Journal: Disease Models & Mechanisms

Article Title: Functional exploration of copy number alterations in a Drosophila model of triple-negative breast cancer

doi: 10.1242/dmm.050191

Figure Lengend Snippet: Altered TP53 and MYC genes are present in TNBC tumors and reduce Drosophila survival. ( A) Hierarchical clustering of TNBC primary tumors as listed in TCGA, based on CNA and mutation of putative driver genes. CNAs are shown in order of genomic location, mutated putative driver genes are listed in alphabetical order. Overall survival of each patient is shown coded at the left, with white representing the longest and black the shortest survival. n= 72. (B) Heads of GMR>w (control) and GMR>p53lh;Myc flies. Eyes were enlarged when targeted by Myc overexpression plus p53 knockdown ( p53 lh ; long hairpin). (C) Quantification of survival (shown as percent eclosion) of Myc-expressing flies in the presence ( ptc>w and ptc>Myc ) and absence ( ptc>p53 sh and ptc>Myc p53 sh ) of p53 knockdown ( p53 sh ; short hairpin). Kruskal–Wallis test: P <0.0001. n= 14. Error bars represent the +standard error of the mean (+s.e.m.) and do not reflect the paired nature of the data. Other P -values: Wilcoxon test. See also Fig. S1 and Table S2 .

Article Snippet: Protein was transferred onto a PVDF membrane, which was then probed against p53 (DSHB p53-H3 s 1:1000), syntaxin (DSHB 8B-3, 1:1000) or Myc (Santa Cruz Myc d1-717, 1:200) overnight at 4°C.

Techniques: Mutagenesis, Control, Over Expression, Knockdown, Expressing

Journal: Disease Models & Mechanisms

Article Title: Functional exploration of copy number alterations in a Drosophila model of triple-negative breast cancer

doi: 10.1242/dmm.050191

Figure Lengend Snippet: Overexpression of Myc promotes tissue expansion and cell translocation in Drosophila wing discs. (A) Representative wing discs of flies expressing combinations of Myc and p53 sh as indicated. Genotypically white ( ptc>w ) flies served as controls. DAPI staining (red) highlights tissue boundary, GFP signal (green) demarcates transgene expression. Some images rotated for comparison with borders indicated by dashed lines. (B) Maximum projections of confocal z -stacks of the lower half of the wing discs as in shown in the respective images in A, dashed lines indicate the region of virtual sectioning shown in lower inset. (C) Maximum projections (upper three rows) and z -stacks (bottom row) of confocal stacks of the lower half of wing discs such as in A stained with antibodies against Mmp1 antibody (red) or cleaved-caspase (white), both indicative of cell translocation ; dashed lines indicate the region of virtual sectioning shown in lower inset. Arrowheads in B and C mark delaminating cells. Brightness and contrast were uniformly increased to improve visualization. In A-C, anterior at left, posterior at right, apical at top, basal at bottom. (D) Quantification of transgenic tissue overgrowth in flies expressing Myc and p53 sh driven by ptc-Gal4 alone or in combination. Kruskal–Wallis test: P <0.0001. Other P -values: Student's t -tests. (E) Quantification of cell translocation in transgenic tissue produced by flies expressing Myc and p53 sh driven by ptc-Gal4 or in combination. Kruskal–Wallis test: P =0.0075. Other P -values: Mann–Whitney test compared to w controls. No significant differences were observed between flies expressing Myc and Myc,p53 sh . See also Fig. S2 .

Article Snippet: Protein was transferred onto a PVDF membrane, which was then probed against p53 (DSHB p53-H3 s 1:1000), syntaxin (DSHB 8B-3, 1:1000) or Myc (Santa Cruz Myc d1-717, 1:200) overnight at 4°C.

Techniques: Over Expression, Translocation Assay, Expressing, Staining, Comparison, Transgenic Assay, Produced, MANN-WHITNEY

Journal: Disease Models & Mechanisms

Article Title: Functional exploration of copy number alterations in a Drosophila model of triple-negative breast cancer

doi: 10.1242/dmm.050191

Figure Lengend Snippet: Integrated computational-functional screen to assess potential TNBC driver genes. (A) Prioritization scheme of potential driver genes from CNAs based on TCGA data. (B) Prioritized groups of genes for functional testing. Computational evidence is weaker for Group 2I (dashed border) than Group 2G. (C-E) Validation of the screening results: reduced activity after using RNA-interference (Pten i , Rbf i ) or increased activity after overexpression (Dp110, Egfr). Four known driver genes in trans to ptc>Myc,p53 sh led to decreased viability (C) ( n =4 for Egfr, n =8 otherwise), increased cell translocation (D) and increased overgrowth of transgenic tissue (Pten i shown as example in E) compared to ptc>Myc,p53 sh alone. P -values reflect Student's t -test where data are normally distributed or, otherwise, Mann–Whitney test, compared to genotypically white (w) control flies ( ptc>w ). (w). See also Fig. S3 and Table S3 .

Article Snippet: Protein was transferred onto a PVDF membrane, which was then probed against p53 (DSHB p53-H3 s 1:1000), syntaxin (DSHB 8B-3, 1:1000) or Myc (Santa Cruz Myc d1-717, 1:200) overnight at 4°C.

Techniques: Functional Assay, Activity Assay, Over Expression, Translocation Assay, Transgenic Assay, MANN-WHITNEY, Control

Journal: Disease Models & Mechanisms

Article Title: Functional exploration of copy number alterations in a Drosophila model of triple-negative breast cancer

doi: 10.1242/dmm.050191

Figure Lengend Snippet: Driver genes produce tissue phenotypes in the background of Myc and p53 sh . (A) Quantification of cell translocation for high-priority genes based on their known link to cancer progression. genes. Altering genes marked in red directed a significant increase in translocation compared to ptc>w controls (arrow), measured as P <0.05 in the original experiment and false discovery rate (fdr)<0.1 in the aggregate analysis shown here. Reducing activity of individual genes from each Group in the context of ptc>Myc p53sh , 16/52 from Group1I and 7/21 from Group 1G were significant. (B) Quantification of transgenic tissue overgrowth for high-priority genes. Altering genes marked in red directed a significant increase compared to w (arrow), measured as P <0.05 in the original experiment and fdr<0.1 in the aggregate analysis shown here. Some genes that are significant in this figure were not significant in their respective experiments due to variation between experiments. i indicates RNA-interference mediated knockdown; * indicates a heterozygous null allele;+indicates a duplication. 15/23 from Group1I and 5/12 from Group 1G were significant. (C) Selected phenotypes produced by specific driver genes: cell translocation (PRL-1, Rbf i , srp), small overt mass (Rbf i ), disruption of morphology (srp), transgenic tissue overgrowth (Myb, Hey), and large overt mass (Hey), all compared to w. DAPI staining (red) highlights tissue boundary, GFP signal (green) demarcates transgene expression. Some images were rotated for comparison; borders are indicated by dashed lines. Translocation and overgrowth were not quantified for Hey (last image on right) because the large overt mass phenotype was 100% penetrant. In all cases, each gene was placed in trans to ptc>Myc,p53 sh and compared to ptc>Myc,p53 sh alone. See also Fig. S4 and Tables S12 , .

Article Snippet: Protein was transferred onto a PVDF membrane, which was then probed against p53 (DSHB p53-H3 s 1:1000), syntaxin (DSHB 8B-3, 1:1000) or Myc (Santa Cruz Myc d1-717, 1:200) overnight at 4°C.

Techniques: Translocation Assay, Activity Assay, Transgenic Assay, Knockdown, Produced, Disruption, Staining, Expressing, Comparison

Journal: Disease Models & Mechanisms

Article Title: Functional exploration of copy number alterations in a Drosophila model of triple-negative breast cancer

doi: 10.1242/dmm.050191

Figure Lengend Snippet: Genetic modifiers abrogate the response of p53 sh Myc to fluorouracil. (A) ptc>Myc,p53 sh Drosophila strains were cultured in medium containing screening-optimized doses of cancer drugs at 27°C. Viability was assessed and eclosion rate for each drug is shown in percent. DMSO was used as a control for drugs dissolved in DMSO and water was used as a control for drugs dissolved in water (black bars). Fluorouracil (red bar) significantly improved viability (Mann–Whitney U test versus DMSO: P =0.03). (B,C) Fluorouracil was tested on the ptc>Myc,p53 sh line at 27°C (B) and the ptc>Myc,p53 sh line plus six selected driver genes (Hey, Ppcs, aPKC, Dp110, Myb, Rop as indicated) at 27°C (C). In each case, addition of an additional driver led to loss of fluorouracil-mediated rescue. ns, not significant. (D) Fluorouracil was tested at two doses (10 or 50 µM) on control ( ptc>w ), ptc>Myc,p53 sh (Myc p53 sh ), ptc>Myc,p53 sh ,Myb (Myc,p53 sh Myb) and ptc>Myc,p53 sh ,Dp110 (Myc,p53 sh Dp11) flies, and transgenic tissue overgrowth was quantified as described in Fig. 4B . Two-way ANOVA results were (C) genotype: P <0.0001, drug: ns, interaction: ns; (D) genotype: P <0.0001, drug: P =0.0054, interaction: P =0.0883. Displayed P -values reflect t -tests (see Materials and Methods). See also Table S8 .

Article Snippet: Protein was transferred onto a PVDF membrane, which was then probed against p53 (DSHB p53-H3 s 1:1000), syntaxin (DSHB 8B-3, 1:1000) or Myc (Santa Cruz Myc d1-717, 1:200) overnight at 4°C.

Techniques: Cell Culture, Control, MANN-WHITNEY, Transgenic Assay

Journal: Nature Communications

Article Title: SKA2 regulated hyperactive secretory autophagy drives neuroinflammation-induced neurodegeneration

doi: 10.1038/s41467-024-46953-x

Figure Lengend Snippet: A SNAP29, SNAP23, STX3, SEC22B, and FKBP5 co-immunoprecipitation (SKA2 IP) and whole cell extract (WCE) in hippocampus (HIP), prefrontal cortex (PFC) and amygdala (AMY) samples of mice ( n = 8). B HIS pull down assay (replicated in 3 independent in vitro experiments). DDK(Flag)-tagged SNAP23, SNAP29, Syntaxin3 or Syntaxin4 was incubated with purified magnetic beads-HIS-tagged SKA2 or magnetic beads-HIS protein alone. After incubation, bead bound proteins were eluted at room temperature (RT) or at 95 °C and subjected to western blot analysis using antibodies against HIS and FLAG. Input lane contains HIS alone (left) or HIS-tagged SKA2 (right). C – M SIM-A9 cells transfected with SKA2, FKBP5 or their respective controls, were harvested 24 h later. After immunoprecipitation (IP) of protein complexes, input and co-IP proteins were quantified by western blotting. C , F , I , K Representative blots of ( D , E , G , H , J , L , M ). Graphs display quantification of SNAP29/SEC22B, STX3/SEC22B, SKA2/SNAP29, FKBP5/SEC22B protein association after SEC22B or SNAP29 IP (unpaired two tailed t-test: ( D ) t 6 = 8.945, p < 0.0001, ( E ) t 6 = 12.94, p < 0.0001, ( G ) t 6 = 6.056, p = 0.0009, ( H ) t 6 = 5.554, p = 0.0014; one-way ANOVA: ( J ) F 2, 9 = 17.28, p = 0.0008, Tukey’s post hoc test: ctrl vs. FKBP5-OE, p = 0.0743, ctrl vs. FKBP5-KO, p = 0.0218, FKBP5-OE vs. FKBP5-KO, p = 0.0006; unpaired two tailed t-test: ( L ) t 6 = 10.27, p < 0.0001, ( M ) t 6 = 8.140, p = 0.0002; n = mean derived from four independent in vitro experiments). * = p < 0.05; ** = p < 0.01; *** = p < 0.001; **** = p < 0.0001. Data are presented as mean + SEM. Source data are provided as a file.

Article Snippet: Purified Syntaxin3-DDK (Origene, TP300658), SNAP29-DDK (Origene, TP302179), Syntaxin4 (Origene, TP300347), SNAP23-DDK (Origene, TP301596) or correspondingly SEC22B-HIS (Origene, AR50533PU-S) (100 ng) was used for the binding reaction.

Techniques: Immunoprecipitation, Pull Down Assay, In Vitro, Incubation, Purification, Magnetic Beads, Western Blot, Transfection, Co-Immunoprecipitation Assay, Two Tailed Test, Derivative Assay