Journal: Cell Death and Differentiation

Article Title: FHL3 links cell growth and self-renewal by modulating SOX4 in glioma

doi: 10.1038/s41418-018-0152-1

Figure Lengend Snippet: FHL3 regulates the target genes SOX4, CAV1, and DDIT3 in glioma cells. a Glioma cell lines (T98G, U87MG, and U251) were transfected with PLVX empty vector (−) or FHL3 overexpression plasmid (+). Lysates were collected 48 h post-transfection and immunoblotted for the indicated proteins. β-Actin was used as a loading control. The bar graph shows cell viability relative to the control groups 96 h post-transfection. b Schematic illustration of the procedure used to screen and refine the set of FHL3-regulated target genes identified by three independent glioma microarray data replicates. c Twenty-eight indicated genes reported to be involved in glioma were assessed by microarray (gray bars) and real-time PCR (black bars). GAPDH was used as a housekeeping gene. d Heatmaps illustrating the expression profiles of the 11 differentially expressed genes verified by microarray experiments (n = 3 for each biological replicate). e Soluble chromatin was subjected to immunoprecipitation with IgG or anti-FHL3 antibodies in T98G cells. We designed two pairs of primers (P1 and P2) to amplify the predicted binding regions upstream of each gene, as peaks were identified in these regions in the ChIP-on-chip assay. The locations of the peaks identified by the ChIP-on-chip assay are denoted as short red bars. Immunoprecipitated DNA was PCR amplified with primers (locations indicated with short green bars) that annealed to the proximal region of the DDIT3, CAV1, or SOX4 promoters. The lengths of the amplified fragments are 247 bp (DDIT3-P1), 237 bp (DDIT3-P2), 263 bp (CAV1-P1), 219 bp (CAV1-P2), 288 bp (SOX4-P1), and 210 bp (SOX4-P2)

Article Snippet: Cell lines and cell culture The human glioma cell lines T98G, U87MG, and A172 were purchased from ATCC and cultured according to the guidelines recommended by the ATCC.

Techniques: Transfection, Plasmid Preparation, Over Expression, Microarray, Real-time Polymerase Chain Reaction, Expressing, Immunoprecipitation, Binding Assay, Amplification

Journal: Cell Death and Differentiation

Article Title: FHL3 links cell growth and self-renewal by modulating SOX4 in glioma

doi: 10.1038/s41418-018-0152-1

Figure Lengend Snippet: FHL3 inhibits glioma cell proliferation mainly through the downregulation of SOX4 expression. a Representative western blot showing CAV1, DDIT3, SOX4, and FHL3 protein levels in FHL3-overexpressing (+) glioma cell lines. b, c Western blot analysis of T98G, U87MG, and U251 glioma cell lines transfected with pcDNA6.0-Flag-CAV1 (b), pcDNA6.0-Flag-DDIT3 (c) or control vector (−). An anti-Flag antibody was used to detect target gene overexpression. Bar graphs show the results of MTS assays in the same three glioma cell lines 96 h after transfection with plasmids. d, e Western blot analysis of SOX4 knockdown and overexpression in T98G, U87MG, and U251 glioma cell lines following lentiviral infection with shSOX4 (d), LV-3Flag-SOX4 (e), or a control (−). Anti-SOX4 and anti-Flag antibodies were separately used to detect SOX4 knockdown and overexpression, respectively. Bar graphs show the results of MTS assays performed 96 h after lentiviral infection in the same three glioma cell lines. f Western blot showing SOX4 and FHL3 protein levels in T98G and U251 glioma cell lines overexpressing either FHL3 or SOX4 alone or co-overexpressing FHL3 and SOX4. g Growth curves in T98G and U251 glioma cells overexpressing either FHL3 or SOX4 alone or co-overexpressing both FHL3 and SOX4. Data are presented as the mean ± SD of three independent experiments. *P < 0.05

Article Snippet: Cell lines and cell culture The human glioma cell lines T98G, U87MG, and A172 were purchased from ATCC and cultured according to the guidelines recommended by the ATCC.

Techniques: Expressing, Western Blot, Transfection, Plasmid Preparation, Over Expression, Infection

Journal: Cell Death and Differentiation

Article Title: FHL3 links cell growth and self-renewal by modulating SOX4 in glioma

doi: 10.1038/s41418-018-0152-1

Figure Lengend Snippet: FHL3 suppresses SOX4 transcriptional activity and TGF-β-responsive transcription in a TGF-β1-independent manner. a Schematic diagram of the SOX4 promoter reporter construct. b, c T98G cells were co-transfected with an FHL3 overexpression construct (b) or FHL3 siRNAs (c), and either the dual luciferase reporter vector pEZX-PG04 or a SOX4 promoter reporter. Cells were treated with (+) or without (−) TGF-β1 and analyzed for Gaussia Luciferase (GLuc) and Secreted Alkaline Phosphatase (SEAP) activities, using the SEAP signal as an internal control. The normalized signals (ratio of GLuc to SEAP activities) are represented as the mean ± SD of three independent experiments. *P < 0.05 versus empty vector (b) or control siRNA (c) without TGF-β1. #P < 0.05 versus empty vector (b) or control siRNA (c) with TGF-β1. d T98G cells were co-transfected with the TGF-β signaling pathway reporter p3TP-Lux and either an FHL3 overexpression construct or empty vector. Cells were treated with (+) or without (–) TGF-β1 and analyzed for luciferase activity. Values are presented as the mean ± SD of three independent experiments. *P < 0.05 versus empty vector without TGF-β1. #P < 0.05 versus empty vector with TGF-β1. e Microarray results showing changes in the expression of TGF-β-responsive genes upon FHL3 overexpression. Red represents upregulated genes, while blue represents downregulated genes. f The correlation between FHL3 and SOX4 mRNA in TCGA GBM samples was analyzed on the LinkedOmics website using a Spearman correlation test. g Relative SOX4 and FHL3 protein levels in 13 grade II, 13 grade II–III, 7 grade III, 5 grade III–IV, and 16 grade IV glioma tissues compared with 7 normal brain tissue controls. Western blotting results were quantified using ImageJ software and are shown as the relative ratios of SOX4/β-Actin or FHL3/β-Actin protein levels (average values shown above the blots). *P < 0.05. P values were generated using an unpaired t-test

Article Snippet: Cell lines and cell culture The human glioma cell lines T98G, U87MG, and A172 were purchased from ATCC and cultured according to the guidelines recommended by the ATCC.

Techniques: Activity Assay, Construct, Transfection, Over Expression, Luciferase, Plasmid Preparation, Microarray, Expressing, Western Blot, Software, Generated

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Hypoxic exosomes facilitate angiogenesis and metastasis in esophageal squamous cell carcinoma through altering the phenotype and transcriptome of endothelial cells

doi: 10.1186/s13046-019-1384-8

Figure Lengend Snippet: Uptake of exosomes derived from ECA109 and KYSE410 by HUVECs at 15 min, 60 min, 2 h and 4 h. HUVECs were cultured with exosomes (25 μg /mL) from ECA109, or exosomes (25 μg /mL) from KYSE410, or in the absence of exosomes (Exosome (−)). Fluorescence microscopy images showing the internalization of exosomes by HUVECs. Blue: Nucleus stained with DAPI. Red: PKH26-labeled exosomes. Green: Phalloidin-iFluor 488 Reagent. Scale bar, 50 μm

Article Snippet: Primary human umbilical vein endothelial cells (HUVECs) were also purchased from American Type Culture Collection and maintained in endothelial cell medium (ECM) (Science cell, USA).

Techniques: Derivative Assay, Cell Culture, Fluorescence, Microscopy, Staining, Labeling

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Hypoxic exosomes facilitate angiogenesis and metastasis in esophageal squamous cell carcinoma through altering the phenotype and transcriptome of endothelial cells

doi: 10.1186/s13046-019-1384-8

Figure Lengend Snippet: The regulatory role of normoxic and hypoxic exosomes in the proliferation, cell cycle distribution, migration and invasion of HUVECs. HUVECs were cultured with exosomes (25 μg /mL) from ECA109 that cultured in normoxic environment (norm-Exo (ECA109)) or hypoxic environment (hypo-Exo (ECA109)), or exosomes (25 μg /mL) from KYSE410 that cultured in normoxic environment (norm-Exo (KYSE410)) or hypoxic environment (hypo-Exo (KYSE410)), or in the absence of exosomes (Exosome (−)). The proliferation of HUVECs was detected by colony formation assay ( a ). The graph summarizes the results of three independent experiments ( b ). The cell cycle of HUVECs were analyzed by flow cytometry. Representative pictures of the cell cycle distributions in HUVECs ( c ). The graph summarizes the results of three independent experiments ( d ). Transwell assays were used to investigate the migratory ( e ) and invasive ( g ) abilities of HUVECs. The graph summarizes the results of three independent experiments of migration ( f ) and invasion assay ( h ). Data was presented as mean ± standard deviation (SD).* P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: Primary human umbilical vein endothelial cells (HUVECs) were also purchased from American Type Culture Collection and maintained in endothelial cell medium (ECM) (Science cell, USA).

Techniques: Migration, Cell Culture, Colony Assay, Flow Cytometry, Invasion Assay, Standard Deviation

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Hypoxic exosomes facilitate angiogenesis and metastasis in esophageal squamous cell carcinoma through altering the phenotype and transcriptome of endothelial cells

doi: 10.1186/s13046-019-1384-8

Figure Lengend Snippet: Hypoxic exosomes promoted angiogenesis in vitro and increased the vessel density in vivo. HUVECs were plated with matrigel and cultured with exosomes (25 μg /mL) or not. Representative pictures of tube formation were taken after stained with Calcein-AM ( a ). The tube formation ability was quantified by measuring the total branching length ( b ). Matrigel containing exosomes, or not, were injected subcutaneously into the nude mice. Representative images of the general observation of matrigel plugs ( c ). In vivo neovascularization induced by exosomes was measured by H&E staining. Representative pictures of neovascularization were shown in ( d ) and quantified for blood vessel density ( e ). Data was presented as mean ± standard deviation (SD). * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: Primary human umbilical vein endothelial cells (HUVECs) were also purchased from American Type Culture Collection and maintained in endothelial cell medium (ECM) (Science cell, USA).

Techniques: In Vitro, In Vivo, Cell Culture, Staining, Injection, Standard Deviation

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Hypoxic exosomes facilitate angiogenesis and metastasis in esophageal squamous cell carcinoma through altering the phenotype and transcriptome of endothelial cells

doi: 10.1186/s13046-019-1384-8

Figure Lengend Snippet: Microarray analysis revealed differentially expressed RNAs between different groups. a Scatter-Plot of differentially expressed RNAs variations between HUVECs in the control group and norm-Exo group. Dots above the top line (red) and below the bottom line (green) indicated the fold change of the RNAs is more than 1.5 between the two groups. Heat map of the dysregulated mRNA, lncRNA and circular RNA expression in control group and norm-Exo group. b Scatter-Plot of differentially expressed RNAs variations between HUVECs in the control group and hypo-Exo group. Heat map of the dysregulated mRNA, lncRNA and circular RNA expression in control group and hypo-Exo group. Eight hundred and thirty nine down-regulated mRNAs ( c ), 113 up-regulated mRNAs ( d ), 232 down-regulated lncRNAs ( e ), 99 up-regulated lncRNAs ( f ), 692 down-regulated circular RNAs ( g ) and 86 up-regulated circular RNAs ( h ) were identified according to the intersection of transcriptome between norm-Exo group and hypo-Exo group

Article Snippet: Primary human umbilical vein endothelial cells (HUVECs) were also purchased from American Type Culture Collection and maintained in endothelial cell medium (ECM) (Science cell, USA).

Techniques: Microarray, Control, RNA Expression

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Mediator complex subunit 1 promotes oral squamous cell carcinoma progression by activating MMP9 transcription and suppressing CD8 + T cell antitumor immunity

doi: 10.1186/s13046-024-03191-9

Figure Lengend Snippet: MED1 is upregulated in OSCC patients and associated with poor prognosis. A Analysis of the mRNA levels of MED1 (cancer vs. normal) in multiple solid cancers from Oncomine Database. B Analysis of the MED1 expression level (tumor vs. normal) in multiple solid cancers from TIMER Database. C mRNA expression of MED1 in 3 samples (GSE30784, GSE25099, and GSE10121) from the GEO database. D Immunohistochemical staining of MED1 in OSCC tissue microarray (TMA). E Quantification of immunostaining results. F Kaplan–Meier analysis of overall survival of OSCC patients from GEPIA database stratified by MED1 levels. G Kaplan–Meier analysis of overall survival of OSCC patients from UALCAN database stratified by MED1 levels. H Kaplan–Meier analysis of overall survival of OSCC patients from TCGA database stratified by MED1 levels. Scale bar = 200 µm (10 ×), and 50 µm (40 ×), Bars = means ± SD. ** P < 0.01, *** P < 0.001, **** P < 0.0001. OSCC, oral squamous cell carcinoma

Article Snippet: Human OSCC cell lines Cal-27, UPCI-SCC-090, SCC-9, UPCI-SCC-154 and mouse OSCC cell line SCC-7 were purchased from the American Type Culture Collection (ATCC, USA).

Techniques: Expressing, Immunohistochemical staining, Staining, Microarray, Immunostaining

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Mediator complex subunit 1 promotes oral squamous cell carcinoma progression by activating MMP9 transcription and suppressing CD8 + T cell antitumor immunity

doi: 10.1186/s13046-024-03191-9

Figure Lengend Snippet: MED1 is highly expressed in human OSCC cell line and has no significant effect on metastatic OSCC cells proliferation. A Cell morphology of human oral keratinocytes (HOK), nonmetastatic oral squamous cell carcinoma UPCI-SCC-090, and metastatic oral squamous cell carcinoma UPCI-SCC-154. B mRNA levels of MED1 in HOK and four OSCC lines tested by qRT-PCR. n = 3 independent experiments. C Protein levels of MED1 in HOK and four OSCC lines tested by WB. D CCK8 to detect SCC-9 cell proliferation change after MED1 knockdown. n = 3 independent experiments. E CCK8 to detect UPCI-SCC-154 cell proliferation change after MED1 knockdown. n = 3 independent experiments. F Plate colony formation assay to detection of clonality in SCC-9 cells following MED1 knockdown. n = 3 independent experiments. G Plate colony formation assay to detection of clonality in UPCI-SCC-154 cells following MED1 knockdown. n = 3 independent experiments. H Immunofluorescence staining and quantitative analysis of proliferation marker Ki67 after MED1 knockdown in SCC-9 cells. n = 3 independent experiments. I Immunofluorescence staining and quantitative analysis of proliferation marker Ki67 after MED1 knockdown in UPCI-SCC-154 cells. n = 3 independent experiments. Scale bar = 100 µm (10 ×), and 50 µm (40 ×). Bars = means ± SD. ns means nonsignificant, *** P < 0.001, **** P < 0.0001

Article Snippet: Human OSCC cell lines Cal-27, UPCI-SCC-090, SCC-9, UPCI-SCC-154 and mouse OSCC cell line SCC-7 were purchased from the American Type Culture Collection (ATCC, USA).

Techniques: Quantitative RT-PCR, Knockdown, Colony Assay, Immunofluorescence, Staining, Marker

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Mediator complex subunit 1 promotes oral squamous cell carcinoma progression by activating MMP9 transcription and suppressing CD8 + T cell antitumor immunity

doi: 10.1186/s13046-024-03191-9

Figure Lengend Snippet: MED1 knockdown inhibits metastatic OSCC cells migration and invasion in vitro. A Cell scratch assay to examine SCC-9 cells migration ability after MED1 knockdown. B Quantitative analysis of the cell migration ratio in ( A ). n = 3 independent experiments. C Cell scratch assay to examine UPCI-SCC-154 cells migration ability after MED1 knockdown. D Quantitative analysis of the cell migration ratio in ( C ). n = 3 independent experiments. E Transwell invasion assay to examine SCC-9 cells invasion ability after MED1 knockdown. F Quantitative analysis of the number of invasive cells in (E). n = 3 independent experiments. G Transwell invasion assay to examine UPCI-SCC-154 cells invasion ability after MED1 knockdown. H Quantitative analysis of the number of invasive cells in ( G ). n = 3 independent experiments. I MMP2 and MMP9 gene expression assessed by qRT-PCR in SCC-9 cells after MED1 knockdown. n = 3 independent experiments. J MMP2 and MMP9 gene expression assessed by qRT-PCR in UPCI-SCC-154 cells after MED1 knockdown. n = 3 independent experiments.Scale bar = 200 µm (10 ×). Bars = means ± SD.* P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001

Article Snippet: Human OSCC cell lines Cal-27, UPCI-SCC-090, SCC-9, UPCI-SCC-154 and mouse OSCC cell line SCC-7 were purchased from the American Type Culture Collection (ATCC, USA).

Techniques: Knockdown, Migration, In Vitro, Wound Healing Assay, Transwell Invasion Assay, Gene Expression, Quantitative RT-PCR

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Mediator complex subunit 1 promotes oral squamous cell carcinoma progression by activating MMP9 transcription and suppressing CD8 + T cell antitumor immunity

doi: 10.1186/s13046-024-03191-9

Figure Lengend Snippet: MED1 regulates metastatic OSCC cells migration and invasion through the modulation of MMP9. A MMP9 gene expression assessed by qRT-PCR. n = 3 independent experiments. B MMP9 protein expression assessed by WB. n = 3 independent experiments. C The luciferase activity of MMP9 promoter detected by luciferase reporter assay. n = 3 independent experiments. D The recruitments of cofactors AP-1(c-Jun/c-Fos) on MMP9 promoter in SCC-9 cells and UPCI-SCC-154 cells by CHIP assay and qRT-PCR. n = 3 independent experiments. E Cell scratch assay to determine the changes in migration abilities of SCC-9 cells after adding exogenous MMP9 and quantitative analysis. n = 3 independent experiments. F Cell scratch assay to determine the changes in migration abilities of UPCI-SCC-154 cells after adding exogenous MMP9 and quantitative analysis. n = 3 independent experiments. G Transwell assay to determine the changes in invasion abilities of SCC-9 cells after adding exogenous MMP9 and quantitative analysis ( H ). n = 3 independent experiments. I Transwell assay to determine the changes in invasion abilities of UPCI-SCC-154 cells after adding exogenous MMP9 and quantitative analysis ( J ). n = 3 independent experiments. Scale bar = 200 µm (10 ×). Bars = means ± SD.* P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001

Article Snippet: Human OSCC cell lines Cal-27, UPCI-SCC-090, SCC-9, UPCI-SCC-154 and mouse OSCC cell line SCC-7 were purchased from the American Type Culture Collection (ATCC, USA).

Techniques: Migration, Gene Expression, Quantitative RT-PCR, Expressing, Luciferase, Activity Assay, Reporter Assay, Wound Healing Assay, Transwell Assay

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Mediator complex subunit 1 promotes oral squamous cell carcinoma progression by activating MMP9 transcription and suppressing CD8 + T cell antitumor immunity

doi: 10.1186/s13046-024-03191-9

Figure Lengend Snippet: Graphic abstract of molecular mechanisms of MED1 promoting tumor progression in OSCC. A For untreated metastatic OSCC cells, MED1 facilitates MMP9 expression by promoting the transcription of MMP9, resulting in heightened migration and invasion of metastatic OSCC cells, just like strengthen the destructive force of "bandits". Additionally, MED1 upregulates PD-L1 expression via activation of the Notch signaling pathway, resulting in diminished cytotoxicity of CD8 + T cells within the tumor microenvironment and consequent attenuation of anti-tumor immunity responses. This behavior is similar to weaken the fighting capacity of "police". B For MED1 knockdown metastatic OSCC cells, the transcription of MMP9 is inhibited, leading to decreased migration and invasion of metastatic OSCC cells, just like weaken the destructive force of "bandits". This inhibition also leads to a decrease in PD-L1 expression through the suppression of Notch signaling pathway, indirectly enhancing the cytotoxic activity of CD8 + T cells in the TME and strengthening antitumor immunity responses. This behavior is similar to strengthen the fighting capacity of "police". MMP9: matrix metallopeptidase 9, POL II: RNA polymerase II, TATA: TATA box, PD-L1: programmed cell death ligand 1, NICD: Notch intracellular domain, PD-1: programmed cell death 1, IFN-γ: interferon-γ, IL-2: Interleukin-2

Article Snippet: Human OSCC cell lines Cal-27, UPCI-SCC-090, SCC-9, UPCI-SCC-154 and mouse OSCC cell line SCC-7 were purchased from the American Type Culture Collection (ATCC, USA).

Techniques: Expressing, Migration, Activation Assay, Knockdown, Inhibition, Activity Assay

Journal: bioRxiv

Article Title: RNA binding protein RBM3 augments kissing loop formation with lncRNAs to enhance translational control

doi: 10.1101/2021.12.14.472669

Figure Lengend Snippet: (A) The Cancer Genome Atlas (TCGA) database analyses demonstrate RBM3 mRNA levels are significantly higher in colon cancer (T) (n=286) than in normal colon (N) (n=41) (p<0.001). (B) RBM3 mRNA expression from a cDNA array of colon tumor (T) samples (n=24) and matched adjacent normal colon tissue (N) (n=24) normalized to β-actin, shows a significant increase of RBM3 expression in tumor tissue (p=0.023). (C) Immunohistochemistry (IHC) of a colon cancer tumor microarray shows that RBM3 is upregulated in colon adenocarcinoma along with lymph node metastasis and liver metastasis as compared to the normal colon, lymph node, and liver. (D) Composite score of colon cancer TMA shows significantly higher expression of RBM3 in tumor (n=28) (p=0.002) and metastasis (n=30) (p=0.036) as compared to normal tissue. RBM3 expression is also increased in the different stages of colon cancer (Stage I (n = 3), Stage II (n = 12) (p=0.015), Stage III (n= 11) (p=0.07), Stage IV (n = 2) and metastasis (n=30) (p=0.036)) as compared to normal colon, liver and lymph node (n=3 each). (E) Western blot analysis of RBM3 protein expression in established colon cancer cell lines HCT116, DLD1, SW480, SW620, HT29, RKO and LST17T as compared to normal colon epithelial cells (FHC cell line). Data in are represented as ± SEM. Also, see Supplementary figure 1

Article Snippet: Human colon cancer cells HCT116, DLD1, RKO and endothelial cell line HUVEC (all cell lines obtained from American Type Culture Collection, at passage 4).

Techniques: Expressing, Immunohistochemistry, Microarray, Western Blot

Journal: bioRxiv

Article Title: RNA binding protein RBM3 augments kissing loop formation with lncRNAs to enhance translational control

doi: 10.1101/2021.12.14.472669

Figure Lengend Snippet: (A) Volcano plots for RNA-sequencing (RNA-seq) showing differentially expressed lncRNA in both HCT116 and DLD1 RBM3 overexpressing (RBM3 O/E) cell lines compared to empty vector (EV) cell lines. (B) Venn diagram depicting the number of co-expressed and uniquely expressed lncRNA found in RNA-seq. (C) The volcano plots for RNA-immunoprecipitation coupled sequencing (RNA-IP seq) showing differentially expressed lncRNA in both HCT116 and DLD1, RBM3 overexpressing cell lines compared to empty vector cell lines. (D) Venn diagram depicting the number of co-expressed and uniquely expressed lncRNA found in RNA-IP seq. (E-F) Plot from REVIGO software showing the Gene Ontology (GO) terms enriched in lncRNA (RNA-seq) for HCT116 RBM3 (E). The graph has been modified to highlight the GO terms and color key. The box plot drawn highlighting enriched GO terms(F). (G) RT-PCR validation of lncRNA identified through RNA-seq in colon cancer cell lines compared to normal FHC cells. (H) RT-PCR validation of lncRNA identified through RNA-seq, in the RBM3 overexpressing (RKO, HCT116, and DLD1) cells, show increased expression of lnc-HOTAIR, lnc-TUG1, lnc-Flii- 1, lnc-LSAMP-3. (I) Western blot analysis for the expression demonstrating levels of RBM3 in the distal colon in representative wild type littermates and RBM3 transgenic mice (RBM3 O/E). (J) RT-PCR analysis demonstrating increased expression of lncRNA lnc-Flii-1 and lnc-LSAMP- 3 compared to GAPDH in representative wild type (WT) littermates and RBM3 overexpressing transgenic mice (RBM3 O/E). Data in are represented as ± SEM. Also, see Supplementary figure 2.

Article Snippet: Human colon cancer cells HCT116, DLD1, RKO and endothelial cell line HUVEC (all cell lines obtained from American Type Culture Collection, at passage 4).

Techniques: RNA Sequencing, Plasmid Preparation, RNA Immunoprecipitation, Sequencing, Software, Modification, Reverse Transcription Polymerase Chain Reaction, Biomarker Discovery, Expressing, Western Blot, Transgenic Assay

Journal: bioRxiv

Article Title: RNA binding protein RBM3 augments kissing loop formation with lncRNAs to enhance translational control

doi: 10.1101/2021.12.14.472669

Figure Lengend Snippet: (A-D) Quantitative PCR shows increased mRNA levels of VEGFA, ZEB1, TWIST, and SNAI2 in RBM3 overexpressing cells compared to empty vector cell lines. (E) Plot for tube length of the endothelial tubular network formed by HUVEC cell in (G). Increased tube formation in HUVEC cells treated by condition media from HCT116 (p=0.038) and DLD1 (p=0.004) RBM3 overexpressing compared to empty vector cells (F) Plot showing increase in migration of HCT116 (p=0.001), DLD1 (p=0.02) and RKO (p=0.04) RBM3 overexpressing cells compared to empty vector performed by wound healing assay. (G) Plot showing increase in migration of HCT116 (p=0.006), DLD1 (p=0.002) and RKO (p=0.006) RBM3 overexpressing cells compared to empty vector performed by transwell migration assay. (H) Plot showing increase in invasion of HCT116 (p<0.001), DLD1 (p<0.01) and RKO (p<0.01) RBM3 overexpressing cells compared to empty vector performed by transwell invasion assay. Data in are represented as ± SEM. Also, see Supplementary figure 3.

Article Snippet: Human colon cancer cells HCT116, DLD1, RKO and endothelial cell line HUVEC (all cell lines obtained from American Type Culture Collection, at passage 4).

Techniques: Real-time Polymerase Chain Reaction, Plasmid Preparation, Migration, Wound Healing Assay, Transwell Migration Assay, Transwell Invasion Assay

Journal: bioRxiv

Article Title: RNA binding protein RBM3 augments kissing loop formation with lncRNAs to enhance translational control

doi: 10.1101/2021.12.14.472669

Figure Lengend Snippet: (A-D) The plot of tumor weights (A, C) and tumor volumes (B, D) from HCT116 and DLD1 empty vector and RBM3 overexpressing xenografts. The tumor volume and weight were significantly increased in RBM3 overexpression compared to control (p<0.05) (E) RT-PCR validation of lncRNA in the tumor xenograft tissues, in both HCT116 and DLD1 xenografts. RBM3 overexpressing xenografts show an increase in lnc-Flii-1 and lnc-LSAMP-3 expression compared to empty vector. (F) RT-PCR validation for increased expression of TWIST1, SNA2 and VEGFA in HCT116 and DLD1 empty vector and RBM3 overexpressing xenograft tumors tissues. (G) Western blot analysis of tumors from HCT116 and DLD1 xenograft tissues for VEGF expression. (H) Increassed RBM3 and PCNA levels in HCT116 and DLD1 RBM3 overexpressing xenograft tissues as compared to empty vector as seen by immunohistochemistry analysis. (I) Increase in percentage of PCNA positive nuclei in RBM3 overexpressing HCT116 (p=0.022) and DLD1 (p=0.025) xenograft tissues immunohistochemistry. The data for are presented as the means ± SEM, (n=10). Also, see Supplementary figure S5.

Article Snippet: Human colon cancer cells HCT116, DLD1, RKO and endothelial cell line HUVEC (all cell lines obtained from American Type Culture Collection, at passage 4).

Techniques: Plasmid Preparation, Over Expression, Control, Reverse Transcription Polymerase Chain Reaction, Biomarker Discovery, Expressing, Western Blot, Immunohistochemistry

Journal: bioRxiv

Article Title: RNA binding protein RBM3 augments kissing loop formation with lncRNAs to enhance translational control

doi: 10.1101/2021.12.14.472669

Figure Lengend Snippet: (A) Protein expression of RBM3 decreases in shRNA knockdown clones (2,3,5) compared to scramble (scr1,scr2) in the HCT116 cell line by Western blot analysis. (B) RT-PCR validation for the decreased expression of lnc-LSAMP-3 and lnc-Flii-1 in HCT116 RBM3 shRNA knocked down clone compared to scramble. (C) Plot for decrease in percentage proliferation of the RBM3 shRNA knockdown clones over empty vector for sh2 (proliferation= 55.06%, p=0.04), sh3 (proliferation= 53.8%, p=0.0023), sh5 (proliferation= 47.45%, p=0.0001). (D) The plot showing decrease in percent migration in HCT116 shRNA knocked down clones (sh2 p=0.044 and sh5 p=0.048) compared to scramble performed by Transwell migration. (E) The plot showing decrease in percent invasion in HCT116 shRNA knocked down clones (sh2 p=<0.001 and sh5 p=0.001) compared to scramble performed by Transwell invasion assay. (F and G) The plot of tumor weights (F) and tumor volumes (G) from HCT116 scramble (scr2) and RBM3 shRNA knockdown (shRNA-2) xenografts. Both tumor volume and weight were significantly reduced in RBM3 knockdown compared to scramble control (p<0.05). (H) Kaplan-Meier analysis showing increased percent survival after the AOM/DSS induced carcinogenesis in cre inducible RBM3 knockout (KO) compared to wild type (WT) mice. (I) Plot showing decreased intestinal permeability in RBM3 knockout mice as compared to the wild type littermates (p=0.04). (J) The plot showing decreased number of tumors in the distal colon observed after the AOM/DSS induced carcinogenesis in cre inducible RBM3 knockout compared to wild type mice (p=0.007). (K) Representative image of H and E staining of the distal colon with tumors after the AOM/DSS induced carcinogenesis in RBM3 knockout and wild type mice. (L) Decrease in expression of lnc-LSAMP-3 and lnc-Flii-1 seen by PCR analysis in RBM3 knockout mice as compared to the wild type mice. The data for are presented as the means ± SEM, (mice n=10). Also, see Supplementary figure 6.

Article Snippet: Human colon cancer cells HCT116, DLD1, RKO and endothelial cell line HUVEC (all cell lines obtained from American Type Culture Collection, at passage 4).

Techniques: Expressing, shRNA, Knockdown, Clone Assay, Western Blot, Reverse Transcription Polymerase Chain Reaction, Biomarker Discovery, Plasmid Preparation, Migration, Transwell Invasion Assay, Control, Knock-Out, Permeability, Staining

Journal: bioRxiv

Article Title: RNA binding protein RBM3 augments kissing loop formation with lncRNAs to enhance translational control

doi: 10.1101/2021.12.14.472669

Figure Lengend Snippet: (A and B) RT-PCR validation of lnc-LSAMP-3 and lnc-Flii-1 knocked down in HCT116 (A) and DLD1 (B) RBM3 O/E and empty vector cells. (C and D) RT-PCR validation of VEGFA, ZEB1 and TWIST in HCT116 (C) and DLD1 (D) RBM3 O/E and empty vector cells, after lncRNA (lnc-Flii-1 and lnc-LSAMP-3) knockdown. (E) Plot showing decreased percent migration of HCT116 empty vector and RBM3 overexpressing cells having knockdown of lncRNA lnc-LSAMP-3 (Empty vector p=0.017, RBM3 O/E p=0.031) and lnc-Flii-1(Empty vector p=ns, RBM3 O/E p=0.011) performed by scratch plate assay. (F) Plot showing decreased tube length of the endothelial tubular network formed by HUVEC cell treated by condition media from knockdown of lncRNA lnc-LSAMP-3 and lnc-Flii-1(Empty vector p=0.012, RBM3 O/E p=0.022) in HCT116 cells. (G-J)The plot showing decrease in tumor weight (G, I) and tumor volumes (H, J) from HCT116 and DLD1 xenografts treated with intratumoral injection of si+ LNA gapmer for lnc-Flii-1 knockdown and lnc-LSAMP-3 knockdown compared to control (p< 0.05). (K and L) RT-PCR validation for the decreased expression of lncRNA (lnc-LSAMP-3 and lnc- Flii-1) in the tumor xenograft of HCT116 (K) and DLD1(L) treated with intratumoral injection of si+ LNA gapmer for lnc-Flii-1 knockdown and lnc-LSAMP-3 knockdown compared to control. (M and N) Western blot showing decreased protein expression of SNAI2 and VEGF in HCT116 (M) and DLD1 (N) tumor xenograft treated with intratumoral injection of si+ LNA gapmer for lnc- Flii-1 knockdown and lnc-LSAMP-3 knockdown compared to control. The data for are presented as the means ± SEM. Also, see figure Supplementary figure

Article Snippet: Human colon cancer cells HCT116, DLD1, RKO and endothelial cell line HUVEC (all cell lines obtained from American Type Culture Collection, at passage 4).

Techniques: Reverse Transcription Polymerase Chain Reaction, Biomarker Discovery, Plasmid Preparation, Knockdown, Migration, Injection, Control, Expressing, Western Blot

Journal: Frontiers in Cell and Developmental Biology

Article Title: Sulfatase 2-Induced Cancer-Associated Fibroblasts Promote Hepatocellular Carcinoma Progression via Inhibition of Apoptosis and Induction of Epithelial-to-Mesenchymal Transition

doi: 10.3389/fcell.2021.631931

Figure Lengend Snippet: SULF2 secreted by HCC cells induced the transformation from HSCs to CAFs. (A) Both RT-PCR and western immunoblotting assays showed that there was more expression of α-SMA, FAP, and POSTN in LX2 cells co-cultured with Hep3B SULF2 cells (LX2 SULF2 cells) than those co-cultured with Hep3B Vector cells (the protein was extracted from total cells). (B) LX2 cells co-cultured with Huh7 Scr shRNA cells expressed more α-SMA, FAP, and POSTN than those with Huh7 SULF2 shRNA cells. Cell fractions: LX2 cells (the protein was extracted from total cells). SULF2, sulfatase 2; HCC, hepatocellular carcinoma; CAF, carcinoma-associated fibroblast.

Article Snippet: The human HCC cell lines (Hep3B, PLC/PRF/5, and SNU398), non-HCC cell line (LO2), and the HSC cell line (LX2 cells) were purchased from American Type Culture Collection (Manassas, VA, United States).

Techniques: Transformation Assay, Reverse Transcription Polymerase Chain Reaction, Western Blot, Expressing, Cell Culture, Plasmid Preparation, shRNA

Journal: Frontiers in Cell and Developmental Biology

Article Title: Sulfatase 2-Induced Cancer-Associated Fibroblasts Promote Hepatocellular Carcinoma Progression via Inhibition of Apoptosis and Induction of Epithelial-to-Mesenchymal Transition

doi: 10.3389/fcell.2021.631931

Figure Lengend Snippet: SULF2 activated HSCs to CAFs via mediating TGFβ1/SMAD3 signaling. (A) LX2 cells co-cultured with Hep3B SULF2 cells (LX2 SULF2 cells) were found to express more expression of both TGFβ1 and p-SMAD3 than those with Hep3B Vector cells (LX2 Vector cells) by western immunoblotting and RT-PCR. Cell fractions: LX2 cells (the protein was extracted from total cells). (B) Microarray profiling of mRNA expression showed that there was a significantly higher mRNA expression of TGFβ1 signaling downstream genes including BMP7, MAPK9, JUN, PMEPA1, MAPK12, SERPINE1, MAPK13, RUNX2, and RUNX3 in LX2 SULF2 cells than LX2 Vector cells. (C) Immunofluorescence staining displayed that more TGFβ1 protein was bound in the cell membrane of LX2 SULF2 cells than LX2 Vector cells. (D) Both luciferase reporter and ChIP assays confirmed that p-SMAD3 was bound with the α-SMA promoter directly in LX2 cells. (E) ChIP assay showed that p-SMAD3 protein was able to bind with the promoter of POSTN in LX2 cells treated with TGFβ1. SULF2, sulfatase 2; HCC, hepatocellular carcinoma; CAF, carcinoma-associated fibroblast; ChIP, chromatin immunoprecipitation.

Article Snippet: The human HCC cell lines (Hep3B, PLC/PRF/5, and SNU398), non-HCC cell line (LO2), and the HSC cell line (LX2 cells) were purchased from American Type Culture Collection (Manassas, VA, United States).

Techniques: Cell Culture, Expressing, Plasmid Preparation, Western Blot, Reverse Transcription Polymerase Chain Reaction, Microarray, Immunofluorescence, Staining, Membrane, Luciferase, Chromatin Immunoprecipitation

Journal: Frontiers in Cell and Developmental Biology

Article Title: Sulfatase 2-Induced Cancer-Associated Fibroblasts Promote Hepatocellular Carcinoma Progression via Inhibition of Apoptosis and Induction of Epithelial-to-Mesenchymal Transition

doi: 10.3389/fcell.2021.631931

Figure Lengend Snippet: CAFs activated SDF-1/CXCR4/PI3K/AKT pathway and consequently inhibited HCC cell apoptosis. (A) Both RT-PCR and western immunoblotting assays showed that LX2 SULF2 cells expressed significantly more SDF-1 than LX2 Vector cells. Cell fractions: LX2 cells. (B) It was found by ELISA assay that there was more SDF-1 protein in conditioned medium from LX2 SULF2 cells than that from LX2 Vector cells (the protein was extracted from total cells). (C) Microarray profiling of mRNA expression showed that most downstream genes of SDF-1/CXCR4 signaling were remarkably upregulated in Hep3B CAFs cells in contrast to Hep3B Vector cells. (D) Co-culture with CAFs was found by western immunoblotting to increase expression of SDF-1, CXCR4, p-PI3K, and p-AKT in Hep3B cells, while treatment of CXCR4 inhibitor (AMD3100) did not alter the expression of phosphorylation of PI3K, AKT, BAD, caspase 9, and FKHRL 1 in Hep3B cells. Cell fractions: Hep 3B cells (the protein was extracted from total cells). (E) By both DAPI staining and caspase 3/7 activity assay, it was found that co-culture with CAFs suppressed cell apoptosis, whereas AMD3100 treatment did not influence Hep3B cell apoptosis apparently. CAF, carcinoma-associated fibroblast; HCC, hepatocellular carcinoma; SULF2, sulfatase 2.

Article Snippet: The human HCC cell lines (Hep3B, PLC/PRF/5, and SNU398), non-HCC cell line (LO2), and the HSC cell line (LX2 cells) were purchased from American Type Culture Collection (Manassas, VA, United States).

Techniques: Reverse Transcription Polymerase Chain Reaction, Western Blot, Plasmid Preparation, Enzyme-linked Immunosorbent Assay, Microarray, Expressing, Co-Culture Assay, Phospho-proteomics, Staining, Activity Assay

Journal: Frontiers in Cell and Developmental Biology

Article Title: Sulfatase 2-Induced Cancer-Associated Fibroblasts Promote Hepatocellular Carcinoma Progression via Inhibition of Apoptosis and Induction of Epithelial-to-Mesenchymal Transition

doi: 10.3389/fcell.2021.631931

Figure Lengend Snippet: CAFs driven by SULF2 induced HCC EMT phenotype via upregulating SNAI1 by activating SDF-1/CXCR4 signaling. (A) By western immunoblotting assay, it was found that Hep3B LX2 SULF2 cells had significantly more expression of SNAI1, N-cadherin, and Vimentin and less E-cadherin expression than Hep3B LX2 Vector cells, while co-culture with LX2 SULF2 cells did not alter the expression of E-cadherin, SNAI1, N-cadherin, and Vimentin after treatment of AMD3100. Cell fractions: Hep 3B cells (the protein was extracted from total cells). (B) As assessed by wound healing assay, the migration ability of Hep3B cells was found to be accelerated by co-culture with LX2 SULF2 cells, which was weakened by treatment of AMD3100. (C) Transwell invasion assay showed that co-culture with LX2 SULF2 cells consolidated the invasion capacity of Hep3B cells and AMD3100 treatment attenuated the regulatory effect of co-culture with LX2 SULF2 cells on the Hep3B cell invasion ability. CAF, carcinoma-associated fibroblast; SULF2, sulfatase 2; HCC, hepatocellular carcinoma; EMT, epithelial-to-mesenchymal transition.

Article Snippet: The human HCC cell lines (Hep3B, PLC/PRF/5, and SNU398), non-HCC cell line (LO2), and the HSC cell line (LX2 cells) were purchased from American Type Culture Collection (Manassas, VA, United States).

Techniques: Western Blot, Expressing, Plasmid Preparation, Co-Culture Assay, Wound Healing Assay, Migration, Transwell Invasion Assay

Journal: Frontiers in Cell and Developmental Biology

Article Title: Sulfatase 2-Induced Cancer-Associated Fibroblasts Promote Hepatocellular Carcinoma Progression via Inhibition of Apoptosis and Induction of Epithelial-to-Mesenchymal Transition

doi: 10.3389/fcell.2021.631931

Figure Lengend Snippet: SDF-1/CXCR4 axis inhibited miR-153-3p expression and then induced EMT of HCC cells via upregulating SNAI1. (A) The data from TargetScan 7.2 database showed the positions 440–447 of SNAI1 3′-UTR had the complementary sequence of miR-153-3p. (B) As assessed by qRT-PCR, it was found that Hep3B LX2 SULF2 cells had significantly less miR-153-3p in contrast to Hep3B LX2 Vector cells. (C) Western immunoblotting showed that enhanced expression of miR-153-3p in SNU398 cells decreased expression of SNAI1, N-cadherin, and Vimentin and upregulated E-cadherin magnificently. Cell fractions: SNU398 cells (the protein was extracted from total cells). (D) Knockdown of miR-153-3p with miRNA inhibitors in Hep3B cells resulted in upregulation of SNAI1, N-cadherin, and Vimentin, while it led to loss of E-cadherin. Cell fractions: Hep 3B cells (the protein was extracted from total cells). (E) Luciferase reporter assay demonstrated that miR-153-3p overexpression by mimics treatment markedly decreased the luciferase activity of plasmid containing wt 3′-UTR of SNAI1 rather than mt 3′-UTR of SNAI1 in SNU398 cells. EMT, epithelial-to-mesenchymal transition; HCC, hepatocellular carcinoma; SULF2, sulfatase 2.

Article Snippet: The human HCC cell lines (Hep3B, PLC/PRF/5, and SNU398), non-HCC cell line (LO2), and the HSC cell line (LX2 cells) were purchased from American Type Culture Collection (Manassas, VA, United States).

Techniques: Expressing, Sequencing, Quantitative RT-PCR, Plasmid Preparation, Western Blot, Knockdown, Luciferase, Reporter Assay, Over Expression, Activity Assay

Journal: Frontiers in Cell and Developmental Biology

Article Title: Sulfatase 2-Induced Cancer-Associated Fibroblasts Promote Hepatocellular Carcinoma Progression via Inhibition of Apoptosis and Induction of Epithelial-to-Mesenchymal Transition

doi: 10.3389/fcell.2021.631931

Figure Lengend Snippet: OIP5-AS1 accelerated the growth of HCC in vivo and induced EMT phenotype of HCC cells via upregulating SNAI1 expression. (A) By measuring tumor growth curves and tumor weight, it was found that knockdown of OIP5-AS1 attenuated the growth of HCC xenografts. (B) qRT-PCR analysis showed that SNU398 OIP5-AS1 cells had significantly less OIP5-AS1 expression than SNU398 Scr cells. (C) SNU398 OIP5-AS1 cells were found by qRT-PCR assay to express more miR-153-3p expression than SNU398 Scr cells. (D) IHC staining was carried out in HCC xenograft tissues, and it was found that there was magnificently lower level of both SNAI1 and Vimentin, and more E-cadherin is detected in SNU398 OIP5-AS1 cells than SNU398 Scr cells. HCC, hepatocellular carcinoma; EMT, epithelial-to-mesenchymal transition; IHC, immunohistochemical.

Article Snippet: The human HCC cell lines (Hep3B, PLC/PRF/5, and SNU398), non-HCC cell line (LO2), and the HSC cell line (LX2 cells) were purchased from American Type Culture Collection (Manassas, VA, United States).

Techniques: In Vivo, Expressing, Knockdown, Quantitative RT-PCR, Immunohistochemistry, Immunohistochemical staining

Journal: Cell Death & Disease

Article Title: LncRNA SNHG14/miR-5590-3p/ZEB1 positive feedback loop promoted diffuse large B cell lymphoma progression and immune evasion through regulating PD-1/PD-L1 checkpoint

doi: 10.1038/s41419-019-1886-5

Figure Lengend Snippet: a Hierarchical clustering showed the differentially expressed lncRNAs in DLBCL tissues compared with the paired para-tumor tissues according to the microarray analysis (Fold change > 2, P < 0.05). b The expressions of top-5 upregulated lncRNAs in DLBCL tissues in TCGA DLBCL samples were analyzed through GEPIA. c RT-qPCR data showed the upregulated expression of SNHG14 in DLBCL cell lines. d Knockdown of SNHG14 in FARAGE and U2932 cells was confirmed by RT-qPCR. e – f Viability and colony generation of DLBCL cells were evaluated by CCK-8 and colony formation assays. g Invasion of DLBCL cells was detected by transwell invasion assay. Scale bar: 100 μm. h – i EMT markers (E-cadherin and N-cadherin) were detected by western blot and IF staining assay in DLBCL cells. Scale bar: 50 μm. * P < 0.05, ** P < 0.01

Article Snippet: Human lymphoblastoid B cell (GM12878), human renal epithelial cell (293T), murine DLBCL cell (A20), and human DLBCL cells (OCI-LY7, DB, U2932, and FARAGE) were purchased from American Type Culture Collection (ATCC; Manassas, VA, USA).

Techniques: Microarray, Quantitative RT-PCR, Expressing, Knockdown, CCK-8 Assay, Transwell Invasion Assay, Western Blot, Staining

Journal: Cell Death & Disease

Article Title: LncRNA SNHG14/miR-5590-3p/ZEB1 positive feedback loop promoted diffuse large B cell lymphoma progression and immune evasion through regulating PD-1/PD-L1 checkpoint

doi: 10.1038/s41419-019-1886-5

Figure Lengend Snippet: a Heat map showed the expressions of 124 miRNAs potentially targeted by SNHG14 in 3 DLBCL tissues compared with the matched non-tumor tissues. b Luciferase reporter assay was conducted to detect the interaction between the top 5 downregulated miRNAs with SNHG14. c RT-qPCR analysis of miR-5590-3p expression in DLBCL cell lines and normal cell line. d RT-qPCR analysis following the RIP assay was conducted to confirm the interaction between miR-5590-3p with SNHG14 in DLBCL cells. e Interacting sequences on SNHG14 for miR-5590-3p were obtained from Starbase3.0 and mutated by altering them with the complementary sequences. Luciferase reporter assay was performed to detect the interaction between SNHG14 with miR-5590-3p. f Pearson’s correlation curve showed the negative relation between SNHG14 and miR-5590-3p in DLBCL tissues. g Overexpression of miR-5590-3p in DLBCL cells was confirmed by RT-qPCR assay. h Expression of SNHG14 upon miR-5590-3p overexpression in DLBCL cells was detected by RT-qPCR. i Expression of miR-5590-3p upon SNHG14 silence in DLBCL was detected by RT-qPCR. * P < 0.05, ** P < 0.01

Article Snippet: Human lymphoblastoid B cell (GM12878), human renal epithelial cell (293T), murine DLBCL cell (A20), and human DLBCL cells (OCI-LY7, DB, U2932, and FARAGE) were purchased from American Type Culture Collection (ATCC; Manassas, VA, USA).

Techniques: Luciferase, Reporter Assay, Quantitative RT-PCR, Expressing, Over Expression

Journal: Cell Death & Disease

Article Title: LncRNA SNHG14/miR-5590-3p/ZEB1 positive feedback loop promoted diffuse large B cell lymphoma progression and immune evasion through regulating PD-1/PD-L1 checkpoint

doi: 10.1038/s41419-019-1886-5

Figure Lengend Snippet: a Expression of ZEB1 in DLBCL tissues versus paired para-tumorous tissues was detected by RT-qPCR. b Expression of ZEB1 in DLBCL cell lines and normal cell line was detected by RT-qPCR. c Pearson’s correlation curve showed that ZEB1 was positively correlated with SNHG14 and negatively correlated with miR-5590-3p in DLBCL tissues. d Interacting sequences on ZEB1 for miR-5590-3p were obtained from Starbase3.0 and mutated by altering them with the complementary sequences. Luciferase reporter assay was performed to detect the interaction between ZEB1 with miR-5590-3p. e RT-qPCR analysis following the RIP assay was conducted to confirm the interaction between miR-5590-3p with ZEB1 and SNHG14 in DLBCL cells. f Expressions of ZEB1 mRNA and protein and PD-L1 protein upon miR-5590-3p overexpression in DLBCL cells were detected by RT-qPCR and western blot. g Expressions of ZEB1 mRNA and protein and PD-L1 protein upon indicated transfection in DLBCL cells were detected by RT-qPCR and western blot. * P < 0.05, ** P < 0.01

Article Snippet: Human lymphoblastoid B cell (GM12878), human renal epithelial cell (293T), murine DLBCL cell (A20), and human DLBCL cells (OCI-LY7, DB, U2932, and FARAGE) were purchased from American Type Culture Collection (ATCC; Manassas, VA, USA).

Techniques: Expressing, Quantitative RT-PCR, Luciferase, Reporter Assay, Over Expression, Western Blot, Transfection

Journal: Cell Death & Disease

Article Title: LncRNA SNHG14/miR-5590-3p/ZEB1 positive feedback loop promoted diffuse large B cell lymphoma progression and immune evasion through regulating PD-1/PD-L1 checkpoint

doi: 10.1038/s41419-019-1886-5

Figure Lengend Snippet: a DNA motif of ZEB1 and the predicted binding sites on PD-L1 promoter were obtained from JASPAR. b Silence of ZEB1 in FARAGE and U2932 cells was confirmed by RT-qPCR. c Expressions of PD-L1 mRNA and protein and ZEB1 protein upon ZEB1 silence in DLBCL cells were detected by RT-qPCR and western blot. d – e ChIP and luciferase reporter assays showed that ZEB1 bound to both sites 1 and 2 on PD-L1 promoter. f The predicted ZEB1 binding sites on SNHG14 promoter were obtained from JASPAR. g – h ChIP and luciferase reporter assays showed that ZEB1 bound to both sites 1 and 2, rather than site 3 on SNHG14 promoter. i Expression of SNHG14 upon ZEB1 silence in DLBCL cells was detected by RT-qPCR. * P < 0.05, ** P < 0.01

Article Snippet: Human lymphoblastoid B cell (GM12878), human renal epithelial cell (293T), murine DLBCL cell (A20), and human DLBCL cells (OCI-LY7, DB, U2932, and FARAGE) were purchased from American Type Culture Collection (ATCC; Manassas, VA, USA).

Techniques: Binding Assay, Quantitative RT-PCR, Western Blot, Luciferase, Expressing

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Targeting PEG10 as a novel therapeutic approach to overcome CDK4/6 inhibitor resistance in breast cancer

doi: 10.1186/s13046-023-02903-x

Figure Lengend Snippet: Characterization of palbociclib-resistant cells with high PEG10 expression and EMT process activation. A Venn diagram representing the number of upregulated genes in MCF7-PR and T47D-PR cells. A gene showing fold change ≥ 2 compared with that in parental cells (MCF7 and T47D) is considered an upregulated gene. B Heat map showing the list of commonly upregulated genes in MCF7-PR and T47D-PR cells. C mRNA expression of PEG10 in the palbociclib-resistant (MCF7-PR and T47D-PR) compared with their corresponding parental (MCF7 and T47D) cells by qRT-PCR. Three independently repeated experiments were performed with similar results. Independent sample t-test: * p < 0.05, ** p < 0.01. D Immunoblots showing PEG10 (RF1) and PEG10 (FR2) protein expression in the palbociclib-resistant (MCF7-PR and T47D-PR) and parental (MCF7 and T47D) cells. E Association of PEG10 expression and palbociclib sensitivity using GDSC database. Palbociclib sensitivity was defined as IC 50 ≤ 3.5 µM. P- value was calculated by independent sample t-test. F A panel of genes associated with EMT process from microarray data analysis in palbociclib-resistant (MCF7-PR and T47D-PR) versus parental (MCF7 and T47D) cells. G mRNA expression of EMT markers in the palbociclib-resistant (MCF7-PR and T47D-PR) cells compared with their corresponding parental (MCF7 and T47D) cells by qRT-PCR. Three independently repeated experiments were performed with similar results. Independent sample t-test: * p < 0.05, ** p < 0.01, *** p < 0.001, Abbreviation: ns, not significant. H Immunoblots showing protein expression of mesenchymal markers (ZEB1 & LAMC2), and epithelial marker (E-cadherin) in MCF7-PR and T47D-PR cells compared with MCF7 and T47D cells, respectively. I Association of ZEB1 expression and palbociclib sensitivity in 11 HR+ breast cancer cell lines from GDSC database. Palbociclib sensitivity was defined as IC 50 ≤ 3.5 µM. P- value was calculated by independent sample t-test

Article Snippet: HR+ breast cancer cell lines MCF7, T47D, and endogenously PEG10-expressing prostate cancer cell line PC3 were purchased from the American Type Culture Collection (Manassas, VA, USA) and maintained in a humidified atmosphere with 5% CO 2 at 37 °C.

Techniques: Expressing, Activation Assay, Quantitative RT-PCR, Western Blot, Microarray, Marker

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Targeting PEG10 as a novel therapeutic approach to overcome CDK4/6 inhibitor resistance in breast cancer

doi: 10.1186/s13046-023-02903-x

Figure Lengend Snippet: Ectopic overexpression of PEG10 augments EMT and leads to palbociclib resistance. A Immunoblot demonstrates ectopic overexpression of different PEG10 protein isoforms in MCF7 and T47D cells. Ectopic overexpression of PEG10 elevated the ZEB1 and suppressed the E-cadherin expression. B , C Representative images from a migration and invasion assay after ectopic overexpression of different PEG10 protein isoforms in MCF7 and T47D cells. The area of migratory and invading cells from three different non-overlapping 100 × microscopic fields is expressed as mean ± SD in the right panel. Independent sample t-test: * p < 0.05, ** p < 0.01, *** p < 0.001. D - I Cell viability (MTT) assay of MCF7 and T47D cells before and after ectopic overexpression of PEG10-RF1 and subsequent treatment with the indicated dose of palbociclib, abemaciclib, and ribociclib for 72 h. Three independently repeated experiments were performed with similar results. Independent sample t-test: * p < 0.05, ** p < 0.01, *** p < 0.001, Abbreviation: ns, not significant. J , K Cell cycle distribution of MCF7 and T47D cell line before and after the ectopic overexpression of different PEG10 protein isoforms and subsequent treatment with the IC 50 concentration of palbociclib for 48 h. Three independently repeated experiments were performed with similar results. Independent sample t-test: ** p < 0.01, Abbreviation: ns, not significant

Article Snippet: HR+ breast cancer cell lines MCF7, T47D, and endogenously PEG10-expressing prostate cancer cell line PC3 were purchased from the American Type Culture Collection (Manassas, VA, USA) and maintained in a humidified atmosphere with 5% CO 2 at 37 °C.

Techniques: Over Expression, Western Blot, Expressing, Migration, Invasion Assay, MTT Assay, Concentration Assay

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Targeting PEG10 as a novel therapeutic approach to overcome CDK4/6 inhibitor resistance in breast cancer

doi: 10.1186/s13046-023-02903-x

Figure Lengend Snippet: PEG10 inhibition suppresses EMT and overcomes palbociclib resistance. A , B Immunoblot showed changes in the ZEB1 and E-cadherin expression after the knockdown of PEG10 using PEG10 siRNA and PEG10-ASO in MCF7-PR and T47D-PR cells. C Representative images from a migration and invasion assay of parental (MCF7 and T47D) versus palbociclib-resistant cells (MCF7-PR and T47D-PR), respectively. The area of migratory and invading cells from three different non-overlapping 100 × microscopic fields is expressed as mean ± SD in the right panel. Independent sample t-test: * p < 0.05, ** p < 0.01, *** p < 0.001. D Representative image from a migration and invasion assay of palbociclib-resistant (MCF7-PR and T47D-PR) cells after the transient PEG10 knockdown by PEG10 siRNA. The area of migratory and invading cells from three different non-overlapping 100 × microscopic fields is expressed as mean ± SD in the right panel. Independent sample t-test: * p < 0.05, ** p < 0.01, *** p < 0.001. E Cell cycle analysis using PI staining after PEG10 knockdown in MCF7-PR and T47D-PR cells. The cell cycle was initially synchronized (syn) at G0/G1 with a double-thymidine block and then released and analyzed at the indicated time points. The bar represents the cell population distribution in each phase of the cell cycle. F Cell viability (MTT) assay of MCF7-PR and T47D-PR cells after treatment with PEG10 siRNA or palbociclib and combination of various concentrations of palbociclib and a fixed concentration of siRNA for 72 h. Three independently repeated experiments were performed with similar results. G Cell viability (MTT) assay of MCF7-PR and T47D-PR cells after treatment with PEG10-ASO or palbociclib and combination of various concentrations of palbociclib and a fixed concentration of ASO for 72 h. Three independently repeated experiments were performed with similar results. H Immunoblot showed induction of cleaved caspase-3 in the combination treatment of PEG10 siRNA and palbociclib. I Immunoblot showed ZEB1 inhibition by ZEB1 shRNA for 48 h. J Cell viability (MTT) assay of MCF7-PR and T47D-PR cells after treatment with ZEB1 shRNA or palbociclib and combination of various concentrations of palbociclib and a fixed concentration of ZEB1 shRNA for 72 h. Three independently repeated experiments were performed with similar results

Article Snippet: HR+ breast cancer cell lines MCF7, T47D, and endogenously PEG10-expressing prostate cancer cell line PC3 were purchased from the American Type Culture Collection (Manassas, VA, USA) and maintained in a humidified atmosphere with 5% CO 2 at 37 °C.

Techniques: Inhibition, Western Blot, Expressing, Knockdown, Migration, Invasion Assay, Cell Cycle Assay, Staining, Blocking Assay, MTT Assay, Concentration Assay, shRNA

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Targeting PEG10 as a novel therapeutic approach to overcome CDK4/6 inhibitor resistance in breast cancer

doi: 10.1186/s13046-023-02903-x

Figure Lengend Snippet: PEG10 suppresses natural cell cycle inhibitor p21 and EMT process inhibitor SIAH1. A Immunoblot showed changes in the cell cycle-related protein p21 and ubiquitin-related protein SIAH1 expression in parental (MCF7 and T47D) versus palbociclib-resistant (MCF7-PR and T47D-PR) cells. B , C Immunoblot showed p21 and SIAH1 protein expression level after the transient PEG10 knockdown by using PEG10 siRNA or PEG10-ASO. D , E Cell viability (MTT) assay of MCF7-PR and T47D-PR cells before and after ectopic overexpression of p21 and subsequent treatment with the various concentrations of palbociclib for 72 h. Three independently repeated experiments were performed with similar results. Independent sample t-test: * p < 0.05, ** p < 0.01, *** p < 0.001, Abbreviation: ns, not significant. F , G Cell viability (MTT) assay of MCF7-PR and T47D-PR cells before and after ectopic overexpression of SIAH1 and subsequent treatment with the various concentrations of palbociclib for 72 h. Three independently repeated experiments were performed with similar results. Independent sample t-test: * p < 0.05, ** p < 0.01, *** p < 0.001, Abbreviation: ns, not significant. H Immunoblot showed the depletion of cyclins (cyclin E, cyclin D1, and cyclin A) and CDK2 after the ectopic overexpression of p21 in MCF7-PR and T47D-PR cells. I Immunoblot showed the differential expression of cyclins (cyclin E, cyclin D1, and cyclin A) and CDK2 in parental cells (MCF7 and T47D) versus palbociclib-resistant cells (MCF7-PR and T47D-PR), respectively. J Immunoblot showed the depletion of cyclins (cyclin E, cyclin D1, and cyclin A) and CDK2 after PEG10 knockdown by PEG10 siRNA in MCF7-PR and T47D-PR cells. K , L Immunoblot showed the expression of ZEB1 and E-cadherin after the ectopic overexpression of p21 and SIAH1 in MCF7-PR and T47D-PR cells

Article Snippet: HR+ breast cancer cell lines MCF7, T47D, and endogenously PEG10-expressing prostate cancer cell line PC3 were purchased from the American Type Culture Collection (Manassas, VA, USA) and maintained in a humidified atmosphere with 5% CO 2 at 37 °C.

Techniques: Western Blot, Ubiquitin Proteomics, Expressing, Knockdown, MTT Assay, Over Expression, Quantitative Proteomics

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Targeting PEG10 as a novel therapeutic approach to overcome CDK4/6 inhibitor resistance in breast cancer

doi: 10.1186/s13046-023-02903-x

Figure Lengend Snippet: Combined treatment of palbociclib and PEG10-ASO regresses the palbociclib-resistant breast cancer synergistically in a xenograft model. A Experimental design for in vivo efficacy test using acquired palbociclib-resistant xenograft model in BALB/c nude mice. When tumor reached 60–80 mm3, mice were randomized to control-ASO ( n = 5), palbociclib ( n = 5), PEG10-ASO ( n = 5), and combination ( n = 5) treatment groups. Control-ASO and PEG10-ASO were administrated intraperitoneally (i.p.) for 3 weeks (15 mg/kg/day for first 5 days loading, followed by 17 days of maintenance dose of 15 mg/kg, 3 times/week). Palbociclib (100 mg/kg/day) was given by oral gavage for 3 weeks. B Gross harvested tumor. The black dotted circles represent the complete regression of the tumor. C Average tumor sizes of the indicated treatment groups before sacrifice. Independent sample t-test: *** p < 0.001, Abbreviation: ns, not significant. D Average tumor growth of MCF7-PR xenograft tumor treated with indicated drugs. Error bars represent SD of 5 tumors per group. Independent sample t-test: *** p < 0.001, Abbreviation: ns, not significant. E Average body weight of mice with indicated groups. Error bars represent the SD of 5 mice per group. F Immunoblots using xenograft tumors showed the suppression of PEG10 in the PEG10-ASO and combination treatment groups, along with altered ZEB1 and E-cadherin expression. G IHC staining of PEG10 in MCF7-PR xenograft tumors of indicated groups. Staining images were taken at 400 × magnification. The bar graph represented the PEG10 protein expression in five random, non-overlapped fields. Data are presented as mean ± SD. Independent sample t-test: *** p < 0.001, Abbreviation: ns, not significant. H IHC staining of Ki67 in MCF7-PR xenograft tumors of indicated groups. Staining images were taken at 400 × magnification. The bar graph represented the Ki67 cells in five random, non-overlapped fields. Data are presented as mean ± SD. Independent sample t-test: * p < 0.05, *** p < 0.001. I TUNEL assay using xenograft tumors at sacrifice. Staining images in each group are shown and bar graphs represent average apoptotic cells in each group in five random, non-overlapped fields at 400 × magnification. Data are presented as mean ± SD. Independent sample t-test: ** p < 0.01, *** p < 0.001, Abbreviation: ns, not significant

Article Snippet: HR+ breast cancer cell lines MCF7, T47D, and endogenously PEG10-expressing prostate cancer cell line PC3 were purchased from the American Type Culture Collection (Manassas, VA, USA) and maintained in a humidified atmosphere with 5% CO 2 at 37 °C.

Techniques: In Vivo, Control, Western Blot, Expressing, Immunohistochemistry, Staining, TUNEL Assay

Journal: Journal of Translational Medicine

Article Title: PTOV1 exerts pro-oncogenic role in colorectal cancer by modulating SQSTM1-mediated autophagic degradation of p53

doi: 10.1186/s12967-025-06179-x

Figure Lengend Snippet: PTOV1 promotes CRC progression in vitro. ( A ) Western blot analysis of PTOV1 expression in PTOV1 stably overexpression HCT116 and DLD1 cells. ( B ) Western blot analysis of PTOV1 expression in PTOV1 stably knockdown LoVo and RKO cells. ( C ) The colony formation assay of PTOV1 stably overexpression HCT116 and DLD1 cells. **, P < 0.01 . ( D ) The colony formation assay of PTOV1 stably knockdown LoVo and RKO cells. **, P < 0.01 . ( E ) The CCK8 assay of PTOV1 stably overexpression HCT116 (left panel) and DLD1 cells (right panel). ***, P < 0.001 . ( F ) The CCK8 assay of PTOV1 stably knockdown LoVo (left panel) and RKO cells (right panel). ***, P < 0.001 . ( G ) The migration and invasion assay of PTOV1 stably overexpression HCT116 (upper panel) and DLD1 cells (lower panel). The average number of cells per field were calculated. n = 3 samples per group, four fields per sample. **, P < 0.01; ***, P < 0.001. ( H ) The migration and invasion assay of PTOV1 stably knockdown RKO (upper panel) and LoVo cells (lower panel). The average number of cells per field were calculated. n = 3 samples per group, four fields per sample. **, P < 0.01; ***, P < 0.001. ( I ) Western blot analysis of c-MYC, CyclinD1, N-cadherin, E-cadherin, and Vimentin expression in PTOV1 stably overexpression HCT116 and DLD1 cells. ( J ) Western blot analysis of c-MYC, CyclinD1, N-cadherin, E-cadherin, and Vimentin expression in PTOV1 stably knockdown LoVo and RKO cells

Article Snippet: HEK293T cells and human colorectal cancer cell lines HCT116, RKO, LoVo and DLD-1 were purchased from the American Type Culture Collection (ATCC).

Techniques: In Vitro, Western Blot, Expressing, Stable Transfection, Over Expression, Knockdown, Colony Assay, CCK-8 Assay, Migration, Invasion Assay

Journal: Journal of Translational Medicine

Article Title: PTOV1 exerts pro-oncogenic role in colorectal cancer by modulating SQSTM1-mediated autophagic degradation of p53

doi: 10.1186/s12967-025-06179-x

Figure Lengend Snippet: PTOV1 promotes CRC progression in vivo. (A-C) PTOV1 stably overexpression or control HCT116 cells were subcutaneously injected into nude mice. Representative tumor sizes ( A ), tumor growth curves ( B ), and tumor weight ( C ) are shown. n = 5 per group; Scale bar, 50 mm. ***, P < 0.001. ( D-F ) PTOV1 stably knockdown or control LoVo cells were subcutaneously injected into nude mice. Representative tumor sizes ( D ), tumor growth curves ( E ), and tumor weight ( F ) are shown. n = 5 per group; Scale bar, 50 mm. ***, P < 0.001. ( G-H ) The representative immunohistochemical staining of Ki67 and Caspase-3 in xenograft tumor from PTOV1 stably overexpression ( G ) or PTOV1 stably knockdown ( H ) groups. Scale bar, 100 μm. ( I ) Kaplan–Meier analysis of tumor-bearing nude mice with subcutaneous injected with PTOV1 stably overexpression and control HCT116 cells. P < 0.01. ( J ) Kaplan–Meier analysis of tumor-bearing nude mice with subcutaneous injected with PTOV1 stably knockdown and control LoVo cells. P < 0.01. ( K-M ) PTOV1 stably overexpression and control HCT116 cells were injected into nude mice via spleen. The representative hematoxylin and eosin (H&E) images ( K ), quantification of metastatic foci ( L ) and liver weight ( M ) were shown. n = 5 per group. Scale bars ( K , upper panel), 1 cm; Scale bars ( K , lower panel), 100 μm. **, P < 0.01. ( N-P ) PTOV1 stably knockdown and control LoVo cells were injected into nude mice via spleen. The representative hematoxylin and eosin (H&E) images ( N ), quantification of metastatic foci ( O ) and liver weight ( P ) were shown. n = 5 per group. Scale bars ( N , upper panel), 1 cm; Scale bars ( N , lower panel), 100 μm. **, P < 0.01; ***, P < 0.001

Article Snippet: HEK293T cells and human colorectal cancer cell lines HCT116, RKO, LoVo and DLD-1 were purchased from the American Type Culture Collection (ATCC).

Techniques: In Vivo, Stable Transfection, Over Expression, Control, Injection, Knockdown, Immunohistochemical staining, Staining

Journal: Journal of Translational Medicine

Article Title: PTOV1 exerts pro-oncogenic role in colorectal cancer by modulating SQSTM1-mediated autophagic degradation of p53

doi: 10.1186/s12967-025-06179-x

Figure Lengend Snippet: POTV1 promotes the autophagic degradation of p53. ( A ) Western blot analysis of p53 expression in PTOV1 stably overexpression HCT116 and DLD1 cells. ( B ) Western blot analysis of p53 expression in HCT116 cells transfected with indicated amount of Flag-PTOV1 plasmid for 48 h. ( C ) Western blot analysis of p53 expression in PTOV1 stably knockdown LoVo and RKO cells. ( D ) Cycloheximide chase analysis of p53 protein half-life in PTOV1 stably overexpression HCT116 and DLD1 cells treated with 10 μm cycloheximide. ( E-F ) Western blot analysis of p53 expression in PTOV1 stably overexpression HCT116 cells treated with 10 µM MG132 for 12 h ( E ) or 50 µM CQ for 4 h ( F ). ( G-H ) PTOV1 stably overexpression HCT116 cells were transfected with siULK1 ( G ) or siATG7 ( H ) for 48 h, western blot analysis of p53 protein levels. ( I ) TEM analysis of PTOV1 stably overexpression HCT116 cells and representative TEM images were shown. The number of autophagic structures per cell was quantified. Scale bar, 2 μm; **, P < 0.01. ( J ) Western blot analysis of LC3 and SQSTM1 protein levels in PTOV1 stably overexpression HCT116 and DLD1 cells. ( K ) Western blot analysis of LC3 and SQSTM1 protein levels in PTOV1 stably knockdown LoVo and RKO cells. ( L ) PTOV1 stably overexpression HCT116 or DLD1 cells were transfected with GFP-mCherry-LC3B for 48 h. GFP-mCherry-LC3B distribution was observed by confocal microscopy. Scale bar, 5 μm

Article Snippet: HEK293T cells and human colorectal cancer cell lines HCT116, RKO, LoVo and DLD-1 were purchased from the American Type Culture Collection (ATCC).

Techniques: Western Blot, Expressing, Stable Transfection, Over Expression, Transfection, Plasmid Preparation, Knockdown, Confocal Microscopy

Journal: Journal of Translational Medicine

Article Title: PTOV1 exerts pro-oncogenic role in colorectal cancer by modulating SQSTM1-mediated autophagic degradation of p53

doi: 10.1186/s12967-025-06179-x

Figure Lengend Snippet: PTOV1 facilitates CRC progression through downregulating p53. ( A ) The CCK8 assay of PTOV1 stably overexpression HCT116 and DLD1 cells with or without p53 overexpression. ( B ) The migration and invasion assay of PTOV1 stably overexpression LoVo and RKO cells with or without p53 overexpression. The average number of cells per field were calculated. n = 3 samples per group, four fields per sample. *, P < 0.05; ***, P < 0.01. ( C ) Western blot analysis of c-MYC, CyclinD1, N-cadherin, E-cadherin and Vimentin expression in PTOV1 stably overexpression HCT116 and DLD1 cells with or without p53 overexpression. ( D-F ) PTOV1 stably overexpression or control HCT116 cells with or without p53 overexpression were subcutaneously injected into nude mice. Representative tumor sizes ( D ), tumor growth curves ( E ), and tumor weight ( F ) are shown. n = 5 per group; Scale bar, 50 mm. ***, P < 0.001. ( G ) The representative immunohistochemical staining of Ki67 and Caspase-3 in xenograft tumor from PTOV1 stably overexpression with or without p53 overexpression groups. Scale bar, 100 μm. ( H ) Western blot analysis of PTOV1 and p53 expression in 18 paired CRC tissues and peritumoral tissues. ( I ) Representative images of PTOV1 and p53 immunohistochemistry staining in tumor ( n = 94) and adjacent normal tissues ( n = 82) in CRC tissue microarray. Scale bars, 200 μm for 5 x and 100 μm for 10 x magnification. ( J ) Pearson correlation analysis of PTOV1 and p53 expression in CRC tissue microarray. P < 0.002. ( K ) Kaplan–Meier survival analysis of overall survival (OS) based on PTOV1 and p53 expression in CRC tissue microarray. P < 0.0001

Article Snippet: HEK293T cells and human colorectal cancer cell lines HCT116, RKO, LoVo and DLD-1 were purchased from the American Type Culture Collection (ATCC).

Techniques: CCK-8 Assay, Stable Transfection, Over Expression, Migration, Invasion Assay, Western Blot, Expressing, Control, Injection, Immunohistochemical staining, Staining, Immunohistochemistry, Microarray

Journal: Journal of Translational Medicine

Article Title: PTOV1 exerts pro-oncogenic role in colorectal cancer by modulating SQSTM1-mediated autophagic degradation of p53

doi: 10.1186/s12967-025-06179-x

Figure Lengend Snippet: PTOV1 promotes p53 autophagic degradation dependent on SQSTM1. ( A ) HCT116 cells were co-transfected with indicated autophagy cargo receptors and HA-p53 for 48 h. Total cell lysates were immunoprecipitated with anti-Flag antibody. Immunoprecipitation complex was detected by anti-HA and anti-Flag antibodies. ( B-C ) HCT116 cells were co-transfected with Flag-SQSTM1 and HA-p53 for 48 h. Total cell lysates were immunoprecipitated with anti-Flag ( B ) or anti-HA ( C ) antibodies. HA-p53 and Flag-SQSTM1 were detected by western blot. ( D ) PTOV1 stably overexpression HCT116 and DLD1 cells or control cells were treated with CQ (50µM) for 4 h. Total cell lysates were immunoprecipitated with anti-SQSTM1 antibodies. p53 and SQSTM1 were detected by western blot. ( E ) PTOV1 stably knockdown LoVo and RKO cells or control cells were treated with CQ (50µM) for 4 h. Total cell lysates were immunoprecipitated with anti-SQSTM1 antibodies. p53 and SQSTM1 were detected by western blot. ( F ) PTOV1 stably overexpression HCT116 and DLD1 cells were infected with SQSTM1 shRNA for 48 h. Western blot analysis of p53 protein levels. ( G ) Immunofluorescence assay of p53 and SQSTM1 in PTOV1 stably overexpression HCT116 and cells treated with CQ (50µM) for 4 h. Representative confocal microscopy images were shown. Scale bars, 5 μm

Article Snippet: HEK293T cells and human colorectal cancer cell lines HCT116, RKO, LoVo and DLD-1 were purchased from the American Type Culture Collection (ATCC).

Techniques: Transfection, Immunoprecipitation, Western Blot, Stable Transfection, Over Expression, Control, Knockdown, Infection, shRNA, Immunofluorescence, Confocal Microscopy