Journal: Frontiers in Oncology

Article Title: PTIP Inhibits Cell Invasion in Esophageal Squamous Cell Carcinoma via Modulation of EphA2 Expression

doi: 10.3389/fonc.2021.629916

Figure Lengend Snippet: Primers used for plasmid construction.

Article Snippet: Lentiviral vectors harboring short hairpin RNA (shRNA) targeting PTIP (shPTIP), Fosl2 (shFosl2) and EphA2 (shEphA2) were synthesized by Genscript (Nanjing, China). cDNAs encoding PTIP were cloned into the expression vector pLenti-EFs-BSD with a blasticidin-resistant gene for N-terminal tagging of MYC epitope (PTIP-MYC) or FLAG epitope (PTIP-FLAG).

Techniques: Plasmid Preparation, Sequencing

Journal: Frontiers in Oncology

Article Title: PTIP Inhibits Cell Invasion in Esophageal Squamous Cell Carcinoma via Modulation of EphA2 Expression

doi: 10.3389/fonc.2021.629916

Figure Lengend Snippet: Primers used for Real-time PCR.

Article Snippet: Lentiviral vectors harboring short hairpin RNA (shRNA) targeting PTIP (shPTIP), Fosl2 (shFosl2) and EphA2 (shEphA2) were synthesized by Genscript (Nanjing, China). cDNAs encoding PTIP were cloned into the expression vector pLenti-EFs-BSD with a blasticidin-resistant gene for N-terminal tagging of MYC epitope (PTIP-MYC) or FLAG epitope (PTIP-FLAG).

Techniques: Sequencing

Journal: Frontiers in Oncology

Article Title: PTIP Inhibits Cell Invasion in Esophageal Squamous Cell Carcinoma via Modulation of EphA2 Expression

doi: 10.3389/fonc.2021.629916

Figure Lengend Snippet: Primers used for ChIP-qPCR.

Article Snippet: Lentiviral vectors harboring short hairpin RNA (shRNA) targeting PTIP (shPTIP), Fosl2 (shFosl2) and EphA2 (shEphA2) were synthesized by Genscript (Nanjing, China). cDNAs encoding PTIP were cloned into the expression vector pLenti-EFs-BSD with a blasticidin-resistant gene for N-terminal tagging of MYC epitope (PTIP-MYC) or FLAG epitope (PTIP-FLAG).

Techniques: Sequencing

Journal: Frontiers in Oncology

Article Title: PTIP Inhibits Cell Invasion in Esophageal Squamous Cell Carcinoma via Modulation of EphA2 Expression

doi: 10.3389/fonc.2021.629916

Figure Lengend Snippet: PTIP inhibits ESCC cell invasion and migration through EphA2. (A) Western blotting analysis against PTIP, EphA2, pS897-EphA2, pY588-EphA2 in PTIP knockdown(shPTIP#1, shPTIP#2) and control (shCtrl) TE1 cells. (B) IHC score for EphA2 in ESCC tumor sections. Unpaired, two-tailed Student’s t-test; ** P < 0.01. (C) Representative IHC images for EphA2 in ESCC tumor sections. Low EphA2 and high EphA2 groups were divided based on the EphA2 immunostaining intensity scores mentioned in method. Cut off for high and low EphA2 expression in ESCC was defined < or > 11. (D) Percentage of invasion and non-invasion in ESCC groups. The differences between rates were tested by χ2; * P < 0.05. (E) Comparative expression between PTIP and EphA2 in ESCC samples from (B) analyzed by Pearson correlation. (F) Knockdown efficiency of shRNAs targeting PTIP and EphA2 in TE1 cells as determined by qRT-PCR. One-way ANOVA; * P < 0.05,** P < 0.01, *** P < 0.001. (G, H) The effect of PTIP and EphA2 double knockdown on the invasiveness of TE1 cells. For invasion assay, six different microscopic fields (magnification, ×10) from at least three independent experiments were examined; Relative intensities of the fields were measured (n ≥3). Representative images and statistical plots are shown; Mean ± s.d. are given for three independent experiments. One-way ANOVA; *** P < 0.001. (I) ChIP-seq density profiles for PTIP in TE1 cells. Gene models are shown below the density profiles. (J, K) ChIP-qPCR primer sets marked with arrows were designed to cover regions present within (EphA2) or outside (EphA2-NC) of the EphA2 gene (C) . ChIP-qPCR analyses of EphA2 binding (D) . Representative images and statistical plots are shown; Mean ± s.d. are given for three independent experiments. Unpaired, two-tailed Student’s t-test; ** P < 0.01.

Article Snippet: Lentiviral vectors harboring short hairpin RNA (shRNA) targeting PTIP (shPTIP), Fosl2 (shFosl2) and EphA2 (shEphA2) were synthesized by Genscript (Nanjing, China). cDNAs encoding PTIP were cloned into the expression vector pLenti-EFs-BSD with a blasticidin-resistant gene for N-terminal tagging of MYC epitope (PTIP-MYC) or FLAG epitope (PTIP-FLAG).

Techniques: Migration, Western Blot, Knockdown, Control, Two Tailed Test, Immunostaining, Expressing, Quantitative RT-PCR, Invasion Assay, ChIP-sequencing, ChIP-qPCR, Binding Assay

Journal: Frontiers in Oncology

Article Title: PTIP Inhibits Cell Invasion in Esophageal Squamous Cell Carcinoma via Modulation of EphA2 Expression

doi: 10.3389/fonc.2021.629916

Figure Lengend Snippet: PTIP inhibits EphA2 expression by competing with Fosl2 for binding to EphA2. (A) PTIP highly enriched binding motifs in bioPTIP ChIPseq were identified by HOMER. P -values are indicated within the boxes. (B, C) Western blot analysis of Fosl2 and EphA2 expression in Fosl2 knockdown (shFosl2#1, shFosl2#2) and control (shCtrl) TE1 (B) and KYSE-150 (C) cells. (D–G) The effect of Fosl2 knockdown on the invasiveness of TE1 (D, E) and KYSE-150 (F, G) cells. For invasion assay, six different microscopic fields (magnification, ×10) from at least three independent experiments were examined; Relative intensities of the fields were measured (n≥3). Representative images and statistical plots are shown; Mean ± s.d. are given for three independent experiments. One-way ANOVA; * P < 0.05; * P < 0.01; *** P < 0.001. (H–M) Western blot showing stable expression of Flag-EphA2 in shFosl2#1 TE1 cells (H) and KYSE-150 cells (K) . Overexpression EphA2 partially restored TE1 cells (I, J) and KYSE-150 cells (L, M) invasive ability, which attenuated by depletion of Fosl2. For invasion assay, six different microscopic fields (magnification, ×10) from at least three independent experiments were examined; Relative intensities of the fields were measured (n≥3). Representative images and statistical plots are shown; Mean ± s.d. are given for three independent experiments. One-way ANOVA; * P < 0.05; *** P < 0.001. (N, O) ChIP-qPCR analyses of the relationship between Fosl2 and PTIP in transcriptional regulation for EphA2. Mean ± s.d. are given for three independent experiments. Unpaired, two-tailed Student’s t-test; * P < 0.05.

Article Snippet: Lentiviral vectors harboring short hairpin RNA (shRNA) targeting PTIP (shPTIP), Fosl2 (shFosl2) and EphA2 (shEphA2) were synthesized by Genscript (Nanjing, China). cDNAs encoding PTIP were cloned into the expression vector pLenti-EFs-BSD with a blasticidin-resistant gene for N-terminal tagging of MYC epitope (PTIP-MYC) or FLAG epitope (PTIP-FLAG).

Techniques: Expressing, Binding Assay, Western Blot, Knockdown, Control, Invasion Assay, Over Expression, ChIP-qPCR, Two Tailed Test

Journal: Matrix biology : journal of the International Society for Matrix Biology

Article Title: Progranulin/EphA2 axis: A novel oncogenic mechanism in bladder cancer

doi: 10.1016/j.matbio.2020.03.009

Figure Lengend Snippet: Progranulin, Ephrin-A1, and EphA2 expression in bladder cancer. (A) mRNA levels of GRN and EFNA1 in normal or urothelial carcinomas of low and high grade (Oncomine). (B) Steady state levels of progranulin (GRN) and ephrin-A1 (EFNA1) in T24 and UMUC-3 cells; mean ±SEM, n = 3. (C) Micrographs from the human protein Atlas depicting representative images of normal bladder and low or high-grade urothelial carcinomas. (D) EphA2 expression levels in bladder cancer tissues using IHC on a bladder cancer TMA. (E) Quantification of EphA2 expression in tissues using ImageJ; bladder, n = 27; T1-T4 urothelial carcinomas, n = 123; ***p<0.001. (F) EphA2 expression in various urothelial cancer cells by immunoblot using anti-EphA2 and anti-β-actin antibodies.

Article Snippet: Generation of EphA2-depleted bladder cancer cells Cell lines (UMUC-3) stably depleted of endogenous EphA2 were generated by transfecting the pRS-shScr (scrambled shRNAs) and pRS/shEphA2 plasmids expressing shRNAs against human EphA2 (OriGene Technologies, Inc.) using the TransIT ® -Prostate Transfection Kit (Mirus).

Techniques: Expressing, Western Blot

Journal: Matrix biology : journal of the International Society for Matrix Biology

Article Title: Progranulin/EphA2 axis: A novel oncogenic mechanism in bladder cancer

doi: 10.1016/j.matbio.2020.03.009

Figure Lengend Snippet: Progranulin evokes EphA2 phosphorylation. Serum-starved T24 cells were exposed to progranulin (150 nM, 15 min) and immunoprecipitated with anti-EphA2 antibodies. Coomassie-stained bands of interest were trypsin-digested and analyzed by LC-MS/MS on a Q Exactive™ Plus mass spec. The mass spec data were probed against the UniProt human database for STY phosphorylation. False discovery rate for peptides/site identifications was set at 1%. (A) Modifications of various residues shown as the sum of MS peptides intensities. (B) EphA2 schematic showing the location of phosphorylated residues. (C-D) Western immunoblots of serum-starved T24 cells exposed to 150 nM progranulin using several Phospho-specific and total antibodies. (E) Western immunoblots of serum-starved 5637 cells after progranulin stimulation ±specific inhibitors for PI3K pathway (LY294002, 20 μM) or Erk1/2 (U0126, 10 μM). (F) Phospho-EphA2 (Ser897) assessed by immunoblot in UMUC-3/shScr (control) and UMUC-3/shPGRN cells. (G) Inhibition of EphA2 kinase activity blocks progranulin-induced phosphorylation. Western immunoblots of serum-starved T24 cells exposed to Ephrin-A1-Fc (2.1 nM) or progranulin (150 nM) for 15 min ±ALW-II-41–27 (1 μM), and probed with Phospho-EphA2 (Ser897) and EphA2 antibodies.

Article Snippet: Generation of EphA2-depleted bladder cancer cells Cell lines (UMUC-3) stably depleted of endogenous EphA2 were generated by transfecting the pRS-shScr (scrambled shRNAs) and pRS/shEphA2 plasmids expressing shRNAs against human EphA2 (OriGene Technologies, Inc.) using the TransIT ® -Prostate Transfection Kit (Mirus).

Techniques: Phospho-proteomics, Immunoprecipitation, Staining, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry, Western Blot, Control, Inhibition, Activity Assay

Journal: Matrix biology : journal of the International Society for Matrix Biology

Article Title: Progranulin/EphA2 axis: A novel oncogenic mechanism in bladder cancer

doi: 10.1016/j.matbio.2020.03.009

Figure Lengend Snippet: EphA2 is required for progranulin-evoked activity and cisplatin sensitivity. (A) EphA2 was depleted in UMUC-3 cells by siRNA approaches and EphA2 expression levels were assessed by immunoblot with anti-EphA2 polyclonal antibodies and normalized over β-actin content. Densitometric analysis was performed using ImageJ (National Institutes of Health) and expressed as arbitrary units. (B) Motility assays in Boyden chambers ±progranulin, ***p<0.001. (C) Anchorage-independent growth in soft-agar was performed as described in Materials and Methods ***p<0.001. (D) Cell survival as assessed by a Colorimetric Cell Cytotoxicity Assay Kit at various cisplatin concentrations. Mean ±SD; n = 3 biological replicates run in duplicates. **p<0.005; ***p<0.001.

Article Snippet: Generation of EphA2-depleted bladder cancer cells Cell lines (UMUC-3) stably depleted of endogenous EphA2 were generated by transfecting the pRS-shScr (scrambled shRNAs) and pRS/shEphA2 plasmids expressing shRNAs against human EphA2 (OriGene Technologies, Inc.) using the TransIT ® -Prostate Transfection Kit (Mirus).

Techniques: Activity Assay, Expressing, Western Blot, Cytotoxicity Assay

Journal: Matrix biology : journal of the International Society for Matrix Biology

Article Title: Progranulin/EphA2 axis: A novel oncogenic mechanism in bladder cancer

doi: 10.1016/j.matbio.2020.03.009

Figure Lengend Snippet: EphA2 interacts with liprinα–1 and vinculin. (A) EphA2 interactome involved in cell motility as identified by proteomics. (B) Co-immunoprecipitation and western immunoblot of serum-starved T24 cells ±progranulin (150 nM) with the indicated antibodies. (C) Immunoblot of Liprinα–1 and vinculin in various urothelial carcinoma cells. (D) Co-localization of EphA2/liprinα–1 assessed by confocal microscopy. Insets = higher magnification of the relative areas within the white boxes. Bar = 10 μm. (E) Western immunoblot of liprinα–1- depleted T24 cells. (F) Quantification of T24 cell lateral motility determined by wound healing assays as previously described [18,19,26]. p = 0.000666.

Article Snippet: Generation of EphA2-depleted bladder cancer cells Cell lines (UMUC-3) stably depleted of endogenous EphA2 were generated by transfecting the pRS-shScr (scrambled shRNAs) and pRS/shEphA2 plasmids expressing shRNAs against human EphA2 (OriGene Technologies, Inc.) using the TransIT ® -Prostate Transfection Kit (Mirus).

Techniques: Immunoprecipitation, Western Blot, Confocal Microscopy

Journal: Matrix biology : journal of the International Society for Matrix Biology

Article Title: Progranulin/EphA2 axis: A novel oncogenic mechanism in bladder cancer

doi: 10.1016/j.matbio.2020.03.009

Figure Lengend Snippet: Mechanism of progranulin-dependent EphA2 activation. Scheme depicting progranulin-induced EphA2 activation at Tyr588 and resultant Akt/MAPK feedback loop to phosphorylate Ser897.

Article Snippet: Generation of EphA2-depleted bladder cancer cells Cell lines (UMUC-3) stably depleted of endogenous EphA2 were generated by transfecting the pRS-shScr (scrambled shRNAs) and pRS/shEphA2 plasmids expressing shRNAs against human EphA2 (OriGene Technologies, Inc.) using the TransIT ® -Prostate Transfection Kit (Mirus).

Techniques: Activation Assay