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

Journal: Frontiers in Pharmacology

Article Title: Pioglitazone, a PPAR-γ Activator, Stimulates BK Ca but Suppresses IK M in Hippocampal Neurons

doi: 10.3389/fphar.2018.00977

Figure Lengend Snippet: Effect of PIO on whole-cell Ca 2+ -activated K + current ( I K(Ca) ) in mHippoE-14 hippocampal neurons. In these experiments, cells were bathed in normal Tyrode’s solution, the composition of which was described under Section “Materials and Methods.” The recording pipette was filled with K + -containing solution. (A) Superimposed I K(Ca) traces obtained in the control (middle part) and during cell exposure to 10 μM PIO (bottom part). The upper part indicates the voltage protocol applied, and arrowheads are zero current level. (B) Averaged I–V relationships of I K(Ca) obtained in the control ( ), during the exposure ( ) to 10 μM PIO and after washout ( ) of PIO (mean ± SEM; n = 11 for each point). ∗ Significantly different from control groups taken at the same level of voltage pulse. (C) Bar graph showing summary of the effect of PIO, PIO plus TRAM-39, PIO plus apamin, and PIO plus paxilline, and PIO plus tolbutamide on I K(Ca) amplitude (mean ± SEM; n = 10–12 for each bar). Current amplitude was measured at +50 mV. (a) Control; (b) 10 μM PIO; (c) 10 μM PIO plus 3 μM TRAM-39; (d) 10 μM PIO plus 200 nM apamin; (e) 10 μM PIO plus 1 μM paxilline; (f) 10 μM PIO plus 30 μM tolbutamide. ∗ Significantly different from control ( P < 0.05) and ∗∗ significantly different from PIO alone group ( P < 0.05) ( n = 9–10 for each bar).

Article Snippet: Embryonic mouse hippocampal cell line (mHippoE-14; CLU198) was obtained from Cedarlane CELLutions Biosystems, Inc. (Burlington, ON, Canada) ( ).

Techniques: Transferring, Control

Journal: Frontiers in Pharmacology

Article Title: Pioglitazone, a PPAR-γ Activator, Stimulates BK Ca but Suppresses IK M in Hippocampal Neurons

doi: 10.3389/fphar.2018.00977

Figure Lengend Snippet: Effect of PIO on BK Ca channel activity in mHippoE-14 hippocampal neurons. (A) Original current traces of BK Ca channels obtained in the absence (left) and presence (right) of 10 μM pioglitazone (PIO). The examined cells were bathed in symmetrical K + solution (145 mM). Under inside-out current recordings, the potential was held at +60 mV and bath medium contained 0.1 μM Ca 2+ . The upward deflection represents the opening event of the channel. The lower part indicates the expanded trace recorded from the uppermost part in the control and during exposure to PIO. (B) BK Ca -channel trace obtained after washout of PIO. (C) Concentration-dependent increase in channel open probability (mean ± SEM; n = 9–11 for each point). Channel activity measured at +60 mV during the exposure to 100 μM PIO was taken to be 100%. The values for EC 50 , Hill coefficient and maximal percentage increase of BK Ca channels in the presence of PIO were 7.6 μM 1.3 and 100%, respectively.

Article Snippet: Embryonic mouse hippocampal cell line (mHippoE-14; CLU198) was obtained from Cedarlane CELLutions Biosystems, Inc. (Burlington, ON, Canada) ( ).

Techniques: Activity Assay, Control, Concentration Assay

Journal: Frontiers in Pharmacology

Article Title: Pioglitazone, a PPAR-γ Activator, Stimulates BK Ca but Suppresses IK M in Hippocampal Neurons

doi: 10.3389/fphar.2018.00977

Figure Lengend Snippet: Effect of PIO on mean open- (A) and closed-time (B) histograms of BK Ca channels recorded from mHippoE-14 hippocampal neurons. The holding potential was set at +60 mV, and inside-out configuration was performed. In control (left side), the open-time histogram of the channel was fitted by a single exponential function (indicated by red smooth line) with a mean open time of 1.9 ms, while the closed-time histogram was by a sum of a two-exponential function with a mean closed time of 3.5 and 47.5 ms. After addition of 10 μM PIO (right side), the mean open time was increased to 2.7 ms, and the slow component of closed time was shortened to 28.7 ms; however, minimal change in the fast component of closed time (i.e., 3.4 ms) in the presence of this compound. Of note, the abscissa and ordinate in each histogram indicate the logarithm of open or closed time (ms) and the square root of even number, respectively. Data were taken from a measurement of 100 channel openings. The vertical black dashed lines are placed at the values of mean open or closed time for BK Ca channels.

Article Snippet: Embryonic mouse hippocampal cell line (mHippoE-14; CLU198) was obtained from Cedarlane CELLutions Biosystems, Inc. (Burlington, ON, Canada) ( ).

Techniques: Control

Journal: Frontiers in Pharmacology

Article Title: Pioglitazone, a PPAR-γ Activator, Stimulates BK Ca but Suppresses IK M in Hippocampal Neurons

doi: 10.3389/fphar.2018.00977

Figure Lengend Snippet: Effect of PIO on the I–V relation of BK Ca channels in mHippoE-14 hippocampal neurons. The experiments on BK Ca channels were conducted with symmetrical K + -rich concentration (145 mM). Under inside-out configuration, the potential was held at +60 mV and bath medium medium contained 0.1 μM Ca 2+ . (A) Original current traces obtained in the control and during exposure to 10 μM PIO. The labels in the rightmost side indicate the holding potential applied. Arrowhead in each trace corresponds to zero current level, and the upper deflection indicates the opening event of the channel. In (B) , the single-channel conductance in the absence ( ) and presence ( ) of 10 μM PIO is nearly identical. Each point represents mean ± SEM ( n = 9–10). The dashed red lines obtained with or without addition of PIO are pointed toward the values of the reversal potential (i.e., 0.0 ± 0.1 mV, n = 8). (C) The relationship between relative open probability of BK Ca channels and membrane potential obtained with or without addition of 10 μM PIO. The ramp pulses were applied from 0 to +80 mV with a duration of 1 s. Under inside-out current recordings, PIO (10 μM) was applied to the intracellular surface of the excised patch. The smooth lines represent the best fit to the Boltzmann equation as detailed in Section “Materials and Methods.”

Article Snippet: Embryonic mouse hippocampal cell line (mHippoE-14; CLU198) was obtained from Cedarlane CELLutions Biosystems, Inc. (Burlington, ON, Canada) ( ).

Techniques: Concentration Assay, Control, Membrane

Journal: Frontiers in Pharmacology

Article Title: Pioglitazone, a PPAR-γ Activator, Stimulates BK Ca but Suppresses IK M in Hippocampal Neurons

doi: 10.3389/fphar.2018.00977

Figure Lengend Snippet: Effect of linopirdine and linopirdine plus flupirtine on the amplitude of M-type K + current [ I K ( M ) ] in mHippoE-14 hippocampal neurons. In this set of experiments, cells were bathed in high K + , Ca 2+ -free solution and the recording pipette was filled with K + -containing solution. (A) Superimposed I K(M) traces obtained in the control (a) and during the exposure to 10 μM linopirdine (b), and 10 μM linopirdine plus 10 μM flupirtine (c). The upper part indicates the voltage protocol used. (B) Bar graph showing the effect of linopirdine and linopirdine plus flupirtine on I K ( M ) amplitude (mean ± SEM; n = 9 for each bar). ∗ Significantly different from control ( P < 0.05). LINO, linopirdine; FLUP, flupirtine.

Article Snippet: Embryonic mouse hippocampal cell line (mHippoE-14; CLU198) was obtained from Cedarlane CELLutions Biosystems, Inc. (Burlington, ON, Canada) ( ).

Techniques: Transferring, Control

Journal: Frontiers in Pharmacology

Article Title: Pioglitazone, a PPAR-γ Activator, Stimulates BK Ca but Suppresses IK M in Hippocampal Neurons

doi: 10.3389/fphar.2018.00977

Figure Lengend Snippet: Effect of PIO on I K ( M ) amplitude in mHippoE-14 hippocampal neurons. These experiments were conducted in cells bathed in high K + , Ca 2+ -free solution and the recording pipette was filled with K + -containing solution. (A) Superimposed I K ( M ) traces obtained in the absence (a) and presence of 3 μM (b), and 10 μM PIO (c). The upper part indicates the voltage protocol used. (B) Bar graph showing the effect of PIO, linopirdine, and PIO plus flupirtine on I K ( M ) amplitude (mean ± SEM; n = 9–11 for each bar). The I K ( M ) amplitude elicited by membrane depolarization from –50 to –10 mV was measured. (a) Control; (b) 10 μM PIO; (c) 10 μM linopirdine; (d) 10 μM PIO plus 10 μM flupirtine. ∗ Significantly different from control ( P < 0.05) and ∗∗ significantly different from PIO (10 μM) alone group ( P < 0.05). LINO, linopirdine; FLUP, flupirtine.

Article Snippet: Embryonic mouse hippocampal cell line (mHippoE-14; CLU198) was obtained from Cedarlane CELLutions Biosystems, Inc. (Burlington, ON, Canada) ( ).

Techniques: Transferring, Membrane, Control