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bsmbi v2 digested plenticrisprv2  (New England Biolabs)


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    New England Biolabs bsmbi v2 digested plenticrisprv2
    Data Supporting Figure 5. A , Immunoblot from lysates of A549 cells or SLC7A11 knockout clones for indicated proteins upon treatment with βMe (50μM). Inferred MW of band indicated. B , Viability (ATP) of cells from C cultured in the presence or absence of cystine (CySS) with indicated concentration of supplemented βMe for 3 days. Data report mean of technical triplicates. C , Viability (ATP) of A549 cells in cystine-free media or upon erastin treatment (10μM), or SLC7A11 knockout clones in standard conditions, treated with the indicated concentrations of reducing agents βMe, dithiothreitol (DTT), or Tris(2-carboxyethyl)phosphine (TCEP), 3 days. Data are the mean of two technical replicates. D , Viability (ATP, solid lines) and GSH content (dashed lines) of A549 cells cultured at indicated CySS concentration for 3 days. Data report mean of two technical replicates. E , Upper left, schematic delineating the contribution of serine-derived carbon to cysteine, glycine, GSH, or GSSG. Right, isotopic labeling of the indicated metabolites derived from A549 cells cultured in U- 13 C-serine (286 μM) as the only source of serine (top, GC-MS detection; bottom, LC-MS detection), in the presence or absence of CySS or βMe (50 μM) for 24 hr. ND = not detected. F , Crystal violet staining of A549 SLC7A11 knockout clones from upon transwell culture with A549 cells expressing <t>pLentiCRISPRv2</t> or an SLC7A11 knockout clone, 3 days. Data are from one experiment. G , Immunoblot from lysates of A549 cells or SLC7A11 knockout clones for indicated proteins in the presence or absence of CySS, βMe (50 μM) of Fer-1 (10 μM) for 2 days. Inferred MW of band indicated. Data are from one experiment. H , Viability (ATP) of A549 cells upon treatment with Na2SeO3 and the indicated concentrations of reducing agents βMe, DTT, or TCEP, 3 days. Data are the mean ± SEM of n=3 biological replicates.
    Bsmbi V2 Digested Plenticrisprv2, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 1621 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/bsmbi+v2+digested+plenticrisprv2/bio_rxiv__64898__2026__03__11__711115-218-9-19?v=New+England+Biolabs
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    1) Product Images from "Systematic Evaluation Defines the Limits of Ferroptosis in Cancer Therapy"

    Article Title: Systematic Evaluation Defines the Limits of Ferroptosis in Cancer Therapy

    Journal: bioRxiv

    doi: 10.64898/2026.03.11.711115

    Data Supporting Figure 5. A , Immunoblot from lysates of A549 cells or SLC7A11 knockout clones for indicated proteins upon treatment with βMe (50μM). Inferred MW of band indicated. B , Viability (ATP) of cells from C cultured in the presence or absence of cystine (CySS) with indicated concentration of supplemented βMe for 3 days. Data report mean of technical triplicates. C , Viability (ATP) of A549 cells in cystine-free media or upon erastin treatment (10μM), or SLC7A11 knockout clones in standard conditions, treated with the indicated concentrations of reducing agents βMe, dithiothreitol (DTT), or Tris(2-carboxyethyl)phosphine (TCEP), 3 days. Data are the mean of two technical replicates. D , Viability (ATP, solid lines) and GSH content (dashed lines) of A549 cells cultured at indicated CySS concentration for 3 days. Data report mean of two technical replicates. E , Upper left, schematic delineating the contribution of serine-derived carbon to cysteine, glycine, GSH, or GSSG. Right, isotopic labeling of the indicated metabolites derived from A549 cells cultured in U- 13 C-serine (286 μM) as the only source of serine (top, GC-MS detection; bottom, LC-MS detection), in the presence or absence of CySS or βMe (50 μM) for 24 hr. ND = not detected. F , Crystal violet staining of A549 SLC7A11 knockout clones from upon transwell culture with A549 cells expressing pLentiCRISPRv2 or an SLC7A11 knockout clone, 3 days. Data are from one experiment. G , Immunoblot from lysates of A549 cells or SLC7A11 knockout clones for indicated proteins in the presence or absence of CySS, βMe (50 μM) of Fer-1 (10 μM) for 2 days. Inferred MW of band indicated. Data are from one experiment. H , Viability (ATP) of A549 cells upon treatment with Na2SeO3 and the indicated concentrations of reducing agents βMe, DTT, or TCEP, 3 days. Data are the mean ± SEM of n=3 biological replicates.
    Figure Legend Snippet: Data Supporting Figure 5. A , Immunoblot from lysates of A549 cells or SLC7A11 knockout clones for indicated proteins upon treatment with βMe (50μM). Inferred MW of band indicated. B , Viability (ATP) of cells from C cultured in the presence or absence of cystine (CySS) with indicated concentration of supplemented βMe for 3 days. Data report mean of technical triplicates. C , Viability (ATP) of A549 cells in cystine-free media or upon erastin treatment (10μM), or SLC7A11 knockout clones in standard conditions, treated with the indicated concentrations of reducing agents βMe, dithiothreitol (DTT), or Tris(2-carboxyethyl)phosphine (TCEP), 3 days. Data are the mean of two technical replicates. D , Viability (ATP, solid lines) and GSH content (dashed lines) of A549 cells cultured at indicated CySS concentration for 3 days. Data report mean of two technical replicates. E , Upper left, schematic delineating the contribution of serine-derived carbon to cysteine, glycine, GSH, or GSSG. Right, isotopic labeling of the indicated metabolites derived from A549 cells cultured in U- 13 C-serine (286 μM) as the only source of serine (top, GC-MS detection; bottom, LC-MS detection), in the presence or absence of CySS or βMe (50 μM) for 24 hr. ND = not detected. F , Crystal violet staining of A549 SLC7A11 knockout clones from upon transwell culture with A549 cells expressing pLentiCRISPRv2 or an SLC7A11 knockout clone, 3 days. Data are from one experiment. G , Immunoblot from lysates of A549 cells or SLC7A11 knockout clones for indicated proteins in the presence or absence of CySS, βMe (50 μM) of Fer-1 (10 μM) for 2 days. Inferred MW of band indicated. Data are from one experiment. H , Viability (ATP) of A549 cells upon treatment with Na2SeO3 and the indicated concentrations of reducing agents βMe, DTT, or TCEP, 3 days. Data are the mean ± SEM of n=3 biological replicates.

    Techniques Used: Western Blot, Knock-Out, Clone Assay, Cell Culture, Concentration Assay, Derivative Assay, Isotopic Labeling, Gas Chromatography-Mass Spectrometry, Liquid Chromatography with Mass Spectroscopy, Staining, Expressing



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    Data Supporting Figure 5. A , Immunoblot from lysates of A549 cells or SLC7A11 knockout clones for indicated proteins upon treatment with βMe (50μM). Inferred MW of band indicated. B , Viability (ATP) of cells from C cultured in the presence or absence of cystine (CySS) with indicated concentration of supplemented βMe for 3 days. Data report mean of technical triplicates. C , Viability (ATP) of A549 cells in cystine-free media or upon erastin treatment (10μM), or SLC7A11 knockout clones in standard conditions, treated with the indicated concentrations of reducing agents βMe, dithiothreitol (DTT), or Tris(2-carboxyethyl)phosphine (TCEP), 3 days. Data are the mean of two technical replicates. D , Viability (ATP, solid lines) and GSH content (dashed lines) of A549 cells cultured at indicated CySS concentration for 3 days. Data report mean of two technical replicates. E , Upper left, schematic delineating the contribution of serine-derived carbon to cysteine, glycine, GSH, or GSSG. Right, isotopic labeling of the indicated metabolites derived from A549 cells cultured in U- 13 C-serine (286 μM) as the only source of serine (top, GC-MS detection; bottom, LC-MS detection), in the presence or absence of CySS or βMe (50 μM) for 24 hr. ND = not detected. F , Crystal violet staining of A549 SLC7A11 knockout clones from upon transwell culture with A549 cells expressing <t>pLentiCRISPRv2</t> or an SLC7A11 knockout clone, 3 days. Data are from one experiment. G , Immunoblot from lysates of A549 cells or SLC7A11 knockout clones for indicated proteins in the presence or absence of CySS, βMe (50 μM) of Fer-1 (10 μM) for 2 days. Inferred MW of band indicated. Data are from one experiment. H , Viability (ATP) of A549 cells upon treatment with Na2SeO3 and the indicated concentrations of reducing agents βMe, DTT, or TCEP, 3 days. Data are the mean ± SEM of n=3 biological replicates.
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    Data Supporting Figure 5. A , Immunoblot from lysates of A549 cells or SLC7A11 knockout clones for indicated proteins upon treatment with βMe (50μM). Inferred MW of band indicated. B , Viability (ATP) of cells from C cultured in the presence or absence of cystine (CySS) with indicated concentration of supplemented βMe for 3 days. Data report mean of technical triplicates. C , Viability (ATP) of A549 cells in cystine-free media or upon erastin treatment (10μM), or SLC7A11 knockout clones in standard conditions, treated with the indicated concentrations of reducing agents βMe, dithiothreitol (DTT), or Tris(2-carboxyethyl)phosphine (TCEP), 3 days. Data are the mean of two technical replicates. D , Viability (ATP, solid lines) and GSH content (dashed lines) of A549 cells cultured at indicated CySS concentration for 3 days. Data report mean of two technical replicates. E , Upper left, schematic delineating the contribution of serine-derived carbon to cysteine, glycine, GSH, or GSSG. Right, isotopic labeling of the indicated metabolites derived from A549 cells cultured in U- 13 C-serine (286 μM) as the only source of serine (top, GC-MS detection; bottom, LC-MS detection), in the presence or absence of CySS or βMe (50 μM) for 24 hr. ND = not detected. F , Crystal violet staining of A549 SLC7A11 knockout clones from upon transwell culture with A549 cells expressing <t>pLentiCRISPRv2</t> or an SLC7A11 knockout clone, 3 days. Data are from one experiment. G , Immunoblot from lysates of A549 cells or SLC7A11 knockout clones for indicated proteins in the presence or absence of CySS, βMe (50 μM) of Fer-1 (10 μM) for 2 days. Inferred MW of band indicated. Data are from one experiment. H , Viability (ATP) of A549 cells upon treatment with Na2SeO3 and the indicated concentrations of reducing agents βMe, DTT, or TCEP, 3 days. Data are the mean ± SEM of n=3 biological replicates.
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    Data Supporting Figure 5. A , Immunoblot from lysates of A549 cells or SLC7A11 knockout clones for indicated proteins upon treatment with βMe (50μM). Inferred MW of band indicated. B , Viability (ATP) of cells from C cultured in the presence or absence of cystine (CySS) with indicated concentration of supplemented βMe for 3 days. Data report mean of technical triplicates. C , Viability (ATP) of A549 cells in cystine-free media or upon erastin treatment (10μM), or SLC7A11 knockout clones in standard conditions, treated with the indicated concentrations of reducing agents βMe, dithiothreitol (DTT), or Tris(2-carboxyethyl)phosphine (TCEP), 3 days. Data are the mean of two technical replicates. D , Viability (ATP, solid lines) and GSH content (dashed lines) of A549 cells cultured at indicated CySS concentration for 3 days. Data report mean of two technical replicates. E , Upper left, schematic delineating the contribution of serine-derived carbon to cysteine, glycine, GSH, or GSSG. Right, isotopic labeling of the indicated metabolites derived from A549 cells cultured in U- 13 C-serine (286 μM) as the only source of serine (top, GC-MS detection; bottom, LC-MS detection), in the presence or absence of CySS or βMe (50 μM) for 24 hr. ND = not detected. F , Crystal violet staining of A549 SLC7A11 knockout clones from upon transwell culture with A549 cells expressing <t>pLentiCRISPRv2</t> or an SLC7A11 knockout clone, 3 days. Data are from one experiment. G , Immunoblot from lysates of A549 cells or SLC7A11 knockout clones for indicated proteins in the presence or absence of CySS, βMe (50 μM) of Fer-1 (10 μM) for 2 days. Inferred MW of band indicated. Data are from one experiment. H , Viability (ATP) of A549 cells upon treatment with Na2SeO3 and the indicated concentrations of reducing agents βMe, DTT, or TCEP, 3 days. Data are the mean ± SEM of n=3 biological replicates.
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    Data Supporting Figure 5. A , Immunoblot from lysates of A549 cells or SLC7A11 knockout clones for indicated proteins upon treatment with βMe (50μM). Inferred MW of band indicated. B , Viability (ATP) of cells from C cultured in the presence or absence of cystine (CySS) with indicated concentration of supplemented βMe for 3 days. Data report mean of technical triplicates. C , Viability (ATP) of A549 cells in cystine-free media or upon erastin treatment (10μM), or SLC7A11 knockout clones in standard conditions, treated with the indicated concentrations of reducing agents βMe, dithiothreitol (DTT), or Tris(2-carboxyethyl)phosphine (TCEP), 3 days. Data are the mean of two technical replicates. D , Viability (ATP, solid lines) and GSH content (dashed lines) of A549 cells cultured at indicated CySS concentration for 3 days. Data report mean of two technical replicates. E , Upper left, schematic delineating the contribution of serine-derived carbon to cysteine, glycine, GSH, or GSSG. Right, isotopic labeling of the indicated metabolites derived from A549 cells cultured in U- 13 C-serine (286 μM) as the only source of serine (top, GC-MS detection; bottom, LC-MS detection), in the presence or absence of CySS or βMe (50 μM) for 24 hr. ND = not detected. F , Crystal violet staining of A549 SLC7A11 knockout clones from upon transwell culture with A549 cells expressing <t>pLentiCRISPRv2</t> or an SLC7A11 knockout clone, 3 days. Data are from one experiment. G , Immunoblot from lysates of A549 cells or SLC7A11 knockout clones for indicated proteins in the presence or absence of CySS, βMe (50 μM) of Fer-1 (10 μM) for 2 days. Inferred MW of band indicated. Data are from one experiment. H , Viability (ATP) of A549 cells upon treatment with Na2SeO3 and the indicated concentrations of reducing agents βMe, DTT, or TCEP, 3 days. Data are the mean ± SEM of n=3 biological replicates.
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    Data Supporting Figure 5. A , Immunoblot from lysates of A549 cells or SLC7A11 knockout clones for indicated proteins upon treatment with βMe (50μM). Inferred MW of band indicated. B , Viability (ATP) of cells from C cultured in the presence or absence of cystine (CySS) with indicated concentration of supplemented βMe for 3 days. Data report mean of technical triplicates. C , Viability (ATP) of A549 cells in cystine-free media or upon erastin treatment (10μM), or SLC7A11 knockout clones in standard conditions, treated with the indicated concentrations of reducing agents βMe, dithiothreitol (DTT), or Tris(2-carboxyethyl)phosphine (TCEP), 3 days. Data are the mean of two technical replicates. D , Viability (ATP, solid lines) and GSH content (dashed lines) of A549 cells cultured at indicated CySS concentration for 3 days. Data report mean of two technical replicates. E , Upper left, schematic delineating the contribution of serine-derived carbon to cysteine, glycine, GSH, or GSSG. Right, isotopic labeling of the indicated metabolites derived from A549 cells cultured in U- 13 C-serine (286 μM) as the only source of serine (top, GC-MS detection; bottom, LC-MS detection), in the presence or absence of CySS or βMe (50 μM) for 24 hr. ND = not detected. F , Crystal violet staining of A549 SLC7A11 knockout clones from upon transwell culture with A549 cells expressing pLentiCRISPRv2 or an SLC7A11 knockout clone, 3 days. Data are from one experiment. G , Immunoblot from lysates of A549 cells or SLC7A11 knockout clones for indicated proteins in the presence or absence of CySS, βMe (50 μM) of Fer-1 (10 μM) for 2 days. Inferred MW of band indicated. Data are from one experiment. H , Viability (ATP) of A549 cells upon treatment with Na2SeO3 and the indicated concentrations of reducing agents βMe, DTT, or TCEP, 3 days. Data are the mean ± SEM of n=3 biological replicates.

    Journal: bioRxiv

    Article Title: Systematic Evaluation Defines the Limits of Ferroptosis in Cancer Therapy

    doi: 10.64898/2026.03.11.711115

    Figure Lengend Snippet: Data Supporting Figure 5. A , Immunoblot from lysates of A549 cells or SLC7A11 knockout clones for indicated proteins upon treatment with βMe (50μM). Inferred MW of band indicated. B , Viability (ATP) of cells from C cultured in the presence or absence of cystine (CySS) with indicated concentration of supplemented βMe for 3 days. Data report mean of technical triplicates. C , Viability (ATP) of A549 cells in cystine-free media or upon erastin treatment (10μM), or SLC7A11 knockout clones in standard conditions, treated with the indicated concentrations of reducing agents βMe, dithiothreitol (DTT), or Tris(2-carboxyethyl)phosphine (TCEP), 3 days. Data are the mean of two technical replicates. D , Viability (ATP, solid lines) and GSH content (dashed lines) of A549 cells cultured at indicated CySS concentration for 3 days. Data report mean of two technical replicates. E , Upper left, schematic delineating the contribution of serine-derived carbon to cysteine, glycine, GSH, or GSSG. Right, isotopic labeling of the indicated metabolites derived from A549 cells cultured in U- 13 C-serine (286 μM) as the only source of serine (top, GC-MS detection; bottom, LC-MS detection), in the presence or absence of CySS or βMe (50 μM) for 24 hr. ND = not detected. F , Crystal violet staining of A549 SLC7A11 knockout clones from upon transwell culture with A549 cells expressing pLentiCRISPRv2 or an SLC7A11 knockout clone, 3 days. Data are from one experiment. G , Immunoblot from lysates of A549 cells or SLC7A11 knockout clones for indicated proteins in the presence or absence of CySS, βMe (50 μM) of Fer-1 (10 μM) for 2 days. Inferred MW of band indicated. Data are from one experiment. H , Viability (ATP) of A549 cells upon treatment with Na2SeO3 and the indicated concentrations of reducing agents βMe, DTT, or TCEP, 3 days. Data are the mean ± SEM of n=3 biological replicates.

    Article Snippet: Purified products were cloned by Golden Gate assembly into BsmBI-v2–digested pLentiCRISPRv2 (Cas9 libraries) or pRDA-550 (enAsCas12a libraries) using the NEBridge Golden Gate Assembly Kit (E1602L, New England Biolabs).

    Techniques: Western Blot, Knock-Out, Clone Assay, Cell Culture, Concentration Assay, Derivative Assay, Isotopic Labeling, Gas Chromatography-Mass Spectrometry, Liquid Chromatography with Mass Spectroscopy, Staining, Expressing