lenticrisprv2 hygro  (Addgene inc)

Journal: Nature Structural & Molecular Biology

Article Title: Vitamin B2 metabolism promotes FSP1 stability to prevent ferroptosis

doi: 10.1038/s41594-026-01759-x

Figure Lengend Snippet: a , Schematic of riboflavin metabolism. b , Distribution for the relative enrichment of RFK and FLAD1 sgRNAs (blue lines) from the batch retest (BFP and GFP:BFP) screens. The gray line indicates the mean of the control sgRNA distribution. c , FENIX assay to determine the RTA activity of oxidized and reduced vitamin B2 at the indicated concentrations compared to the uninhibited (black) and positive control PMC (gray). d , Fluorescence histograms of FSP1 KO cells expressing GFP-tagged WT FSP1 in the indicated cell lines. e , Immunoblot of lysates from d . f , FAD quantification of lysates from d . Data are shown as the mean ± s.e.m. of four independent experiments. g , Fluorescence histograms of FSP1 KO cells expressing WT FSP1–GFP in the indicated cell lines supplemented with 50 µM exogenous flavin metabolites (vitamin B2, FMN or FAD) or treated with 1 µM MG132 for 24 h. h , Quantification of MFI change in GFP from g . Data are shown as the median ± s.e.m. of three independent experiments. i , Kinetics of GFP fluorescence decay in FSP1 KO cells expressing WT FSP1–GFP in the indicated cell lines following Dox washout with or without the presence of 1 µM MG132. j – l , Dose response of RSL3-induced cell death in FSP1 KO cells expressing WT FSP1–GFP in the indicated cell lines (Cas9 control, j ; RFK KO , k ; FLAD1 KO , l ) supplemented with or without 50 µM exogenous flavin metabolites (vitamin B2, FMN or FAD).

Article Snippet: FSP1–GFP-expressing cells were enriched by two rounds of FACS of the GFP-positive populations upon induction with 1 μg ml −1 Dox for 24 h. RFK KO and FLAD1 KO lines were generated by transduction with lentiCRISPRv2 hygro virus (Addgene, plasmid 98291) and selection in medium containing 375 μg ml −1 hygromycin.

Techniques: Control, Activity Assay, Positive Control, Fluorescence, Expressing, Western Blot

Journal: Nature Structural & Molecular Biology

Article Title: Vitamin B2 metabolism promotes FSP1 stability to prevent ferroptosis

doi: 10.1038/s41594-026-01759-x

Figure Lengend Snippet: a-b , Cloud plots indicating count numbers corresponding to RFK ( a ) or FLAD1 ( b ) (color scale) and control (gray scale) sgRNAs. c-d , Fluorescence histograms of FSP1 GFP-P2A-BFP cells in the indicated cell lines showing changes in GFP:BFP ratio ( c ) or GFP and BFP fluorescence ( d ). Data are shown as the median ± SEM of 3 independent experiments. e , Immunoblot of FSP1 GFP-P2A-BFP lysates from ( c-d ). f , FAD quantification of lysates from ( c-d ). Data are shown as the mean ± SEM of 4 independent experiments. g , Cloud plots indicating count numbers corresponding to SLC25A vitamin B2 transporters (color scale) and control (gray scale) sgRNAs. h , Distribution for the relative enrichment of SLC25A vitamin B2 transporters sgRNAs (blue lines). The gray line indicates the mean of the control sgRNA distribution. i-j , Fluorescence histograms of FSP1 GFP-P2A-BFP cells grown in normal or vitamin B2-deficient medium for 72 h showing changes in GFP:BFP ratio ( i ) or GFP and BFP fluorescence ( j ). Data are shown as the median ± SEM of 3 independent experiments. k , Immunoblot of FSP1 GFP-P2A-BFP lysates from ( i-j ). l , Dose response of RSL3-induced cell death in FSP1 GFP-P2A-BFP cells grown in normal or vitamin B2-deficient medium for 72 h.

Article Snippet: FSP1–GFP-expressing cells were enriched by two rounds of FACS of the GFP-positive populations upon induction with 1 μg ml −1 Dox for 24 h. RFK KO and FLAD1 KO lines were generated by transduction with lentiCRISPRv2 hygro virus (Addgene, plasmid 98291) and selection in medium containing 375 μg ml −1 hygromycin.

Techniques: Control, Fluorescence, Western Blot

Journal: Nature Structural & Molecular Biology

Article Title: Vitamin B2 metabolism promotes FSP1 stability to prevent ferroptosis

doi: 10.1038/s41594-026-01759-x

Figure Lengend Snippet: a , FAD quantification of FSP1 KO cells expressing FSP1-GFP supplemented with 50 µM exogenous flavin metabolites (vitamin B2, FMN, or FAD) for 5 days. Data are shown as the mean ± SEM of 4 independent experiments. b , Immunoblot of lysates from ( a ). c , FSP1 turnover in FSP1/RFK DKO and FSP1/FLAD1 DKO cells at indicated times after inhibiting protein synthesis with 75 µM emetine in the presence or absence of 10 µM MG132. d , Fluorescence histograms after 12 h of doxycycline washout from FSP1 KO cells expressing FSP1-GFP in the indicated cell lines. e , Characterization of in-vitro translated FSP1 visualized by immunoblot (top) and Coomassie gel (bottom). f , NADH oxidation by in-vitro translated FSP1 in the presence of flavin metabolites (vitamin B2, FMN, or FAD) and coenzyme Q 1 . Data are shown as the mean ± SEM of 3 independent experiments. g-h , RFK ( g ) or FLAD1 ( h ) expression positively correlates to ferroptosis inducers, RSL3 and ML162, in cancer cells. Plotted data were mined from the CTRP database, which contains correlation coefficients between gene expression and drug sensitivity for 860 cancer cell lines treated with 481 compounds. i , Percent cell death of FSP1 KO cells expressing FSP1-GFP in the indicated cell lines in the presence of various cell death inhibitors (1 µM RSL3, 2 µM Fer-1, 20 µM ZVAD, and 1 µM Nec-1). Data are shown as the mean ± SEM of 3 independent experiments. j-l , Dose response of RSL3-induced cell death in FSP1 KO cells expressing FSP1-GFP in the indicated cell lines either untreated ( j ), treated with 2 µM FSEN1 ( k ), or without 24 h treatment of doxycycline ( l ).

Article Snippet: FSP1–GFP-expressing cells were enriched by two rounds of FACS of the GFP-positive populations upon induction with 1 μg ml −1 Dox for 24 h. RFK KO and FLAD1 KO lines were generated by transduction with lentiCRISPRv2 hygro virus (Addgene, plasmid 98291) and selection in medium containing 375 μg ml −1 hygromycin.

Techniques: Expressing, Western Blot, Fluorescence, In Vitro, Gene Expression

Journal: Nature Structural & Molecular Biology

Article Title: Vitamin B2 metabolism promotes FSP1 stability to prevent ferroptosis

doi: 10.1038/s41594-026-01759-x

Figure Lengend Snippet: a-b , Summary of FSP1 gene expression and RFK ( a ) or FLAD1 ( b ) gene expression in various non-small cell lung cancer cell lines, data mined from the CTRP database. c-f , Dose response of RSL3-induced cell death in the indicated cell lines: H520 ( c ), H460 ( d ), H441 ( e ), and H1703 ( f ). Data are shown as the mean ± SEM of 3 independent experiments. g , Immunoblots of FSP1 and select flavoproteins in U-2 OS KO cell lines. h , Immunoblots of FSP1 and select flavoproteins in non-small cell lung cancer cell lines transfected with the indicated control and RFK and FLAD targeted siRNAs (n = 3) (Also see Supplementary Information Fig. ).

Article Snippet: FSP1–GFP-expressing cells were enriched by two rounds of FACS of the GFP-positive populations upon induction with 1 μg ml −1 Dox for 24 h. RFK KO and FLAD1 KO lines were generated by transduction with lentiCRISPRv2 hygro virus (Addgene, plasmid 98291) and selection in medium containing 375 μg ml −1 hygromycin.

Techniques: Gene Expression, Western Blot, Transfection, Control