Journal: eLife

Article Title: TAK1-mediated phosphorylation of PLCE1 represses PIP2 hydrolysis to impede esophageal squamous cancer metastasis

doi: 10.7554/eLife.97373

Figure Lengend Snippet: ( A–B ) Knockdown of TAK1 by Map3k7 shRNA. ECA-109 cells were transduced with lentivirus bearing Map3k7 shRNA (LV- Map3k7 shRNA) or NC shRNA (LV-NC). 48 hr post-transduction, cells were harvested for analyzing TAK1 expression by quantitative real time-PCR (qRT-PCR) ( A ) and western blot ( B ). n = 3 biologically independent replicates. ( C–E ) Decreased expression of TAK1 facilitates cell migration and invasion. ECA-109 cells were transduced with LV- Map3k7 shRNA or LV-NC. 48 hr post-transduction, cells were subjected to transwell ( C, D ) or wound healing ( E ) assay. n=5 biologically independent replicates. Scale bar = 500 µm ( C ); scale bar = 100 µm ( E ). ( F – G ) Reduced expression of TAK1 increases mesenchymal marker expression and decreases epithelial marker expression. n=3 biologically independent replicates. ( H ) Reduced expression of TAK1 in ECA-109 cells affects epithelial-mesenchymal transition (EMT) related gene expression. Protein levels were analyzed by western blot, and Actin was used as a loading control. Gene expression was detected by qRT-PCR, and Gapdh was used as a house-keeping gene. n=4 biologically independent replicates. Data are presented as mean ± SD. Statistical significance was tested by unpaired Student’s t-test. *p<0.05, **p<0.01, and ***p<0.001. Figure 1—figure supplement 3—source data 1. TAK1 knockdown facilitates esophageal squamous cell carcinoma (ESCC) migration and invasion. Figure 1—figure supplement 3—source data 2. PDF file containing original western blots for , indicating the relevant bands. Figure 1—figure supplement 3—source data 3. Original files for western blot analysis displayed in .

Article Snippet: Rabbit monoclonal antibodies against TAK1 (#5206), phospho-TAK1 (Ser412, #9339), PKCα (#2056), phospho-PKC (pan) (gamma Thr514) (#38938), GSK-3β (#9315), phospho-GSK-3β (Ser9) (#5558), β-Catenin (#8480), phospho-β-Catenin (Ser33/37/Thr41) (#9561), MMP-2 (#87809), mouse monoclonal antibodies against Actin (#3700), Myc-Tag (Sepharose Bead Conjugate) (#55464), Epithelial-Mesenchymal Transition (EMT) antibody sample kit (#9782), phosphor-IKK (#2078), IKK (#61294), phosphor-JNK (#4668), JNK (#9252), phospho-ERK (#4370), ERK (#9102), phospho-P38 MAPK (#9211), P38 MPAK (#9212), and normal rabbit IgG (#2729) were from Cell Signaling Technology (Beverley, MA, USA).

Techniques: Knockdown, shRNA, Transduction, Expressing, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Western Blot, Migration, Marker, Gene Expression, Control

Journal: eLife

Article Title: TAK1-mediated phosphorylation of PLCE1 represses PIP2 hydrolysis to impede esophageal squamous cancer metastasis

doi: 10.7554/eLife.97373

Figure Lengend Snippet: ( A–B ) TAK1 expression was decreased by Map3k7 gRNA in ECA-109 cells. TAK1 knockout was achieved by CRISPR-Cas9. (B–D) Knockout of TAK1 expression in ECA-109 cells accelerates cell migration and invasion as analyzed by transwell ( B, C ) and wound healing ( D ) assays. Scale bar = 500 µm ( B ); scale bar = 100 µm ( D ). ( E – F ) Loss of TAK1 increases mesenchymal protein marker expression and reduces epithelial protein marker expression. ECA-109 cells were treated with Map3k7 gRNA, and then cells were harvested for western blot analysis. n=3 biologically independent replicates. ( G ) Knockout of TAK1 expression in ECA-109 cells alters epithelial-mesenchymal transition (EMT) related gene expression as analyzed by quantitative real time-PCR (qRT-PCR). Gapdh was used as a house-keeping gene. n=4 biologically independent replicates. Protein levels were analyzed by western blot, and Actin was used as a loading control. Data are presented as mean ± SD. Statistical significance was tested by unpaired Student’s t-test. *p<0.05, **p<0.01, and ***p<0.001. Figure 1—figure supplement 4—source data 1. TAK1 knockout accelerates esophageal squamous cell carcinoma (ESCC) migration and invasion. Figure 1—figure supplement 4—source data 2. PDF file containing original western blots for , indicating the relevant bands. Figure 1—figure supplement 4—source data 3. Original files for western blot analysis displayed in .

Article Snippet: Rabbit monoclonal antibodies against TAK1 (#5206), phospho-TAK1 (Ser412, #9339), PKCα (#2056), phospho-PKC (pan) (gamma Thr514) (#38938), GSK-3β (#9315), phospho-GSK-3β (Ser9) (#5558), β-Catenin (#8480), phospho-β-Catenin (Ser33/37/Thr41) (#9561), MMP-2 (#87809), mouse monoclonal antibodies against Actin (#3700), Myc-Tag (Sepharose Bead Conjugate) (#55464), Epithelial-Mesenchymal Transition (EMT) antibody sample kit (#9782), phosphor-IKK (#2078), IKK (#61294), phosphor-JNK (#4668), JNK (#9252), phospho-ERK (#4370), ERK (#9102), phospho-P38 MAPK (#9211), P38 MPAK (#9212), and normal rabbit IgG (#2729) were from Cell Signaling Technology (Beverley, MA, USA).

Techniques: Expressing, Knock-Out, CRISPR, Migration, Marker, Western Blot, Gene Expression, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Control

Journal: eLife

Article Title: TAK1-mediated phosphorylation of PLCE1 represses PIP2 hydrolysis to impede esophageal squamous cancer metastasis

doi: 10.7554/eLife.97373

Figure Lengend Snippet: ( A–D ) IP3R blocking inhibits PLCE1 promoted cell migration and invasion. ECA-109 cells were transfected with the plasmid expressing Plce1 for 6 hr and then cells were treated with 2-APB (10 µM) for additional 18 hr. Cell migration and invasion were analyzed by transwell ( A, B ) or wound healing ( C, D ) assay. n=5 biologically independent replicates. Scale bar = 500 µm ( A ) and 100 µm ( C ). (E) IP3R blocking counteracts PLCE1-induced changes in epithelial-mesenchymal transition (EMT) gene expression. The levels of mRNA were analyzed by quantitative real time-PCR (qRT-PCR), and Gapdh was used as a house-keeping gene. n=3 biologically independent replicates. Data are presented as mean ± SD. Statistical significance was tested by two-tailed one-way ANOVA test. * p<0.05, **p<0.01 , and *** p < 0.001. Figure 5—figure supplement 2—source data 1. 2-APB treatment counteracts PLCE1-induced cell migration.

Article Snippet: Rabbit monoclonal antibodies against TAK1 (#5206), phospho-TAK1 (Ser412, #9339), PKCα (#2056), phospho-PKC (pan) (gamma Thr514) (#38938), GSK-3β (#9315), phospho-GSK-3β (Ser9) (#5558), β-Catenin (#8480), phospho-β-Catenin (Ser33/37/Thr41) (#9561), MMP-2 (#87809), mouse monoclonal antibodies against Actin (#3700), Myc-Tag (Sepharose Bead Conjugate) (#55464), Epithelial-Mesenchymal Transition (EMT) antibody sample kit (#9782), phosphor-IKK (#2078), IKK (#61294), phosphor-JNK (#4668), JNK (#9252), phospho-ERK (#4370), ERK (#9102), phospho-P38 MAPK (#9211), P38 MPAK (#9212), and normal rabbit IgG (#2729) were from Cell Signaling Technology (Beverley, MA, USA).

Techniques: Blocking Assay, Migration, Transfection, Plasmid Preparation, Expressing, Gene Expression, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Two Tailed Test

Journal: eLife

Article Title: TAK1-mediated phosphorylation of PLCE1 represses PIP2 hydrolysis to impede esophageal squamous cancer metastasis

doi: 10.7554/eLife.97373

Figure Lengend Snippet: Cells were transfected with plasmid expressing Plce1 for 6 hr and then treated with 2-APB (10 µM) for additional 18 hr. ( A–B ) Transwell assay showing the application of 2-APB attenuates cell migration and invasion induced by PLCE1. Scale bar = 500 µm. n=5 biologically independent replicates. ( C–D ) Wound healing assay showing the treatment of 2-APB represses cell migration induced by PLCE1. Scale bar = 100 µm. n=5 biologically independent replicates. ( E ) 2-APB counteracts PLCE1-induced changes in epithelial-mesenchymal transition (EMT) gene expression. The mRNA levels were detected by quantitative real time-PCR (qRT-PCR), and Gapdh was used as a house-keeping gene. n=3 biologically independent replicates. Data are presented as mean ± SD. Statistical significance was tested by two-tailed one-way ANOVA test. * p < 0.05, ** p<0.01, and *** p<0.001. Figure 5—figure supplement 3—source data 1. IP3R inhibition represses PLCE1-stimulated cell migration and invasion in KYSE-150 cells.

Article Snippet: Rabbit monoclonal antibodies against TAK1 (#5206), phospho-TAK1 (Ser412, #9339), PKCα (#2056), phospho-PKC (pan) (gamma Thr514) (#38938), GSK-3β (#9315), phospho-GSK-3β (Ser9) (#5558), β-Catenin (#8480), phospho-β-Catenin (Ser33/37/Thr41) (#9561), MMP-2 (#87809), mouse monoclonal antibodies against Actin (#3700), Myc-Tag (Sepharose Bead Conjugate) (#55464), Epithelial-Mesenchymal Transition (EMT) antibody sample kit (#9782), phosphor-IKK (#2078), IKK (#61294), phosphor-JNK (#4668), JNK (#9252), phospho-ERK (#4370), ERK (#9102), phospho-P38 MAPK (#9211), P38 MPAK (#9212), and normal rabbit IgG (#2729) were from Cell Signaling Technology (Beverley, MA, USA).

Techniques: Transfection, Plasmid Preparation, Expressing, Transwell Assay, Migration, Wound Healing Assay, Gene Expression, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Two Tailed Test, Inhibition

Journal: eLife

Article Title: TAK1-mediated phosphorylation of PLCE1 represses PIP2 hydrolysis to impede esophageal squamous cancer metastasis

doi: 10.7554/eLife.97373

Figure Lengend Snippet: Cells were transfected with plasmid expressing Plce1 for 6 hr and then treated with 2-APB (10 µM) for additional 18 hr. ( A–D ) Cell migration and invasion induced by PLCE1 were repressed by the application of 2-APB in TE-1 cells. Cell migration and invasion were analyzed by transwell assay (A–B, scale bar = 500 µm) and wound healing assay (C–D, scale bar = 100 µm). n=5 biologically independent replicates. ( E ) 2-APB abolishes PLCE1-induced changes in epithelial-mesenchymal transition (EMT) gene expression in TE-1 cells. Gene expression was analyzed by quantitative real time-PCR (qRT-PCR), and Gapdh was used as a house-keeping gene. n=3 biologically independent replicates. Data are presented as mean ± SD. Statistical significance was tested by two-tailed one-way ANOVA test. ** p<0.01 and *** p<0.001 . Figure 5—figure supplement 4—source data 1. IP3R inhibition reduces PLCE1-stimulated cell migration and invasion in TE-1 cells.

Article Snippet: Rabbit monoclonal antibodies against TAK1 (#5206), phospho-TAK1 (Ser412, #9339), PKCα (#2056), phospho-PKC (pan) (gamma Thr514) (#38938), GSK-3β (#9315), phospho-GSK-3β (Ser9) (#5558), β-Catenin (#8480), phospho-β-Catenin (Ser33/37/Thr41) (#9561), MMP-2 (#87809), mouse monoclonal antibodies against Actin (#3700), Myc-Tag (Sepharose Bead Conjugate) (#55464), Epithelial-Mesenchymal Transition (EMT) antibody sample kit (#9782), phosphor-IKK (#2078), IKK (#61294), phosphor-JNK (#4668), JNK (#9252), phospho-ERK (#4370), ERK (#9102), phospho-P38 MAPK (#9211), P38 MPAK (#9212), and normal rabbit IgG (#2729) were from Cell Signaling Technology (Beverley, MA, USA).

Techniques: Transfection, Plasmid Preparation, Expressing, Migration, Transwell Assay, Wound Healing Assay, Gene Expression, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Two Tailed Test, Inhibition

Journal: eLife

Article Title: TAK1-mediated phosphorylation of PLCE1 represses PIP2 hydrolysis to impede esophageal squamous cancer metastasis

doi: 10.7554/eLife.97373

Figure Lengend Snippet:

Article Snippet: Rabbit monoclonal antibodies against TAK1 (#5206), phospho-TAK1 (Ser412, #9339), PKCα (#2056), phospho-PKC (pan) (gamma Thr514) (#38938), GSK-3β (#9315), phospho-GSK-3β (Ser9) (#5558), β-Catenin (#8480), phospho-β-Catenin (Ser33/37/Thr41) (#9561), MMP-2 (#87809), mouse monoclonal antibodies against Actin (#3700), Myc-Tag (Sepharose Bead Conjugate) (#55464), Epithelial-Mesenchymal Transition (EMT) antibody sample kit (#9782), phosphor-IKK (#2078), IKK (#61294), phosphor-JNK (#4668), JNK (#9252), phospho-ERK (#4370), ERK (#9102), phospho-P38 MAPK (#9211), P38 MPAK (#9212), and normal rabbit IgG (#2729) were from Cell Signaling Technology (Beverley, MA, USA).

Techniques: Transfection, Construct, shRNA, Recombinant, Plasmid Preparation, Sequencing, Activity Assay, Enzyme-linked Immunosorbent Assay, Software

Journal: Cancer Reports

Article Title: Transmembrane Protein TMEM59L Modulates 5‐ FU Resistance via PTPRN ‐Mediated DNA Damage Repair in Colorectal Cancer

doi: 10.1002/cnr2.70448

Figure Lengend Snippet: TMEM59L regulates colorectal cancer cells proliferation, migration, and invasion. (A) Western blotting confirmed expression of TMEM59L in different CRC cell lines. (B) Western blotting detects the knockdown of TMEM59L by shRNA and overexpression of TMEM59L by plasmid. (C) Downregulation of TMEM59L suppresses cell proliferation in HCT116 cells; overexpression of TMEM59L in SW480 promotes cell proliferation. (D) The function of TMEM59L on the migration and invasion ability of CRC cells was detected by Transwell assay. (E) E‐cadherin and Vimentin were evaluated through immunofluorescence staining in TMEM59L knockdown and overexpression CRC cells.

Article Snippet: Non‐specific binding sites were blocked with 3% BSA prior to overnight incubation at 4°C with primary antibodies targeting DNA damage marker p‐γ‐H2AX (ab81299, Abcam) and EMT regulators E‐cadherin (20874‐1‐AP)/Vimentin (10366‐1‐AP, Proteintech).

Techniques: Migration, Western Blot, Expressing, Knockdown, shRNA, Over Expression, Plasmid Preparation, Transwell Assay, Immunofluorescence, Staining

Journal: Cancer Reports

Article Title: Transmembrane Protein TMEM59L Modulates 5‐ FU Resistance via PTPRN ‐Mediated DNA Damage Repair in Colorectal Cancer

doi: 10.1002/cnr2.70448

Figure Lengend Snippet: The effect of TMEM59L and PTPRN on DNA damage, apoptosis, stemness and EMT in vivo. (A) Images of Xenograft tumors from HCT116/FU cells. (B) Average tumor volumes are measured in xenograft mice every 2 days. (C) Representative images of γ‐H2AX, TUNEL, KI67, CD133, E‐cadherin and Vimentin in tumor tissues.

Article Snippet: Non‐specific binding sites were blocked with 3% BSA prior to overnight incubation at 4°C with primary antibodies targeting DNA damage marker p‐γ‐H2AX (ab81299, Abcam) and EMT regulators E‐cadherin (20874‐1‐AP)/Vimentin (10366‐1‐AP, Proteintech).

Techniques: In Vivo, TUNEL Assay

Journal: Frontiers in Immunology

Article Title: TOX Acts as a Tumor Suppressor by Inhibiting mTOR Signaling in Colorectal Cancer

doi: 10.3389/fimmu.2021.647540

Figure Lengend Snippet: TOX represses the EMT process. IHC shows assay E-cadherin (A) and vimentin (B) expression in CRC and para-CRC tissues ( n = 40). (C) Western blot shows EMT-related proteins ZEB1, E-cadherin, vimentin, and Snail expression in PLVX-Flag-TOX and PLVX-Flag SW1116 cells. (D) Western blot assay shows ZEB1, E-cadherin, vimentin, and Snail in shCK HCT116, shTOX-1 (sh1), and shTOX-2 (sh2) HCT116 cells. (E) qRT-PCR shows ZEB1, E-cadherin, vimentin, and Snail expression in PLVX-Flag-TOX, PLVX-Flag SW1116, shCK HCT116, shTOX-1 (sh1), and shTOX-2 (sh2) HCT116 cells. Each experiment was repeated three times. * p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: Resolved proteins were transferred to polyvinylidene fluoride membranes and incubated with antibodies against TOX (#ab155768, 1:1,000; Abcam, USA), EMT markers (E-cadherin #3195, Snail #3879, and vimentin #5741, 1:1,000, CST, USA; ZEB1 #21544-1-AP, 1:1000 Proteintech, China), and PI3K/AKT/mTOR pathway molecules (p-PI3K #4228, PI3K #4249, p-AKT, AKT, mTOR #2983, p-mTOR #2974, 1:1,000; CST, USA).

Techniques: Expressing, Western Blot, Quantitative RT-PCR

Journal: Frontiers in Immunology

Article Title: TOX Acts as a Tumor Suppressor by Inhibiting mTOR Signaling in Colorectal Cancer

doi: 10.3389/fimmu.2021.647540

Figure Lengend Snippet: TOX acts as a tumor suppressor by inhibiting mTOR signaling in colorectal cancer. TOX acts as a CRC suppressor with decreasing expression and longer OS. TOX partly suppressed the mTOR signaling to inhibit cell proliferation, migration, invasion, and EMT process. Rapamycin alone or combined with PD1 inhibitor has therapeutic potential in CRC.

Article Snippet: Resolved proteins were transferred to polyvinylidene fluoride membranes and incubated with antibodies against TOX (#ab155768, 1:1,000; Abcam, USA), EMT markers (E-cadherin #3195, Snail #3879, and vimentin #5741, 1:1,000, CST, USA; ZEB1 #21544-1-AP, 1:1000 Proteintech, China), and PI3K/AKT/mTOR pathway molecules (p-PI3K #4228, PI3K #4249, p-AKT, AKT, mTOR #2983, p-mTOR #2974, 1:1,000; CST, USA).

Techniques: Expressing, Migration

Journal: Scientific Reports

Article Title: Dynamic changes of phenotypically different circulating tumor cells sub-populations in patients with recurrent/refractory small cell lung cancer treated with pazopanib

doi: 10.1038/s41598-018-20502-1

Figure Lengend Snippet: CK + /Ki67 + ( a ), CK + /M30 + ( b ) and CK + /Vim + ( c ) CTCs by double immunofluorescense staining.

Article Snippet: The presence of CTCs in PBMCs’ cytospins was investigated using monoclonal antibodies against Ki67 (a proliferation marker; Abcam, Cambridge, UK), M30 (an apoptosis marker; CytoDEATH fluorescein, Roche, Manheim, Germany) and Vimentin (an EMT marker; Santa Cruz, Santa Cruz, CA, USA).

Techniques: Staining