Journal: Nature communications

Article Title: Regulation of local GTP availability controls RAC1 activity and cell invasion.

doi: 10.1038/s41467-021-26324-6

Figure Lengend Snippet: Fig. 3 Localization of regions with high RAC1 activity and high local GTP concertation. a Schematic representation of the RAC1 FRET (Förster resonance energy transfer) biosensor. CRIB Cdc42/Rac interactive binding motif. b MDA-MB-231 cells co-expressed the RAC1 biosensor and GEVAL30 or GEVALNull. The signal of each individual biosensor was imaged and rendered as described in Methods. c MDA-MB-231 cells co-expressed the RAC1 biosensor and GEVAL30 or GEVALNull as in (b) and assayed for the correlation between biosensor activities. For each cell, biosensor data were collected in a series of images taken at 1-min intervals over the course of 30 min and analyzed as described in Methods. Pixel-wide Pearson correlation between RAC1 activity (measured as FRET (Förster resonance energy transfer) index for the RAC1 biosensor) and GTP index (measured as the activity of the indicated GEVAL variant) was calculated for each image. The correlation values for the image series corresponding to each individual cell were summarized as “bar-and-whiskers” plots, with “whiskers” indicating the first and the fourth quartiles, the horizontal line (median) splitting the bars into the second and the third quartiles, and “X” indicating quartiles, as well as the mean correlation coefficient (r) for each series. The mean correlation coefficients were compared by Mann–Whitney test. d Cells expressing the indicated constructs were probed in RAC1 activity assay as described in Methods. Note that the difference in migration of RAC1T17N compared to other proteins is likely due to the fact that RAC1T17N is fused to one Myc-tag, whereas other RAC1 proteins—to two Myc tags. Shown are representative images of at least two independent experiments. e Quantification of (d); n = 2 biologically independent samples (left panel); n = 3 biologically independent samples (right panel) The data represents average ± SEM.

Article Snippet: Expression vectors for RAC1WT, RAC1P29S, RAC1Q61L, and RAC1T17N were purchased from Addgene (#128580, #128581, #128582, and #12984, respectively).

Techniques: Activity Assay, Förster Resonance Energy Transfer, Binding Assay, Variant Assay, MANN-WHITNEY, Expressing, Construct, Migration

Journal: Cancer Research

Article Title: Loss of ARF Sensitizes Transgenic BRAFV600E Mice to UV-Induced Melanoma via Suppression of XPC

doi: 10.1158/0008-5472.can-12-4454

Figure Lengend Snippet: Figure 2. p53 is functional in BRAFV600E;ARF/ melanocytes and melanoma cells despite ARF deficiency. A, immunoblots detecting p53 from total skin and tumor lysates. B, immunoblots detecting p21 and phospho-p53 at Ser 15 (p-p53) from total BRAFV600E;ARF/ tumor lysates. Control lane was lysate from a spontaneous BRAFV600E melanoma (470 strain, the same sample with that used in Fig. 1A) serving as a positive control for ARF. C, immunoblots showing p53 knockdown efficiency and reduced level of p21, a downstream target of p53, in isolated primary BRAFV600E;ARF/ melanoma cells. Control sample for ARF was the same as in B. D, immunoblots showing induction of phospho-p53 in primary BRAFV600E;ARF/ melanoma cells with or without ARF restoration. p53 can be rapidly activated upon UVR, regardless of ARF status, confirming that the p53 present in the cells is functional. E and F, growth curve and soft agar colony-formation ability of BRAFV600E;ARF/ primary melanoma cells. Cells (#4228) with dominant-negative p53 (DNp53) grew faster (E) and formed more colonies (F) than control. Colonies that were bigger than 30 mm were quantified. Student t test; , P < 0.05; , P < 0.005.

Article Snippet: The retroviral vector pBabe-puro-p19ARF was generously provided by N. Sharpless (University of North Carolina, Chapel Hill, NC), pBabe-hygro-dominant-negative-p53 was obtained from Addgene (#9058; ref. 28).

Techniques: Functional Assay, Western Blot, Control, Positive Control, Knockdown, Isolation, Dominant Negative Mutation

Journal: Cancer Research

Article Title: Loss of ARF Sensitizes Transgenic BRAFV600E Mice to UV-Induced Melanoma via Suppression of XPC

doi: 10.1158/0008-5472.can-12-4454

Figure Lengend Snippet: Figure 3. Loss of ARF cannot suppress BRAFV600E-induced senescence in vivo. A, SA-b-gal staining on skin sections from age- matched mice of the indicated genotypes. Hematoxylin was used to counterstain the nucleus. Red- boxed areas in the top row are enlarged in the bottom row. B, qRT-PCR showing the mRNA expression of selected members of the senescence-associated secretome in skins of different genotypes. Student t test, compared with WT skin; , P < 0.05; , P < 0.005. C, IHC of IL-6 conducted on representative skin sections from BRAFV600E and BRAFV600E;ARF/ mice. Boxed areas are enlarged as indicated and arrowheads represent the positive- staining cells. Sections were counterstained with hematoxylin. D, IHC of p16INK4a and p53 on representative skin sections from BRAFV600E;ARF/ mice. IgG is negative control and nucleus was counterstained by hematoxylin.

Article Snippet: The retroviral vector pBabe-puro-p19ARF was generously provided by N. Sharpless (University of North Carolina, Chapel Hill, NC), pBabe-hygro-dominant-negative-p53 was obtained from Addgene (#9058; ref. 28).

Techniques: In Vivo, Staining, Quantitative RT-PCR, Expressing, Negative Control

Journal: Cancer Research

Article Title: Loss of ARF Sensitizes Transgenic BRAFV600E Mice to UV-Induced Melanoma via Suppression of XPC

doi: 10.1158/0008-5472.can-12-4454

Figure Lengend Snippet: Figure 4. Loss of ARF sensitizes BRAFV600E mice to UV-induced melanoma. A, schematic representation of the neonatal UVB irradiation protocol. Mice of different genotypes were irradiated with 750 mJ/cm2 UVB at postnatal day 3.5 (P3.5). B, Kaplan–Meier curve showing melanoma-free survival of mice receiving neonatal UVB irradiation. Triangle in the ARF/ curve indicates mouse died of sarcoma. C, H&E and immunostaining of melanoma markers Trp2, S100, Melan A, and HMB45 on representative sections from UVB-induced BRAFV600E;ARF/ tumors. Arf staining confirms the absence of the protein in tumors. Arrowheads in the p53 and phospho-p53 (Ser15) images indicate the positive-staining cells.

Article Snippet: The retroviral vector pBabe-puro-p19ARF was generously provided by N. Sharpless (University of North Carolina, Chapel Hill, NC), pBabe-hygro-dominant-negative-p53 was obtained from Addgene (#9058; ref. 28).

Techniques: Irradiation, Immunostaining, Staining

Journal: Cancers

Article Title: Menin and Menin-Associated Proteins Coregulate Cancer Energy Metabolism

doi: 10.3390/cancers12092715

Figure Lengend Snippet: Identification of MEN1- modulated genes in breast cancer cells. ( A ) RT-qPCR of MEN1 in T47D or MCF-7 cells treated with vehicle or MEN1 shRNA lentivirus ( n = 3). ( B ) Quantitative Western immunoassays (WES) of menin expression in T47D or MCF-7 cells treated with vehicle or MEN1 shRNA lentivirus ( n = 3). ( C ) Venn diagrams of differentially expressed genes (fold change ≥1.5 or ≤0.66) in T47D or MCF-7 cells after sh MEN1 knockdown compared with vehicle controls ( n = 2). ( D ) Pathway annotation analysis of MEN1 -upregulated and MEN1 -downregulated genes in T47D or MCF-7 cells using DAVID including cancer hallmark pathways. ( E ) Schematic illustration of five major metabolic pathways. ( F ) Expression heat maps of oxidative phosphorylation (OXPHOS) and glycolytic genes in both MEN1 knockdown T47D and MCF-7 cells (fold changes relative to vehicle controls). ( G ) Bar charts of the expression levels of representative OXPHOS and glycolytic genes affected by MEN1 knockdown in T47D or MCF-7 cells using RT-qPCR. Data are presented as mean ± S.D. Unpaired two-tailed Student’s t -test was used for statistics. * p < 0.05, ** p < 0.01, and *** p < 0.001.

Article Snippet: The full-length human MEN1 cDNA from pBABE hygro MEN1 WT (Addgene plasmid #11024) was cloned into pRetroX-mycBioID-MCS at Not1 and Mlu I site by the Gibson reaction (New England Biolabs, Ipswich, MA, USA) to obtain pRetroX-mycBirA-MEN1.

Techniques: Quantitative RT-PCR, shRNA, Western Blot, Expressing, Knockdown, Phospho-proteomics, Two Tailed Test

Journal: Cancers

Article Title: Menin and Menin-Associated Proteins Coregulate Cancer Energy Metabolism

doi: 10.3390/cancers12092715

Figure Lengend Snippet: Identification of menin-associated proteins (MAPs) in breast cancer cells. ( A ) and ( B ) WES of BirA-Menin fusion proteins ( A ) and biotin-labeled proteins ( B ) in total lysates of BirA-MEN1 BioID engineered T47D or MCF-7 cells after incubating with or without doxycycline and biotin. ( C ) Schematic purification and proteomic identification of MAPs using LC–MS/MS. ( D ) Heatmap of the quantification of 35 MAPs commonly shared in T47D and MCF-7 cells. MAPs further verified by WES immunoassays were indicated by arrows. ( E ) Network analysis of 35 MAPs in MCF-7 cells. The distance between menin and MAPs represented the quantitative ratio of each MAP and menin. MAPs marked in blue were further assayed by WES. ( F ) Nuclear or cytoplasmic lysates of BirA-MEN1 BioID engineered T47D or MCF-7 cells after streptavidin beads pull-down were detected by WES with antibodies against menin, KMT2A, MED12, WAPL, GATA3, LaminA/C, or GAPDH. FL, full length; SP, spliced form.

Article Snippet: The full-length human MEN1 cDNA from pBABE hygro MEN1 WT (Addgene plasmid #11024) was cloned into pRetroX-mycBioID-MCS at Not1 and Mlu I site by the Gibson reaction (New England Biolabs, Ipswich, MA, USA) to obtain pRetroX-mycBirA-MEN1.

Techniques: Labeling, Purification, Liquid Chromatography with Mass Spectroscopy

Journal: Cancers

Article Title: Menin and Menin-Associated Proteins Coregulate Cancer Energy Metabolism

doi: 10.3390/cancers12092715

Figure Lengend Snippet: Expression correlation relationship of menin/MAPs genes and OXPHOS/glycolytic genes. ( A ) Workflow of the in silico correlation analysis of gene expression in The Cancer Genome Atlas (TCGA) breast cancer cohort. ( B ) Heatmaps of the expression correlation between MEN1 /selected 4 MAP genes and OXPHOS genes (upper) or glycolytic genes (lower) in normal (N) and tumor (T) samples. The genes are arranged from the highest to the lowest according to gene expression correlation coefficients of MEN1 –OXPHOS genes or MEN1 –glycolytic genes in breast tumors. ( C ) and ( D ) Scatter plots and linear regression analyses of MEN1/selected MAPs expression and mean expression of OXPHOS genes ( C ) or glycolytic genes ( D ) in normal and tumor samples. ( E ) Violin plots (lower panel) shows the average expressions of the genes of OXPHOS complexes I-V and glycolysis in the samples of each of the corresponding 4 groups are shown as violin plots. Based on the median values (where ≥median is “high” and

Article Snippet: The full-length human MEN1 cDNA from pBABE hygro MEN1 WT (Addgene plasmid #11024) was cloned into pRetroX-mycBioID-MCS at Not1 and Mlu I site by the Gibson reaction (New England Biolabs, Ipswich, MA, USA) to obtain pRetroX-mycBirA-MEN1.

Techniques: Expressing, In Silico, Gene Expression

Journal: Cancers

Article Title: Menin and Menin-Associated Proteins Coregulate Cancer Energy Metabolism

doi: 10.3390/cancers12092715

Figure Lengend Snippet: Bioenergetic dynamics are regulated by menin and MAPs in T47D and MCF-7 cells. ( A ) and ( B ) Glycolytic and OXPHOS ATP productions in T47D ( A ) or MCF-7 ( B ) cells infected with vehicle, sh MEN1 , sh KMT2A , sh MED12 , sh WAPL , or sh GATA3 lentivirus. Statistics represented the difference of glycolytic or OXPHOS ATP production between shRNA knockdown and vehicle controls. ( C ) and ( D ) Bar charts representing mitochondrial functions in the single knockdown of MEN1 , KMT2A , MED12 , WAPL , or GATA3 and their vehicle control in T47D ( C ) or MCF-7 ( D ) cells. ( E ) Schematic summary of mitochondrial dynamics affected by the knockdown of MEN1 or MAPs. ( F , G ) Bar charts represented the glycolytic functions in T47D ( F ) or MCF-7 ( G ) cells subject to gene knockdown by sh MEN1 , sh KMT2A , sh MED12 , sh WAPL , or sh GATA3 lentivirus. ( H ) Schematic summary of glycolytic functions affected by the knockdown of MEN1 or MAPs. Data are presented as mean ± S.D. ( n = 15–20 technical-replicate wells). Statistical significance was performed by an unpaired two-tailed Student’s t -test between treated groups and corresponding controls. * p < 0.05, ** p < 0.01, and *** p < 0.001.

Article Snippet: The full-length human MEN1 cDNA from pBABE hygro MEN1 WT (Addgene plasmid #11024) was cloned into pRetroX-mycBioID-MCS at Not1 and Mlu I site by the Gibson reaction (New England Biolabs, Ipswich, MA, USA) to obtain pRetroX-mycBirA-MEN1.

Techniques: Infection, shRNA, Knockdown, Control, Two Tailed Test

Journal: Cancers

Article Title: Menin and Menin-Associated Proteins Coregulate Cancer Energy Metabolism

doi: 10.3390/cancers12092715

Figure Lengend Snippet: Integrity of the menin–KMT2A complex is required for OXPHOS functions. ( A ) WES of T47D or MCF-7 cells treated with DMSO or 1 μM MI-503 for 3 days (left). Relative protein expression normalized to the average of LaminA/C in WES (right). FL, full length; SP, spliced form. ( B ) Nuclear lysates of T47D or MCF-7 cells treated with DMSO or 1 μM of MI-503 for 3 days were immunoprecipitated with the menin antibody or IgG, and assayed by WES (upper). Relative protein expression in WES (lower). The protein expression in DMSO treated input was normalized as 1. FL, full length; SP, spliced form. ( C ) Glycolytic or OXPHOS ATP production in T47D or MCF-7 cells treated with 1 μM MI-503 for 0, 1, 3, 6, and 72 h, or DMSO control for 72 h. ( D , E ) Bar charts of the Seahorse mitochondrial stress test ( D ) and glycolytic stress test ( E ) on T47D or MCF-7 cells treated with 1 μM MI-503 for 0, 1, 3, 6, and 72 h, or DMSO for 72 h. Data are presented as mean ± S.D. ( n = 10–15 technical-replicate wells). An unpaired two-tailed Student’s t -test was used to determine statistical significance for the difference between MI-503-treated groups and its controls. * p < 0.05, ** p < 0.01, and *** p < 0.001.

Article Snippet: The full-length human MEN1 cDNA from pBABE hygro MEN1 WT (Addgene plasmid #11024) was cloned into pRetroX-mycBioID-MCS at Not1 and Mlu I site by the Gibson reaction (New England Biolabs, Ipswich, MA, USA) to obtain pRetroX-mycBirA-MEN1.

Techniques: Expressing, Immunoprecipitation, Control, Two Tailed Test

Journal: Cancers

Article Title: Menin and Menin-Associated Proteins Coregulate Cancer Energy Metabolism

doi: 10.3390/cancers12092715

Figure Lengend Snippet: MEN1 and OXPHOS expression are increased in breast circulating tumor cells (CTCs). ( A ) t-SNE profile plots and cell clustering of 93 CTCs from 5 breast cancer patients based on the single cell RT-qPCR expression profiling of 11 OXPHOS genes ( NDUFA7 , NDUFA11 , NDUFA13 , NDUFB7 , NDUFS7 , NDUFS8 , NDUFV1 , SDHA , SDHB , SDHC , and SDHD ). ( B ) Violin plots of MEN1 or selected MAPs expression, mean expression of 7 glycolytic genes ( ALDOA , ALDOC , ENO1 , PFKL , PFKP , PGK1 , and TPI1 ) or mean expression of 11 OXPHOS genes (aforementioned) in the five cell clusters. Statistical significance among clusters was carried out using the Duncan multi-range test. ( C ) Mean expression of 7 glycolytic genes and 11 OXPHOS genes in these 93 breast CTCs or in the TCGA primary breast cancer cohort. ( D ) Glycolytic and OXPHOS ATP productions of T47D or MCF-7 cells after circulation ( n = 6–10 technical replicates). ( E , F ) Mitochondrial ( E ) and glycolytic ( F ) functions of T47D or MCF-7 cells after circulation ( n = 5–9 technical-replicate wells). Statistics represented the difference between no circulating control and each treatment. Data are presented as mean ± S.D. An unpaired two-tailed Student’s t -test was used for statistical significance determination. * p < 0.05, ** p < 0.01, and *** p < 0.001.

Article Snippet: The full-length human MEN1 cDNA from pBABE hygro MEN1 WT (Addgene plasmid #11024) was cloned into pRetroX-mycBioID-MCS at Not1 and Mlu I site by the Gibson reaction (New England Biolabs, Ipswich, MA, USA) to obtain pRetroX-mycBirA-MEN1.

Techniques: Expressing, Quantitative RT-PCR, Control, Two Tailed Test