Journal: Nucleic Acids Research

Article Title: IGF2BP1 promotes mesenchymal cell properties and migration of tumor-derived cells by enhancing the expression of LEF1 and SNAI2 (SLUG)

doi: 10.1093/nar/gkt410

Figure Lengend Snippet: IGF2BP1 knockdown promotes epithelial-like cell properties in HEK293 cells. ( A and B ) HEK293 cells were transfected with control (siC) or IGF2BP1-directed (siI1-2 or siI1-3) siRNAs for 72 h. Cell morphology was monitored by light microscopy (A). The size of adherent cells was analyzed on immunostaining for CTNNB1 as well as F-actin labeling by phalloidin and is depicted as box plots (B). Images were acquired by LSM microscopy. Adherent cells were traced by manual labeling using CTNNB1-defined cell borders to determine the cell area (µm 2 ) using the Leica-SP5× software (also see Supplementary Figure S1A ). ( C ) HEK293 cells were transfected with control (siC) or three distinct IGF2BP1-directed siRNAs (siI1-1, siI1-2 or siI1-3) for 72 h. IGF2BP1 paralogue-specific knockdown was analyzed by western blotting using IGF2BP1-, IGF2BP2- or IGF2BP3-directed monoclonal antibodies. VCL served as a loading control. ( D and E ) HEK293 cells were transfected with indicated siRNAs as in (A). The F-actin cytoskeleton and cell–cell contact formation was analyzed by phalloidin labeling and immunostaining for CTNNB1 (D) or CDH1 (E). Where indicated nuclei were stained by DAPI. Enlargements of boxed regions (left panels) are shown in the right panels (enlargement). Note the enrichment of CTNNB1 and CDH1 at adherens junctions and a knockdown-induced enhancement of cortical F-Actin (also see Supplementary Figure S1F ). Representative images were acquired by LSM microscopy; bars, 10 µm. ( F ) HEK293 cells were transfected with indicated siRNAs as in (A). CDH1, CTNNB1 and IGF2BP1 protein abundance was analyzed by western blotting with indicated antibodies. Protein levels on IGF2BP1 knockdown were determined relative to controls (siC) by normalization to VCL, as indicated above panels. Representative western blots of three independent analyses are shown. ( G ) Soluble FN1 levels were analyzed by ELISA in HEK293 cells transfected with indicated siRNAs for 72 h. Statistical significance was validated by Student’s t -test: ** P < 0.005. Error bars indicate standard deviation (SD) of at least three independent analyses.

Article Snippet: Soluble FN1 protein levels secreted by HEK293 cells were determined using a human FN1 enzyme-linked immunosorbent assay (ELISA) (Boster Biological Technology).

Techniques: Knockdown, Transfection, Control, Light Microscopy, Immunostaining, Labeling, Microscopy, Software, Western Blot, Bioprocessing, Staining, Quantitative Proteomics, Enzyme-linked Immunosorbent Assay, Standard Deviation

Journal: Nucleic Acids Research

Article Title: IGF2BP1 promotes mesenchymal cell properties and migration of tumor-derived cells by enhancing the expression of LEF1 and SNAI2 (SLUG)

doi: 10.1093/nar/gkt410

Figure Lengend Snippet: IGF2BP1 promotes LEF1 expression by preventing LEF1 mRNA degradation. ( A and B ) HEK293 cells were transfected with control (siC) or indicated IGF2BP1-directed (siI1-1, siI1-2) siRNAs for 72 h. Protein abundance on IGF2BP1 knockdown was determined relative to controls (siC) by western blotting using VCL and TUBA4A for cross-normalization, as indicated above panels. Representative western blots of three independent analyses are shown. ACTB and LEF1 mRNA levels were analyzed by qRT-PCR. Changes in RNA abundance on IGF2BP1 knockdown (siIGF2BP1) were determined relative to controls (siC) by the ΔΔC t -method using PPIA for normalization. ( C ) RNA decay was monitored in HEK293 cells transfected with indicated siRNAs for 72 h by blocking mRNA synthesis using ActD (5 µM) for indicated times. RNA levels were determined by qRT-PCR using normalization to PPIA by the ΔΔC t -method. RPLP0 served as a control. RNA decay is depicted in semi-logarithmic scale. Statistical significance determined over three independent analyses was analyzed by Student’s t -test, as shown in panels ( P -values). ( D and E ) The association of indicated mRNAs with IGF2BP1 in HEK293 cells was analyzed by RIP using formaldehyde fixation to stabilize mRNPs prior purification. Endogenous IGF2BP1 was immunopurified (I1) by a monoclonal antibody, as indicated by western blotting in the lower panel (IB). Co-purification of indicated mRNAs was analyzed relative to the input fraction (I, 10% of cell lysates) by semi-quantitative (D) as well as qRT-PCR (E). IgG-agarose served as a control (C) for unspecific mRNA binding. The enrichment of mRNAs by immunopurification of IGF2BP1 (I1) was determined relative to the input fraction by using the ΔC t -method (E). ( F ) Upper panel: Scheme of used Firefly reporters comprising the two alternative LEF1 3′-UTRs (A: Acc.No., NM_016269 /001130713/ 001166119; B: Acc.No., NM_001130714) or the vector-encoded BGH-3′UTR (C). Lower panel: HEK293 cells were transfected with control or indicated IGF2BP1-directed siRNAs for 48 h before the co-transfection of Firefly luciferase reporters (A–C: see scheme in upper panel) and Renilla luciferase control reporters for 24 h. Changes in Firefly luciferase reporter activities on IGF2BP1 knockdown (siIGF2BP1) were determined relative to controls (siC) on normalization by Renilla activities. Statistical significance was validated by Student’s t -test: * P < 0.05; ** P < 0.005; *** P < 0.0005. Error bars indicate SD of at least three independent analyses.

Article Snippet: Soluble FN1 protein levels secreted by HEK293 cells were determined using a human FN1 enzyme-linked immunosorbent assay (ELISA) (Boster Biological Technology).

Techniques: Expressing, Transfection, Control, Quantitative Proteomics, Knockdown, Western Blot, Quantitative RT-PCR, Blocking Assay, Purification, Copurification, Binding Assay, Immu-Puri, Plasmid Preparation, Cotransfection, Luciferase

Journal: Nucleic Acids Research

Article Title: IGF2BP1 promotes mesenchymal cell properties and migration of tumor-derived cells by enhancing the expression of LEF1 and SNAI2 (SLUG)

doi: 10.1093/nar/gkt410

Figure Lengend Snippet: IGF2BP1 modulates FN1 and SNAI2 (SLUG) transcription via LEF1. ( A ) Schematic of luciferase reporters comprising the full-length in silico predicted (FN-839) or 5′-truncated fragments of the human FN1 promoter. The proposed transcription start is indicated by +1 with a reported 5′-UTR of 266 nt. Putative LEF1-binding sites predicted by ‘PROMO’ are depicted as white boxes with labels ‘1-5’ in 5′-to-3′ direction. ( B ) The Firefly luciferase activity of indicated promoter fragments or empty pGL4 vector was monitored in HEK293 cells on transient co-transfection with RFP or LEF1 for 30 h. Firefly activities were normalized by Renilla activities [relative luciferase units (RLU)], serving as internal controls. All reporters comprising the putative LEF1-binding site four showed promoter activity and were activated by LEF1. ( C and D ) Binding of endogenous LEF1 protein to the human FN1 promoter in HEK293 cells was assessed by ChIP. The association of endogenous LEF1 or histone H3 to the FN1 promoter was monitored by semi-quantitative (C) as well as quantitative PCR (D) using to FN1 promoter specific amplicons (P1 and P2, indicated in lower panel). An intergenic probe served as positive control. IgG-agarose was used to monitor unspecific binding (C, negative control). In (D), the enrichment of indicated genomic DNA fragments (P1 and P2) or the intergenic control (intergenic) was determined relative to the diluted input fraction (I) normalized by IgG-controls using the ΔC t -method. ( E ) HEK293 cells were co-transfected with FN-839 luciferase reporter and IGF2BP1-directed (shI1-1), LEF1-directed (shL1-1) or control shRNA encoding vectors for 48 h. RLUs were determined as described in (B). ( F ) HEK293 cells were transfected with IGF2BP1-directed (siI1-2) or control siRNAs (siC) for 72 h. The abundance of SNAI2 mRNA in response to IGF2BP1 knockdown was analyzed by qRT-PCR using the ΔΔC t -method and PPIA for normalization. ACTB served as control. ( G ) HEK293 cells transfected as in (F) were treated with ActD (5 µM) to block transcription for indicated times. SNAI2 mRNA turnover was analyzed by qRT-PCR using the ΔΔC t -method and PPIA for normalization. RNA decay is depicted in semi-logarithmic scale revealing no significant difference in mRNA turnover ( P -value not shown). ( H ) HEK293 cells were transfected with LEF1-directed (siL1-1) or control siRNAs (siC) for 72 h. The abundance of SNAI2 mRNA in response to LEF1 depletion was analyzed by qRT-PCR using the ΔΔC t -method and PPIA for normalization. RPLP0 served as control. ( I ) Schematic of Firefly luciferase reporters comprising the SNAI1 or SNAI2 promoter sequences, as previously reported ( , ). Indicated putative LEF1-binding sites within the SNAI1 or SNAI2 promoter were predicted [white boxes; as described in (A)] or as previously reported [gray boxes, only for SNAI2; ]. ( J ) The Firefly activity of SNAI1 or SNAI2 promoter fragments cloned in pGL4 as well as the activity of empty pGL4 vector was monitored in HEK293 cells on transient co-transfection with RFP or LEF1 for 30 h. RLUs were determined as described in (B). LEF1 only enhanced the activity of the SNAI2 promoter. ( K ) HEK293 cells were co-transfected with SNAI1 or SNAI2 promoter reporters and indicated shRNA-encoding vectors for 48 h. RLUs were determined as described in (B). SNAI2 promoter activity was reduced by IGF2BP1 as well as LEF1 knockdown, whereas the SNAI1 reporter activity remained largely unaffected and was barely elevated compared with the empty control reporter. Statistical significance was validated by Student’s t -testing: * P < 0.05; *** P < 0.0005. Error bars indicate SD of at least three independent analyses.

Article Snippet: Soluble FN1 protein levels secreted by HEK293 cells were determined using a human FN1 enzyme-linked immunosorbent assay (ELISA) (Boster Biological Technology).

Techniques: Luciferase, In Silico, Binding Assay, Activity Assay, Plasmid Preparation, Cotransfection, Real-time Polymerase Chain Reaction, Positive Control, Negative Control, Control, Transfection, shRNA, Knockdown, Quantitative RT-PCR, Blocking Assay, Clone Assay