human gene knockout kit kn209144  (OriGene)

Journal: Cancer Research Communications

Article Title: A Checkpoint Reversal Receptor Mediates Bipartite Activation and Enhances CAR T-cell Function

doi: 10.1158/2767-9764.CRC-24-0125

Figure Lengend Snippet: PD-L1 expression in GBM and PD-1 recruitment to the CARIS with GBM. A, Constitutive (UPN01) and inducible (UPN06) surface expression of PD-L1 in primary GBM cells after 24–48 hours of IFN-γ exposure. Representative results from two samples shown. UPN, unique patient number. B, PD-L1 median fluorescent intensity (MFI) on primary GBM ( n = 14) before and at 48 hours of IFN-γ (10 ng/mL) exposure; ***, P < 0.001, Wilcoxon signed-rank test. C, Fold-change in PD-L1 MFI in GBM cells from baseline, at 24, 48, and 72 hours. Each color represents a single patient donor, with some measured at multiple time points. Data are shown as individual values with the mean ± SD. D, PD-L1 expression in WT LN229-GBM cells and LN229 with PD-L1 deletion (LN229-PD-L1 KO) at baseline and at 24 hours of exposure to IFN-γ (10 ng/mL) or CAR28ζ T cells. E, MFI of PD-1 in the immune synapse between CARζ cells and WT LN229-GBM cells at 15, 30, and 60 minutes (****, P < 0.0001; ns, P > 0.5, Kruskal–Wallis and Wilcox pairwise); ≥20,000 events were captured, and 300–1,000 CAR + conjugates were examined for PD-1 recruitment to CARIS. The white box represents the IQR with horizontal lines at 25%, 50%, and 75%. F, Representative image capture showing the CARIS with tumor cell (WT LN229-GBM) and other parameters evaluated. Gating strategy is shown in the Supplementary material. G, Spearman correlation between PD-1 intensity and CAR intensity in the immune synapse at 15, 30, and 60 minutes. H, Spearman correlation between PD-1 intensity and actin intensity in the CARIS at 15, 30, and 60 minutes. ns, not significant.

Article Snippet: PD-L1 knockout (KO) LN229 cells were generated using the CRISPR-mediated PD-L1 (CD274) Human Gene Knockout Kit (KN213071, OriGene) according to the manufacturer’s instructions.

Techniques: Expressing

Journal: Cancer Research Communications

Article Title: A Checkpoint Reversal Receptor Mediates Bipartite Activation and Enhances CAR T-cell Function

doi: 10.1158/2767-9764.CRC-24-0125

Figure Lengend Snippet: Design and functional screening of PD-1 TR and CPR28 molecules. A, Schematic representation of the bicistronic vectors encoding for the HER2-CAR with truncated PD-1 (PD-1 TR ) or CPR28. The CPR28 dimer included a membrane proximal extracellular cysteine residue (C141) required for CD28 homodimerization, as indicated. B, Flow cytometry analysis showing coexpression of HER2-CAR and PD-1 CPR on T cells. CAR28ζ and NT T cells from the same donor were used as controls for assessment of CPR expression determined by surface PD-1 detection. C, Long-term cytolytic function of CAR28ζ/PD-1 TR , compared with CAR28ζ cells, against U373-GBM cells at effector to target ratios of 1:5 and 1:10 assessed using a cell-impedance based assay (xCELLigence). D, Comparison of the cytolytic ability of patient-derived CAR28ζ cells ( n = 3) coexpressing CPR28 dimer or CPR28 monomer against autologous HER2 + GBM cells at an effector to target ratio of 1:10 by assessment of tumor cell viability in an xCELLigence assay. In C and D , error bars represent the mean ± SD at each time point. ****, P < 0.0001, two-way ANOVA with the Tukey multiple comparisons test. E, IL-2 and ( F ) IFN-γ release by CAR28ζ, CAR28ζ/PD-1 TR , and CAR28ζ/CPR28 cells (100,000 T cells/well) upon stimulation with Fc-conjugated HER2 (0–2 μg/mL) and PD-L1 (0–5 μg/mL) proteins. Median values from a representative donor shown. G, CAR28ζ/CPR28 cell–induced lysis of Raji cells modified to express HER2 or HER2 and PD-L1 across different T-cell to tumor cell ratios but not the WT Raji cells (HER2 − /PD-L1 − ) or those expressing PD-L1 alone in a 51 Cr-release assay. T cells with CPR28 alone had no cytotoxic effect against HER2 + or PD-L1 + Raji cells, like NT T cells from the same donor. A, Created in BioRender. Navai, S. (2024) BioRender.com/l86h941 .

Article Snippet: PD-L1 knockout (KO) LN229 cells were generated using the CRISPR-mediated PD-L1 (CD274) Human Gene Knockout Kit (KN213071, OriGene) according to the manufacturer’s instructions.

Techniques: Functional Assay, Membrane, Residue, Flow Cytometry, Expressing, Comparison, Derivative Assay, Lysis, Modification, Release Assay

Journal: Cancer Research Communications

Article Title: A Checkpoint Reversal Receptor Mediates Bipartite Activation and Enhances CAR T-cell Function

doi: 10.1158/2767-9764.CRC-24-0125

Figure Lengend Snippet: Effect of decoupling signal 2 from the CAR on T-cell function. A, Illustration depicting the bipartite T-cell activation through signal 1 delivery from CAR engaging the HER2 antigen and signal 2 from binding of CPR with PD-L1 (or PD-L2). B, Percent increase in IFN-γ production by CAR28ζ/CPR28 and CARζ/CPR28 compared with CAR28ζ cells (50,000 T cells/well) at 24 hours of coculture with autologous GBM cells ( n = 5 patients). Effector (100,000 T cells) to target ratio of 1:1. **, P < 0.01; ****, P < 0.0001, one-way ANOVA with the Tukey multiple comparisons test. C, Cytolytic function of CAR28ζ/CPR28 and CARζ/CPR28 cells assessed using 4-hour 51 Cr-release assay at baseline and at 7 days of persistent T-cell stimulation through the CAR. NT T cells had poor viability after prolonged stimulation without added homeostatic cytokines and were not evaluable. ****, P < 0.0001, two-way ANOVA with the Tukey multiple comparisons test. D, Percent of total T cells expressing HER2-CAR on cell surface, and percent of CAR + T cells detected with PD-1 (surrogate marker for CPR), respectively, amongst different CPR/CART groups ( n = 4 patients) using flow cytometry. E, Median fluorescent intensity (MFI) of HER2-CAR detected in transduced T cells shown in D . In D and E , only statistically significant differences are shown, *, P < 0.05; **, P < 0.01; ****, P < 0.0001, one-way ANOVA with the Tukey multiple comparisons test. F, Multiplex analysis for proinflammatory cytokines (IL-2, MIP-1α, TNF-α, and GM-CSF) in autologous T-cell and GBM coculture ( n = 4 patients) supernatants at 24 hours. UPN, unique patient number. *, P < 0.05; **, P < 0.01; ****, P < 0.0001, two-way ANOVA with the Tukey multiple comparisons test. G, Assessment of long-term cytolytic function of CPR/CART against autologous GBM cells ( n = 3 patients) using cell impedance–based xCELLigence assay. Statistical differences (denoted by the color key) shown are in comparison to control treatment CAR28ζ cells overtime. ****, P < 0.0001, two-way ANOVA with the Tukey multiple comparisons test. In G, black arrow indicates addition of T cells. A, Created in BioRender. Navai, S. (2019) BioRender.com/p40z816 .

Article Snippet: PD-L1 knockout (KO) LN229 cells were generated using the CRISPR-mediated PD-L1 (CD274) Human Gene Knockout Kit (KN213071, OriGene) according to the manufacturer’s instructions.

Techniques: Cell Function Assay, Activation Assay, Binding Assay, Release Assay, Cell Stimulation, Expressing, Marker, Flow Cytometry, Multiplex Assay, Comparison, Control

Journal: Cancer Research Communications

Article Title: A Checkpoint Reversal Receptor Mediates Bipartite Activation and Enhances CAR T-cell Function

doi: 10.1158/2767-9764.CRC-24-0125

Figure Lengend Snippet: Phenotypic and functional profile of CART receiving bipartite activation signals through CPR41BB costimulation. A, In vivo functional screening of CARζ and CAR28ζ cells coexpressing CPR28 or CPR41BB against orthotopic xenografts of HER2 + PD-L1 + U373-GBM in SCID mice ( n = 5 per group). B, Fold change in tumor burden after treatment relative to the tumor volume before intratumoral injection of T cells (day 0), quantified by serial BLI. *, P < 0.05; **, P < 0.01, two-way ANOVA with the Tukey multiple comparisons test. Data are shown as the mean ± SD. C, CD8 + : CD4 + ratio in CAR-expressing T cells among CPR/CART from patients with GBM ( n = 4) as assessed by flow cytometry. Box plots show minimum to maximum with individual values. ns, P > 0.5, one-way ANOVA with the Tukey multiple comparisons test. D, Pie graph demonstrating immunophenotype distribution of CARζ/CPR41BB cells ( n = 4 patients) at baseline. Percentages shown represent the mean value. Immunophenotype is defined as follows: naïve, CCR7 + /CD45RA + ; central memory, CCR7 + /CD45RA − ; effector memory, CCR7 − /CD45RA − ; and terminal effector, CCR7 − /CD45RA + . E, The percentage CAR + CD8 + central memory (CCR7 + /CD45RA − ) T cells did not significantly differ amongst different CPR/CART groups ( n = 4 patients). The mean ± SEM is shown. ns, P > 0.5, one-way ANOVA with the Dunnett multiple comparisons test. F, Pie graph demonstrating immunophenotype distribution of CARζ/CPR41BB cells ( n = 4 patients) after 7 days of continued stimulation by repeat cocultures with autologous GBM cells. Percentages shown represent the mean value. On day 7 of repeated stimulation, CARζ/CPR41BB cells ( n = 4 patients) demonstrated ( G ) a significantly higher proportion of CAR + /CD8 + cells with the central memory phenotype compared with other CPR/CART groups and CAR28ζ, and ( H ) a significantly lower proportion of CAR + /CD8 + cells with the effector memory phenotype compared with CAR28ζ/CPR28 cells. The mean ± SEM is shown. *, P < 0.05; **, P < 0.01, one-way ANOVA with the Dunnett multiple comparisons test. I, Log-change in the median fluorescent intensity (MFI) of T-cell surface PD-1, LAG3, and TIM3 at 7 days of repeat coculture with autologous GBM cells ( n = 4 patients) from baseline. Box plots show minimum to maximum with individual values. *, P < 0.05; **, P < 0.01, two-way ANOVA with the Tukey multiple comparisons test. ns, not significant.

Article Snippet: PD-L1 knockout (KO) LN229 cells were generated using the CRISPR-mediated PD-L1 (CD274) Human Gene Knockout Kit (KN213071, OriGene) according to the manufacturer’s instructions.

Techniques: Functional Assay, Activation Assay, In Vivo, Injection, Expressing, Flow Cytometry

Journal: Cancer Research Communications

Article Title: A Checkpoint Reversal Receptor Mediates Bipartite Activation and Enhances CAR T-cell Function

doi: 10.1158/2767-9764.CRC-24-0125

Figure Lengend Snippet: Dynamics of CARζ/CPR41BB T-cell activation and CARIS formation in comparison with CAR41BBζ cells. A, CARζ/CPR41BB cells ( n = 3 donors) demonstrated significantly lower IL-2 and IFN-γ release compared with CAR41BBζ cells at 24 hours of coculture with LN229-GBM cells, with CAR28ζ cells consistently showing the higher Th1 cytokine production. Data are shown as the mean ± SD. **, P < 0.000; ****, P < 0.0001; two-way ANOVA with the Tukey multiple comparisons test. B, Western blot analysis for CAR-phosphoCD3ζ (pCD3) in T cells in a resting state (maintained in culture with IL-7/IL-15). The pCD3 to CD3 ratio is normalized to CARζ in each donor. *, P < 0.05; one-way ANOVA with the Tukey multiple comparisons test. C, Representative image capture showing the CARIS with tumor cell (LN229-GBM) and different CARζ/CPR41BB immune synapse parameters evaluated. Gating strategy is shown in the Supplementary Material. Spearman correlation ( D ) between the intensity of CAR and CPR at the CARIS and ( E ) between the intensity of actin and CPR at the CARIS, both assessed by imaging flow cytometry at 15, 30, and 60 minutes. F, CARζ/CPR41BB and CAR41BBζ cells show significantly higher percent (%) of F-actin at the CARIS compared with CARζ cells. ns, P > 0.5; **, P < 0.01; ***, P < 0.0001, two-way ANOVA with the Tukey post hoc test. Data are shown as the mean with 95% confidence interval (CI). G, CARζ/CPR41BB cells show significantly higher CPR intensity in the CARIS with WT LN229 GBM cells at 15, 30, and 60 minutes compared with conjugates with LN229-PD-L1 KO (Kruskal–Wallis and Wilcox pairwise). CPR intensity increased over time in both conditions. ns, not significant.

Article Snippet: PD-L1 knockout (KO) LN229 cells were generated using the CRISPR-mediated PD-L1 (CD274) Human Gene Knockout Kit (KN213071, OriGene) according to the manufacturer’s instructions.

Techniques: Activation Assay, Comparison, Western Blot, Imaging, Flow Cytometry

Journal: Cancer Research Communications

Article Title: A Checkpoint Reversal Receptor Mediates Bipartite Activation and Enhances CAR T-cell Function

doi: 10.1158/2767-9764.CRC-24-0125

Figure Lengend Snippet: Metabolomic parameters of CARζ/CPR41BB cells in comparison with CAR41BBζ cells. A, OCR measurements of resting (cultured in media containing in IL-7 and IL-15) CARζ/CPR41BB and CAR41BBζ cells ( n = 3 donors; 200,000 T cells per well) under basal metabolic conditions and after the addition of mitochondrial inhibitors. B, Comparison of maximal respiration between CARζ/CPR41BB cells and CAR41BBζ cells at baseline (day 0). P = 0.059, Student two-tailed t test. C, Basal OCR, maximal respiration, and SRC between CAR41BBζ and CARζ/CPR41BB cells after 7 days of stimulation with Fc-conjugated HER2 and PD-L1 proteins. ns, P > 0.05, Student two-tailed t test. D, The ECAR in resting CARζ/CPR41BB and CAR41BBζ cells. E, The ECAR of CARζ/CPR41BB and CAR41BBζ at baseline and at 7 days of continued stimulation with plate-bound HER2 and PD-L1 proteins. ns, P > 0.05; **, P < 0.01, Student two-tailed t test. F, Basal OCR to ECAR ratio in CARζ/CPR41BB compared with CAR41BBζ cells at rest and at 7 days of continued stimulation with HER2 and PD-L1 proteins. ns, P > 0.05; *, P < 0.05, Student two-tailed t test. G, The OCR in CD8 + CARζ/CPR41BB and CAR41BBζ cells ( n = 3 donors; 150,000 T cells per well) following 48-hour coculture with LN229-GBM cells (effector to tumor ratio 1:2). H, The SRC of CD8 + CARζ/CPR41BB and CAR41BBζ cells after 48 hours of coculture with LN229 GBM cells. ns, P > 0.05, Student two-tailed t test. The OCR and ECAR measurements are shown as the mean ± SEM. Data shown denote the IQR in the violin plot and the mean ± SEM in the bar graphs. ns, not significant.

Article Snippet: PD-L1 knockout (KO) LN229 cells were generated using the CRISPR-mediated PD-L1 (CD274) Human Gene Knockout Kit (KN213071, OriGene) according to the manufacturer’s instructions.

Techniques: Comparison, Cell Culture, Two Tailed Test

Journal: bioRxiv

Article Title: Stable lentiviral-mediated expression of Cytochrome P450 2D6 in HepaRG cells: New means for in vitro assessment of xenobiotic biotransformation and cytotoxicity

doi: 10.1101/2025.01.25.634872

Figure Lengend Snippet: A) Parental (WT) and CYP2D6 + progenitor cells were thawed and plated to monitor the cell morphology in phase contrast microscopy at days 3, 7 14 and 30 days after plating. B) Immunoblotting of CYP2D6 and CYP3A4 in parental (WT) and CYP2D6 + cells at different times after plating until days 30 in absence (-) or presence (+) of DMSO. HSC70 protein was used as loading control. C) Dextrometorphan O -demethylase activity (Cl int values expressed in μL.min -1 .mg -1 ) measured in human hepatocytes (PHH), HepaSH cells, parental (WT) and CYP2D6 + HepaRG cells. Statistics: *p < 0.05 and **p< 0.01 between Parental and CYP2D6 + HepaRG cells. D) Inhibition of dextrometorphan O -demethylase activity in CYP2D6 + HepaRG cells by quinidine at 1 and 10 μM. Statistics: *p < 0.05 between parental and CYP2D6 + HepaRG cells (a), and quinidine-treated cells versus untreated CYP2D6 + HepaRG cells (b).

Article Snippet: The Gene Knockout kit (CRISPR-Cas9 kit) targeting human CYP2D6 purchased form Synthego (Redwood, CA, USA) contained three different sgRNA ( Supporting Information-Table 1 ) and a Cas9 protein batch. sgRNA were resuspended at 30μM in buffer R while Cas9 was ready to use at 20μM.

Techniques: Microscopy, Western Blot, Control, Activity Assay, Inhibition

Journal: bioRxiv

Article Title: Stable lentiviral-mediated expression of Cytochrome P450 2D6 in HepaRG cells: New means for in vitro assessment of xenobiotic biotransformation and cytotoxicity

doi: 10.1101/2025.01.25.634872

Figure Lengend Snippet: In situ detection of GFP by fluorescence microscopy (A) and by flow cytometry (B) in hepatocyte- and cholangiocyte-enriched cell populations. For flow cytometry data (B), results are expressed in percentages of GFP positive (Pos) cells and means of fluorescence (FITC-A mean) in both hepatocyte- and cholangiocyte-enriched cell populations. C) Dextrometorphan O -demethylase, phenacetin O-deethylase, bupropion hydroxylase and midazolam 1’-hydroxylase activities probing CYP2D6, CYP1A2, CYP2B66 and CYP3A4/5, respectively, in both hepatocyte- and cholangiocyte-enriched cell populations. All data were significantly different between the two cell populations: ***p < 0.001, ****p < 0.0001. D) In situ detection of CYP2D6 (red staining) and ERAP2 (green staining) by immunofluorescence and mitochondria staining using mitotracker.

Article Snippet: The Gene Knockout kit (CRISPR-Cas9 kit) targeting human CYP2D6 purchased form Synthego (Redwood, CA, USA) contained three different sgRNA ( Supporting Information-Table 1 ) and a Cas9 protein batch. sgRNA were resuspended at 30μM in buffer R while Cas9 was ready to use at 20μM.

Techniques: In Situ, Fluorescence, Microscopy, Flow Cytometry, Staining, Immunofluorescence

Journal: bioRxiv

Article Title: Stable lentiviral-mediated expression of Cytochrome P450 2D6 in HepaRG cells: New means for in vitro assessment of xenobiotic biotransformation and cytotoxicity

doi: 10.1101/2025.01.25.634872

Figure Lengend Snippet: A) Schematic representation of the biotransformation of tramadol via CYP2D6, CYP2B6 and CYP3A4 enzymatic activities and the main metabolites of tramadol: M1: O -desmethyltramadol, M2: N -desmethyltramadol, M3: N , N -bidesmethyltramadol, M4: O -desmethyl- N , N -bisdesmethyltramadol, M5: N , O -didesmethyltramadol (M1, M2, M3, M4 and M5). B) Chromatograms of M1-M2 metabolites detected in parental (HepaRG WT) and CYP2D6 + transgenic HepaRG cells. C) Table presenting the quantification LC–MS/MS analysis of metabolites M1 to M5 in progenitor and differentiated parental (WT) and CYP2D6 + HepaRG cells. Results were expressed in µg/L.

Article Snippet: The Gene Knockout kit (CRISPR-Cas9 kit) targeting human CYP2D6 purchased form Synthego (Redwood, CA, USA) contained three different sgRNA ( Supporting Information-Table 1 ) and a Cas9 protein batch. sgRNA were resuspended at 30μM in buffer R while Cas9 was ready to use at 20μM.

Techniques: Transgenic Assay, Liquid Chromatography with Mass Spectroscopy

Journal: bioRxiv

Article Title: Stable lentiviral-mediated expression of Cytochrome P450 2D6 in HepaRG cells: New means for in vitro assessment of xenobiotic biotransformation and cytotoxicity

doi: 10.1101/2025.01.25.634872

Figure Lengend Snippet: A) Relative ATP contents (left panel) and LDH release (right panel) and B) IC 50 in parental (WT) and CYP2D6 + differentiated HepaRG cells exposed to various concentration of perhexiline. C) Oxygen Consumption Rate (OCR) in parental (WT) and CYP2D6 + differentiated HepaRG cells exposed to perhexiline at 10μM and quantifications of proton leak, ATP production and coupling efficiency (left panel) and D) levels of mitochondrial β-oxidation of linoleic acid. All data were significantly different between the two cell lines: *p < 0.05 and **p< 0.01

Article Snippet: The Gene Knockout kit (CRISPR-Cas9 kit) targeting human CYP2D6 purchased form Synthego (Redwood, CA, USA) contained three different sgRNA ( Supporting Information-Table 1 ) and a Cas9 protein batch. sgRNA were resuspended at 30μM in buffer R while Cas9 was ready to use at 20μM.

Techniques: Concentration Assay

Journal: bioRxiv

Article Title: Stable lentiviral-mediated expression of Cytochrome P450 2D6 in HepaRG cells: New means for in vitro assessment of xenobiotic biotransformation and cytotoxicity

doi: 10.1101/2025.01.25.634872

Figure Lengend Snippet: A) UMAP representation of parental (WT) and CYP2D6 + HepaRG cells at different time points after plating (2, 4, 7, 14, 32, 37 and 44 days). The first dimension is mainly associated to the time of culture. B) Heatmap of the top 100 most differentially expressed genes during differentiation, with consistent expression patterns observed between parental (WT and CYP2D6 + HepaRG cells. C) Volcano plot of genes differentially expressed at D37 highlighting significant downregulation of various CYP450 genes in transgenic cells compared to parental cells. D) Radar plot showing enrichment of gene ontology (GO) terms related to xenobiotic metabolism at D37 and D44. The size of the points is the ratio r/R and the scale corresponds to the log10 of the adjusted p-value, where r = number of genes in the list associated with the term of interest and R = total number of genes associated with one term of interest.

Article Snippet: The Gene Knockout kit (CRISPR-Cas9 kit) targeting human CYP2D6 purchased form Synthego (Redwood, CA, USA) contained three different sgRNA ( Supporting Information-Table 1 ) and a Cas9 protein batch. sgRNA were resuspended at 30μM in buffer R while Cas9 was ready to use at 20μM.

Techniques: Expressing, Transgenic Assay

Journal: bioRxiv

Article Title: Stable lentiviral-mediated expression of Cytochrome P450 2D6 in HepaRG cells: New means for in vitro assessment of xenobiotic biotransformation and cytotoxicity

doi: 10.1101/2025.01.25.634872

Figure Lengend Snippet: A) Detection of GFP by fluorescence microscopy and by flow cytometry in CYP2D6 + and KO CYP2D6/GFP HepaRG cells. Results are expressed in percentages of GFP positive (Pos) or negative (Neg) cells and means of fluorescence (FITC-A mean). B) RT-qPCR evaluating the relative mRNA expression of CYP2D6, 3A4, 2C8, 2C9, 2A7, AhR, FXR, PXR, HNF4α and SHP in parental (HRP WT), CYP2D6 + and KO CYP2D6/GFP (HRP KO) HepaRG cells. Statistics: *p < 0.05 between parental (HRP WT = a), CYP2D6 + (b) and KO CYP2D6/GFP (HRP KO = c) HepaRG cells.

Article Snippet: The Gene Knockout kit (CRISPR-Cas9 kit) targeting human CYP2D6 purchased form Synthego (Redwood, CA, USA) contained three different sgRNA ( Supporting Information-Table 1 ) and a Cas9 protein batch. sgRNA were resuspended at 30μM in buffer R while Cas9 was ready to use at 20μM.

Techniques: Fluorescence, Microscopy, Flow Cytometry, Quantitative RT-PCR, Expressing

Journal: bioRxiv

Article Title: Stable lentiviral-mediated expression of Cytochrome P450 2D6 in HepaRG cells: New means for in vitro assessment of xenobiotic biotransformation and cytotoxicity

doi: 10.1101/2025.01.25.634872

Figure Lengend Snippet: A) Schematic representation of the lentiviral derived mRNA transcribed from the transgene inserted into the genome of HepaRG cells and CRISPR/Cas9 mediated alteration of the lentiviral transgene in KO CYP2D6/GFP (HRP KO) HepaRG cells. B) RT-qPCR evaluating the relative mRNA expression of the CYP2D6-GFP-WPRE lentiviral derived transcript and NXF3 and TRIM63 in CYP2D6 + and KO CYP2D6/GFP HepaRG cells. Statistics: *p < 0.05 between parental (HRP WT = a), CYP2D6 + (b) and KO CYP2D6/GFP (HRP KO = c) HepaRG cells.

Article Snippet: The Gene Knockout kit (CRISPR-Cas9 kit) targeting human CYP2D6 purchased form Synthego (Redwood, CA, USA) contained three different sgRNA ( Supporting Information-Table 1 ) and a Cas9 protein batch. sgRNA were resuspended at 30μM in buffer R while Cas9 was ready to use at 20μM.

Techniques: Derivative Assay, CRISPR, Quantitative RT-PCR, Expressing

Journal: Journal of Translational Medicine

Article Title: PD-L1 and IFN-γ modulate Non-Small Cell Lung Cancer (NSCLC) cell plasticity associated to immune checkpoint inhibitor (ICI)-mediated hyperprogressive disease (HPD)

doi: 10.1186/s12967-024-06023-8

Figure Lengend Snippet: Clinical features of patient’s tumor before and after ICI treatment. Time points: Tdx, time of the diagnosis; Tb, time of baseline; Thy, time of hyperprogression. A Hematoxylin and eosin staining (H&E) and CD44 and PD-L1 immunohistochemical staining of tumor samples. Tdx: pleural effusion; Tb: pleural biopsy; Thy: subcutaneous lesion. Left: H&E, black line from top to bottom: 313.30 µm, 204.97 µm and 92.60 µm; middle: membrane CD44 expression on neoplastic cells, black line from top to bottom: 313.30 µm, 120.01 µm, 80.01 µm; right: PD-L1 expression on tumor cells by double stain for PDL1 and CD68 (PD-L1: brown, CD68: red), black line from top to bottom: 139.24 µm, 300 µm and 61.73 µm. B Imaging findings at the time of diagnosis (Tdx) (i. – iv.) and of progression to immunotherapy (Thy) (v. – viii.) . Small right paratracheal lymph node (i.) progressed on immunotherapy (v., red arrow) . Left pleural effusion (ii.) and left pleurodesis signs associated with the appearance of subcutaneous metastatic site (vi., red arrow) . Left pleural effusion (iii.) and left pleurodesis signs associated with the appearance of a metastatic site at the left thoracic wall (vii., red arrow) . Small right paraortic lymph node (iv.) progressed on immunotherapy (viii., red arrow) . ICI: immune checkpoint inhibitors

Article Snippet: The silencing of PD-L1 in NSCLC-B cells was performed by using PD-L1 (CD274) Human Gene non-homology mediated CRISPR knock-out kit (KN413071; Origene, Rockville, MD, USA), according to the manufacturer’s instructions.

Techniques: Staining, Immunohistochemical staining, Membrane, Expressing, Imaging

Journal: Journal of Translational Medicine

Article Title: PD-L1 and IFN-γ modulate Non-Small Cell Lung Cancer (NSCLC) cell plasticity associated to immune checkpoint inhibitor (ICI)-mediated hyperprogressive disease (HPD)

doi: 10.1186/s12967-024-06023-8

Figure Lengend Snippet: Comparison of immune-related genes and proteins between NSCLC-B and NSCLC-H cell lines. A Normalized expression (RLOG) of PD-L1 (CD274) gene, as identified by RNAseq analysis (Log2FC: −0.589, p-adj: 0.0003), and validation by flow cytometry (NSCLC-B, grey: unstained control, black: stained sample; NSCLC-H, orange: unstained control, red: stained sample). B GSEA analysis of curated list related to inflammatory response. C Heatmap showing the Log2FC of a subgroup of enriched score genes associated to GSEA analysis and reported in Additional file : Figure S1C, which are involved in immune-related cellular responses. Log2FC of differentially expressed genes in NSCLC-H compared to NSCLC-B is reported in the box and p-adj is reported on the right. D Western blot analysis for proteins involved in response to inflammatory stimuli and cell proliferation (n = 3, except for p-JAK2/t-JAK2 and p-IRF3/t-IRF3, in which n = 2). *p < 0.05, **p < 0.01, ***p < 0.001 by One Sample t test (each group vs theoretical mean of 100). Each bar represents mean with SEM. SEM: standard error of the mean

Article Snippet: The silencing of PD-L1 in NSCLC-B cells was performed by using PD-L1 (CD274) Human Gene non-homology mediated CRISPR knock-out kit (KN413071; Origene, Rockville, MD, USA), according to the manufacturer’s instructions.

Techniques: Comparison, Expressing, Flow Cytometry, Control, Staining, Western Blot

Journal: Journal of Translational Medicine

Article Title: PD-L1 and IFN-γ modulate Non-Small Cell Lung Cancer (NSCLC) cell plasticity associated to immune checkpoint inhibitor (ICI)-mediated hyperprogressive disease (HPD)

doi: 10.1186/s12967-024-06023-8

Figure Lengend Snippet: Response of NSCLC-B cell line to PD-L1 modulation in 2D and 3D culture conditions. A NSCLC-B soft-agar colony formation in the presence of atezolizumab 10 μg/mL (n = 6, three experiments–circle, triangle, square–, each one with two technical replicates). Each bar represents mean with SEM. Each dot represents a replicate. B Morphological and phenotypical characterization of NSCLC-B “bulk agar” cell lines obtained from 2D-subcultered agar colonies grown in the presence of atezolizumab 10 μg/mL. First line: representative pictures of NSCLC-B soft-agar colonies grown without any treatment or in the presence of atezolizumab, observed through an inverted microscope in dark-field. To improve the visual appearance of the images, photos were converted into grayscale; second line: morphologies of NSCLC-B “bulk agar” cell lines cultured under 2D-adherent conditions, observed through an inverted microscope. White line corresponds to 100 µm. To improve the visual appearance of the images, photos were converted into grayscale and contrast and brightness were enhanced (+ 40% and + 10%, respectively); expression of CD44 ( third line ) and percentage of CD44 + cells ( bottom ) on NSCLC-B “bulk agar” cell lines cultured under 2D-adherent conditions, measured by cytofluorimetric analysis (M1: marker) (n = 4). **p < 0.01 by Student’s t -test. Each bar represents mean with SEM. C NSCLC-B sphere formation in the presence of atezolizumab 10 μg/mL (n = 18, nine experiments–different symbols–, each one with two technical replicates). ***p < 0.001 by One sample t test (group vs theoretical mean of 100). Each bar represents mean with SEM. Each dot represents a replicate. D Assessment of MAPK activation in NSCLC-B cells treated with atezolizumab (5 μg/mL) in 2D-adherent culture conditions, as measured by Western blot analysis (n = 4). Each bar represents mean with SEM. E Comparison of morphological and phenotypical features between NSCLC-B CRISPR-engineered cell lines (NSCLC-B CL1 and NSCLC-B CL2), NSCLC-B and NSCLC-H cell lines. Up: representative photos of NSCLC-B, NSCLC-B CL1, NSCLC-B CL2 and NSCLC-H cell lines, observed through an inverted microscope in dark-field. White line corresponds to 100 µm; bottom: expression of CD44 on NSCLC-B (black), NSCLC-B CL1 (green), NSCLC-B CL2 (dark green) and NSCLC-H (red) cell lines cultured under 2D-adherent conditions, measured by cytofluorimetric analysis. F Comparison of 2D-clonal efficiency between NSCLC-B, NSCLC-B CL1, NSCLC-B CL2 and NSCLC-H cell lines (n = 6). ***p < 0.001 by Student’s t -test. Each bar represents mean with SEM. 2D: two-dimensional, SEM: standard error of the mean

Article Snippet: The silencing of PD-L1 in NSCLC-B cells was performed by using PD-L1 (CD274) Human Gene non-homology mediated CRISPR knock-out kit (KN413071; Origene, Rockville, MD, USA), according to the manufacturer’s instructions.

Techniques: Inverted Microscopy, Cell Culture, Expressing, Marker, Activation Assay, Western Blot, Comparison, CRISPR

Journal: Glycoconjugate Journal

Article Title: MUC1 expression is associated with ST3GAL2 and negatively correlated with the androgen receptor in castration-resistant prostate cancer

doi: 10.1007/s10719-024-10173-8

Figure Lengend Snippet: Knockout of ST3GAL2 and changes of gene expression. a High-performance thin-layer chromatography immunostaining by mAb RM1 specific to SSEA-4. b Orcinol staining of glycolipids extracted from DU145 and ST3GAL2 -knockout (KO) clones. Clone 83 and 13 are complete KO clones, and clone 2 and 5 are partial KO clones. c and d Volcano plot of expression level of mRNAs of ST3GAL2 -KO clone 13 and 81, respectively, as compared to that of control (DU145 cells)

Article Snippet: ST3GAL2 -knockout clones were generated by using a ST3GAL2 Human Gene Knockout Kit (CRISPR) (OriGene Technologies, Inc., Rockville, MD), which contained two gRNA vectors and one donor vector including the GFP-puromycin functional cassette.

Techniques: Knock-Out, Gene Expression, High Performance Thin Layer Chromatography, Immunostaining, Staining, Clone Assay, Expressing, Control

Journal: Glycoconjugate Journal

Article Title: MUC1 expression is associated with ST3GAL2 and negatively correlated with the androgen receptor in castration-resistant prostate cancer

doi: 10.1007/s10719-024-10173-8

Figure Lengend Snippet: Examples of RNA-seq analysis of cancer-associated genes in ST3GAL2 -KO clones

Article Snippet: ST3GAL2 -knockout clones were generated by using a ST3GAL2 Human Gene Knockout Kit (CRISPR) (OriGene Technologies, Inc., Rockville, MD), which contained two gRNA vectors and one donor vector including the GFP-puromycin functional cassette.

Techniques:

Journal: Glycoconjugate Journal

Article Title: MUC1 expression is associated with ST3GAL2 and negatively correlated with the androgen receptor in castration-resistant prostate cancer

doi: 10.1007/s10719-024-10173-8

Figure Lengend Snippet: Western blot analysis of MUC1 in prostate cancer cell lines. a Down-regulation of MUC1 expression in ST3GAL2 -KO clones. b Marked expression of MUC1 in AR-negative DU145 and AICaP1 cells. NCI-H660 and KUCaP13 are neuroendocrine prostate cancer (NEPC) cell lines. Clone 2 and 5 are partial KO clones. The other clones are complete KO clones

Article Snippet: ST3GAL2 -knockout clones were generated by using a ST3GAL2 Human Gene Knockout Kit (CRISPR) (OriGene Technologies, Inc., Rockville, MD), which contained two gRNA vectors and one donor vector including the GFP-puromycin functional cassette.

Techniques: Western Blot, Expressing, Clone Assay