Journal: Bioengineered

Article Title: Long non-coding RNA TINCR promotes hepatocellular carcinoma proliferation and invasion via STAT3 signaling by direct interacting with T-cell protein tyrosine phosphatase (TCPTP).

doi: 10.1080/21655979.2021.1930336

Figure Lengend Snippet: Figure 1. LncRNA TINCR correlated with TCPTP protein. (a) Schematic diagram of RNA pull-down assay/mass spectrometry procedures. (b) RNA pull-down assay was conducted by using antisense synthesized TINCR RNA (Antisense) or full-length sense synthesized TINCR RNA (TINCR). The purified interacting proteins from Hep3B was detected by using SDS-PAGE and silver staining. Red box indicates position of gel slice for mass spectrometry. (c) Annotated MS/MS spectrum assigned to the TCPTP peptide CAQYWPTDDQEMLFK from TINCR vs. TINCR antisense RNA pull-down assay. (d) Western blot experiment demonstrated the interaction between TCPTP and TINCR. The purified interacting proteins from Hep3B was obtained from TINCR vs. TINCR antisense RNA pull-down assay. AS, TINCR antisense RNA. (e) The interaction fragments between LncRNA TINCR (NR_027064.3) and protein TCPTP (isoform 2, NP_536347) were predicted by using catRAPID omics algorithm. (f) Visualization of interaction between TCPTP and TINCR was simulated by Protein Data Bank and mfold web server. The active site (cysteine 216) of TCPTP is shown in red. (g) Schematic diagram for generating in vitro transcription of biotinylated TINCR fragment constructions (Antisense 1–3733 nt, FL 1–3733 nt, D1 360–3733 nt, D2 1100–3733 nt, D3 1800–3733 nt, D4 1–1800 nt). The Hep3B cellular protein was extracted for RNA pull down assay. The different TINCR in vitro transcriptions were employed to perform RNA pull-down assay, and western blotting was performed to detect TCPTP protein in purified proteins and Input. (h) Western blot for TCPTP and real-time PCR for TINCR using RNA immunoprecipitation (RIP) enriched samples of Hep3B and HCCLM3 cells. Data are given as mean ± SEM (n = 3, *P < 0.05, by Student’s t-test).

Article Snippet: The siRNA duplexes against human TCPTP (#sc-76635) was supplied by Santa Cruz Biotechnology (Santa Cruz, USA).

Techniques: Pull Down Assay, Mass Spectrometry, Synthesized, Purification, SDS Page, Silver Staining, Tandem Mass Spectroscopy, Western Blot, In Vitro, Real-time Polymerase Chain Reaction, RNA Immunoprecipitation

Journal: Bioengineered

Article Title: Long non-coding RNA TINCR promotes hepatocellular carcinoma proliferation and invasion via STAT3 signaling by direct interacting with T-cell protein tyrosine phosphatase (TCPTP).

doi: 10.1080/21655979.2021.1930336

Figure Lengend Snippet: Figure 2. LncRNA TINCR promotes hepatocellular carcinoma (HCC) cell growth and inhibits apoptosis through TCPTP. (a) Real-time PCR assay of TINCR expression in different TINCR siRNA-silenced Hep3B and HCCLM3 cells. Data are indicated as mean ± SEM (n = 3, *P < 0.05, by ANOVA). (b) Western blot assay of TCPTP in TCPTP siRNA-silenced Hep3B and HCCLM3 cells. Data are indicated as mean ± SEM (n = 3, *P < 0.05, by Student’s t-test). (c) Real-time PCR assay of TCPTP in TCPTP siRNA-silenced Hep3B and HCCLM3 cells. Data are shown as mean ± SEM (n = 3, *P < 0.05, by Student’s t-test). The CCK-8 (d), colony formation (e and f), EdU (g and h), and apoptosis (i and j) assays were performed in Hep3B and HCCLM3 cells transfected with siRNA-NC, siRNA-TINCR, or co- transfected with siRNA-TINCR and siRNA-TCPTP. Data are given as mean ± SEM (n = 3, *P < 0.05, by ANOVA).

Article Snippet: The siRNA duplexes against human TCPTP (#sc-76635) was supplied by Santa Cruz Biotechnology (Santa Cruz, USA).

Techniques: Real-time Polymerase Chain Reaction, Expressing, Western Blot, CCK-8 Assay, Transfection

Journal: Bioengineered

Article Title: Long non-coding RNA TINCR promotes hepatocellular carcinoma proliferation and invasion via STAT3 signaling by direct interacting with T-cell protein tyrosine phosphatase (TCPTP).

doi: 10.1080/21655979.2021.1930336

Figure Lengend Snippet: Figure 3. LncRNA TINCR promotes HCC cell invasion and STAT3 signaling through TCPTP. The wound healing (a and b) and transwell (c and d) assays were conducted in Hep3B and HCCLM3 cells transfected with siRNA-NC, siRNA-TINCR, or co-transfected with siRNA-TINCR and siRNA-TCPTP. Data are given as mean ± SEM (n = 3, *P < 0.05, by ANOVA). (e and f) Real-time PCR assay of STAT3 target genes expression in Hep3B and HCCLM3 cells transfected with siRNA-NC, siRNA-TINCR, or co-transfected with siRNA- TINCR and siRNA-TCPTP. Data are indicated as mean ± SEM (n = 3, *P < 0.05, by ANOVA). (G, H and I) Western blot assay of STAT3 phosphorylation and STAT3 target genes in HCCLM3 cells transfected with siRNA-NC, siRNA-TINCR, or co-transfected with siRNA- TINCR and siRNA-TCPTP. Data are given as mean ± SEM (n = 3, *P < 0.05, by ANOVA).

Article Snippet: The siRNA duplexes against human TCPTP (#sc-76635) was supplied by Santa Cruz Biotechnology (Santa Cruz, USA).

Techniques: Transfection, Real-time Polymerase Chain Reaction, Expressing, Western Blot, Phospho-proteomics

Journal: Bioengineered

Article Title: Long non-coding RNA TINCR promotes hepatocellular carcinoma proliferation and invasion via STAT3 signaling by direct interacting with T-cell protein tyrosine phosphatase (TCPTP).

doi: 10.1080/21655979.2021.1930336

Figure Lengend Snippet: Figure 4. LncRNA TINCR regulates HCC progression and STAT3 signaling via direct interacting with TCPTP. (a) Schematic illustration of the plasmids encoding full-length (WT, 1–3733 nt) or the fragments (Δ1-360, 360–3733 nt) of TINCR. (b) Real-time PCR assay of TINCR expression in Hep3B and HCCLM3 cells transfected with WT or Δ1-360 TINCR. Data are given as mean ± SEM (n = 3, *P < 0.05, by ANOVA). The CCK-8 (c), EdU (d and e), apoptosis (f and g), and transwell (h and i) assays were conducted in Hep3B and HCCLM3 cells transfected with WT or Δ1-360 TINCR. Data are indicated as mean ± SEM (n = 3, *P < 0.05, by ANOVA). (j and k) Real- time PCR assay of STAT3 target genes expression in Hep3B and HCCLM3 cells transfected with WT or Δ1-360 TINCR. Data are given as mean ± SEM (n = 3, *P < 0.05, by ANOVA).

Article Snippet: The siRNA duplexes against human TCPTP (#sc-76635) was supplied by Santa Cruz Biotechnology (Santa Cruz, USA).

Techniques: Real-time Polymerase Chain Reaction, Expressing, Transfection, CCK-8 Assay

Journal: Journal of Biological Chemistry

Article Title: Benzoquinone, a leukemogenic metabolite of benzene, catalytically inhibits the protein tyrosine phosphatase PTPN2 and alters STAT1 signaling

doi: 10.1074/jbc.ra119.008666

Figure Lengend Snippet: Figure 5: Exposure of HEK293T cells to 1,4-benzoquinone (BQ) leads to PTPN2 inhibition through arylation mechanism. (a) HEK293T cells were transfected with a flag-PTPN2 plasmid and exposed to BQ or H2O2 for 30 min. Cells were lysed and PTPN2 was isolated by immunoprecipitation using an anti PTPN2 antibody. PTPN2 activity of immunoprecipitates was measured by RP-UFLC. Error bars indicate S.D. values. *p<0.05 compared with control (Ctrl). (b) Immunoprecipitated PTPN2 was incubated with IAF for 20 min at room temperature. Immunobeads were separated and analysed by western blot using anti-oxidized PTP (PTPox). (c) Detection of IAF labeling was done by fluorescence. Anti-PTPN2 antibody was used as loading control.

Article Snippet: Cells (4x106 cells) were seeded in a 10 cm dish and transfected with 5 μg of FLAG-tagged human PTPN2 plasmid (Origene, USA) using Metafectene (Biontex, Germany).

Techniques: Inhibition, Transfection, Plasmid Preparation, Isolation, Immunoprecipitation, Activity Assay, Control, Incubation, Western Blot, Labeling, Fluorescence

Journal: Journal of Biological Chemistry

Article Title: Benzoquinone, a leukemogenic metabolite of benzene, catalytically inhibits the protein tyrosine phosphatase PTPN2 and alters STAT1 signaling

doi: 10.1074/jbc.ra119.008666

Figure Lengend Snippet: Figure 6: Exposure of Jurkat cells to 1,4-benzoquinone (BQ) leads to inhibition of endogenous PTPN2 and subsequent over-activation of the STAT1 pathway. (a) After a 30 min treatment with BQ (1 µM) or vehicle (Ctrl)(corresponding to time tN.I.), Jurkat cells were incubated with IFNγ for 20 min (corresponding to time t0). Cells were then washed and cultured in fresh medium to remove IFNγ and harvested every 15 min up to 60 min (corresponding to times t15, t30, t45 and t60). (b) Jurkat cells treated as described in (a) were taken at different times : tN.I. (30 min after BQ treatment), t0, t15, t30, t45 and t45 min (after IFN treatment) were analyzed by Western blot using anti-pSTAT1, anti- STAT1, anti-PTPN2 and anti-tubulin. Quantification of STAT1 phosphorylation in western blots was carried out using ImageJ software. Error bars indicate S.D. values. *p<0.05 compared with control (Ctrl). (c) Jurkat cells treated with 1 µM BQ for 30 min (corresponding to time tN.I.) were washed, lysed and endogenous PTPN2 was isolated by immunoprecipitation using an anti-PTPN2 antibody. PTPN2 activity of immunoprecipitates was measured by RP-UFLC. Error bars indicate S.D. values. *p<0.05 compared with control (Ctrl). (d) Jurkat cells were treated with BQ (1 µM) for 30 min, then incubated or not with IFNγ for 180 min. Relative gene expression of GBP1, IRF1 and APOL1 were analyzed by RT-qPCR. Error bars indicate S.D. values. *p<0.05 compared with control (Ctrl).

Article Snippet: Cells (4x106 cells) were seeded in a 10 cm dish and transfected with 5 μg of FLAG-tagged human PTPN2 plasmid (Origene, USA) using Metafectene (Biontex, Germany).

Techniques: Inhibition, Activation Assay, Incubation, Cell Culture, Western Blot, Phospho-proteomics, Software, Control, Isolation, Immunoprecipitation, Activity Assay, Gene Expression, Quantitative RT-PCR

Journal: JCI Insight

Article Title: The HIV latency reversing agent HODHBt inhibits the phosphatases PTPN1 and PTPN2

doi: 10.1172/jci.insight.179680

Figure Lengend Snippet: ( A ) Compressed CETSA-MS results indicating changes in thermal stability of protein in both PBMCs and K562. The x axis represents amplitude (log 2 fold change), and the y axis represents effect significance (–log 10 [ P value]). Statistically significant ( P < 0.01) proteins in common between PBMCs and K562 cells are indicated. ( B ) Thermal melting curves for PTPN1, PTPN2, CRKL, STAT5, RPL7A, and β-actin in CD4 T cell lysates after treatment with 100 μM HODHBt versus 100 μM HBt ( n = 2–4). ( C ) Purified PTPN1 catalytic domain and purified commercial PTPN2 catalytic domain CETSA after treatment with DMSO, 100 μM HODHBt, or 100 μM HBt ( n = 2–3).

Article Snippet: Fluorogenic PTP1B catalytic domain assay kit (catalog 79764) and recombinant GST-tag TC-PTP (PTPN2, 30013) were purchased from BPSBioscience.

Techniques: Purification

Journal: JCI Insight

Article Title: The HIV latency reversing agent HODHBt inhibits the phosphatases PTPN1 and PTPN2

doi: 10.1172/jci.insight.179680

Figure Lengend Snippet: ( A ) HODHBt directly inhibits the catalytic activity of the catalytic domain of PTPN1and the catalytic domain of PTPN2 using a fluorogenic assay ( n = 2). Data are shown as mean ± SD. IC 50 values calculated for 2 independent experiments. ( B ) Effect of HODHBt on PTPN1- and PTPN2-catalyzed fluorogenic PTPN1 substrate. ( C ) Lineweaver-Burk plots. Lineweaver-Burk plots represent the Michaelis-Menten equation of enzyme kinetics.

Article Snippet: Fluorogenic PTP1B catalytic domain assay kit (catalog 79764) and recombinant GST-tag TC-PTP (PTPN2, 30013) were purchased from BPSBioscience.

Techniques: Activity Assay

Journal: JCI Insight

Article Title: The HIV latency reversing agent HODHBt inhibits the phosphatases PTPN1 and PTPN2

doi: 10.1172/jci.insight.179680

Figure Lengend Snippet: ( A ) Total CD4s were pretreated with 100 μM HODHBt, 10 μM AC-484, and 5 μM AC-484 for 2 hours prior to the addition of IL-2. pSTAT5 levels were measured by flow cytometry 1, 24, and 48 hours after stimulation with IL-2 ( n = 6). Wilcoxon matched-pairs signed-rank test was used to calculate P values (* P < 0.05; ** P < 0.01). ( B ) Total CD4 T cells were pretreated with 100 μM HODHBt or 10 μM AC-484 for 2 hours prior to the addition of IL-15. pSTAT5 levels were measured by flow cytometry 48 hours after stimulation with IL-2 ( n = 3). Paired 2-tailed t test was used to calculate P values (* P < 0.05; ** P < 0.01). ( C and D ) Protein levels of PTPN1, PTPN2, pSTAT5, and STAT5 were measured by Western blot in CD4 T cells pretreated with 100 μM HODHBt or 20 μM AC-484 for 2 hours before the addition of IL-2 ( C ) and IL-15 ( D ) for 24 hours ( n = 3). Paired 2-tailed t test was used to calculate P values (* P < 0.05).

Article Snippet: Fluorogenic PTP1B catalytic domain assay kit (catalog 79764) and recombinant GST-tag TC-PTP (PTPN2, 30013) were purchased from BPSBioscience.

Techniques: Flow Cytometry, Western Blot