Journal: EMBO Reports

Article Title: KIAA1429 / VIRMA promotes breast cancer progression by m 6 A ‐dependent cytosolic HAS2 stabilization

doi: 10.15252/embr.202255506

Figure Lengend Snippet: Western blot analysis of KIAA1429, METTL3, and YTHDC1 expression in the cytosolic (30 μg protein) and nuclear (15 μg protein) fractions of MDA‐MB‐231 cells. Lamin A/C and β‐actin (ACTB) were probed as nuclear and cytosolic marker proteins, respectively. Representative blots are shown. n = 3, biological replicates. Heat map of proteins co‐immunoprecipitated with anti‐KIAA1429 antibody or control IgG from the nuclear or cytosolic fractions of MDA‐MB‐231 cells followed by mass spectrometry. Protein profile heatmap shows the log 2 (ratio) of each sample to the average area across different samples. IGF2BP3/IF2B3, KIAA1429/VIR(MA), and WTAP/FL2D are indicated by red boxes. Western blot analysis of IGF2BP3 and KIAA1429 expression in cytosolic (30 μg protein) and nuclear (15 μg protein) fractions of MDA‐MB‐231 cells. Lamin A/C and α‐Tubulin were probed as nuclear and cytosolic markers, respectively. Representative blots are shown. n = 3, biological replicates. Western blot analysis of KIAA1429 and IGF2BP3 present in anti‐KIAA1429 or control IgG immunoprecipitates of nuclear and cytosolic fractions of MDA‐MB‐231 cells. Representative blots are shown. n = 3, biological replicates. Western blot analysis of IGF2BP3 co‐immunoprecipitated with anti‐KIAA1429 antibody or control IgG in the cytosolic fraction of MDA‐MB‐231 cells. Immunoprecipitates were incubated with or without 100 μg/ml RNase I at 37°C for 15 min before analysis. Representative blots are shown. n = 3, biological replicates. RNA immunoprecipitation (RIP) assays in MDA‐MB‐231 cells expressing control or KIAA1429‐targeting shRNA. Anti‐IGF2BP3 or control IgG immunoprecipitates were subjected to western blot analysis of IGF2BP3 protein (left panel) or RT–qPCR analysis of HAS2 mRNA present in the immunoprecipitates (right panel). * P < 0.05, ** P < 0.01, *** P < 0.001. Representative blots are shown. n = 3, biological replicates. Error bars show mean ± standard error (SEM). P ‐values were calculated by Student's t ‐test. Immunofluorescence images of KIAA1429 staining in MDA‐MB‐231 cells treated with vehicle (DMSO) or 40 μM importazole for 1 h, or with 40 μM importazole for 1 h followed by washout for importazole. Representative blots are shown. n = 3, biological replicates. Scale bar = 100 μm. Dot blot analysis of cytosolic or nucleus m 6 A in DMSO‐treated or importazole‐treated MDA‐MB‐231 cells. Methylene blue (MB) was probed as a loading control. Representative blots are shown. n = 3, biological replicates. Cytosolic RNA immunoprecipitation (RIP) assays in MDA‐MB‐231 cells treated with DMSO or importazole. Anti‐IGF2BP3 or control IgG immunoprecipitates were subjected to western blot analysis of IGF2BP3 protein (left panel) or RT–qPCR analysis of KIAA1429, HAS2 and JUN mRNA present in the immunoprecipitates (right panel). * P < 0.05. Representative blots are shown. n = 3, biological replicates. Error bars show mean ± standard error (SEM). P ‐value was calculated by Student's t ‐test. See also Dataset .

Article Snippet: The membranes were blocked with 5% nonfat milk for 1 h at room temperature and then incubated overnight at 4°C with primary antibodies against KIAA1429/VIRMA (Bethyl Laboratories, A302‐124A and A302‐123A), METTL3 (Abcam, ab195352), m 6 A (Abcam, ab151230), HAS2 (Invitrogen, MA5‐37783), YTHDC1 (GeneTex, GTX32976), α‐Tubulin (Proteintech, HRP‐66031), laminA/C (Proteintech, 10298‐1‐AP), β‐actin (Proteintech, HRP‐60008), phospho‐AKT (Ser473; Cell Signaling Technology, 4058T), IGF2BP3 (Bethyl Laboratories, A303‐425A), or GAPDH (14C10, Cell Signaling Technology).

Techniques: Western Blot, Expressing, Marker, Immunoprecipitation, Control, Mass Spectrometry, Incubation, RNA Immunoprecipitation, shRNA, Quantitative RT-PCR, Immunofluorescence, Staining, Dot Blot

Journal: EMBO Reports

Article Title: KIAA1429 / VIRMA promotes breast cancer progression by m 6 A ‐dependent cytosolic HAS2 stabilization

doi: 10.15252/embr.202255506

Figure Lengend Snippet: Western blotting of KIAA1429 and HAS2 levels in triple‐negative MDA‐MB‐231 and MDA‐MB‐157 cells expressing control plasmid or KIAA1429/VIRMA overexpressing plasmids. GAPDH was probed as a loading control. Representative blots are shown. n = 3, biological replicates. Western blot analysis of KIAA1429 and IGF2BP3 expression in the cytosolic and nuclear fractions of KIAA1429/VIRMA overexpressing MDA‐MB‐231 cells. Lamin A/C and α‐Tubulin were probed as nuclear and cytosolic marker proteins, respectively. Representative blots are shown. n = 3, biological replicates. Dot blot analysis of cytosolic or nucleus m 6 A in control plasmid or KIAA1429/VIRMA overexpressing plasmid‐treated MDA‐MB‐231 cells. Methylene blue (MB) was probed as a loading control. Representative blots are shown. n = 3, biological replicates. Representative images of a cell migration assay of control and KIAA1429 overexpression MDA‐MB‐231 and MDA‐MB‐157 cells. n = 3, biological replicates. Scale bar = 100 μm. Representative images of three‐dimensional (3D) cell invasion assay of control and KIAA1429 overexpressing MDA‐MB‐231 cells 48 or 96 h after planting. n = 3, biological replicates. The red boxes highlight the enhanced invasiveness in KIAA1429 overexpressed cells. Scale bar = 100 μm. RT–qPCR analysis of KIAA1429 and HAS2 mRNA in MDA‐MB‐231 cells expressing control or KIAA1429‐targeting shRNAs with or without HAS2 overexpression. mRNA levels are expressed relative to the levels in shControl cells. * P < 0.05, ** P < 0.01, *** P < 0.001. n = 3, biological replicates. Error bars show mean ± standard error (SEM). P ‐values were calculated by Student's t ‐test. Enumeration of MDA‐MB‐231 cells expressing control or KIAA1429‐targeting shRNAs 24 h with or without HAS2 overexpression. Color coding is the same as for (G). * P < 0.05, ** P < 0.01. n = 3, biological replicates. Error bars show mean ± standard error (SEM). P ‐values were calculated by Student's t ‐test. HAS2, KIAA1429/VIRMA, ERBB2/HER2, ESR1/ER, PGR1/PR gene alteration in BCa datasets from METABRIC (Curtis et al , ; Ferrari et al , ), TCGA, and MSK (Razavi et al , ). Spearman's correlation analysis of HAS2 and KIAA1429/VIRMA mRNA levels in various cancer types from the TCGA database. All correlations are significant ( P < 0.05) except for adrenocortical carcinoma (ACC) and testicular germ cell tumors (TGCT). P ‐value was calculated using a t‐distribution test with n −2 degrees of freedom ( n stands for sample number in each tumor type). See also Fig .

Article Snippet: The membranes were blocked with 5% nonfat milk for 1 h at room temperature and then incubated overnight at 4°C with primary antibodies against KIAA1429/VIRMA (Bethyl Laboratories, A302‐124A and A302‐123A), METTL3 (Abcam, ab195352), m 6 A (Abcam, ab151230), HAS2 (Invitrogen, MA5‐37783), YTHDC1 (GeneTex, GTX32976), α‐Tubulin (Proteintech, HRP‐66031), laminA/C (Proteintech, 10298‐1‐AP), β‐actin (Proteintech, HRP‐60008), phospho‐AKT (Ser473; Cell Signaling Technology, 4058T), IGF2BP3 (Bethyl Laboratories, A303‐425A), or GAPDH (14C10, Cell Signaling Technology).

Techniques: Western Blot, Expressing, Control, Plasmid Preparation, Marker, Dot Blot, Cell Migration Assay, Over Expression, Invasion Assay, Quantitative RT-PCR

Journal: EMBO Reports

Article Title: KIAA1429 / VIRMA promotes breast cancer progression by m 6 A ‐dependent cytosolic HAS2 stabilization

doi: 10.15252/embr.202255506

Figure Lengend Snippet: Western blot analysis of KIAA1429 and IGF2BP3 expression in the cytosolic and nuclear fractions of KIAA1429/VIRMA overexpressing MDA‐MB‐157 cells. Lamin A/C and α‐Tubulin were probed as nuclear and cytosolic marker proteins, respectively. Representative blots are shown. n = 3, biological replicates. MTS cell proliferation assay of MDA‐MB‐231 and MDA‐MB‐157 cells expressing control plasmid or KIAA1429/VIRMA overexpressing plasmids. * P < 0.05. n = 3, biological replicates. The P ‐value was calculated using Student's t ‐test. Representative images of a cell migration assay of control and KIAA1429 overexpression MDA‐MB‐231 and MDA‐MB‐157 cells. Scale bar = 100 μm. Representative images of three‐dimensional (3D) cell invasion assay of control and KIAA1429 overexpressing MDA‐MB‐231 cells 48 h after planting. Scale bar = 100 μm. Representative images from a cell migration assay of MDA‐MB‐231 cells expressing control or KIAA1429‐targeting shRNAs with or without HAS2 overexpression. Images were captured after 16–18‐h incubation. Scale bar = 215 px.

Article Snippet: The membranes were blocked with 5% nonfat milk for 1 h at room temperature and then incubated overnight at 4°C with primary antibodies against KIAA1429/VIRMA (Bethyl Laboratories, A302‐124A and A302‐123A), METTL3 (Abcam, ab195352), m 6 A (Abcam, ab151230), HAS2 (Invitrogen, MA5‐37783), YTHDC1 (GeneTex, GTX32976), α‐Tubulin (Proteintech, HRP‐66031), laminA/C (Proteintech, 10298‐1‐AP), β‐actin (Proteintech, HRP‐60008), phospho‐AKT (Ser473; Cell Signaling Technology, 4058T), IGF2BP3 (Bethyl Laboratories, A303‐425A), or GAPDH (14C10, Cell Signaling Technology).

Techniques: Western Blot, Expressing, Marker, Proliferation Assay, Control, Plasmid Preparation, Cell Migration Assay, Over Expression, Invasion Assay, Incubation