Journal: bioRxiv

Article Title: Spatial chromosome folding and active transcription drive DNA fragility and formation of oncogenic MLL translocations

doi: 10.1101/485763

Figure Lengend Snippet: (A) Verification of the engineered TOP2A-mAID and TOP2A-mAID-TOP2B −/- HCT116 cells by assessing TOP2 isoform-specific levels upon auxin treatment by western blotting (left panel). Right panel: percentage of HCT116 cells with MLL breakage upon ETO treatment (50μM or 100 μM for 3h) assessed by C-Fusion 3D upon depletion of TOP2A by auxin (500μM, 4h pretreatment) or in cells deficient for TOP2B (1700 to 7300 cells were analyzed per sample in four independent experiments, means ± SD). Representative images of cells upon indicated conditions is shown (lower panel, green probe is MLL 5’, red probe is MLL 3’ see also , scale bar equal to 10μm). (B) Percentage of HT1080 cells deficient for TOP2A or TOP2B with MLL breakage (left) and MLL-AF4 translocations (right) upon ETO (2μM or 5μM for 4h, 24h release) assessed by C-Fusion 3D (1500 to 5600 cells were analyzed per sample, two independent experiments, means ± SD). (C) Both TOP2A and TOP2B isoforms contribute to ETO-induced MLL breakage and MLL translocations in human Cal51 cells upon silencing of the respective isoforms by siRNA an ETO (20μM or 30μM for 4h, 24h release) (1500 to 8100 cells were analyzed per sample, three independent experiments, means ± SD). (D) ETO-induced DSBs in CD34+ progenitor cells are both TOP2A and TOP2B-dependent. CD34+ cells were transduced with different shRNAs for the two TOP2 isoforms and ETO-induced DSBs were quantified by γH2AX staining upon ETO treatment (4h) at the indicated doses. GFP positive cells were selected by image analysis; at least 700 GFP positive cells per condition were quantified for γH2AX mean intensity. Representative box plot of two independent experiments is shown. Statistical significance was calculated with One Way ANOVA test and Tukey compared to Scramble. (E) TK6 cells were arrested in G1 or G2/M phases by treatment with mimosine or the Cdk1 inhibitor (RO3306) respectively, and were released into the next cell cycle phase (S and G1, respectively) in the presence of ETO (20 or 30μM for 4h). After ETO treatment, cells were maintained in the second inhibitor for 24h. Cell cycle distribution at indicated time-points was assessed by measuring DNA content from Hoechst staining . (F) Cell cycle regulation of MLL breakage and (G) MLL-AF4 translocations. Cells were treated as in (E) and upon release from ETO for one day (in the presence of RO3306 or mimosine, prohibiting cells to leave G2/M or G1, respectively), the percentage of cells with MLL breakage and MLL-AF4 translocations was assessed by C-Fusion 3D (1500 to 14,500 cells were analyzed per sample, three experiments, means ± SD). (A-G) *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, Student’s t test comparison to respective treatment in wild type cells (A to C) or to asynchronous cells (F, G).

Article Snippet: Vectors expressing shRNAs were purchased from Origene: shRNA TOP2A-1 (pGFP-C-shLenti TL308699A), shRNA TOP2A-2 (pGFP-C-shLenti TL308699C), shRNA TOP2B-1, pGFP-C-shLenti TL308698A), shRNA Top2B-2, (pGFP-C-shLenti TL308698C), shRNA scramble, (pGFP-C-shLenti shRNA Vector TR30021).

Techniques: Western Blot, Transduction, Staining

Journal: bioRxiv

Article Title: Spatial chromosome folding and active transcription drive DNA fragility and formation of oncogenic MLL translocations

doi: 10.1101/485763

Figure Lengend Snippet: (A) Quantification of ETO-induced DSB-signaling in HCT116 cells by γH2AX immunostaining in the absence of TOP2 isoforms (4500 to 8400 cells were analyzed per sample, one representative out of three independent experiments is shown. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, One Way ANOVA and Tukey test comparison to wildtype cells). (B) TOP2A and/or TOP2B expression levels in HT1080 fibrosarcoma cells (HTETOP) deficient for TOP2A and/or TOP2B by western blotting. Quantification of γH2AX or pRPA (phopho-Ser4/Se8) immunostaining levels upon etoposide are shown in the absence of the respective TOP2 isoforms. (C) ETO-induced breakage was assessed by quantifying γH2AX phosphorylation levels in the indicated cell lines upon siRNA-mediated knock down of TOP2A and/ or TOP2B expression. Time and concentration of ETO-treatment was optimized for each cell line (Cal51 cells: etoposide 10μM and 20μM for 4h, MCF-7 cells: etoposide 15μM and 20μM for 3h). Statistical significance was measured by One Way ANOVA test and Tukey test. Quantification of the number of γH2AX foci led to similar results (data not shown). (D) Expression levels of TOP2A and TOP2B in various human cell lines and peripheral hematopoietic CD34+ stem cells and progenitors. (E) The efficiency of knock down was evaluated by western blotting 3days after siRNAs transfection. (F) Cell cycle distributions of cells released from G1 (Mimosine) or G2/M (RO3306) in the presence of 20μM ETO (4h release) and after ETO treatment for an additional 24h (28h release) in the presence of the second inhibitor.

Article Snippet: Vectors expressing shRNAs were purchased from Origene: shRNA TOP2A-1 (pGFP-C-shLenti TL308699A), shRNA TOP2A-2 (pGFP-C-shLenti TL308699C), shRNA TOP2B-1, pGFP-C-shLenti TL308698A), shRNA Top2B-2, (pGFP-C-shLenti TL308698C), shRNA scramble, (pGFP-C-shLenti shRNA Vector TR30021).

Techniques: Immunostaining, Expressing, Western Blot, Concentration Assay, Transfection

Journal: bioRxiv

Article Title: Sprouty4 is required for Mdm2 regulation of invasion, focal adhesion formation and metastasis in cells lacking p53

doi: 10.1101/2023.05.08.539890

Figure Lengend Snippet: HT1080 p53KO cells were transfected with siRNAs against Mdm2 or siCtrl for 24 h; or treated with 7 µM MEL23 or DMSO (vehicle) for 24 h. (A) Volcano plots show all peptides identified by mass spectrometry. Colored dots represent significantly differentially expressed proteins that were downregulated (blue dots) and upregulated (red dots) in each condition shown at left of plot. Gray dots represent non-significant changes. MEL23 treated cells were compared to DMSO treated cells. Cells transfected with siMdm2#1 or #2 were compared to siCtrl-transfected cells. (B-C) Spry4 expression in HT1080 p53KO cells in response to Mdm2 knockdown or treatment with MEL23 for 24 h by (B) mass spectrometry or (C) immunoblotting. β-actin and α-tubulin were used as loading control for immunoblot. (D) Co-immunoprecipitation of Mdm2 and Spry4 in the presence or absence of MG132. β-actin was used as loading control. (E) Quantification of Spry4, Mdm2 and MdmX protein levels after treatment with MG132 (or vehicle, DMSO) for 4 h. (F) Spry4 mRNA levels in response to Mdm2 knockdown or treatment with MEL23 for 24 h. RPL32 was used as housekeeping. (G) Spry4 luciferase promoter assay in cells silenced for Mdm2 using siRNA. (H) Spry4 mRNA levels in cells treated with MEL23 or vehicle at the indicated times after treatment with DMSO, ActinomycinD (10 µg/ml), or DRB (100 µM) as indicated. Experiments shown represent mean ±SD of at least 3 biological replicates, *p<0.05, **p<0.01, ***p<0.001.

Article Snippet: For shSpry4 stable cell lines, cells were transduced with a set of 4 shRNAs against Spry4 (OriGene, cat.#HC108594) or shRNA negative control (OriGene, cat.#TR30033), and using the appropriate drug for cell selection.

Techniques: Transfection, Mass Spectrometry, Expressing, Western Blot, Immunoprecipitation, Luciferase, Promoter Assay

Journal: bioRxiv

Article Title: Sprouty4 is required for Mdm2 regulation of invasion, focal adhesion formation and metastasis in cells lacking p53

doi: 10.1101/2023.05.08.539890

Figure Lengend Snippet: H1299 cells were transfected with siRNAs against Mdm2 and siCtrl . (A) Protein levels of Mdm2, MdmX and p53 after. β-actin was used as loading control. (B) Cell migration assay. Representative images (top) and quantification (bottom) of wound scratch migration assay. Scale bar equivalent to 1 mm. (C) Quantification and representative micrographs showing attachment to ECM component, collagen I. Scale bar equivalent to 1 mm. (D) Representative images of immunofluorescence showing FA foci by vinculin staining (red), stress fiber formation by phalloidin staining of F-actin (green), and nuclei (blue) detected by DAPI staining of DNA. Scale bar equivalent to 20 µm. (E) Protein levels of Spry4 as well as Mdm2, MdmX and p53 after Mdm2 silencing using siRNAs. β-actin was used as loading control. (F) mRNA levels of Spry4 after Mdm2 silencing using siRNAs. RPL32 was used as housekeeping. Experiments shown represent mean ±SD of at least 3 biological replicates, *p<0.05, **p<0.01, ****p<0.0001.

Article Snippet: For shSpry4 stable cell lines, cells were transduced with a set of 4 shRNAs against Spry4 (OriGene, cat.#HC108594) or shRNA negative control (OriGene, cat.#TR30033), and using the appropriate drug for cell selection.

Techniques: Transfection, Cell Migration Assay, Migration, Immunofluorescence, Staining

Journal: bioRxiv

Article Title: Sprouty4 is required for Mdm2 regulation of invasion, focal adhesion formation and metastasis in cells lacking p53

doi: 10.1101/2023.05.08.539890

Figure Lengend Snippet: (A-E) HT1080 p53KO cells were transfected with siRNA against Mdm2 alone or both siRNA against Mdm2 and a pool of Spry4 siRNAs. (A) Protein levels of Mdm2, MdmX and Spry4 after transfection with indicated siRNAs. β-actin was used as loading control. (B) Quantification of wound scratch migration assay comparing migration into wound scratches in cells treated with the indicated siRNAs as in . (C) Quantification of cell area after attachment to collagen coated coverslips as in . (D) Immunofluorescence showing FA foci by vinculin staining (red), stress fiber formation by phalloidin staining of F-actin (green), and nuclei (blue) as detected by DAPI staining of DNA. Representative images shown at left and quantification of FA parameters shown at right. Scale bar equivalent to 20 µm. (E) Immunoblot (left) and densitometry (right) of levels of total and phospho-cofilin-1(Ser3) in HT1080 p53KO cells silenced for Mdm2 alone or with double KD of Mdm2 and Spry4. (F-H) H1299 cells were transfected with siRNA against Mdm2 alone or both siRNA against Mdm2 and a pool of Spry4 siRNAs. (F) Protein levels of Mdm2, MdmX and Spry4 after transfection with indicated siRNAs. β-actin was used as loading control. (G) Quantification of wound scratch migration assay comparing migration into wound scratches in cells treated with the indicated siRNAs as in . (H) Immunoblot of levels of total and phospho-cofilin-1(Ser3) in H1299 cells silenced for Mdm2 alone or with double KD of Mdm2 and Spry4. (I-J) HT1080 p53KO cell lines were established stably expressing a pool of shRNAs against Mdm2 alone or Mdm2 and Spry4 together. (I) Protein levels of Mdm2, MdmX and Spry4 in stable cells lines. β-actin was used as loading control. (J) Analysis of metastatic burden in vivo using tail-vein injection model as in and . Representative images above and quantification below of metastatic foci in the lungs after 8 weeks of injection. Experiments shown represent mean ±SD of at least 3 biological replicates, *p<0.05, **p<0.01, ****p<0.0001, n.s.: not significant.

Article Snippet: For shSpry4 stable cell lines, cells were transduced with a set of 4 shRNAs against Spry4 (OriGene, cat.#HC108594) or shRNA negative control (OriGene, cat.#TR30033), and using the appropriate drug for cell selection.

Techniques: Transfection, Migration, Immunofluorescence, Staining, Western Blot, Stable Transfection, Expressing, In Vivo, Injection

Journal: Journal of cancer therapy

Article Title: Oncomorphic TP 53 Mutations in Gynecologic Cancers Lose the Normal Protein:Protein Interactions with the microRNA Microprocessing Complex

doi: 10.4236/jct.2014.56058

Figure Lengend Snippet: WT p53 is functional in the UCI-107 cell line. (a) The expression of p53 targets (p21 and puma) and p53 itself was measured 10 hours post 8 Gy radiation. (b) WT p53 was stably knocked down using a shRNA in UCI-107 cell lines. (c) The clonogenic potential of UCI-107 cells was examined by measuring colony formation after treatment with the chemotherapy Cisplatin.

Article Snippet: WT p53 was knocked down using a shRNA plasmid vector or scrambled control (Origene).

Techniques: Functional Assay, Expressing, Stable Transfection, shRNA