EMBO/soda-nel huggingface dataset:
Mutated axon guidance gene PLXNB2 sustains growth and invasiveness of stem cells isolated from Cancers of Unknown Primary (2023)
Figure 6

Figure 6. Over-expression of G842C-mutated PlxnB2 in wild type CUP cells enhanced AS viability and tumor growth in mice.

(A) Time course analysis of the cellular viability of AS901, stably overexpressing either wild-type or G842C-mutated PlxnB2, or controls transduced with an empty vector. Plotted values are the mean ± SD of n =3 independent experiments (six technical replicates for each). The statistical significance was assessed by two-way ANOVA with Tukey's correction for multiple comparisons. PlxnB2-G842C vs. controls (CTRL): at 24h **p = 0.0097, 48h **p = 0.0026, 72h **p = 0.0079, 96h *p = 0.0226. PlxnB2-G842C vs. PlxnB2-WT: at 48h $p = 0.0418, 72h $p = 0.0318. (B) Tumor growth curves of ADX 901 over-expressing either PlxnB2-G842C or PlxnB2-WT, or mock control. Plotted values are the mean ± SD (n = 8 each groups). The statistical significance was assessed by two-way ANOVA with Tukey's correction for multiple comparisons. PlxnB2-G842C vs control (CTRL): at 25 days **p = 0.0029, 28 days **p = 0.0028, 32 days **p = 0.0033. PlxnB2-G842C vs PlxnB2-WT: at 25 days $p = 0.0221, 28 days $p = 0.0234, 32 days $$p = 0.0024.
Mutated axon guidance gene PLXNB2 sustains growth and invasiveness of stem cells isolated from Cancers of Unknown Primary (2023)
Figure 7

Figure 7. Overexpression of G842C-PlxnB2 promotes cancer cell migration/invasion.

(A) Western blotting showing basal endogenous or overexpressed PlxnB2, either WT or G842C-mutated isoforms, in MCF7 cells. In this wide view of the hybridized membrane, it is appreciable the presence of full length molecules at 250 kDa, as well as the processed 80 kDa fragments (reported in ref. 64). Vinculin staining provided protein loading controls. (B) The graph shows the mean ± SD of relative increase in 24-hour spontaneous migration across Transwell inserts of MCF7 cells (analysed above) over-expressing either wild-type or G842C-mutated PlxnB2, versus mock-transfected controls (n=4 biological replicates). The statistical significance was verified by one-way ANOVA with Bonferroni correction for multiple comparisons: PlxnB2-WT vs. mock *p=0.0145; PlxnB2-G842C vs. PlxnB2-WT *p=0.0277; PlxnB2-G842C vs. mock ***p=0.0002. Representative images of the cells migrated across the semipermeable membrane are shown in appendix Figure S4A. (C) Wound healing scratch assays on monolayers of MCF7 cells engineered as described above. The graph shows the mean ± SD of values obtained in N=3 independent experiments (with at least three technical replicates for each). The statistical significance was verified by one-way ANOVA with Tukey's correction for multiple comparisons: *p = 0.0350; **p = 0.0029; ***p = 0.0002. Representative phase contrast images of the wounded monolayers are further shown in larger size in Appendix Figure S4B. (D) Western blotting analysis of endogenous and overexpressed PlxnB2 (either WT or G842C-mutated isoforms) in CUP cells AS906 (both full length molecules at 250 kDa and processed 80 kDa fragments). Vinculin staining provided protein loading controls. (E) The invasive capacity of CUP cells AS906 (shown in previous panel), overexpressing WT or G842-mutated PlxnB2 (or mock control), was assessed across Matrigel-coated transwell inserts. The graph shows the mean ± SD of values obtained in N=3 independent experiments (including technical duplicates, in two cases). Below the graph, are shown representative fields of the semipermeable membranes (additional larger size images can be found in appendix Figure S4C); scale bar: 100μm. The statistical significance was verified by one-way ANOVA with Bonferroni correction for multiple comparisons: *p=0.0406; **p=0.0045; ***p=0.0003.
Mutated axon guidance gene PLXNB2 sustains growth and invasiveness of stem cells isolated from Cancers of Unknown Primary (2023)
Figure 8

Figure 8. The expression of G842C-PlxnB2 controls EGFR phosphorylation and intracellular signaling

(A) Phospho-EGFR analysis by western blotting in control and PlxnB2-silenced AS43 cells. Images show representative results of n=3 independent experiments, while the graph at the bottom shows the mean ± SD of normalized band quantification. The statistical significance across replicates was verified by unpaired t-test: ****p<0.0001. (B) Phospho-EGFR analysis by western blotting in AS901 and AS906 cells either mock, or overexpressing WT or G842C-mutated PlxnB2. Images show representative results of n=3 independent experiments, while the graphs at the bottom show the mean ± SD of normalized band quantification. The statistical significance across independent replicas was verified by one-way ANOVA followed by Tukey test: AS901 PlxnB2-G842C vs. PlxnB2-WT *p=0.0434, PlxnB2-G842C vs. mock *p=0.0211; AS906 PlxnB2-G842C vs. PlxnB2-WT *p=0.0436, PlxnB2-G842C vs. mock *p=0.0481. (C) Western blotting analysis of pERK and pS6 intracellular signal transducers in the indicated agnospheres, after 6-hour treatment with the EGFR inhibitor cetuximab (50μg/ml). Images show representative results of n=3 independent experiments, while the graphs at the bottom show the mean ± SD of normalized band quantification. The statistical significance across independent replicas was verified by Sidak's multiple comparison test between cetuximab-treated and untreated conditions, per each group: AS43 *p=0.0153, ***p=0.0001, ****p<0.0001; AS901 *p=0.0201.
Mutated axon guidance gene PLXNB2 sustains growth and invasiveness of stem cells isolated from Cancers of Unknown Primary (2023)
Figure 9

Figure 9. The viability and invasive capacity of CUP cells expressing G842C-mutated, but not wild-type, PlxnB2 is dependent on EGFR

(A) The cellular viability of the indicated agnospheres was assessed after six days in presence or absence of the EGFR inhibitor cetuximab (50μg/ml). In the plot, each box represents values from 25th to 75th percentiles, the central band indicates the mean, and the whiskers expand from maximum to minimum values of individual cetuximab-treated experimental points (n≥9), normalized to the average value of untreated cells within each group. The statistical significance across three independent experiments, each comprising ≥3 technical replicates, was verified by unpaired t-test multiple comparisons of cetuximab-treated and untreated conditions, per each group: ***p=0.0002; ****p<0.0001. (B) The viability of AS43, in presence (or absence) of cetuximab (50μg/ml), was assessed over the indicated time course, compared to values at T0. The graph shows the mean ± SD of two independent experiments (with quadruplicate technical replicates each). The statistical significance was verified through multiple comparisons by two-way ANOVA with Bonferroni correction of cetuximab-treated and untreated samples, per each time point: ****p<0.0001. (C) Limiting dilution assays (LDA) were used to assess the clonogenic capacity of dissociated CUP cells derived from the indicated agnospheres in presence or absence of cetuximab (50μg/ml). Analyses generated by the ELDA software are shown below, reporting the estimated stem cell frequency (percentage of clonogenic cells) with confidence intervals (C.I.) and statistical analysis by chi-square test. (D) The invasive capacity of AS906 cells (overexpressing WT or G842C-mutated PlxnB2, or mock transfected) was assessed as in Fig. 7E, in the presence or absence of cetuximab 50μg/ml. Images show representative results (scale bar: 100μm), while the graph on the right shows the mean ± SD of n=3 independent experiments. The statistical significance across replicates was verified by one-way ANOVA with Bonferroni correction: **p= 0.0010; ***p=0.0005; ****p<0.0001.

EXPANDED VIEW - FIGURE LEGENDS
Apg9p/Cvt7p is an integral membrane protein required for transport vesicle formation in the Cvt and autophagy pathways. (2000)
Figure 2

Cloning and characterization of APG9. A, Wild-type (WT; SEY6210), apg9 (THY154), apg9Δ (JKY007), and the apg9Δ strain transformed with single copy (CEN; pAPG9(414)) or multicopy (2μ; pAPG9(424)) plasmids encoding APG9 were grown to log phase in SMD. Protein extracts were prepared and analyzed by immunoblot using antiserum to API as described in Materials and Methods. The positions of precursor and mature API are indicated. The APG9 gene complements the prAPI accumulation phenotype of the apg9Δ mutant. B, WT (SEY6210), apg9Δ (JKY007), and the apg9Δ strain transformed with pAPG9(414) centromeric plasmid were grown in SMD and transferred to SD(−N) as described in Materials and Methods. Aliquots were removed at the indicated times and spread onto YPD plates in triplicate. Numbers of viable colonies were determined after two to three days. The APG9 gene complements the starvation-sensitive phenotype of the apg9Δ mutant.
Apg9p/Cvt7p is an integral membrane protein required for transport vesicle formation in the Cvt and autophagy pathways. (2000)
Figure 3

Apg9p is an unglycosylated, integral membrane protein. A, Wild-type (WT; SEY6210), apg9Δ (JKY007), and the apg9Δ strain transformed with single copy (CEN) or multicopy (2μ) plasmids encoding APG9 were grown to log phase in SMD. Protein extracts were prepared and analyzed by immunoblot using antiserum to Apg9p. Apg9p is detected as an ∼125-kD protein. B, Hydrophobicity analysis of Apg9p based on the Kyte-Doolittle algorithm (Kyte and Doolittle 1982) predicts six to eight transmembrane domains. C, Cells of wild-type strain YW5-1B were grown in YPD, converted to spheroplasts, and osmotically lysed as described in Materials and Methods. After a preclearing step to remove cell debris, the lysates were incubated with or without 1% Triton X-100 on ice for 30 min, as indicated. Centrifugation at 10,000 g for 1 h separated the lysate into supernatant (S) and pellet (P) fractions, which were analyzed by immunoblots with antiserum to Apg9p. Apg9p is found in the low-speed pellet in the absence of Triton X-100. Detergent extraction completely solubilizes Apg9p and causes it to appear in the supernatant fraction. D, Strain CTD1 harboring a multicopy 3×HA APG9 plasmid was grown in YPD and converted to spheroplasts as described in Materials and Methods. A crude lysate was prepared and treated with or without endo H as indicated overnight at 37°C and analyzed by immunoblots with anti-HA antibodies or anti-CPY serum. Endo H treatment removes the N-linked glycosyl residues of CPY, causing a shift in its electrophoretic mobility. In contrast, Apg9p is not glycosylated and remains unchanged in the presence or absence of endo H treatment.
Apg9p/Cvt7p is an integral membrane protein required for transport vesicle formation in the Cvt and autophagy pathways. (2000)
Figure 4

Apg9p does not comigrate with typical endomembrane markers in sucrose density gradients. A and B, Strain STY1 (pep4Δ) was grown in YPD to log-phase, and osmotically lysed. Lysates were precleared, loaded on a sucrose density gradient ranging from 18–55% (wt/wt), and centrifuged for 2.5 h at 174,000 g as described in Materials and Methods. Fractions were collected from the top (fraction number 1) to the bottom (fraction number 16). Fractions were subjected to SDS-PAGE and immunoblot with antiserum against Apg9p and: in A, Kex2p (trans Golgi network), Pho8p (vacuole), and Pma1p (plasma membrane); and in B, Sec12p (ER) and Pep12p (endosome). The graphs in panels A and B are from the same gradient, but are presented separately for clarity.
Apg9p/Cvt7p is an integral membrane protein required for transport vesicle formation in the Cvt and autophagy pathways. (2000)
Figure 6

Apg9p localizes to large perivacuolar punctate structures. Immunofluorescence microscopy of strains CTD1 (apg9Δ), KSL12C (vps4Δ), and SKD6-1D (apg6Δ) transformed with the multicopy plasmid 3×HA APG9. Cells were grown in YPD to log phase (vegetative), and incubated further in SD(−N) medium with 1 mM PMSF for 3 h (starvation). The cells were fixed with formaldehyde and examined by immunofluorescence microscopy as described in Materials and Methods. Anti-HA and FITC-conjugated antibodies mark Apg9p (left), and the vacuole can be visualized by Nomarski optics (middle). An overlay is shown in the right panels.
Apg9p/Cvt7p is an integral membrane protein required for transport vesicle formation in the Cvt and autophagy pathways. (2000)
Figure 5

Apg9p does not comigrate with prAPI in a strain that accumulates Cvt/autophagic vesicles. Strain TK415 (ypt7Δ) was grown in YPD to log-phase, and osmotically lysed. Lysates were precleared, centrifuged at 100,000 g for 1 h, and the pellet fraction resuspended and centrifuged a second time. The resulting pellet fraction was subsequently loaded on a sucrose density gradient ranging from 18–55% (wt/wt), and centrifuged for 16 h at 174,000 g as described in Materials and Methods. 16 fractions were collected starting from the top and subjected to SDS-PAGE and immunoblot with antisera against Apg9p and API. The graph represents an average of two experiments.
Apg9p/Cvt7p is an integral membrane protein required for transport vesicle formation in the Cvt and autophagy pathways. (2000)
Figure 7

In vivo examination of the Apg9pGFP fusion protein. Wild-type (SEY6210) cells expressing Apg9pGFP from a multicopy plasmid were grown in SMD to midlog phase and labeled with FM 4-64 to stain the vacuoles as described in Materials and Methods. The labeled cells were viewed directly with a Leica DM IRB confocal microscope. Large, punctate Apg9pGFP structures appear juxtaposed to the FM 4-64–labeled vacuole membranes. In addition, smaller punctate structures appear throughout the cytoplasm.
Apg9p/Cvt7p is an integral membrane protein required for transport vesicle formation in the Cvt and autophagy pathways. (2000)
Figure 8

Ultrastructural localization of Apg9p for immunoelectron microscopy. Strain STY1 (pep4Δ) expressing 3×HA Apg9p was grown in YPD to log phase and transferred to SD(−N) medium for 2 h. The cells were fixed and stained with anti-HA antibody and 5-nm colloidal gold-conjugated goat anti–mouse IgG as described in Materials and Methods. A, Section showing autophagic bodies in the vacuole lumen. B, Section showing a cytosolic autophagosome. Apg9p is seen in patches near the vacuole, but not on autophagic bodies or autophagosomes. AB, autophagic body; AP, autophagosome; N, nucleus; V, vacuole; VM, vacuolar membrane. Arrows point to areas of concentrated Apg9p.
Apg9p/Cvt7p is an integral membrane protein required for transport vesicle formation in the Cvt and autophagy pathways. (2000)
Figure 9

Vesicle accumulation test and kinetic analysis of prAPI import in the temperature conditional Apg9p mutant. A, Morphological analysis of apg9Δ indicates an import defect at an early step in the pathway. Wild-type (YW5-1B) and apg9Δ (CTD1) cells were grown in YPD (vegetative) or transferred to SD(−N) (starvation) for 3 h in the presence of PMSF (starvation) and examined by DIC (Nomarski) microscopy (Zeiss Axioplan). PMSF inhibits the degradation of autophagic bodies. Under starvation conditions, wild-type cells accumulate autophagic bodies when vesicle breakdown is blocked. However, under the same conditions, apg9Δ cells do not accumulate autophagic bodies, indicating that Apg9p is required for a step before vesicle fusion and release of the autophagic body into the vacuolar lumen. The apg9Δ strain transformed with the APG9 plasmid shows the same result as wild-type (data not shown). B, The apg9ts strain is tightly blocked for prAPI import at nonpermissive temperature. Wild-type (SEY6210) and apg9Δ (JKY007) cells transformed with the apg9ts centromeric plasmid were incubated at 24 and 38°C for 5 min, pulse-labeled for 10 min, and then subjected to nonradioactive chase reactions for the indicated times. Samples at each time point were immunoprecipitated with antiserum to API and resolved by SDS-PAGE as described in Materials and Methods. API-immunoprecipitated bands were quantified by a Molecular Dynamics STORM PhosphorImager and the results are presented in the graph. The percent mature API was determined by dividing the mature API value over the sum of the precursor and mature API values for each time point.
Apg9p/Cvt7p is an integral membrane protein required for transport vesicle formation in the Cvt and autophagy pathways. (2000)
Figure 10

Analysis of the apg9ts strain indicates Apg9p is directly required for the vesicle formation step. A, The nature of prAPI binding and pelleting in the apg9ts strain. Spheroplasts of apg9ts were labeled for 10 min and chased for 30 min at 38°C. The spheroplasts were osmotically lysed in a buffer containing no salt (20 mM Pipes, pH 6.8) or a physiological concentration of salt (100 mM KOAc, 50 mM KCl, 5 mM MgCl2, 20 mM Pipes, pH 6.8) and pelleted at 5,000 g. All samples were immunoprecipitated with antiserum to API and the cytosolic marker PGK. apg9ts retains the salt dependent binding and pelleting of prAPI. B, prAPI in the apg9ts strain accumulates in both a membrane-associated state and a large pelletable complex. Spheroplasts of apg9ts and ypt7Δ were labeled for 10 min and chased for 30 min at 38°C. The labeled spheroplasts were then osmotically lysed and separated into supernatant (S) and pellet (P) fractions by centrifugation at 5,000 g for 5 min. An aliquot was removed for the total lysate control (T). The pellet fraction (P) was resuspended in 15% Ficoll-400 in gradient buffer (20 mM Pipes, 5 mM MgCl2, complete EDTA-free protease inhibitor cocktail) in the presence or absence of Triton X-100 and overlaid with 13 and 2% Ficoll-400 in gradient buffer. The step gradients were centrifuged at 13,000 g for 10 min. Membrane-containing float (F), nonfloat (NF), and pellet (P2) fractions were immunoprecipitated with antiserum to API as described in Materials and Methods. The position of prAPI is indicated. C, prAPI in the apg9ts strain is protease accessible. Spheroplasts isolated from apg9ts and ypt7Δ cells were pulse-labeled for 10 min and chased for 30 min at 38°C. The labeled spheroplasts were then osmotically lysed and separated into low-speed supernatant (S) and pellet (P) fractions after a 5,000 g centrifugation step. The pellet fractions were subjected to protease treatment in the absence or presence of 0.2% Triton X-100 as described in Materials and Methods. The resulting samples were immunoprecipitated with antiserum to API and PGK. The immunoprecipitated bands were quantified by a Molecular Dynamics STORM PhosphorImager. The percent protease-protected prAPI was determined by dividing the value for prAPI in the protease-treated sample by the value for total prAPI before protease treatment. The percent PGK was determined by dividing the supernatant (S) or pellet (P) value over the sum of the S and P values.
Active immunotherapy reduces NOTCH3 deposition in brain capillaries in a CADASIL mouse model (2022)
Figure 2

Figure 2. Schematic representation of NOTCH3 and NOTCH3 EGF1-5 proteins and NOTCH3 EGF1-5 antigen purification.

A) Schematic representation of NOTCH3 and NOTCH3 EGF1-5. NOTCH3 represents the full-length protein, and NOTCH3 EGF1-5 represents the NOTCH3 protein with exon 1 to 5 fused with a myc-His-Tag at the C-terminus used for purification of the aggregated protein.

B) Western blot of the NOTCH3 EGF1-5 WT and R133C purified proteins. The eluate fractions were visualized by western blot using an α-myc antibody.

C) Western blot of NOTCH3 EGF1-5 WT and R133C aggregated proteins. The incubated fractions of NOTCH3 EGF1-5 WT and R133C were visualized on a western blot using an α-myc antibody. The purified proteins and the aggregates were visualized after 1-5 days incubation by western blot using α-myc antibody under non-reducing conditions.
Active immunotherapy reduces NOTCH3 deposition in brain capillaries in a CADASIL mouse model (2022)
Figure 4

Figure 4. NOTCH3 ECD and alpha-smooth muscle actin (ASMA) expression in cerebral arteries/arterioles.

A) Representative images of TgN3R182C150, sham- and NOTCH3 EGF1-5-immunized mice at 7 months of age and TgN3R182C150 at 18 months of age. Representative images show brain arteries of TgN3R182C150 (7 and 18 months), sham and NOTCH3 EGF1-5-immunized mice stained with a monoclonal antibody against NOTCH3 ECD (1E4, red) and an α-SMA antibody (green). Scale bar =20µm.

B) Quantification of NOTCH3 ECD deposits (numbers per 1,000 μm2) and NOTCH3 ECD stained area and average size per vessel revealed no decrease in NOTCH3 ECD deposition in brain arteries between NOTCH3 EGF1-5 -immunized (n=10), sham (n=8) and non-vaccinated (n=6) TgN3R182C150 mice at 7 months of age. NOTCH3 ECD deposits (numbers per 1,000 μm2) and NOTCH3 ECD stained area and average size per vessel increases significantly in the TgN3R182C150 mice at 18 months (n=3) of age versus NOTCH3 EGF1-5 -immunized (n=10), sham (n=8) and non-vaccinated (n=6) TgN3R182C150 mice at 7 months of age. Statistical significance was assessed using a Brown-Forsythe and Welch ANOVA tests followed by Dunnett's T3 multiple comparisons. (NOTCH3 ECD deposits (% of vessel area): 7 m.o. vs. Sham, ns P=0.9424; 7 m.o. vs. Vaccinated, ns P=0.9809; 7 m.o. vs. 18 m.o.**P=0.0046; Sham vs. Vaccinated, ns P=0.9999; Sham vs. 18 m.o. **P=0.0032; Vaccinated vs. 18 m.o. **P=0.0032). NOTCH3 ECD deposits (number/1000 um2): 7 m.o. vs. Sham, ns P=0.7877; 7 m.o. vs. Vaccinated, ns P=0.8358; 7 m.o. vs. 18 m.o.**P=0.0069; Sham vs. Vaccinated, ns P=0.9995; Sham vs. 18 m.o. **P=0.0044; Vaccinated vs. 18 m.o. *P=0.0104). NOTCH3 ECD deposits size: 7 m.o. vs. Sham, ns P=0.1169; 7 m.o. vs. Vaccinated, ns P>0.9999; 7 m.o. vs. 18 m.o.***P=0.0009; Sham vs. Vaccinated, ns P=0.0577; Sham vs. 18 m.o. **P=0.007; Vaccinated vs. 18 m.o. ****P<0.0001, ns=non-significant). Error bars indicate standard error of the mean (SEM).
Active immunotherapy reduces NOTCH3 deposition in brain capillaries in a CADASIL mouse model (2022)
Figure 5

Figure 5. NOTCH3 ECD and perlecan expression in cerebral capillaries.

A) Representative images of TgN3R182C150, sham- and NOTCH3 EGF1-5-immunized mice at 3, 7 and 18 months of age. Representative images show brain arteries and capillaries of TgN3R182C150, sham and NOTCH3 EGF1-5-immunized mice stained with a monoclonal antibody against NOTCH3 ECD (1E4, red) and an anti-perlecan antibody (green). Scale bar =20µm.

B) Quantification of NOTCH3 ECD deposits (numbers per 1,000 μm2) and NOTCH3-ECD stained area and average size per vessel revealed a significant increase in NOTCH3 ECD deposition in brain arteries and capillaries between non-vaccinated 3 months old TgN3R182C150 (n=3) and 7 months old TgN3R182C150 (n=6) mice and 18 months old TgN3R182C150 (n=3). Quantification of NOTCH3-ECD deposits (numbers per 1,000 μm2) and NOTCH3-ECD stained area and average size per vessel revealed a significant decrease in NOTCH3-ECD deposition in brain arteries and capillaries between NOTCH3 EGF1-5 -immunized (n=11), sham (n=8) and non-vaccinated TgN3R182C150 (n=6) mice. Statistical significance was assessed using a Brown-Forsythe and Welch ANOVA tests followed by Dunnett's T3 multiple comparisons. (NOTCH3 ECD deposits (% of vessel area): 3 m.o. vs. 18 m.o. **P=0.0029; 3 m.o. vs. 7 m.o. ***P=0.0004; 7 m.o. vs. Sham, ns P=0.7326; 7 m.o. vs. Vaccinated, **P=0.003; Sham vs. Vaccinated, ****P<0.0001). NOTCH3 ECD deposits (number/1000 um2): 3 m.o. vs. 18 m.o. **P=0.0038; 3 m.o. vs. 7 m.o. ***P=0.0002; 7 m.o. vs. Sham, ns P=0.9913; 7 m.o. vs. Vaccinated, *P=0.021; Sham vs. Vaccinated, ***P=0.001). NOTCH3 ECD deposits size: 3 m.o. vs. 18 m.o. *P=0.0215; 3 m.o. vs. 7 m.o. **P=0.0029; 7 m.o. vs. Sham, ns P=0.3622; 7 m.o. vs. Vaccinated, *P=0.0428; Sham vs. Vaccinated, **P=0.0038, ns= non-significant). Dotted lines indicate quartiles and dashed thicker lines are the median.