sh scramble control  (OriGene)

Journal: Nature Communications

Article Title: Mdm2 requires Sprouty4 to regulate focal adhesion formation and metastasis independent of p53

doi: 10.1038/s41467-024-51488-2

Figure Lengend Snippet: A , B Tumor spheroid invasion in collagen matrix after ( A ) transfection of HT1080 p53KO cells with siRNAs against Mdm2 or ( B ) treatment with MEL23. Representative images above and quantification below of the area invaded and the number of cells invading 24 h after implantation. C , D Tumor spheroid invasion in the collagen-BME matrix after ( C ) transfection of HT1080 p53KO cells with siRNAs against Mdm2 or ( D ) treatment with MEL23. Representative images and quantification of the area invaded 24 h after implantation. Graphs show three pooled independent experimental replicates, the total number of spheroids in each condition is: siCtrl = 10, siMdm2#1 = 15, siMdm2#2 = 12 in panel ( A ); DMSO = 25, MEL23 = 24 in panel ( B ); siCtrl = 12, siMdm2#1 = 14, siMdm2#2 = 15 in panel ( C ), and DMSO = 8, MEL23 = 9 in panel ( D ). E Representative images of cell morphology of HT1080 p53KO cells transfected with siRNAs against Mdm2 or siCtrl in collagen matrix, 6 h after implantation. F Quantification of more circular cells (circularity of 0.75 or higher) in each condition shown in ( E ), n = 3 groups. The graph represents the average fold change of more circular morphology in Mdm2 silenced cells compared to control in three independent experimental replicates. More details about the quantification can be found in the method’s session. G – I HT1080 p53KO cells stably expressing shRNA scramble (shScramble) or a pool of Mdm2 shRNAs (shMdm2) were used to analyze metastatic burden using mouse models. G Protein levels of Mdm2 and MdmX in HT1080 shScramble and shMdm2 stable cell lines. β-actin was used as a loading control. H , I Representative images above and quantification below of metastatic foci in the lungs after implantation of shScramble or shMdm2 cells using ( H ) orthotropic model, n = 4 mice/group or ( I ) tail-vein model, n = 8 mice/group. In all box and whisker plots the boxes extend from 25 to 75 percentiles and whiskers show min and max values, The line in the center of each box represents the median. Graphs shown in panels ( F , H , I ) represent the mean ± SD of independent experimental replicates. More details about the statistical tests used can be found in the Source Data file.

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

Techniques: Transfection, Control, Stable Transfection, Expressing, shRNA, Whisker Assay

Journal: Nature Communications

Article Title: Mdm2 requires Sprouty4 to regulate focal adhesion formation and metastasis independent of p53

doi: 10.1038/s41467-024-51488-2

Figure Lengend Snippet: A – D 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, n = 3 samples. β-actin was used as a loading control. B Quantification of wound scratch migration assay in cells treated with the indicated siRNAs as in Fig. , n = 3 samples. C Quantification of cell area after attachment to collagen-coated coverslips as in Fig. , n = 3 samples. D Immunofluorescence showing FA foci by vinculin staining (red), cell surface was outlined by phalloidin staining (green), and nuclei (blue) as detected by DAPI staining, n = 3 groups. Representative images are shown on the left, and the quantification of FA parameters is shown on the right. In ( C , D ) the graphs shown represent mean ± SD of independent experimental replicates and in each replicate all parameters were quantified in at least 20 events/condition for total of at least 60 events/condition. E , F H1299 cells were transfected with siRNA against Mdm2 alone or both siRNA against Mdm2 and a pool of Spry4 siRNAs. E Protein levels of Mdm2, MdmX, and Spry4 after transfection with indicated siRNAs. β-actin was used as a loading control, n = 3 samples. F Quantification of wound scratch migration assay comparing migration into wound scratches in cells treated with the indicated siRNAs as in Fig. , n = 3 samples. G , H HT1080 p53KO cell lines were established stably expressing a pool of shRNAs against Mdm2 alone or Mdm2 and Spry4 together. G Protein levels of Mdm2, MdmX, and Spry4 in stable cell lines. β-actin was used as a loading control, n = 3 samples. H Analysis of metastatic burden in vivo using tail-vein injection model as in Fig. H, . Representative images above and quantification below of metastatic foci in the lungs after 8 weeks of injection, n = 7 mice/group. The graphs shown represent the mean ± SD of independent experimental replicates. More details about the statistical tests used can be found in the Source Data file.

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

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

Journal: Nature Communications

Article Title: Mdm2 requires Sprouty4 to regulate focal adhesion formation and metastasis independent of p53

doi: 10.1038/s41467-024-51488-2

Figure Lengend Snippet: A , B Tumor spheroid invasion in collagen matrix after ( A ) transfection of HT1080 p53KO cells with siRNAs against Mdm2 or ( B ) treatment with MEL23. Representative images above and quantification below of the area invaded and the number of cells invading 24 h after implantation. C , D Tumor spheroid invasion in the collagen-BME matrix after ( C ) transfection of HT1080 p53KO cells with siRNAs against Mdm2 or ( D ) treatment with MEL23. Representative images and quantification of the area invaded 24 h after implantation. Graphs show three pooled independent experimental replicates, the total number of spheroids in each condition is: siCtrl = 10, siMdm2#1 = 15, siMdm2#2 = 12 in panel ( A ); DMSO = 25, MEL23 = 24 in panel ( B ); siCtrl = 12, siMdm2#1 = 14, siMdm2#2 = 15 in panel ( C ), and DMSO = 8, MEL23 = 9 in panel ( D ). E Representative images of cell morphology of HT1080 p53KO cells transfected with siRNAs against Mdm2 or siCtrl in collagen matrix, 6 h after implantation. F Quantification of more circular cells (circularity of 0.75 or higher) in each condition shown in ( E ), n = 3 groups. The graph represents the average fold change of more circular morphology in Mdm2 silenced cells compared to control in three independent experimental replicates. More details about the quantification can be found in the method’s session. G – I HT1080 p53KO cells stably expressing shRNA scramble (shScramble) or a pool of Mdm2 shRNAs (shMdm2) were used to analyze metastatic burden using mouse models. G Protein levels of Mdm2 and MdmX in HT1080 shScramble and shMdm2 stable cell lines. β-actin was used as a loading control. H , I Representative images above and quantification below of metastatic foci in the lungs after implantation of shScramble or shMdm2 cells using ( H ) orthotropic model, n = 4 mice/group or ( I ) tail-vein model, n = 8 mice/group. In all box and whisker plots the boxes extend from 25 to 75 percentiles and whiskers show min and max values, The line in the center of each box represents the median. Graphs shown in panels ( F , H , I ) represent the mean ± SD of independent experimental replicates. More details about the statistical tests used can be found in the Source Data file.

Article Snippet: For the double knock-down, cells expressing shMdm2 or shScramble were transduced with lentivirus carrying a pool of shRNAs against Sprouty4 (four different shRNAs purchased from OriGene, cat.#HC108594) or shScramble sequence as control (OriGene, cat.#TR30033).

Techniques: Transfection, Control, Stable Transfection, Expressing, shRNA, Whisker 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: 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