, tortuous blood vessels in the tumor are significantly more dilated than in the healthy brain (n=8 mice). E. Branchpoint quantification during glioma growth (n=8 mice). F. Two-photon live imaging of vessel perfusion using FITC-dextran IV injection in ROSAmT/mG mice (300µm depth stack). G. Quantification of unperfused vessel segments (asterisk) shows increase during glioma growth (n=5 mice). H. Tumor bearing brain overview images after Miles assay. I. Late stage glioma growth vessels are leaky compared to early tumor growth (n=5 mice per group). Statistical analysis: D.E.G.I. one-way ANOVA followed by multiple comparisons Tukey's test. Error bars: meanSD. Scale bars: A: 250µm; B: 50µm; C-F: 150µm; G: 0,5cm.
Dynamic stroma reorganization drives blood vessel dysmorphia during glioma growth. (2017)
Figure 2

Figure 2: Macrophages invading growing glioma are bone marrow derived. A. F4/80 immunohistochemistry on a section of 5 weeks growth glioma implanted in ROSAmT/mG::Pdgfb-iCre mouse (50µm depth stack). B. Two-photon live imaging of LifeAct-GFP bone marrow transplantation in 5 weeks implanted glioma in ROSAmT/mG mice (100µm depth stack). C. Two-photon live imaging of LifeAct-GFP bone marrow transplantation in 3 and 5 weeks implanted glioma in ROSAmT/mG. Macrophages relocate close to blood vessels during glioma growth (50µm depth stack) (n=4 mice). Statistical analysis: C: t-test. Error bars: meanSD. Scale bar: A: 150 µm; B upper panels: 70µm; B lower panels: 280µm; C: 100µm.
Dynamic stroma reorganization drives blood vessel dysmorphia during glioma growth. (2017)
Figure 3

Figure 3: Concomitant macrophage accumulation and in situ switch from M1 to M2 phenotype with blood vessel abnormalities during glioma growth. A. MHCII and MRC1 immunohistochemistry on a section of 3-5 weeks growth glioma implanted in ROSAmT/mG::Pdgfb-iCre mouse. M1 macrophages, preferentially located in hypoxic area, are abundant at 3 weeks and their number decreases at 5 weeks. M2 macrophages, preferentially located around blood vessels, are present at a low level at 3 weeks and their number increases at 5 weeks. B. Macrophage quantification during glioma growth (n=5 mice per group). C. MHCII and MRC1 macrophage population FACs analysis in 2, 3, 4 and 5 weeks glioma (subsets in gated CD11b+ cells) (n=3 mice per group). D. Correlation analysis of M2 like macrophages accumulation (MRC1 positive) with vessel dysmorphia over glioma growth (n=12 mice). E. MRC1 immunohistochemistry on 3 weeks growth glioma in ROSAmTmG::Pdgfb-iCre mouse following MHCII-FITC labeled IV injection at 6 or 24h (5µm depth stack). Note the segregation of MRC1 and MHCII cell labeling at 6 hours and the overlapping of these two markers at 24 hours (n=5 mice per group). Statistical analysis: B. one-way ANOVA followed by multiple comparisons Tukey's test; D. Spearman correlation test; E. t-test. Error bars: meanSD. Scale bars: A: 200µm; D: 70µm; E: 50µm.
Dynamic stroma reorganization drives blood vessel dysmorphia during glioma growth. (2017)
Figure 4

Figure 4: Blood vessel dysmorphia is associated with macrophage polarization in progressive grades of human glioma. A. Glut1 immunohistochemistry on human grade II and grade IV glioma samples (50µm depth stack). Blood vessel diameter increases and network complexity decreases with increasing grade. B. CD68, MHCII and MRC1 immunohistochemistry on human grade II and grade IV glioma samples (50µm depth stack). C. Macrophage polarization quantification. Low-grade glioma is characterized by high number of M1 polarized macrophages. High-grade glioma is characterized by high number of M2 polarized macrophages. D. Correlation analysis of M2 like macrophages accumulation (MRC1 positive) with vessel dysmorphia depending on the tumor grade. E. Quantification of macrophage distance to the nearest blood vessel. Macrophages relocate close to blood vessels in late stage gliomas. For n of patients per group refer to TableEV1. Statistical analysis: A.C.D. one-way ANOVA followed by multiple comparisons Tukey's test; E. Spearman correlation test. Error bars: meanSD. Scale bars: A-B: 100µm.
Dynamic stroma reorganization drives blood vessel dysmorphia during glioma growth. (2017)
Figure 5

Figure 5: Inhibition of macrophage recruitment restores patterning and functionality of the blood vessel network in glioma. A. Glut1 immunohistochemistry on sections of 4 weeks growth glioma in ROSAmT/mG mice treated with anti-CSF1 or control antibodies. Anti-CSF1 antibody treatment induces a two-fold decrease in vessel caliber compared to control antibody (n=6 mice per group) (50µm depth stack). B. Glut1 and MRC1 immunohistochemistry on 4 weeks growth glioma in ROSAmT/mG mice treated with anti-CSF1 or control antibodies. Anti-CSF1 antibody induces a decrease in macrophage recruitment in the tumor microenvironment associated with an improved blood brain barrier functionality assessed by a better Glut1 blood vessel coverage compared to control antibody treated mice (50µm depth stack). C. Macrophage quantification in response to anti-CSF1 or control antibody treatment (n=6 mice per group). D. Tumor volume quantification in response to anti-CSF1 or control antibodies (n=6 mice per group). E. Two-photon live imaging of vessel perfusion in 4 weeks implanted glioma growth using FITC-dextran IV injection in ROSAmT/mG mice treated with anti-CSF1 or control antibodies. F. Quantification of unperfused vessel segments. Anti-CSF1 antibody treatment induces a reduction in the number of unperfused vessel segments (asterisk). (n=5 mice per group) (300µm depth stack). Statistical analysis: A.F. t-test; C. one-way ANOVA followed by multiple comparisons Tukey's test; D. Mann-Whitney u-test. Error bars: A-C-F: meanSD; D: median interquartile. Scale bars: A-B-E: 150µm.
Dynamic stroma reorganization drives blood vessel dysmorphia during glioma growth. (2017)
Figure 6

Figure 6: Promoting macrophage recruitment in glioma drives early dysmorphia of the tumor blood vessel network. A. MRC1 immunohistochemistry on sections of 3 weeks growth glioma in ROSAmT/mG mice treated with recombinant CSF1 or carrier. Recombinant CSF1 treatment induces blood vessel enlargement in early tumors (50µm depth stack). B. Quantification of Tomato positive macrophages. Recombinant CSF1 treatment induces increased macrophage recruitment compared to control (n=6 mice per group). C. Vessel diameter quantification. Recombinant CSF1 treatment drives blood vessel diameter increase compared to control (n=6 mice per group). D. Tumor volume quantification in 3 weeks glioma implanted mice treated with recombinant CSF1, anti-CSF1 antibody or control antibody. Recombinant CSF1 treatment reduces tumor growth compared to control (n=6 mice per group). Statistical analysis: B.D. one-way ANOVA followed by multiple comparisons Tukey's test; C. t-test. Error bars: B-C: meanSD; D: median interquartile. Scale bar: 100µm.
Dynamic stroma reorganization drives blood vessel dysmorphia during glioma growth. (2017)
Figure 7

Figure 7: Macrophage-derived VEGF depletion restores blood vessel architecture in glioma. A. qPCR analysis of CCR7, IL12, CCL19, CXCL10, MRC1, TGFβ, CD209 and VEGF on RNA samples from MHCII or MRC1 tumor extracted macrophages (n=5 isolation per group). B. sFlt1 binding assay on 5 weeks growth glioma section implanted in ROSAmT/mG::Pdgfb-iCre mouse. At late stage tumor growth, M2 macrophages surrounding blood vessels present very high amount of VEGF (binding sFlt1) to the neighboring endothelial cells (5µm depth stack). C. F4/80 immunohistochemistry and sFlt1 binding on sections of 4 weeks growth glioma in Vegfafl/fl::LysMCre+ or Vegfafl/fl ::LysMCre- mice (50µm depth stack). D. Quantification of F4/80 positive macrophages. VEGF depletion in myeloid cells does not alter macrophages recruitment (n=7 mice per group). E. Quantification of F4/80 positive macrophages binding sFlt1. VEGF depletion is effective in 61% of the macrophages invading the tumor (n=7 mice per group). F. Vessel diameter quantification. Myeloid cell derived VEGF depletion induces blood vessel "normalization" compared to control (n=7 mice per group). G. Tumor volume quantification in 4 weeks growth glioma in Vegfafl/fl::LysMCre+or Vegfafl/fl::LysMCre- mice (n=7 mice per group). Statistical analysis: D.E.F. t-test; A. G. Mann-Whitney u-test. Error bars: A-D-E-F: meanSD; G: median interquartile. Scale bars: 100µm.
Dynamic stroma reorganization drives blood vessel dysmorphia during glioma growth. (2017)
Figure 8

Figure 8: Combining macrophage depletion with chemotherapy increases survival of glioma bearing mice. A. Experimental design for the survival trial: 8 weeks old wild type mice were injected with GL261 cells to induce glioma formation and were assigned randomly to vehicle (n = 6), anti-CSF1 monoclonal antibody treatment (0.5mg ; n=6), temozolomide treatment (40mg/kg ; n=6) or temozolomide and anti-CSF1 mAb combination treatment (n = 6). Mice were weighed once daily until they developed symptoms or reached the trial endpoint. B. Survival curves of the mice described in A. Combination therapy is significantly more efficient than chemotherapy alone. (n=6 mice per group). C. phospho-H2AX and Glut1 immunohistochemistry on 3 weeks growth glioma in wild-type mice treated as described in A. Anti-CSF1 antibody in combination with temozolomide chemotherapeutic agent induces a wider tumor cell death efficiency with a significant decrease of blood vessel caliber (D.) and an increase in caspase3 positive tumor cells (E.) (50µm depth stack) (n=6 mice per group). Statistical analysis: B. Multiple comparisons Mantel-Cox log-rank; D.E. one-way ANOVA followed by multiple comparisons Tukey's test. Error bars: meanSD. Scale bars: 300µm
Role of Bcl-2 family proteins in a non-apoptotic programmed cell death dependent on autophagy genes (2004)
figf1

(a) Representative photographs of Bax/Bak−/− MEFs treated with etoposide. Bax/Bak−/− MEFs were treated with 20 μM etoposide in the presence of PI (2 μM) for the indicated times, and were analysed under a phase-contrast and fluorescence (PI) microscope. (b, c) Reduced viability of Bax/Bak−/− MEFs after exposure to etoposide. WT (circles) and Bax/Bak−/− (squares) MEFs were treated with 20 μM etoposide in the presence (closed symbols) or absence (open symbols) of 100 μM zVAD-fmk. Then cell viability was measured by the PI staining (b) and CTB assay (c), and expressed as a percentage of the initial value without etoposide. Data are shown as mean ± s.d. (n = 4). (d) Reduced viability of etoposide-treated Bax/Bak−/− MEFs as assessed by proliferation assay. Bax/Bak−/− MEFs were either not treated (0 h) or were treated with 20 μM etoposide for 12 and 24 h, then all cells were recovered and 5,000 cells were reseeded. Viable cell numbers were measured on the indicated days by the CTB assay. Results were normalized by adjusting the day 0 value. n = 4. (e) Reduced viability of Bax/Bak−/− MEFs after exposure to etoposide and staurosporine, as assessed by clonogenicity assay. MEFs were treated with etoposide (20 μM) or staurosporine (1 μM) at the indicated times, collected, and 2,000 cells were seeded in the normal medium. After 1 week, colonies were counted. n = 4. (f–i) Induction of autophagy in Bax/Bak−/− MEFs by etoposide. (f) Electron micrograph (×14,800) of Bax/Bak−/− MEF treated with etoposide (20 μM) for 18 h. Representative features of Bax/Bak−/− MEFs that have received no treatment (NT; that is, are healthy) are also shown (NT: ×14,000). (g) In magnified photographs, a large number of autolysosomes/autophagosomes (left: ×26,000) were observed, but the mitochondria were unaffected (right: ×17,000). (h, i) Punctate GFP–LC3 fluorescence in Bax/Bak−/− MEFs treated with etoposide. GFP–LC3-transfected cells were incubated with and without etoposide (20 μM) for 24 h (h) or the indicated times (i), and then examined by fluorescent microscopy. (h) Representative photographs of healthy (NT) and etoposide-treated Bax/Bak−/− MEFs are shown. (i) The percentage of cells with punctate GFP–LC3 fluorescence was calculated relative to all GFP-positive cells. Data are shown as mean ± s.d. (n = 4).
Role of Bcl-2 family proteins in a non-apoptotic programmed cell death dependent on autophagy genes (2004)
figf2

(a, b) Bax/Bak−/− MEFs were treated with 20 μM etoposide in the absence or presence of 10 mM 3-MA for 24 h, and then were examined by phase-contrast microscopy (a) and electron microscopy (×8,400) (b). Autolysosomes/autophagosomes were hardly observed. (c, d) Reduction of punctate GFP–LC3 fluorescence in Bax/Bak−/− MEFs by 3-MA. Bax/Bak−/− MEFs that were transfected with GFP–LC3 were incubated without (NT) or with 20 μM etoposide in the presence or absence of 10 mM 3-MA for 24 h, and then were examined by confocal fluorescent microscopy. (c) Representative photograph of etoposide-treated Bax/Bak−/− MEFs in the presence of 3-MA. (d) The percentage of cells with punctate GFP–LC3 fluorescence was calculated relative to all GFP–LC3-positive cells. Data are shown as mean ± s.d. (n = 4). (e–g) Inhibition of etoposide- and staurosporine-induced cell death by 3-MA. Bax/Bak−/− MEFs were treated with 20 μM etoposide (e–g) or 1 μM staurosporine (g) in the absence (open symbols) or presence (closed symbols) of 10 mM 3-MA for the indicated time. Cell viability was measured by PI staining (e), CTB assay (f) and clonogenicity assay (g) (as in Fig. 1e). Upper panels in g: viable cells were visualized by staining with calcein-AM. n = 4.
Role of Bcl-2 family proteins in a non-apoptotic programmed cell death dependent on autophagy genes (2004)
figf3

(a, b) Bax/Bak−/− MEFs were treated with the indicated siRNAs (10 μg) for 24 h and then incubated with 20 μM etoposide for the indicated times. Expression of APG5–APG12 complex (a), Beclin 1 (b) and VDAC (loading control) was analysed by Western blot analysis. (c) Inhibition of etoposide-induced autophagy by silencing of Beclin 1. Bax/Bak−/− MEFs with silencing of Beclin 1 were treated with 20 μM etoposide at 18 h and were analysed by electron microscopy (×8,500). (d) Reduction of punctate GFP–LC3 fluorescence in Bax/Bak−/− MEFs by silencing of Beclin 1 and APG5. Bax/Bak−/− MEFs that were transfected with GFP–LC3 together with the indicated siRNAs, were incubated without (NT) or with 20 μM etoposide for 24 h, and then examined by confocal fluorescent microscopy. The percentage of cells with punctate GFP–LC3 fluorescence was calculated relative to all GFP–LC3-positive cells. Data are shown as mean ± s.d. (n = 4). (e, f) Inhibition of etoposide- and staurosporine-induced cell death by silencing of Beclin 1 and APG5. Bax/Bak−/− MEFs that were treated with the indicated siRNAs were incubated with 20 μM etoposide (e, f) or 1 μM staurosporine (f) for the indicated time. Cell viability was measured by CTB assay (e) and clonogenicity assay (f). n = 4.
Role of Bcl-2 family proteins in a non-apoptotic programmed cell death dependent on autophagy genes (2004)
figf4

(a, b) APG5-dependent punctate GFP–LC3 fluorescence in Bcl-2-transfected MEFs after etoposide treatment. APG5+/+ and APG5−/− MEFs that were transfected with both GFP–LC3 and the indicated plasmids (Bcl-2 or vector) were incubated without or with 20 μM etoposide for 24 h, then examined by confocal fluorescent microscopy (a). The percentage of cells with punctate GFP–LC3 fluorescence was calculated relative to all GFP–LC3-positive cells (b). (c) Accumulation of Beclin 1 and APG5–APG12 complex in Bcl-2-transfected MEFs after etoposide treatment. WT MEFs were transfected with the indicated plasmids, and were incubated without (NT) or with 20 μM etoposide for 24 h. (d, e) Occurrence of etoposide-induced 3-MA-inhibitable non-apoptotic death in Bcl-xL-overexpressing APG5+/+ MEFs, but not APG5−/− MEFs. APG5+/+ (open columns) and APG5−/− (closed columns) MEFs were transfected with the indicated plasmids (2 μg). After 24 h, transfected cells were incubated with 20 μM of etoposide in the presence or absence of 100 μM of zVAD or 10 mM of 3-MA for 24 h, and cell viability was measured by the CTB assay (d) and apoptotic nuclear morphology (e). Data are shown as mean ± s.d. (n = 4). (f) Overexpression of Bcl-xL does not sensitize Beclin 1-silenced MEFs to etoposide-induced 3-MA-inhibitable non-apoptotic death. WT MEFs with silencing of control GFP (open columns) or Beclin 1 (closed columns) were transfected with the indicated plasmids (1 μg). After 24 h, transfected cells were incubated with 20 μM of etoposide in the presence or absence of 10 mM of 3-MA for 24 h, and cell viability was measured by the CTB assay. n = 3.