third generation packaging combination pmdlg prre  (Addgene inc)

Journal: Human Genetics and Genomics Advances

Article Title: Bi-allelic variants in BCAT1 impair mitochondrial function and are associated with a candidate neurometabolic disorder

doi: 10.1016/j.xhgg.2025.100525

Figure Lengend Snippet: Loss of BCAT1 impacts hiPSC-derived cortical neuron differentiation (A) Schematic of hiPSC lentiviral transduction for Ngn2-iN and experimental design. (B) Representative western blot analysis of BCAT1 E/K F/L and BCAT1 −/− Ngn2-iNs collected at day 2 and day 14 of differentiation (left). Quantification of vinculin expression in day 14 neurons (right). Results are mean ± SEM ( n = 3 independent experiments per group in all 3 clonal lines) and analyzed by one-way ANOVA with Dunnett’s post hoc test. (C) qRT-PCR analysis of BCAT1 , BCAT2 , and NeuroD1 transcript levels in day 14 Ngn2-iNs normalized to RPLP0 . Results represented as means ± SEM ( n ≥ 3 independent experiments per group in all 3 clonal lines), analyzed by two-way ANOVA with Dunnett’s post hoc analysis test for multiple comparisons. (D) Representative confocal images and (E) fluorescence intensity measurements of developing neurons 2, 7, and 14 days post-differentiation (dpd) showing increased Tuj1 and Map2 expression. Scale bars, 100 μm. Immunocytochemistry was performed in all three clonal lines and two cell culture replicates per time point, with similar results. (F) Reconstructions of sparse-labeled Ngn2-iNs derived from BCAT1 E/K F/L and isogenic control lines. Experiments were performed in all two cell culture replicates per time point. Results are shown in (G). BCAT1 neurons exhibit reduced growth properties and were significantly shorter at 2 dpd (left) compared with 7 dpd (right) (two-way ANOVA with Sidak correction; 2 dpd: ∗∗ p < 0.005; 7 dpi: n.s., not significant). Values show means ± SEM.; BCAT1 E/K F/L ( n = 1; 33 technical tracing replicates in total); control ( n = 1; 29 technical tracing replicates in total); n refers to biologically independent clonal lines. A.U., arbitrary units.

Article Snippet: Ngn2-iN lentiviruses were produced as previously described in HEK293T cells (ATCC, VA) using polyethylenimine (Polysciences, PEI, 23966-2) and third-generation lentiviral packaging plasmids pMDLg/pRRE (Addgene, 12251), pRSV-RV (Addgene, 12253), and VSC-G (Addgene, 12259), along with pLVX-UbC-rtTA-Ngn2:2A (Addgene, 127288).

Techniques: Derivative Assay, Transduction, Western Blot, Expressing, Quantitative RT-PCR, Fluorescence, Immunocytochemistry, Cell Culture, Labeling, Control

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