third generation lentivirus packaging system pmdlg prre  (Addgene inc)

Journal: Cell Death & Disease

Article Title: IFIT3 promotes lymph node metastasis by interacting with LASP1 to activate FAK-ERK signaling in esophageal squamous cell carcinoma

doi: 10.1038/s41419-025-08327-z

Figure Lengend Snippet: A The IFIT3 protein levels were examined by western blotting in KYSE30 and KYSE150 cells after transfection with shRNA-IFIT3 lentivirus (sh-IFIT3-#1 and sh-IFIT3-#2). The impact of silencing IFIT3 on the invasive and migratory capabilities of KYSE30 ( B ) and KYSE150 ( C ) cells was determined via a Transwell assay. The right panel illustrates the quantification of the invaded cells (scale bar, 250 μm, n = 3). D A time-limited fibronectin adhesion assay was implemented to quantify the level of FA formation in IFIT3-knockdown KYSE30 and KYSE150 cells. The right panel displays a statistical study of adherent cell quantity (scale bar, 250 μm, n = 3). E The E-cadherin and N-cadherin expression levels in KYSE30 and KYSE150 cells were analyzed using western blotting after silencing IFIT3. F Schematic representation of the protocol used to create a nude mouse model of popliteal lymph node metastasis and a representative image of the popliteal lymph node metastasis model. G Bioluminescence images of popliteal LN metastases following IFIT3 silencing using shRNA-IFIT3 lentivirus. H Percentage of LN metastases in all groups ( n = 8). Representative images of popliteal lymph nodes ( I ) and statistical analysis ( J ) of the lymph node volume of all groups ( n = 8). K Representative images of HE staining of popliteal lymph nodes in all groups. Note: black scale bar, 500 μm, red scale bar, 26 μm; black arrows indicate metastasized tumor cells, and red arrows indicate normal cells. Data information: Graphs report the mean ± SD. C , D , J Student’s t-test; H Fisher’s exact test. *** p < 0.001; ** p < 0.01; * p < 0.05.

Article Snippet: ESCC cell lines with IFIT3 and LASP1 overexpressed or knocked down were established using the third-generation lentivirus packaging system pMDLg/pRRE (Addgene, Cat no. #12251), pRSV-Rev (Addgene, Cat no. #12253), and pMD2.

Techniques: Western Blot, Transfection, shRNA, Transwell Assay, Cell Adhesion Assay, Knockdown, Expressing, Staining

Journal: Cell Death & Disease

Article Title: IFIT3 promotes lymph node metastasis by interacting with LASP1 to activate FAK-ERK signaling in esophageal squamous cell carcinoma

doi: 10.1038/s41419-025-08327-z

Figure Lengend Snippet: A Differentially expressed protein levels in ESCC cells overexpressing IFIT3 were quantified by label-free proteomics and are presented in a volcano plot. The ERK, p-ERK, FAK, and p-FAK (Tyr397) protein levels were analyzed in ESCC cells with IFIT3 overexpression ( B ) or knockdown ( C ) using western blotting. D , E The invasion and migration ability of KYSE410 and KYSE150shIFIT3 cells was determined via a Transwell assay after co-treatment with IFIT3-overexpressing lentivirus and defactinib. E provides the quantification of invasive cells (scale bar, 250 μm, n = 3). F , G FA formation levels in KYSE410 and KYSE150shIFIT3 cells were quantified by a time-limited fibronectin adhesion assay after co-treatment with IFIT3-overexpressing lentivirus and defactinib. G The statistical analysis of adherent cell quantity (scale bar, 250 μm, n = 3). H The p-FAK, FAK, p-ERK, ERK, E-cadherin, and N-cadherin protein levels in KYSE410 and KYSE150shIFIT3 cells were measured by western blotting after co-treatment with IFIT3-overexpressing lentivirus and defactinib. I , J The invasion and migratory ability of KYSE410 and KYSE150shIFIT3 cells were assessed via a Transwell assay after co-treatment with IFIT3-overexpressing lentivirus and U0126. Quantification of invasive cells is displayed in ( J ) (scale bar, 250 μm, n = 3). K , L FA formation levels in KYSE410 and KYSE150shIFIT3 cells were quantified by a time-limited fibronectin adhesion assay after co-treatment with IFIT3-overexpressing lentivirus and U0126. L The statistical analysis of adherent cell count (scale bar 250 μm, n = 3). M The p-FAK, FAK, p-ERK, ERK, E-cadherin, and N-cadherin protein levels in KYSE410 and KYSE150shIFIT3 cells were measured by western blotting after co-treatment with IFIT3-overexpressing lentivirus and U0126. Data information: Graphs report the mean ± SD. E , G , J , L one-way ANOVA. *** p < 0.001; ** p < 0.01; * p < 0.05.

Article Snippet: ESCC cell lines with IFIT3 and LASP1 overexpressed or knocked down were established using the third-generation lentivirus packaging system pMDLg/pRRE (Addgene, Cat no. #12251), pRSV-Rev (Addgene, Cat no. #12253), and pMD2.

Techniques: Over Expression, Knockdown, Western Blot, Migration, Transwell Assay, Cell Adhesion Assay, Cell Characterization