importin4  (Proteintech)

Journal: Cancer Cell International

Article Title: IMP2 drives chemoresistance by repressing cisplatin-induced apoptosis and ferroptosis via activation of IPO4 and SLC7A11 under hypoxia in bladder cancer

doi: 10.1186/s12935-024-03570-4

Figure Lengend Snippet: IMP2 augments the mRNA stability of IPO4 and SLC7A11 in BC. A , Schematic diagram of the workflow for multiomics studies. B , m6A motifs detected with HOMMER in IMP2 knockdown and shNC cells with m6A-seq data. C , Venn diagram identifying the overlapping targets by RIP-seq, m6A-seq, RNA-seq and correlation analysis. D - G , Relative IPO4, HMGB2 and SLC7A11 mRNA and protein expression after IMP2 knockdown or overexpression in UM-UC-3 ( D ), T24 ( E ) and 5637 ( F ) cells, as determined by qRT-PCR and Western blot ( G ). H - J , mRNA decay assay showing the half-life (t 1/2 ) of IPO4 and SLC7A11 mRNA expression after IMP2 silencing or overexpression in UM-UC-3 ( H ), T24 ( I ) and 5637 ( J ) cells treated with actinomycin D. K , Predicted m6A sites in IPO4 mRNA according to the SRAMP program. L and M , Image showing the secondary structure of the position of m6A sites with high confidence located in IPO4. N and O , RIP-qPCR validation of m6A modification in IPO4 and SLC7A11 mRNA with anti- m6A ( N ) or anti-IMP2 ( O ) antibodies. Two-tailed Student’s t test was performed. The error bars represent SD. * p < 0.05, ** p < 0.01, *** p < 0.001

Article Snippet: Subsequently, the bead-lysate complexes were incubated with anti-IPO4 antibodies (5 μg, 11679-1-AP, Proteintech).

Techniques: Knockdown, RNA Sequencing, Expressing, Over Expression, Quantitative RT-PCR, Western Blot, Mrna Decay Assay, Biomarker Discovery, Modification, Two Tailed Test

Journal: Cancer Cell International

Article Title: IMP2 drives chemoresistance by repressing cisplatin-induced apoptosis and ferroptosis via activation of IPO4 and SLC7A11 under hypoxia in bladder cancer

doi: 10.1186/s12935-024-03570-4

Figure Lengend Snippet: IPO4 stabilizes and augments nuclear translocation of C/EBPδ in BC cells. A , Cell lysates were subjected to immunoprecipitated using either an IgG control or an anti-IPO4 antibody. The interaction between the IPO4 and C/EBPδ proteins was evaluated by Co-IP, and immunoblotting was then performed. B and C , Correlations between the expression levels of PRKDC and IPO4 ( B ) or IMP2 ( C ) in TCGA database. D and E , PRKDC expression was detected in BC tissues from patients with different chemotherapy responses ( D ) and clinical stages ( E ) in the TCGA cohort. F , Kaplan–Meier curves for the DFS of BC patients with low and high PRKDC expression. G and H , Cytoplasmic (CE, Right) and nuclear (NE, Left) cell extracts were extracted using a Nuclear and Cytoplasmic Extraction Kit and protein fractions were identified by western blot in UM-UC-3 ( G ) and T24 ( H ) cells. GAPDH, cytoplasmic reference; Histone H3, nuclear reference. I , C/EBPδ distribution between the cytoplasm and nucleus was detected using IF upon changing the expression of IMP2 and IPO4 in BC cells. Scale bars, 50 μm. J and K , Immunoblotting assay detecting cell extracts treated with CHX (100 mg/ml) for the specified durations in control or IPO4 overexpression group in UM-UC-3 ( J ) and T24 ( K ) cells. CR, Complete response; PR, Partial response; SD, Stable disease; PD, Progressive disease. Two-tailed Student’s t test was performed. The error bars represent SD. * p < 0.05, *** p < 0.001

Article Snippet: Subsequently, the bead-lysate complexes were incubated with anti-IPO4 antibodies (5 μg, 11679-1-AP, Proteintech).

Techniques: Translocation Assay, Immunoprecipitation, Control, Co-Immunoprecipitation Assay, Western Blot, Expressing, Extraction, Over Expression, Two Tailed Test

Journal: Cancer Cell International

Article Title: IMP2 drives chemoresistance by repressing cisplatin-induced apoptosis and ferroptosis via activation of IPO4 and SLC7A11 under hypoxia in bladder cancer

doi: 10.1186/s12935-024-03570-4

Figure Lengend Snippet: IPO4 partially restores the suppressive effect of IMP2 knockdown in BC cells. A , Immunoblotting assay of IPO4 and IMP2 expression levels in BC cells after IMP2 knockdown or IPO4 overexpression. B , The IC50 values of the indicated groups treated with cisplatin determined by the CCK-8 assay. C and D , Flow cytometry was used to analyze apoptosis in the indicated groups ( C ), and the percentage of apoptotic cells was determined ( D ). E , Tumor growth curves of mice in different groups treated with cisplatin. F , Images and weights of tumor tissues at the end of the indicated treatment. G , Representative IHC images of NATs, sensitive, and resistant bladder cancer tissues. Scale bar: 100 μm. H , Positive correlation of the IPO4 IHC score with IMP2 IHC score ( R = 0.6143). I - L , Kaplan–Meier curves for OS and DFS or PFS of BC patients with low vs. high IPO4 expression in the GSE169455 ( I and J ) and TCGA ( K and L ) cohorts. Two-tailed Student’s t test was performed. The error bars represent SD. * p < 0.05, ** p < 0.01

Article Snippet: Subsequently, the bead-lysate complexes were incubated with anti-IPO4 antibodies (5 μg, 11679-1-AP, Proteintech).

Techniques: Knockdown, Western Blot, Expressing, Over Expression, CCK-8 Assay, Flow Cytometry, Two Tailed Test

Journal: Cancer Cell International

Article Title: IMP2 drives chemoresistance by repressing cisplatin-induced apoptosis and ferroptosis via activation of IPO4 and SLC7A11 under hypoxia in bladder cancer

doi: 10.1186/s12935-024-03570-4

Figure Lengend Snippet: Schematic illustration of the role of the IMP2-IPO4/SLC7A11 signaling axis in the chemoresistance of BC. LINC00941, which is induced by HIF-1α-mediated transcriptional activation, specifically binds to the IMP2 protein. IMP2 enhanced the mRNA stability of IPO4 and SLC7A11 in a m6A-dependent manner. IPO4 subsequently augmented the nuclear translocation of C/EBPδ to activate PRKDC-mediated DNA damage repair in response to cisplatin. Moreover, IMP2 upregulated SLC7A11 level and suppressed cisplatin-induced ferroptosis

Article Snippet: Subsequently, the bead-lysate complexes were incubated with anti-IPO4 antibodies (5 μg, 11679-1-AP, Proteintech).

Techniques: Activation Assay, Translocation Assay

Journal: Journal of Cellular Physiology

Article Title: Dynamic mRNA Stability Buffer Transcriptional Activation During Neuronal Differentiation and Is Regulated by SAMD4A

doi: 10.1002/jcp.31477

Figure Lengend Snippet: SAMD4A Regulates Neuron's Behaviors by Tuning mRNA stability. SAMD4A KD undifferentiated cells were used to analyze the effect of SAMD4A on mRNA stability. (A) The volcano plot showing the log2 FC and ‐log10 adj. p ‐value of DEGs in SAMD4A KD versus Mock. (Cutoff: |Log 2 FC | ≥ 1 and adj. p < 0.05). Top 10 up and down DEGs with the lowest adj. p ‐value were labeled by gene symbol. (B) GSEA identifying Top 10 enriched GO pathways of Up‐DEGs (Red) and Down‐DEGs (Blue), respectively. (C) The volcano plot showing the log2 FC and ‐log10 adj. p ‐value of DSGs in SAMD4A KD versus Mock. (Cutoff: |Log 2 FC | ≥ 1 and adj. p < 0.05). Top 10 up and down DSGs with the lowest adj. p ‐value were labeled by gene symbol. (D) GSEA identifying Top 10 enriched GO pathways of Stabilized genes (Red) and Destabilized genes (Blue), respectively. (E) Heatmap of Log 2 FC of RIP‐seq genes in the data set of transcriptome, stability and translatome. (F) Venn diagram showing four overlapped genes between the published RNA Immunoprecipitation sequencing (RIP‐seq) and DSGs datasets from Diff wild‐type cells. (G–J) representing the mRNA expression and stability of PPA1, IPO4, IQGAP1, and DENR. SAMD4A KD could stabilize PPA1, IPO4 and IQGAP1 while destabilize DENR. The relationship between mRNA stability and gene expression followed the different two patterns: negative correlation (IQGAP1 and DENR) and positive correlation (PPA1 and IPO4).

Article Snippet: The full‐length 3'UTR of IPO4 and PPA1 genes was synthesized (Thermo Fisher Scientific) and then cloned into the pmirGLO Dual‐Luciferase miRNA Target Expression Vector (Promega, Cat# E1330) following the manufacturer's instructions.

Techniques: Labeling, RNA Immunoprecipitation, Sequencing, Expressing