Journal: Cancers

Article Title: Metallothionein 2A Expression in Cancer-Associated Fibroblasts and Cancer Cells Promotes Esophageal Squamous Cell Carcinoma Progression

doi: 10.3390/cancers13184552

Figure Lengend Snippet: The high expression of MT2A in ESCC cells promoted their growth, migration, and invasiveness, possibly due to the downregulation of E-cadherin. ( A ) Expression of MT2A mRNA in five ESCC cell lines, TE-8, TE-9, TE-10, TE-11, and TE-15, evaluated by RT-PCR. GAPDH was used as the internal control. ( B ) Western blotting to confirm the expression of MT2A protein in the five ESCC cell lines. ( C , D ) TE-10 and TE-11 were transfected with siRNA against MT2A (siMT2A) and control siRNA (siNC). MT2A knockdown was confirmed by qRT-PCR ( C ) and Western blotting ( D ) in TE-10 and TE-11. After Western blotting, the normalized relative expression fold-change was calculated using the ImageJ software, and the values were arbitrarily set as 1.00 for the cells transfected with siNC. ( E ) Changes in the cell morphology of ESCC cell lines transfected with siNC or siMT2A under a phase contrast microscope. ( F ) Western blotting for expression levels of E-cadherin and phosphorylation levels of β-catenin in siMT2A-treated TE-10 and TE-11 cells, compared with those in cells treated with siNC. The normalized relative expression fold-change was calculated using the ImageJ software, and the values were arbitrarily set as 1.00 for cells transfected with siNC. ( G ) MTS assay for cell growth in TE-10 and TE-11 cells treated with siMT2A or siNC. Each ESCC cell line transfected with siNC and siMT2A was seeded in 96-well plates at 5 × 10 3 cells per well with RPMI-1640 + 1% FBS. The cell growth was evaluated after 24, 48, and 72 h. ( H , I ) Transwell migration ( H ) and invasion ( I ) assay of ESCC cell lines transfected with siNC and siMT2A. ( J ) Double immunofluorescence was performed using anti-β-catenin (green) and anti-E-cadherin (red) antibodies in the ESCC cell lines transfected with siNC and siMT2A. Cell nuclei were stained with DAPI (blue). Data are presented as mean ± SEM (* p < 0.05, ** p < 0.01, *** p < 0.001).

Article Snippet: The rest were all obtained from Cell Signaling Technology (Beverly, MA, USA): rabbit IGFBP2 (#3922), rabbit phosphorylated Akt (Ser473, #4060), rabbit phosphorylated Akt (Thr308, #2965), rabbit total Akt (#9272), rabbit phosphorylated Erk1/2 (Thr202/Tyr204, #9101), rabbit total Erk1/2 (#9102), rabbit phosphorylated NFκB (#3033), rabbit total NFκB (#8242), rabbit E-cadherin (#3195), rabbit β-catenin (#8480), rabbit phosphorylated β-catenin (Ser33/Ser37/Thr41, #9561 and Thr41/Ser45, #9565), and rabbit β-actin (#4970) antibodies.

Techniques: Expressing, Migration, Reverse Transcription Polymerase Chain Reaction, Western Blot, Transfection, Quantitative RT-PCR, Software, Microscopy, MTS Assay, Immunofluorescence, Staining

Journal: Cell reports

Article Title: ALK upregulates POSTN and WNT signaling to drive neuroblastoma

doi: 10.1016/j.celrep.2024.113927

Figure Lengend Snippet: (A) MYCN and ALK / MYCN tumor lines were analyzed by western blot for expression of POSTN, active form of β-catenin (non-phosphorylated at S33/S37/T41), active SMAD2 (phosphorylated at S465/467), and inactive YAP (phosphorylated at S127) expression and WNT signaling. (B and C) ALK / MYCN with control sgRNA or POSTN sgRNA tumor lines were analyzed by western blot for (B) activation of WNT and YAP signaling and (C) activation of FAK and inhibition of GSK3β. (D) ALK / MYCN tumor cells were treated with or without the FAK inhibitor defactinib (2 μM) for 48 h and analyzed by western blot for FAK and GSK3β activity. See also – .

Article Snippet: Primary antibodies for western blot were obtained commercially for POSTN (Thermo Fisher, 1:1000), FN1 (Cell Signaling Technology, 1:1000), GAPDH (Sigma Aldrich, 1:10000), FLAG (Sigma Aldrich, 1:2000), ALK (Cell Signaling Technology, 1:1000), a-tubulin (Cell Signaling Technologies, 1:1000), FAK (Abcam, 1:1000), pFAK Y397 (Abcam, 1:1000), GSK3β (Cell Signaling Technology, 1:1000), pGSK3β S9 (Cell Signaling Technology, 1:1000), β-catenin (Cell Signaling Technology, 1:1000), non-phosphorylated β-catenin (Cell Signaling Technology, 1:1000), SMAD2 (Cell Signaling Technology, 1:1000), pSMAD2/3 (Cell Signaling Technology, 1:1000), YAP (Santa Cruz, 1:1000), pYAP (Cell Signaling Technology, 1:1000).

Techniques: Western Blot, Expressing, Control, Activation Assay, Inhibition, Activity Assay

Journal: Cell reports

Article Title: ALK upregulates POSTN and WNT signaling to drive neuroblastoma

doi: 10.1016/j.celrep.2024.113927

Figure Lengend Snippet: (A) ALK / MYCN tumor cells are treated with DMSO or 10 μM WNT-C59 for 24 h and analyzed by western blot for POSTN , non-phosphorylated β-catenin, and β-catenin. (B) MYCN tumor cells were treated with DMSO or 3 μM CHIR99021 for 24 h and analyzed by western blot for POSTN , non-phosphorylated β-catenin, and β-catenin. (C) MYCN and ALK / MYCN tumor cells were treated with DMSO or 10 μM WNT C59 for 48 h and analyzed for EdU incorporation. *p < 0.05, n = 3, error bars represent standard error mean. (D) ALK / MYCN tumor cells with POSTN sgRNA were treated with DMSO or 3 μM CHIR99021 for 24 h and analyzed for EdU incorporation. *p < 0.05, n = 3, error bars represent standard error mean. See also .

Article Snippet: Primary antibodies for western blot were obtained commercially for POSTN (Thermo Fisher, 1:1000), FN1 (Cell Signaling Technology, 1:1000), GAPDH (Sigma Aldrich, 1:10000), FLAG (Sigma Aldrich, 1:2000), ALK (Cell Signaling Technology, 1:1000), a-tubulin (Cell Signaling Technologies, 1:1000), FAK (Abcam, 1:1000), pFAK Y397 (Abcam, 1:1000), GSK3β (Cell Signaling Technology, 1:1000), pGSK3β S9 (Cell Signaling Technology, 1:1000), β-catenin (Cell Signaling Technology, 1:1000), non-phosphorylated β-catenin (Cell Signaling Technology, 1:1000), SMAD2 (Cell Signaling Technology, 1:1000), pSMAD2/3 (Cell Signaling Technology, 1:1000), YAP (Santa Cruz, 1:1000), pYAP (Cell Signaling Technology, 1:1000).

Techniques: Western Blot

Journal: Cell reports

Article Title: ALK upregulates POSTN and WNT signaling to drive neuroblastoma

doi: 10.1016/j.celrep.2024.113927

Figure Lengend Snippet:

Article Snippet: Primary antibodies for western blot were obtained commercially for POSTN (Thermo Fisher, 1:1000), FN1 (Cell Signaling Technology, 1:1000), GAPDH (Sigma Aldrich, 1:10000), FLAG (Sigma Aldrich, 1:2000), ALK (Cell Signaling Technology, 1:1000), a-tubulin (Cell Signaling Technologies, 1:1000), FAK (Abcam, 1:1000), pFAK Y397 (Abcam, 1:1000), GSK3β (Cell Signaling Technology, 1:1000), pGSK3β S9 (Cell Signaling Technology, 1:1000), β-catenin (Cell Signaling Technology, 1:1000), non-phosphorylated β-catenin (Cell Signaling Technology, 1:1000), SMAD2 (Cell Signaling Technology, 1:1000), pSMAD2/3 (Cell Signaling Technology, 1:1000), YAP (Santa Cruz, 1:1000), pYAP (Cell Signaling Technology, 1:1000).

Techniques: Recombinant, Membrane, Saline, Western Blot, Blocking Assay, Staining, SYBR Green Assay, Bicinchoninic Acid Protein Assay, Flow Cytometry, Plasmid Preparation, Software