anti human lin28b  (Cell Signaling Technology Inc)

Journal: Oncogenesis

Article Title: LIN28B inhibition sensitizes cells to p53-restoring PPI therapy through unleashed translational suppression.

doi: 10.1038/s41389-022-00412-8

Figure Lengend Snippet: Fig. 1 LIN28B inhibition enhances p53 activity. A qPCR of LIN28B, TP53, BAX, GADD45, and CDKN1A in control (siCtrl) and LIN28B knockdown (siLIN28B) A2780 cells. Representative data of three independent experiments (mean ± s.e.m.). *p < 0.05. n.s. = not significant. B Relative mRNA levels of TP53, NOXA1, CDKN1A, BAX, and GADD45 in control (−Dox) and LIN28B-induced (+Dox) BE2C neuroblastoma cells by analyzing the GSE138741 dataset [47]. *p < 0.05, **p < 0.01. n.s. = not significant. C p53 luciferase activity (pTA-p53) in control (siCtrl) and LIN28B knockdown (siLIN28B) A2780 cells. Representative data of three independent experiments (mean ± s.e.m.). *p < 0.05. D Luciferase activity in control (siCtrl) and LIN28B knockdown (siLIN28B) A2780 cells utilizing TP53 3′UTR (pmirGLO) or 5′UTR (pGL3) reporters. Representative data of three independent experiments (mean ± s.e.m.). *p < 0.05. n.s. = not significant. E Representative western blots of p53 and cleaved caspase-3 in control (siCtrl) and p53 knockdown (siTP53) cells under camptothecin (2 μM, 8 h) treatment. F FACS analysis of apoptosis (Annexin V labeling) in control (siCtrl) and p53 knockdown (siTP53) cells under camptothecin (2 μM, 8 h) treatment. Representative data of three independent experiments (mean ± s.e.m.). *p < 0.05.

Article Snippet: The following primary antibodies were used in this study: rabbit anti-human LIN28B (1:400) and mouse anti-human p53 (1:1800) (both from Cell Signaling Technology).

Techniques: Inhibition, Activity Assay, Control, Knockdown, Luciferase, Western Blot, Labeling

Journal: Oncogenesis

Article Title: LIN28B inhibition sensitizes cells to p53-restoring PPI therapy through unleashed translational suppression.

doi: 10.1038/s41389-022-00412-8

Figure Lengend Snippet: Fig. 2 LIN28B inhibits p53 protein but not mRNA level independent of MDM2/4. A Representative western blots of p53 and LIN28B expression in A2780 and TOV-112D cells with LIN28B knockdown (shLIN28B) and overexpression (LIN28B) from three independent experiments. B Representative western blots of p53 and LIN28B expression in LOXI and CRMM2 cells with LIN28B knockdown (shLIN28B) from three independent experiments. C Correlation of LIN28B and p53 protein expression in ovarian cancer cell lines (n = 29) based on Supplementary Fig. 3 and our previous publication [23]. D Representative immunohistochemistry staining image of p53 in LIN28B knockdown (shLIN28B) and overexpression (LIN28B) xenograft tumors. Bar, 100 μm. E Representative western blots of MDM2 and MDM4 expression in A2780 and TOV-112D cells with LIN28B knockdown (shLIN28B) and overexpression (LIN28B) from three independent experiments. F qPCR of TP53 mRNA level in LIN28B knockdown (shLIN28B) and overexpression (LIN28B) samples. Ctrl = control. Representative data of three independent experiments (mean ± s.e.m.). n.s. = not significant. G, qPCR of TP53 mRNA in LIN28B knockdown (shLIN28B) and overexpression (LIN28B) cells treated by actinomycin D (10 μg/ml). Representative data of three independent experiments (mean ± s.e.m.). n.s. = not significant.

Article Snippet: The following primary antibodies were used in this study: rabbit anti-human LIN28B (1:400) and mouse anti-human p53 (1:1800) (both from Cell Signaling Technology).

Techniques: Western Blot, Expressing, Knockdown, Over Expression, Immunohistochemistry, Staining, Control

Journal: Oncogenesis

Article Title: LIN28B inhibition sensitizes cells to p53-restoring PPI therapy through unleashed translational suppression.

doi: 10.1038/s41389-022-00412-8

Figure Lengend Snippet: Fig. 3 LIN28B suppresses p53 protein translation. A Representative western blots of p53 in control (shCtrl) and LIN28B knockdown (shLIN28B) A2780 cells treated with cycloheximide (CHX, 100 μg/ml). B Representative western blots of p53 in control (shCtrl) and LIN28B knockdown (shLIN28B) TOV-112D cells treated with cycloheximide (CHX, 100 μg/ml). C Quantification of p53 protein levels in (A) and (B). Representative data of three independent experiments (mean ± s.e.m.). n.s. = not significant. D Representative images of western blots (left) and 35S methionine labeling and autoradiography (right) in the indicated cells with LIN28B knockdown (shLIN28B) and overexpression (LIN28B). n.s. = not significant.

Article Snippet: The following primary antibodies were used in this study: rabbit anti-human LIN28B (1:400) and mouse anti-human p53 (1:1800) (both from Cell Signaling Technology).

Techniques: Western Blot, Control, Knockdown, Labeling, Autoradiography, Over Expression

Journal: Oncogenesis

Article Title: LIN28B inhibition sensitizes cells to p53-restoring PPI therapy through unleashed translational suppression.

doi: 10.1038/s41389-022-00412-8

Figure Lengend Snippet: Fig. 4 LIN28B interacts with TP53 mRNA at the 5′ untranslated region (UTR). A Schematic diagram of RNA immunoprecipitation (RNA-IP) and qPCR detection of TP53 mRNA enrichment in A2780 and TOV-112D cells using isotype control IgG or anti-LIN28B antibody. Representative data of three independent experiments (mean ± s.e.m.). *p < 0.05, **p < 0.01. B Schematic of RNA-pulldown and representative western blots of LIN28B and nucleolin in input and RNA-pulldown samples. CDS coding sequence. C Representative interaction heatmap between TP53 mRNA (5′UTR) and LIN28B protein by catRAPID prediction. D Secondary structure of TP53 mRNA (5′UTR) by RNAfold prediction. E Schematic diagram of truncation mutations of TP53 5′UTR and representative western blots of LIN28B, nucleolin and β-actin in input and RNA-pulldown samples. Exp. = exposure time. *p < 0.05.

Article Snippet: The following primary antibodies were used in this study: rabbit anti-human LIN28B (1:400) and mouse anti-human p53 (1:1800) (both from Cell Signaling Technology).

Techniques: RNA Immunoprecipitation, Control, Western Blot, Sequencing

Journal: Oncogenesis

Article Title: LIN28B inhibition sensitizes cells to p53-restoring PPI therapy through unleashed translational suppression.

doi: 10.1038/s41389-022-00412-8

Figure Lengend Snippet: Fig. 5 LIN28B competes with RPL26 at the TP53 mRNA 5′UTR and inhibits its translation enhancer function. A Representative western blot of LIN28B and RPL26 in A2780 cells with RNA pulldown using control RNA (no biotin) and biotin-labeled TP53 5′UTR sequence. B Representative western blots of RPL26 immunoprecipitation in control (shCtrl) and knockdown (shLIN28B) A2780 cells (left). qPCR of TP53 mRNA after RNA-IP using isotype control IgG or anti-RPL26 antibody in control (shCtrl) and knockdown (shLIN28B) A2780 cells (right). 1, TP53 5′ UTR primer #1; 2, TP53 5′UTR primer #2. 18 S served as an internal control. Representative data of three independent experiments (mean ± s.e.m.). *p < 0.05. C Representative western blots of RPL26 immunoprecipitation in TOV-112D cells transfected with vector control or with LIN28B overexpression (left). qPCR of TP53 mRNA after RNA-IP using isotype control IgG or anti-RPL26 antibody in TOV-112D cells transfected with vector control or with LIN28B overexpression (right). 1, TP53 5′UTR primer #1; 2, TP53 5′UTR primer #2. 18S served as an internal control. Representative data of three independent experiments (mean ± s.e.m.). *p < 0.05. D Luciferase activity of the TP53 5′UTR reporter with RPL26 and/or LIN28B overexpression in HeLa cells. Representative data of three independent experiments (mean ± s.e.m.). *p < 0.05. E Representative western blots of p53, RPL26, and LIN28B in RPL26 and/or LIN28B-overexpressing HeLa cells. Camp. camptothecin (2 μM, 8 h); endo. endogenous. Listed numbers represent the relative expression of p53.

Article Snippet: The following primary antibodies were used in this study: rabbit anti-human LIN28B (1:400) and mouse anti-human p53 (1:1800) (both from Cell Signaling Technology).

Techniques: Western Blot, Control, Labeling, Sequencing, Immunoprecipitation, Knockdown, Transfection, Plasmid Preparation, Over Expression, Luciferase, Activity Assay, Expressing

Journal: Oncogenesis

Article Title: LIN28B inhibition sensitizes cells to p53-restoring PPI therapy through unleashed translational suppression.

doi: 10.1038/s41389-022-00412-8

Figure Lengend Snippet: Fig. 6 LIN28B inhibition sensitizes cells to p53–MDM2 inhibitors. A Relative mRNA level of LIN28B in control (DMSO) and p53–MDM2 inhibitor Nutlin3a, RG7388 (RG, Idasanutlin) treated cells by analyzing the GSE154065 (HCT116) and GSE104917 (NB1691 neuroblastoma) datasets. *p < 0.05, **p < 0.01. B Relative cell viability as determined by CCK8 in ocular melanoma cells treated by various concentrations of Nutlin3a and MI773. C Correlation between relative cell viability and LIN28B protein level in ocular melanoma cells treated by Nutlin3a (22.2 μM). D Correlation between Nutlin3a IC50 and LIN28B protein level in ocular melanoma cells. E Correlation between relative cell viability and LIN28B protein level in ocular melanoma cells treated by MI773 (22.2 μM). F Correlation between MI773 IC50 and LIN28B protein level in ocular melanoma cells treated. G Relative cell viability as determined by CCK8 in ocular melanoma cells treated by various concentrations of Siremadin and Idasanutlin (RG7388). Representative data of three independent experiments (mean ± s.e.m.). H Relative cell viability of control (shCtrl) and knockdown (shLIN28B) LOXI cells treated by various concentrations of Nutlin3a. The IC50 of each group was shown. Representative data of three independent experiments (mean ± s.e.m.). I Schematic diagram of the working model.

Article Snippet: The following primary antibodies were used in this study: rabbit anti-human LIN28B (1:400) and mouse anti-human p53 (1:1800) (both from Cell Signaling Technology).

Techniques: Inhibition, Control, Knockdown

Journal: Cell reports

Article Title: LIN28B Underlies the Pathogenesis of a Subclass of Ewing Sarcoma LIN28B Control of EWS-FLI1 Stability.

doi: 10.1016/j.celrep.2019.12.053

Figure Lengend Snippet: Figure 2. Primary LIN28B+ and LIN28B EwS Cells Display Different Growth Kinetics In Vitro and In Vivo (A) qRT-PCR and western blot analyses of LIN28B expression in 4 primary EwS cell cultures (EwS1–EwS4) analyzed. Asterisks represent values that were set to 0 based on late amplification (means ± SEMs, n = 3 technical replicates). (B) LIN28B+ tumors (EwS1 and EwS2) display reduced expression levels of let-7 family members compared to their LIN28B counterparts, as assessed by qRT- PCR analysis. Mean values ± SEMs of 3 technical replicates are shown. The statistical analysis was performed by 2-way ANOVA.

Article Snippet: Rabbit polyclonal anti-human LIN28B antibody (Cell Signaling Technology) was diluted 1:50, as recommended by the manufacturer.

Techniques: In Vitro, In Vivo, Quantitative RT-PCR, Western Blot, Expressing