Journal: Cell Biology and Toxicology

Article Title: Immortalization-upregulated protein promotes pancreatic cancer progression by regulating NPM1/FHL1-mediated cell-cycle-checkpoint protein activity

doi: 10.1007/s10565-022-09695-4

Figure Lengend Snippet: Exploring downstream gene regulated by IMUP. a Heatmap and volcano plot analyzed using RNA-seq of BxPC-3 cells infected with shNC, IMUP-sh1, sh2, and sh3. b Gene ontology analysis of IMUP-correlated genes from TCGA database by LinkedOmics. c The intersection of negatively correlated genes (NCGs) from RNA-seq (fold-change > 2), NCGs from GeneChip, and NCGs from TCGA. d , e Pearson correlation of FHL1 and IMUP expression according to d the results of GeneChip and e TCGA data. f Kaplan–Meier analysis of OS by FHL1 expression data from TCGA

Article Snippet: Antibodies against IMUP (1:100; #ab221063) from Abcam (Cambridge, UK) and FHL1 (1:50; #10,991–1-AP) from Proteintech (Wuhan, China) were used for IHC to verify their expression in PDAC and xenograft tumor tissues.

Techniques: RNA Sequencing, Infection, Expressing

Journal: Cell Biology and Toxicology

Article Title: Immortalization-upregulated protein promotes pancreatic cancer progression by regulating NPM1/FHL1-mediated cell-cycle-checkpoint protein activity

doi: 10.1007/s10565-022-09695-4

Figure Lengend Snippet: IMUP regulates cyclin A2/cyclin E1/CDK2 proteins via FHL1. a WB analysis of BxPC-3 and SW1990 cells infected with lentivirus carrying IMUP-sh1 or sh2. GAPDH and tubulin were used as a loading control. b WB analysis of BxPC-3 and SW1990 cells infected with lentivirus carrying IMUP-sh or co-infected with IMUP-sh and FHL1-siRNA. c Representative IHC staining of IMUP and FHL1 in 57 human PDAC samples. Case 1 and case 2 were two representative specimens analyzed as high and low expression of IMUP, respectively. (case 1, high IMUP and low FHL1; Case 2, low IMUP and high FHL1). Scale bars: 100 μm. Correlation between IMUP and FHL1 analyzed by Pearson correlation tests

Article Snippet: Antibodies against IMUP (1:100; #ab221063) from Abcam (Cambridge, UK) and FHL1 (1:50; #10,991–1-AP) from Proteintech (Wuhan, China) were used for IHC to verify their expression in PDAC and xenograft tumor tissues.

Techniques: Infection, Control, Immunohistochemistry, Expressing

Journal: Cell Biology and Toxicology

Article Title: Immortalization-upregulated protein promotes pancreatic cancer progression by regulating NPM1/FHL1-mediated cell-cycle-checkpoint protein activity

doi: 10.1007/s10565-022-09695-4

Figure Lengend Snippet: Knockdown of FHL1 rescues the phenotype inhibited by IMUP depletion. a , b , c , g BxPC-3 and SW1990 cells infected with shNC or IMUP-sh, or co-infected with IMUP-sh and FHL1-sh were used to analyze the a proliferation by cell Counting Kit-8, b tumorigenicity by mice xenograft ( n = 5/group; male), c colony formation capability by colony formation assay, and g cell cycle by flow cytometry. d Xenograft tumor volume, e colony formation, and h cell cycle were analyzed by two-way ANOVA test. f Tumor weight was analyzed by unpaired Student’s t test. * P < 0.05, ** P < 0.01, *** P < 0.0001

Article Snippet: Antibodies against IMUP (1:100; #ab221063) from Abcam (Cambridge, UK) and FHL1 (1:50; #10,991–1-AP) from Proteintech (Wuhan, China) were used for IHC to verify their expression in PDAC and xenograft tumor tissues.

Techniques: Knockdown, Infection, Cell Counting, Colony Assay, Flow Cytometry

Journal: Cell Biology and Toxicology

Article Title: Immortalization-upregulated protein promotes pancreatic cancer progression by regulating NPM1/FHL1-mediated cell-cycle-checkpoint protein activity

doi: 10.1007/s10565-022-09695-4

Figure Lengend Snippet: IMUP inhibits FHL1 transcription by NPM1-induced promoter methylation. a BxPC-3 cells were transfected with GFP-tagged IMUP. Exogenous co-immunoprecipitation was performed by GFP beads (left). Endogenous interaction was tested in BxPC-3 cells by anti-NPM1 (right). b WB analysis of proteins extracted from BxPC-3 infected with control shRNAs, IMUP sh1, or sh2. c Immunofluorescence double-staining of BxPC-3 cells showed the location of IMUP and NLM1. Cells were stained with anti-IMUP (green) and anti-NPM1 (red). The nuclei were stained with DAPI (blue). Scale bars: 50 μm. d BxPC-3 cells transfected with IMUP or control siRNAs were treated with 100 μg/mL CHX at the indicated time. Proteins were analyzed with anti-IMUP, anti-NPM1, and anti-tubulin. e The densitometric quantitation of NPM1 protein at the indicated time was normalized by tubulin. Statistical differences were analyzed by two-tailed Student’s t test (*** P < 0.0001). f Structure of the human FHL1 gene. Dotted lines indicate two exon fragments containing CpG islands and corresponding sequences. g The pyrosequencing maps of FHL1 promoter CpG islands. DNA was collected from BxPC-3 cells infected with control shRNAs, IMUP-shRNAs, or co-transfected with IMUP-shRNAs and NPM1 vectors. The methylation rates of FHL1 promoter CpG islands. Fragment 1 (left histogram) and fragment 2 (right histogram). Statistical differences were analyzed using two-way ANOVA test. *** P < 0.0001. h Translational factor SP1-binding motif and putative SP1-binding sequences of FHL1 promoter. i ChIP-qPCR revealed SP1 enrichment on the FHL1 promoter in BxPC-3 cells infected with either control shRNAs, or IMUP-shRNAs or co-transfected with IMUP-shRNAs and NPM1 vectors. The upstream of FHL1 promoter and actin promoter were used as negative controls. Statistical differences were analyzed by two-tailed Student’s t test (** P < 0.001, *** P < 0.0001)

Article Snippet: Antibodies against IMUP (1:100; #ab221063) from Abcam (Cambridge, UK) and FHL1 (1:50; #10,991–1-AP) from Proteintech (Wuhan, China) were used for IHC to verify their expression in PDAC and xenograft tumor tissues.

Techniques: Methylation, Transfection, Immunoprecipitation, Infection, Control, Immunofluorescence, Double Staining, Staining, Quantitation Assay, Two Tailed Test, Binding Assay, ChIP-qPCR

Journal: Cell Biology and Toxicology

Article Title: Immortalization-upregulated protein promotes pancreatic cancer progression by regulating NPM1/FHL1-mediated cell-cycle-checkpoint protein activity

doi: 10.1007/s10565-022-09695-4

Figure Lengend Snippet: FHL1 interacts with CHK1/CDC25A/14–3-3ξ and promotes the phosphorylation of CDC25A via CHK1. a WB analysis of input and anti-Flag IP derived from BxPC-3 cells transfected with Flag-FHL1 vectors. b Endogenous proteins from BxPC-3 were immunoprecipitated using anti-CDC25A, CDC25C, CHK1, 14–3-3ξ antibody, or rabbit IgG as a negative control. c WB analysis of BxPC-3 and SW1990 cells treated with control vectors, Flag-FHL1 vectors, or/and treated with 25 μmol CHK1 inhibitor (GDC-0575 analog). d BxPC-3 cells were treated with control vectors or Flag-FHL1 vectors. WB analysis of IP by anti-CHK1 or IgG

Article Snippet: Antibodies against IMUP (1:100; #ab221063) from Abcam (Cambridge, UK) and FHL1 (1:50; #10,991–1-AP) from Proteintech (Wuhan, China) were used for IHC to verify their expression in PDAC and xenograft tumor tissues.

Techniques: Phospho-proteomics, Derivative Assay, Transfection, Immunoprecipitation, Negative Control, Control

Journal: Cell Biology and Toxicology

Article Title: Immortalization-upregulated protein promotes pancreatic cancer progression by regulating NPM1/FHL1-mediated cell-cycle-checkpoint protein activity

doi: 10.1007/s10565-022-09695-4

Figure Lengend Snippet: FHL1 causes CDC25A to become sequestered in the cytoplasm via binding to 14–3-3ξ. a BxPC-3 and SW1990 cells were treated with control vectors, Flag-FHL1 vectors, or co-transfected with Flag-FHL1 vectors and 14–3-3ξ siRNAs. Nuclear-cytoplasmic isolated proteins were used for WB analysis. C, cytoplasm; N, nucleus. b BxPC-3 and SW1990 cells were treated with control siRNAs, IMUP-siRNAs, or co-transfected with IMUP siRNAs and 14–3-3ξ siRNAs. Nuclear-cytoplasmic isolated proteins were used for WB analysis. Right panels show the densitometric analysis of CDC25A distributed in the cytoplasm or nucleus in three independent experiments. Statistical differences (the ratio of nucleus to cytoplasm) were analyzed by unpaired Student’s t test. *** P < 0.0001. c BxPC-3 cells were transfected with control vectors or Flag-FHL1 vectors. WB analysis of IP by anti-14–3-3ξ or IgG. d Representative immunofluorescence analysis of xenograft mouse tumors infected with control shRNA and IMUP-sh1. Cells were stained with anti-CDC25A (red). Nuclei are stained with DAPI (blue). Scale bars: 100 μm. e Schematic of the mechanism underlying IMUP-regulated S phase progression through NPM1/FHL1-mediated cell-cycle kinase protein activation. IMUP enhances the stability of NPM1 by direct binding. NPM1 indirectly facilitates promoter CpG island methylation of FHL1 and inhibits the transcription of FHL1 . FHL1 promotes the phosphorylation of CDC25A by CHK1 and sequesters CDC25A in the cytoplasm by forming CDC25A/14–3-3ξ complexes

Article Snippet: Antibodies against IMUP (1:100; #ab221063) from Abcam (Cambridge, UK) and FHL1 (1:50; #10,991–1-AP) from Proteintech (Wuhan, China) were used for IHC to verify their expression in PDAC and xenograft tumor tissues.

Techniques: Binding Assay, Control, Transfection, Isolation, Immunofluorescence, Infection, shRNA, Staining, Activation Assay, Methylation, Phospho-proteomics