plex307 vector  (Addgene inc)

Journal: EMBO Molecular Medicine

Article Title: DUSP6 inhibition overcomes neuregulin/HER3-driven therapy tolerance in HER2+ breast cancer

doi: 10.1038/s44321-024-00088-0

Figure Lengend Snippet: ( A ) Development of lapatinib resistance in HER2+ breast cancer cells. BT474 and BT474Br cells were treated with 1 μM of lapatinib for 9 days (d), 6 months (mos) and 9 mos to yield drug-tolerant persister (DTP), drug-tolerant expanding perister (DTEP), and long-term resistant (LR) clones, respectively. The cells were stained with crystal violet (0.5% w/v) and the images were acquired with an inverted microscope. The transcriptional profiles from each functional state of lapatinib drug tolerance and resistance development from three parallel BT474 cell plates were surveyed by RNA-sequencing. ( B ) The number of the genes with a significant change in their expression during the resistance acquisition (see Dataset for individual gene names). Transcription factor (TF) binding motifs significantly enriched in significantly regulated genes in each transition are indicated. Bolding indicates shared TF binding sites between DTP downregulated and DTEP upregulated genes. ( C ) Differentially expressed pathways were identified using the R package limma and hallmark gene sets were used for GSVA analysis to reveal hallmarks and signal transduction pathways involved in each step of the resistance acquisition. Red indicates those hallmarks and pathways that are overlapping with processes regulated by DUSP6 depletion in Fig. . .

Article Snippet: To rescue the DUSP6 KO MDA-MB-453 clones, the pLEX307-hygro-DUSP6 lentiviral vector was co-transfected with virus-packaging plasmids pMLDg/pRRE (Addgene #12251), pMD2.G (Addgene #12259), and pRSV-Rev (Addgene #12253) into HEK293T cells using FuGENE6 transfection reagent.

Techniques: Clone Assay, Staining, Inverted Microscopy, Functional Assay, RNA Sequencing Assay, Expressing, Binding Assay, Transduction

Journal: EMBO Molecular Medicine

Article Title: DUSP6 inhibition overcomes neuregulin/HER3-driven therapy tolerance in HER2+ breast cancer

doi: 10.1038/s44321-024-00088-0

Figure Lengend Snippet: ( A ) Expression of DUSP6 protein in different stages of lapatinib resistance development by Western blot analysis. ( B ) Relative expression of DUSP6 mRNA in indicated cell lines either at the untreated control situation, or in the DTP state after following treatments: BT474Br, Lapatinib 1 μM for 9 days; EFM192A and HCC1419, Lapatinib 2.5 μM for 14 days; HCC827 (EGFRmut NSCLC), 1 µM Osimertinib for 10 days; A375(BRAFV600E mutant malignant melanoma (MM)), 1 µM dabrafenib+100 nM trametinib for 10 days; and HT-29 (BRAFV600E mutant colorectal cancer (CRC)), 1 µM dabrafenib+10 µg/ml cetuximab for 10 days. Data for EFM192 and HCC1419 cells was obtained from Dataref: (Chang et al, ) (GSE155342), and for other cells by qRT-PCR analysis of the de novo treated samples. Shown is data from two-three repeat samples. ( C ) Transcriptional profile of MDA-MB-453 cells after DUSP6 knockdown by 3 different siRNA and compared with 3 different scramble controls, followed by the GSVA analysis of the Hallmark gene sets. The Hallmark gene sets overlapping with the gene sets regulated to same direction in DUSP6 low expressing DTEP cells (Fig. ) are indicated with red. ( D , E ) Ectopic overexpression of DUSP6 in BT474 cells inhibits lapatinib effects on cell viability ( D ) and apoptosis ( E ), as measured by WST1 cell viability assay and caspase 3/7 activity, respectively. Data was analyzed by two-way ANOVA followed by Tukey’ post hoc test. Statistically significant values of ** p < 0.01 and **** p < 0.0001 were determined. n = 3. ( F ) Ectopic overexpression of either wild-type DUSP6 (DUSPWT) or ERK binding deficient KIM mutant of DUSP6 (DUSP6MUT) in BT474 cells inhibits lapatinib effects on apoptosis. Shown is a result from a representative experiment from three repeats with similar results. Data were analyzed by two-way ANOVA followed by Tukey’ post hoc test. Statistically significant values of ** p < 0.01 and **** p < 0.0001 were determined. ( G ) DUSP6 inhibitor BCI preempts DTEP development in BT474 cells treated with either lapatinib or neratinib for 6 months. The cells were stained and fixed with crystal violet in methanol (0.5% w/v) and the images were acquired with an inverted microscope. .

Article Snippet: To rescue the DUSP6 KO MDA-MB-453 clones, the pLEX307-hygro-DUSP6 lentiviral vector was co-transfected with virus-packaging plasmids pMLDg/pRRE (Addgene #12251), pMD2.G (Addgene #12259), and pRSV-Rev (Addgene #12253) into HEK293T cells using FuGENE6 transfection reagent.

Techniques: Expressing, Western Blot, Control, Mutagenesis, Quantitative RT-PCR, Knockdown, Over Expression, Viability Assay, Activity Assay, Binding Assay, Staining, Inverted Microscopy

Journal: EMBO Molecular Medicine

Article Title: DUSP6 inhibition overcomes neuregulin/HER3-driven therapy tolerance in HER2+ breast cancer

doi: 10.1038/s44321-024-00088-0

Figure Lengend Snippet: ( A , B ) Volcano plots visualizing differentially expressed genes in ( A ) Control-DTP and ( B ) DTP-DTEP transitions. The volcano blots indicate all genes that were significantly regulated during these transitions (|logFC| < 2 and FDR < 0.05), whereas only the phosphatase genes among these are indicated by names. The four phosphatase genes significantly regulated in both transitions ( DUSP6, CDC25A , CDC25C , and SYNJ1) are indicated in bold. Differentially expressed genes were identified using the R package limma ( n = 3). ( C ) Changes in the DUSP6, CDC25A , CDC25C , and SYNJ1 mRNA levels during the acquisition of lapatinib resistance in BT474 cells. Data is based on RNA sequencing analysis (Dataset ) and was analyzed by one-way ANOVA followed by Tukey’s multiple comparisons test. Statistically significant values of * p < 0.05, ** p < 0.01, and *** p < 0.001 were determined ( n = 3). ( D ) Differential expression of DUSP6, CDC25A , CDC25C , and SYNJ1 in different breast cancer subtypes. Data were extracted from the METABRIC dataset and categorized into five molecular subtypes according to the PAM50 gene expression subtype classification (basal, claudin-low, HER2+, Luminal A, and Luminal B). Data were analyzed by one-way ANOVA followed by Tukey’s multiple comparisons test and shown as mean ± standard deviation (SD). Statistically significant values of * p < 0.05, ** p < 0.01, and **** p < 0.0001 were determined (basal = 209, claudin-low = 218, HER2+ = 224, LumA = 700 and LumB = 475). ( E ) Breast cancer patients from the TCGA-BRCA dataset were divided into DUSP6 high (LogFC>1, FDR < 0.05) and low expression (LogFC < −1, FDR < 0.05) groups and the clinical breast cancer subtypes were compared between the two groups. NA; not available. ( F , G ) Subgroup of 113 patient cases with high tumor ERBB2 mRNA expression (LogFC>1, FDR < 0.05) were divided into DUSP6 high and DUSP6 low groups and their overall survival (OS) ( G ) (Log-rank Test p value = 0.0220) and disease-specific progression-free survival (PFS) ( H ) (Log-rank Test p value = 0.0259) was tested according to DUSP6 status. .

Article Snippet: To rescue the DUSP6 KO MDA-MB-453 clones, the pLEX307-hygro-DUSP6 lentiviral vector was co-transfected with virus-packaging plasmids pMLDg/pRRE (Addgene #12251), pMD2.G (Addgene #12259), and pRSV-Rev (Addgene #12253) into HEK293T cells using FuGENE6 transfection reagent.

Techniques: Control, RNA Sequencing Assay, Expressing, Standard Deviation

Journal: EMBO Molecular Medicine

Article Title: DUSP6 inhibition overcomes neuregulin/HER3-driven therapy tolerance in HER2+ breast cancer

doi: 10.1038/s44321-024-00088-0

Figure Lengend Snippet: ( A , B ) Ectopic overexpression of DUSP6 in BT474 cells inhibits neratinib-elicited effects on cell viability and apoptosis, as measured by WST1 cell viability assay and caspase 3/7 activity, respectively. Data were collected from three independent experiments each performed in triplicate and analyzed by two-way ANOVA followed by Tukey’ post hoc test. Statistically significant values of **** p < 0.0001 were determined. ( C ) Ectopic overexpression of DUSP6 in BT474 cells inhibits the afatinib-elicited effects on cell viability, as measured by WST1 assay. Data were collected from three independent experiments each performed in triplicate and analyzed by two-way ANOVA followed by Tukey’ post hoc test. Statistically significant values of **** p < 0.0001 were determined. ( D ) Ectopic overexpression of DUSP6, but not of DUSP1, inhibits the Tucatinib-elicited effects on cell viability, as measured by WST1 assay. Data were collected from three independent experiments each performed in triplicate and analyzed by two-way ANOVA followed by Tukey’ post hoc test. Statistically significant values of **** p < 0.0001 were determined.

Article Snippet: To rescue the DUSP6 KO MDA-MB-453 clones, the pLEX307-hygro-DUSP6 lentiviral vector was co-transfected with virus-packaging plasmids pMLDg/pRRE (Addgene #12251), pMD2.G (Addgene #12259), and pRSV-Rev (Addgene #12253) into HEK293T cells using FuGENE6 transfection reagent.

Techniques: Over Expression, Viability Assay, Activity Assay

Journal: EMBO Molecular Medicine

Article Title: DUSP6 inhibition overcomes neuregulin/HER3-driven therapy tolerance in HER2+ breast cancer

doi: 10.1038/s44321-024-00088-0

Figure Lengend Snippet: ( A ) RNAi-mediated DUSP6 knockdown, but not of DUSP1 , induces apoptotic cell death in MDA-MB-453 cells, as shown by Western blotting for PARP-1 cleavage. ( B ) CRISPR/CAS9-mediated DUSP6 knockout hinders the clonogenic growth of MDA-MB-453 cells, as compared to the CAS9 expressing controls. Shown are three independent single-cell clones created with two independent gRNAs (g1 and g2). The cells were seeded at low density and maintained for 10 d. The colonies were stained/fixed with 0.5% crystal violet in methanol and imaged using an inverted microscope. ( C ) DUSP6 siRNA knockdown increases sensitivity of HER2i resistant MDA-MB-453 cells to HER2-targeted therapies. Cell viability was measured by WST-1 assay after 48 h of drug treatment. Data were collected from three independent experiments each performed in triplicate and analyzed by one-way ANOVA followed by Tukey’s multiple comparisons test. Statistically significant values of ** p < 0.01, *** p < 0.001, and **** p < 0.0001 were determined. ( D ) A 2D synergy map of neratinib-BCI combination in MDA-MB-453 cells calculated by Bliss SynergyFinder (Ianevski et al, ). Higher score (in red) indicates for higher degree of drug synergy. The cultures were treated with increasing concentrations of the compounds for 48 h and cell viability was measured by WST-1 assay. ( E ) DUSP6 siRNA knockdown increases sensitivity of HER2i resistant MDA-MB-453 cells to combination with capecitabine and neratinib. Cell viability was measured by WST-1 assay after 48 h of drug treatment. Data were collected from three independent experiments each performed in triplicate and analyzed by one-way ANOVA followed by Tukey’s multiple comparisons test. Statistically significant values of **** p < 0.0001 were determined. .

Article Snippet: To rescue the DUSP6 KO MDA-MB-453 clones, the pLEX307-hygro-DUSP6 lentiviral vector was co-transfected with virus-packaging plasmids pMLDg/pRRE (Addgene #12251), pMD2.G (Addgene #12259), and pRSV-Rev (Addgene #12253) into HEK293T cells using FuGENE6 transfection reagent.

Techniques: Knockdown, Western Blot, CRISPR, Knock-Out, Expressing, Clone Assay, Staining, Inverted Microscopy, WST-1 Assay

Journal: EMBO Molecular Medicine

Article Title: DUSP6 inhibition overcomes neuregulin/HER3-driven therapy tolerance in HER2+ breast cancer

doi: 10.1038/s44321-024-00088-0

Figure Lengend Snippet: ( A ) Subcutaneous xenograft growth of two independent DUSP6 single cell knockout clones of MDA-MB-453 cells targeted with two different gRNA guides. One-way ANOVA followed by Tukey’s multiple comparisons test ** p < 0.01 comparing each clone to CAS9 expressing control cells. Data are shown as mean ± SD ( n = 5). ( B – E ) The effect of BCI in combination with lapatinib or neratinib in two HER2 inhibitor resistant xenograft models: HCC1954 ( B , C ) or MDA-MB-453 ( D , E ). Data are shown as mean ± SD ( n = 10 in each treatment group). Mice with tumor size ~100 mm 3 were randomized into the experimental and the control groups and tumor volumes were measured every 3 d. Data were analyzed by one-way ANOVA followed by Tukey’s multiple comparisons test. Statistically significant values of **** p < 0.0001 were determined. ( C ) and ( E ) panels display percentual change in the tumor volume from the start of the therapy as water-fall blots in HCC1954 and MDA-MB453 models, respectively. ( F ) H&E staining of the representative MDA-MB-453 xenograft tumors from the control, lapatinib, BCI, and lapatinib+BCI groups at day 24. Scale bar 200 µm. .

Article Snippet: To rescue the DUSP6 KO MDA-MB-453 clones, the pLEX307-hygro-DUSP6 lentiviral vector was co-transfected with virus-packaging plasmids pMLDg/pRRE (Addgene #12251), pMD2.G (Addgene #12259), and pRSV-Rev (Addgene #12253) into HEK293T cells using FuGENE6 transfection reagent.

Techniques: Knock-Out, Clone Assay, Expressing, Control, Staining

Journal: EMBO Molecular Medicine

Article Title: DUSP6 inhibition overcomes neuregulin/HER3-driven therapy tolerance in HER2+ breast cancer

doi: 10.1038/s44321-024-00088-0

Figure Lengend Snippet: ( A ) A 2D synergy map of lapatinib+MK2206(AKTi) or ( B ) lapatinib+BCI(DUSP6i) combination in MDA-MB-453 cells calculated by the Bliss SynergyFinder. Higher score (in red) indicates for higher degree of drug synergy. The cultures were treated with increasing concentrations of the compounds for 48 h and cell viability was measured by WST-1 assay. ( C ) Comparison of the effects of lapatinib+BCI and lapatinib+MK2206 on apoptosis induction (PARP-1 cleavage), and HER2 or HER3 protein levels by Western blot analysis. The cells were treated with lapatinib (1 μM), MK2206 (2.5 µM), and BCI (2.5 µM) and their combinations for 48 h. ( D ) Quantification of PARP1 cleavage from three repeats of ( C ). Data were analyzed by one-way ANOVA followed by Tukey’s multiple comparisons test. Statistically significant values of ** p < 0.01 and **** p < 0.0001 were determined. ( E , F ) The dose-dependent effects of MK2206 ( E ) and BCI ( F ) on the expression of HER2 and HER3 protein levels in MDA-MB-453 cells by Western blot analysis after 48 h of treatment. ( G ) The time-dependent effects of BCI (2.5 µM) on the expression of HER2 and HER3 protein levels and apoptosis induction (PARP cleavage) in MDA-MB-453 cells by Western blot analysis. Increase in phosphorylated ERK (p-ERK1/2) and inhibition of DUSP6 both indicate for early target engagement by BCI. ( H ) DUSP6 knockdown by siRNA inhibits HER2 and HER3 protein expression in MDA-MB-453 cells. ( I ) Effects of BCI (50 mg/kg) therapy on HER2 and HER3 protein levels in the MDA-MB-453 xenograft tissue on day 24. Shown is immunohistochemical analysis of HER2 and HER3 from the adjacent paraffin embedded tissue slices from Fig. . Scale bar 200 µm. ( J ) Breast cancer patients from the TCGA-BRCA dataset were divided into DUSP6 high (LogFC>1, FDR < 0.05) and low expression (LogFC < −1, FDR < 0.05) profiles and expression of phosphorylated HER3 (p-HER3 Y1298 ) was compared between the two groups. Data were analyzed by two-tailed t test; ** p < 0.01 (DUSP6high = 142, DUSP6low = 149). .

Article Snippet: To rescue the DUSP6 KO MDA-MB-453 clones, the pLEX307-hygro-DUSP6 lentiviral vector was co-transfected with virus-packaging plasmids pMLDg/pRRE (Addgene #12251), pMD2.G (Addgene #12259), and pRSV-Rev (Addgene #12253) into HEK293T cells using FuGENE6 transfection reagent.

Techniques: WST-1 Assay, Comparison, Western Blot, Expressing, Inhibition, Knockdown, Immunohistochemical staining, Two Tailed Test

Journal: EMBO Molecular Medicine

Article Title: DUSP6 inhibition overcomes neuregulin/HER3-driven therapy tolerance in HER2+ breast cancer

doi: 10.1038/s44321-024-00088-0

Figure Lengend Snippet: ( A ) Comparison of the potential of BCI or MK2206 treatment to overcome neuregulin (NRG)-mediated rescue from the anti-proliferative activity of neratinib. The cells were treated with NRG (10 ng/mL), lapatinib (1 µM), MK2206 (1, 2.5, and 5 µM), and BCI (1, 2.5, and 5 µM) for 48 h and cell viability was measured by WST-1 assay. Data were collected from three independent experiments each performed with three technical repeat samples. ( B ) Comparison of the effects of BCI and MK2206 on NRG-mediated evasion from neratinib-induced apoptotic cell death, as measured by Western blot analysis for cleaved PARP-1. The cells were treated with NRG (10 ng/mL), lapatinib (1 μM), MK2206 (2.5 µM), and BCI (2.5 µM) for 48 h. ( C ) HER3 overexpression rescues MDA-MB-453 cells from BCI-elicited inhibition of cell viability. The control or HER3 overexpressing MDA-MB-453 were treated with BCI (3 µM) for 48 h and cell viability was measured by WST-1 assay. Shown is data from four technical replicate samples from a representative of three experiments with similar results. The data was analyzed by two-way ANOVA + Tukey’s post hoc test, **** p < 0.0001. ( D ) Comparison of the effects of lapatinib+BCI and lapatinib+MK2206 on NRG-mediated rescue from the anti-growth activity of lapatinib, as shown by crystal violet staining. The cells were treated with NRG (10 ng/mL), lapatinib (1 μM), MK2206 (2.5 µM), and BCI (2.5 µM) for 48 h, stained/fixed with 0.5% crystal violet in methanol and imaged by an inverted microscope (images acquired at ×10 magnification). ( E ) Breast cancer patients from the TCGA-BRCA dataset were divided into DUSP6 high (LogFC>1, FDR < 0.05) and low expression (LogFC < −1, FDR < 0.05) profiles and the neuregulin ( NRG1 ) mRNA levels were compared between the two groups. Data were analyzed by two-tailed t test; **** p < 0.0001 (DUSP6high = 167, DUSP6low = 181). ( F ) The effect of DUSP6 knockdown on the brain metastatic outgrowth of MDA-MB-361 cells in a zebrafish model. GFP-positive MDA-MB-361 cells transfected either with control scrambled siRNA or DUSP6 siRNA were injected into zebrafish embryo brain and the GFP intensity was measured 3 days after by microscopy. Data were analyzed by two-tailed t test; **** p < 0.0001. Scale bar 100 µm (siSCR = 37, siDUSP6 = 43). ( G ) Improved overall survival of mice with intracranially injected DUSP6 KO MDA-MB-453 cells as compared to the CAS9 positive control cell injected mice. Survival data were analyzed by log-rank Mantel–Cox test, ** p < 0.01. .

Article Snippet: To rescue the DUSP6 KO MDA-MB-453 clones, the pLEX307-hygro-DUSP6 lentiviral vector was co-transfected with virus-packaging plasmids pMLDg/pRRE (Addgene #12251), pMD2.G (Addgene #12259), and pRSV-Rev (Addgene #12253) into HEK293T cells using FuGENE6 transfection reagent.

Techniques: Comparison, Activity Assay, WST-1 Assay, Western Blot, Over Expression, Inhibition, Control, Staining, Inverted Microscopy, Expressing, Two Tailed Test, Knockdown, Transfection, Injection, Microscopy, Positive Control

Journal: EMBO Molecular Medicine

Article Title: DUSP6 inhibition overcomes neuregulin/HER3-driven therapy tolerance in HER2+ breast cancer

doi: 10.1038/s44321-024-00088-0

Figure Lengend Snippet: ( A ) DUSP1 and DUSP6 mRNA levels were determined by qRT-PCR analysis after treatment with increasing concentrations of neratinib for 48 h in indicated cell lines. Red denotes for HER2i resistant cell lines and green HER2i sensitive cells. Data were collected from three independent experiments each performed in triplicate. ( B , C ) Comparison of the effect of neratinib treatment (48 h) on DUSP6, p-AKT, and p-ERK1/2 between HER2i sensitive BT474Br ( B ) and HER2i resistant MDA-MB-361 ( C ) cells, respectively. ( D ) The effect of siRNA-mediated HER3 knockdown on DUSP6 expression in MDA-MB-453 cells by Western blot analysis. ( E ) NRG-mediated induction of DUSP6 mRNA via MEK activation as measured by qRT-PCR analysis after treatment with NRG (10 ng/mL), MK2206(AKTi) (2.5 µM), and trametinib(MEKi) (100 nM) for 48 h. Data were analyzed by one-way ANOVA followed by Tukey’s multiple comparisons test. Statistically significant values of * p < 0.05 and **** p < 0.0001 were determined ( n = 3). ( F ) The effect of NRG on DUSP6 protein expression BT474 cells via MEK activation. The cells were serum-starved for 24 h, followed by treatment with NRG (10 ng/mL), MK2206 (2.5 µM) and trametinib (100 nM) for 48 h. ( G ) Inhibition of DUSP6 expression in HER2i resistant MDA-MB-453 cells by MEKi Trametinib. The cells were treated with MK2206 (2.5 µM) or trametinib (100 nM) for 48 h. ( H ) A schematic illustration of the discovered HER3/DUSP6 feed forward loop in HER2+ breast cancer cells. NRG binding to HER3 induces MEK/ERK-mediated DUSP6 expression which feeds back to increased HER2 and HER3 expression (left panel). In HER2i sensitive cells (middle panel) inhibition of HER3 results in DUSP6 inhibition and loss of DUSP6 driven cancer hallmarks. In HER2i-resistant cells (right panel), MEK is not inhibited by HER2i but its constitutive activity (MEKca.) drives DUSP6-HER2/3 positive feed-back loop resulting in HER3-mediated multitherapy resistance and cancer progression. .

Article Snippet: To rescue the DUSP6 KO MDA-MB-453 clones, the pLEX307-hygro-DUSP6 lentiviral vector was co-transfected with virus-packaging plasmids pMLDg/pRRE (Addgene #12251), pMD2.G (Addgene #12259), and pRSV-Rev (Addgene #12253) into HEK293T cells using FuGENE6 transfection reagent.

Techniques: Quantitative RT-PCR, Comparison, Knockdown, Expressing, Western Blot, Activation Assay, Inhibition, Binding Assay, Activity Assay

Journal: EMBO Molecular Medicine

Article Title: DUSP6 inhibition overcomes neuregulin/HER3-driven therapy tolerance in HER2+ breast cancer

doi: 10.1038/s44321-024-00088-0

Figure Lengend Snippet: ( A , B ) The effects of tucatinib on DUSP6 expression and the signaling pathway activities in ( A ) HER2i sensitive (green) BT474 cells or ( B ) HER2i resistant (red) MDA-MB-453 and BT474BrLR cells. The cells were treated with increasing concentrations of tucatinib for 48 h, followed by Western blot analysis. ( C ) DUSP6 expression in MDA-MB-453 cells is resistant to multiple HER2is including antibody therapy with trastuzumab. The cells were treated with indicated drugs for 48 h, followed by Western blot analysis.

Article Snippet: To rescue the DUSP6 KO MDA-MB-453 clones, the pLEX307-hygro-DUSP6 lentiviral vector was co-transfected with virus-packaging plasmids pMLDg/pRRE (Addgene #12251), pMD2.G (Addgene #12259), and pRSV-Rev (Addgene #12253) into HEK293T cells using FuGENE6 transfection reagent.

Techniques: Expressing, Western Blot

Journal: eLife

Article Title: Rapid cloning-free mutagenesis of new SARS-CoV-2 variants using a novel reverse genetics platform

doi: 10.7554/eLife.89035

Figure Lengend Snippet: ( A ) Schematic representation of the workflow to generate clonal virus populations. Right after transfection, cells were diluted to less than 0.5 virus-producing cells/well in 96-well plates. Then, 7 d post-transfection, the supernatant was transferred onto Vero E6-TMPRSS2 cells. Plates were observed until a cytopathic effect (CPE) became apparent. ( B ) Clonal virus populations arising from a single virus-producing cell were identified after supernatant transfer onto Vero E6-TMPRSS2 cells. CPE of infectious virus was assessed by microscopy and virus was collected for further analysis. Thereafter, plates were fixed and stained for the fast enumeration of positive wells (in light blue).

Article Snippet: Vero E6-TMPRSS2 cells were generated by transduction with a second-generation lentiviral vector pLEX307-TMPRSS2-blast (Addgene plasmid #158458) and selected for 2 wk in DMEM containing 20 µg/mL of Blasticidin (Cat# SBR00022, Sigma-Aldrich).

Techniques: Virus, Transfection, Microscopy, Staining

Journal: eLife

Article Title: Rapid cloning-free mutagenesis of new SARS-CoV-2 variants using a novel reverse genetics platform

doi: 10.7554/eLife.89035

Figure Lengend Snippet:

Article Snippet: Vero E6-TMPRSS2 cells were generated by transduction with a second-generation lentiviral vector pLEX307-TMPRSS2-blast (Addgene plasmid #158458) and selected for 2 wk in DMEM containing 20 µg/mL of Blasticidin (Cat# SBR00022, Sigma-Aldrich).

Techniques: Variant Assay, Virus, Recombinant, Plasmid Preparation, Expressing, Sequencing, Nucleic Acid Purification, Software, Microscopy, Real-time Polymerase Chain Reaction