Journal: Cancer Discovery

Article Title: Synthetic Essentiality of Tryptophan 2,3-Dioxygenase 2 in APC-Mutated Colorectal Cancer

doi: 10.1158/2159-8290.cd-21-0680

Figure Lengend Snippet: Figure 1. TDO2 as a synthetic essential gene for the mutant APC gene in colorectal cancer (CRC). A, Venn diagram analysis using three different data sets identified TDO2 as a top potential SE gene. B, TDO2 mRNA expression is significantly correlated with the expression of WNT pathway signature genes in TCGA colorectal cancer (COAD + READ, provisional) patients (n = 433). ****, P < 0.0001. C, Representative images of IHC staining for TDO2 in serial sectioned human colorectal cancer tumors with negative (n = 34) and positive nuclear β-catenin (n = 47). Scale bars, × 10 (200 μm) and × 40 (50 μm). D, Colorectal cancer tumors with nuclear β-catenin showed higher TDO2 expression (TDO2 staining score 0–3). Pearson correlation coefficient = 42.342; ****, P < 0.0001. Chi-squared test. E, IHC analysis of colorectal cancer tumors from iAP and iKAP mice showed increased nuclear β-catenin, Ki-67, and TDO2 compared with normal colon tissue. Dotted lines separate normal and tumor regions; arrows indicate Ki-67/β-catenin–posi- tive tumor regions. Scale bar, 100 μm for iAP and 500 μm for iKAP. F, Immunoblotting for TDO2 in organoids isolated from C57BL/6J ileum and ApcMin/+ mice. SI, small intestine. G, Immunoblotting for TDO2 in colonoids isolated from C57BL/6J mice. APC-KO colonoids were APC-deleted by CRISPR/Cas9. LI, large intestine.

Article Snippet: The mutated TDO2 ORF gene insert was subcloned into PS100102 (pLenti-C-mGFP-P2A-BSD Tagged Cloning Vector; Origene; cat. #PS10094).

Techniques: Mutagenesis, Expressing, Immunohistochemistry, Staining, Western Blot, Isolation, CRISPR

Journal: Cancer Discovery

Article Title: Synthetic Essentiality of Tryptophan 2,3-Dioxygenase 2 in APC-Mutated Colorectal Cancer

doi: 10.1158/2159-8290.cd-21-0680

Figure Lengend Snippet: Figure 2. TCF4/TCF7L2 mediates the upregulation of TDO2 in APC-mutated colorectal cancer (CRC) cells. A, Immunoblots for TDO2 and β-catenin in colorectal cancer cell lines RKO (human) and MC38 (mouse) with their isogenic APC-KO counterparts. At least three independent experiments were performed. B, RT-qPCR showed APC-deleted RKO and MC38 cell lines exhibit increased Tdo2 mRNA expression. At least three independent experiments were performed. *, P < 0.05; **, P < 0.01; ****, P < 0.0001. FC, fold change. C, DNA sequence binding motif for the transcription factor TCF4/TCF7L2. Pro- moter regions of human and mouse TDO2 genes harbor TCF4 binding motifs near the transcription start site. The motif sequence is conserved in human and mouse genes. D, ChIP-seq in APC-WT and APC-KO MC38 cells showed binding peaks for TCF4 on the promoters of the TDO2 gene. E, ChIP-PCR using the TCF4 antibody showed enriched binding to the promoter regions of the TDO2 gene in DLD-1 cells. GAPDH as a negative control; MYC and AXIN2 as positive controls. F, Luciferase activity of the human TDO2 (hTDO2) promoter in HEK 293T cells with a constitutively active form of β-catenin (Δ90) when cotransfected with dominant-negative (DN) TCF4. ***, P < 0.001. Two independent experiments were performed. G, Luciferase activity of the hTDO2 pro- moter in HEK 293T cells with a constitutively active form of β-catenin (Δ90) and dominant-negative TCF4. **, P < 0.01. H, Luciferase activity of the TCF4 binding motif–mutated hTDO2 promoter in HEK 293T cells with a constitutively active form of β-catenin (Δ90) and dominant-negative TCF4. n.s., P > 0.05. I, Immunoblots for TDO2 and TCF4 in APC-KO MC38 cell lysates after transfecting with siControl (siCon) or three different siTCF4s.

Article Snippet: The mutated TDO2 ORF gene insert was subcloned into PS100102 (pLenti-C-mGFP-P2A-BSD Tagged Cloning Vector; Origene; cat. #PS10094).

Techniques: Western Blot, Quantitative RT-PCR, Expressing, Sequencing, Binding Assay, ChIP-sequencing, Negative Control, Luciferase, Activity Assay, Dominant Negative Mutation

Journal: Cancer Discovery

Article Title: Synthetic Essentiality of Tryptophan 2,3-Dioxygenase 2 in APC-Mutated Colorectal Cancer

doi: 10.1158/2159-8290.cd-21-0680

Figure Lengend Snippet: Figure 3. TDO2–Kyn–AhR signaling is essential for cell survival in APC-mutated colorectal cancer (CRC) cells. A, Representative images of colony forma- tion assays of APC-WT and APC-KO MC38 cell lines expressing shTDO2. Three independent experiments were performed. B, Quantification of A. n.s., P > 0.05; ***, P < 0.001. FC, fold change; shCont, shControl. C, Immunoblots of cleaved caspase-3 in APC-WT and APC-KO MC38 cell lines with inducible shTDO2 after doxycycline (Dox) treatment. D, Immunoblots for TDO2 and cleaved caspase-3 in ApcMin/+ ishControl and ishTDO2 organoid cell lysates after Dox treatment for 48 hours. Three independent experiments were performed. E, Brightfield images of APC-WT and APC-KO colonoids treated with DMSO or 20 μm TDO2 inhibi- tor (680C91) for 48 hours. Scale bars, × 40 (50 μm). F, Total flux measurement of tumors in Supplementary Fig. S4E. n.s., P > 0.05; *, P < 0.05; **, P < 0.01. G, Survival curves of C57BL/6J mice orthotopically implanted with ishTDO2 APC-WT and APC-KO MC38 cell lines (2 × 105 cells). Dox food was supplied at day 5 after orthotopic injection to induce TDO2 knockdown in vivo. n.s., P > 0.05; **, P < 0.01; ***, P < 0.001. Log-rank (Mantel–Cox) test. H, Survival curves of C57BL/6J mice orthotopically implanted with APC-WT and APC-KO MC38 cell lines (2 × 105 cells). TDO2 inhibitor (TDO2i) treatment (100 mg/kg) was initiated at day 5 after injection twice a day by oral gavage. n.s., P > 0.05; **, P < 0.01; ***, P < 0.001. Log-rank (Mantel–Cox) test. I, Survival curves of iAP mice after tamoxifen induction in the distal colon. Vehicle or TDO2 inhibitor treatment (100 mg/kg) was initiated at day 24 after induction once a day by oral gav- age. Log-rank (Mantel–Cox) test.

Article Snippet: The mutated TDO2 ORF gene insert was subcloned into PS100102 (pLenti-C-mGFP-P2A-BSD Tagged Cloning Vector; Origene; cat. #PS10094).

Techniques: Expressing, Western Blot, Injection, Knockdown, In Vivo

Journal: Cancer Discovery

Article Title: Synthetic Essentiality of Tryptophan 2,3-Dioxygenase 2 in APC-Mutated Colorectal Cancer

doi: 10.1158/2159-8290.cd-21-0680

Figure Lengend Snippet: Figure 4. TDO2 mediates tumor growth by regulating macrophage infiltration. A, GSEA (Hallmark gene sets) on genes that overlap between RNA-seq data sets of ishTDO2 APC-KO MC38 cell lines (no Dox vs. 48-hour Dox, n = 3) and microarray data sets of allograft tumors established with ishTDO2 APC- KO MC38 cell lines (no Dox vs. Dox treated, n = 3). The blue bars indicate immune response–related pathways. RNA-seq data from APC-KO MC38 ishTDO2 cell lines and tumor microarray data sets from the tumors established by the cell lines in C57BL/6J mice were overlapped and further narrowed down the list using the pathways that are upregulated by APC deletion to identify the pathways regulated by both the WNT pathway and TDO2. B, GSEA correla- tion of TNFA signaling and inflammatory response with alternatively expressed genes in TDO2-depleted APC-KO MC38 cells. Normalized enrichment scores (NES) and nominal P values are shown. C, viSNE analysis of F4/80+ and CD206+ immune cells assessed by CyTOF from colorectal cancer orthotopic ishTDO2 APC-WT and APC-KO MC38 tumors. D, Quantification of macrophages (CD11b+ F4/80+) and M2 macrophages (CD11b+ F4/80+ CD206hi) in CD45+ cells from tumors shown in C. CyTOF data were analyzed by FlowJo. Data, mean ± SD. n.s., P > 0.05; *, P < 0.05. n = 3 per group. E, TDO2 mRNA expression significantly correlates with the expression of total macrophage markers and M2 macrophage markers in TCGA colorectal cancer (COAD + READ, pro- visional) patients (n = 433). ****, P < 0.0001. F, Representative images of IHC staining for CD163 in serial sectioned human colorectal cancer tumors with negative (n = 42) and positive (n = 50) nuclear β-catenin. Scale bars, × 10 (200 μm) and × 20 (100 μm). G, Colorectal cancer tumors with nuclear β-catenin showed higher CD163 expression. Pearson correlation coefficient = 5.074, P = 0.0243. Chi-squared test. *, P < 0.05.

Article Snippet: The mutated TDO2 ORF gene insert was subcloned into PS100102 (pLenti-C-mGFP-P2A-BSD Tagged Cloning Vector; Origene; cat. #PS10094).

Techniques: RNA Sequencing, Microarray, Expressing, Immunohistochemistry

Journal: bioRxiv

Article Title: Nuclear m6A Reader YTHDC1 Promotes Muscle Stem Cell Activation/Proliferation by Regulating mRNA Splicing and Nuclear Export

doi: 10.1101/2022.08.07.503064

Figure Lengend Snippet: A. Schematic illustration of SC collection from Pax7-nGFP mice. Fixed quiescent SCs (QSC), freshly isolated SCs (FISC) and cultured SCs (ASC) were subject to RNA-seq, western blotting (WB), and Immunofluorescence (IF) analyses. B. The expression dynamics of m6A writer, reader and eraser proteins in the above cells from analyzing the RNA-seq data. C. Representative RNA-seq tracks showing the expression dynamics of the selected m6A regulators. D. The expression dynamic of YTHDC1 mRNA (FPKM) from RNA-seq. E. WB showing the induction of YTHDC1, YTHDF1 and YTHDF2 proteins upon SC activation and proliferation. *denotes the correct position of YTHDC1. Histone H3 was used as a loading control. F. IF staining showing the induction of YTHDC1 protein upon SC activation and proliferation. Scale bar=20μm. G. WB showing the predominant location of YTHDC1 in nuclear portion of C2C12 myoblasts.

Article Snippet: Two donor plasmids were generated by overlap PCR ∼500bp Ythdc1 genomic sequence flanking BSD/HygR-P2A-mAID-mCherry2 sequence (Addgene plasmid #121180, #121183) and cloning into pMD20-T vectors by T-A ligation.

Techniques: Isolation, Cell Culture, RNA Sequencing, Western Blot, Immunofluorescence, Expressing, Activation Assay, Control, Staining

Journal: bioRxiv

Article Title: Nuclear m6A Reader YTHDC1 Promotes Muscle Stem Cell Activation/Proliferation by Regulating mRNA Splicing and Nuclear Export

doi: 10.1101/2022.08.07.503064

Figure Lengend Snippet: A. Breeding scheme for generating YTHDC1 inducible knockout (iKO) and control (Ctrl) mice. B. Schematic outline of the tamoxifen (TMX) administration used in the study and experimental design for testing the effect of YTHDC1 deletion on barium chloride (BaCl 2 ) induced muscle regeneration process. C. Left: WB showing the deletion of YTHDC1 in ASC-48h from iKO but not Ctrl mice. Right: no obvious morphological difference was detected in iKO vs. Ctrl mice. D. H&E staining of the above injured muscles at 0, 5 and 7 dpi. Scale bar=100μm. E. Left: Immunostaining of eMyHC (red) and laminin (green) of the above injured TA muscles at 5 and 7 dpi. Scale bar=100μm. Right: Quantification of eMyHC positive fibers per field. n =3 mice per group. F. Immunostaining of Pax7 (red) and laminin (green) on TA muscle sections at 0, 5 and 7 dpi. Scale bar=100μm. G. Quantification of Pax7 positive SCs per field at 0, 5 and 7 dpi. n =4 mice per group for 0dpi, n =3 mice per group for 5 and 7dpi. Bars represent mean ± s.d. for all graphs. Statistical significance was determined using a two-tailed Student’s t test.

Article Snippet: Two donor plasmids were generated by overlap PCR ∼500bp Ythdc1 genomic sequence flanking BSD/HygR-P2A-mAID-mCherry2 sequence (Addgene plasmid #121180, #121183) and cloning into pMD20-T vectors by T-A ligation.

Techniques: Knock-Out, Control, Staining, Muscles, Immunostaining, Two Tailed Test

Journal: bioRxiv

Article Title: Nuclear m6A Reader YTHDC1 Promotes Muscle Stem Cell Activation/Proliferation by Regulating mRNA Splicing and Nuclear Export

doi: 10.1101/2022.08.07.503064

Figure Lengend Snippet: A. Left: EdU (red) staining of ASC-24h and ASC-48h from iKO and Ctrl mice. Scale bar=200μm. Right: Quantification of percentage of EdU+ cells. n =3 mice per group for ASC-24h, n =4 mice per group for ASC-48h. B. Left: EdU (red) staining of EDL myofibers isolated from Ctrl or iKO and cultured for 48h. Scale bar=100μm. Right: Quantification of the percentage of Pax7+EdU+ SCs. n =3 mice per group. C. Top: IF staining of Pax7 (green) and MyoD (red) of FISC (0h), ASC-24h and ASC-48h from Ctrl and iKO mice. Scale bar=100μm. Bottom: Quantification of the percentage of Pax7+MyoD+ cells. n =3 mice per group. D. Top: IF staining of Pax7 (green) and MyoD (red) on EDL myofibers at 0h (freshly isolated), 24h and 48h. Scale bar=100μm. Bottom: Quantification of the number of Pax7+MyoD+ cells per fiber. ( n =4 mice per group for 0 and 24h, n =5 mice per group for 48h) E. Schematic illustration of in vivo EdU assay in Ctrl and iKO mice. F. Top: EdU staining of the above freshly isolated and fixed SCs at 3 dpi. Scale bar=200μm. Bottom: Quantification of the percentage of EdU+ cells in iKO vs. Ctrl. n =3 mice per group. G-H . Left: RNA-seq was performed in ASC-24h or −48h from iKO and Ctrl. Volcano plot showing the down- and up-regulated genes in iKO vs. Ctrl. Right: GO analysis for the down-regulated genes. I. Schematic illustration of generating a C2C12 cell line with inducible YTHDC1 degradation using the auxin-inducible degron (AID2) system. J. Two independent mAID-YTHDC1 cell lines were treated with DMSO or 5-Ph-IAA(IAA) for the indicated time. EdU assay was performed and the percentage of EdU+ cells were quantified at the designated time points. n =3 replicates. Bars represent mean ± s.d. for all graphs. Statistical significance was determined using a two-tailed Student’s t test.

Article Snippet: Two donor plasmids were generated by overlap PCR ∼500bp Ythdc1 genomic sequence flanking BSD/HygR-P2A-mAID-mCherry2 sequence (Addgene plasmid #121180, #121183) and cloning into pMD20-T vectors by T-A ligation.

Techniques: Staining, Isolation, Cell Culture, In Vivo, EdU Assay, RNA Sequencing, Two Tailed Test

Journal: bioRxiv

Article Title: Nuclear m6A Reader YTHDC1 Promotes Muscle Stem Cell Activation/Proliferation by Regulating mRNA Splicing and Nuclear Export

doi: 10.1101/2022.08.07.503064

Figure Lengend Snippet: A. Schematic illustration of the experimental design for performing LACE-seq and subsequent combination with MeRIP-seq, bulk RNA-seq, subcellular RNA-seq for defining and elucidating YTHDC1 splicing/export targets and post-transcriptional regulation. B. LACE-seq was performed in both ASC-48h and C2C12 myoblasts and the number of identified peaks in each technical (T) or biological replicate (B) and the shared number of peaks (genes) between the replicates are shown. C. Left: Go analysis for the identified YTHDC1 targets in ASCs. Right: genome tracks for three selected genes. D. Left: Go analysis for the identified YTHDC1 targets in C2C12. Right: genome tracks for three selected genes. E. Top: Overlapping between the above identified C2C12 and ASC peaks. Bottom: Genome tracks for two selected genes. F-G. Left: the genome distribution of YTHDC1 binding peaks in C2C12 or ASC. Right: detailed distribution of the YTHDC1 binding peaks on repeat regions. H. Left: Integrating C2C12 MeRIP-seq data with the C2C12 LACE-seq identified 5356 regions (1771 mRNAs) as m6A-YTHDC1 targets. Right: Distribution of YTHDC1 binding on the above targets. I. Left: GO analysis of the above 1771 targets. Right: Genomic tracks of a selected target, Phlda3. J. Left: Integrating the C2C12 MeRIP-seq data with the ASC LACE-seq identified 56 regions (50 mRNAs) as m6A-YTHDC1 targets. Right: Distribution of YTHDC1 binding on the above identified target mRNAs. K. Left: GO analysis of the above 50 targets. Right: Genomic tracks of a selected target, Ybx1.

Article Snippet: Two donor plasmids were generated by overlap PCR ∼500bp Ythdc1 genomic sequence flanking BSD/HygR-P2A-mAID-mCherry2 sequence (Addgene plasmid #121180, #121183) and cloning into pMD20-T vectors by T-A ligation.

Techniques: RNA Sequencing, Binding Assay

Journal: bioRxiv

Article Title: Nuclear m6A Reader YTHDC1 Promotes Muscle Stem Cell Activation/Proliferation by Regulating mRNA Splicing and Nuclear Export

doi: 10.1101/2022.08.07.503064

Figure Lengend Snippet: A. Splicing analysis based on bulk RNA-seq data from ASC-24h or −48h defined five types of splicing events. The total number of each event in Ctrl, and the differential spliced events (DSE) in iKO vs. Ctrl are shown. B. Left: Combining the above ASC-48h DSEs with the ASC LACE-seq targets identified a total of 189 YTHDC1 splicing target mRNAs. Right: genome tracks of three selected targets. C . Top: RT-PCR assay was performed in ASC-48h from YTHDC1-iKO and Ctrl to verify altered splicing of the three selected target mRNAs, Palb2, Lrp8 and Scn5a. GAPDH was used as a control. Bottom: Quantification of exon inclusion level. Exon inclusion level was defined as the percentage of transcripts which includes the specific exon. Included / (Included + Skipped). n =3 mice per group for Lrp8 and Scn5a. D. Left: combining the above ASC-48h DSE with m6A-YTHDC1 targets uncovered 11 m6A-YTHDC1 splicing targets. Right: genome tracks of two selected targets. E. Top: RT-PCR assay was performed in ASC-48 from YTHDC1-iKO and Ctrl to verify altered splicing of the two selected target mRNA, Itgb3bp, and Nek1. GAPDH was used as a control. Bottom: Quantification of exon inclusion level. n =3 mice per group. F. Left: Splicing analysis based on bulk RNA-seq data from C2C12-mAID-YTHDC1 cells with or without YTHDC1 degradation. Right: Combining the above C2C12 DSEs with the C2C12 LACE-seq targets identified a total of 1040 YTHDC1 splicing target mRNAs. G. Overlapping between the above identified YTHDC1 splicing targets in ASC-48h and C2C12. H. Combining the above C2C12 DSEs with m6A-YTHDC1 targets uncovered 350 m6A-YTHDC1 splicing targets. I . Overlapping of m6A-YTHDC1 splicing targets in C2C12 and ASC-48h. Bars represent mean ± s.d. for all graphs. Statistical significance was determined using a two-tailed Student’s t test.

Article Snippet: Two donor plasmids were generated by overlap PCR ∼500bp Ythdc1 genomic sequence flanking BSD/HygR-P2A-mAID-mCherry2 sequence (Addgene plasmid #121180, #121183) and cloning into pMD20-T vectors by T-A ligation.

Techniques: RNA Sequencing, Reverse Transcription Polymerase Chain Reaction, Control, Two Tailed Test

Journal: bioRxiv

Article Title: Nuclear m6A Reader YTHDC1 Promotes Muscle Stem Cell Activation/Proliferation by Regulating mRNA Splicing and Nuclear Export

doi: 10.1101/2022.08.07.503064

Figure Lengend Snippet: A. Subcellular RNA-seq was performed using cytoplasmic and nuclear fractions isolated from ASC-48h of Ctrl and YTHDC1 iKO. The log2 (cyto/nuc) expression change was calculated for YTHDC1 targets. On the top and right, the density plot of log2 (cyto/nuc) expression changes is depicted. B. Quantification of log2 (cyto/nuc) expression changes in iKO vs. Ctrl. C. Overlapping between YTHDC1 mRNA export targets and splicing targets in ASCs. D. Genome tracks of two selected export targets. E-I. The above assay/analysis was performed in DMSO or IAA treated mAID-YTHDC1 C2C12 myoblasts to identify YTHDC1 regulated export targets in C2C12. J-N. The above analyses were conducted using m6A-YTHDC1 targets to identify m6A-YTHDC1 mRNA export targets in C2C12.

Article Snippet: Two donor plasmids were generated by overlap PCR ∼500bp Ythdc1 genomic sequence flanking BSD/HygR-P2A-mAID-mCherry2 sequence (Addgene plasmid #121180, #121183) and cloning into pMD20-T vectors by T-A ligation.

Techniques: RNA Sequencing, Isolation, Expressing

Journal: bioRxiv

Article Title: Nuclear m6A Reader YTHDC1 Promotes Muscle Stem Cell Activation/Proliferation by Regulating mRNA Splicing and Nuclear Export

doi: 10.1101/2022.08.07.503064

Figure Lengend Snippet: A-B. Schematic illustration of the Co-IP/MS procedure. An empty vector or Flag tagged YTHDC1 plasmid was expressed in C2C12 myoblasts followed by pull down with Flag beads; the retrieved proteins were subject to MS with Bruker timsTOF Pro. B. Overexpression of the Flag tagged YTHDC1 was confirmed by WB using anti-Flag antibody. C. 912 proteins were uniquely retrieved in YTHDC1 but not Vector expressing cells. D. GO functions of the above proteins are shown. E-G. The above identified interacting proteins with RNA splicing, mRNA export, or potential transcriptional regulatory functions are shown in the lists. H. Flag tagged YTHDC1 was overexpressed in C2C12 and Flag-beads based IP was performed followed by WB to verify the retrieved hnRNPG protein. I. IP of endogenous YTHDC1 protein in C2C12 myoblasts followed by WB to examine retrieved hnRNPG protein. J. Flag-hnRNPG and HA-YTHDC were overexpressed in 293T cells; IP with YTHDC1 or hnRNPG protein flowed by WB to confirm the interaction between the two proteins.

Article Snippet: Two donor plasmids were generated by overlap PCR ∼500bp Ythdc1 genomic sequence flanking BSD/HygR-P2A-mAID-mCherry2 sequence (Addgene plasmid #121180, #121183) and cloning into pMD20-T vectors by T-A ligation.

Techniques: Co-Immunoprecipitation Assay, Plasmid Preparation, Over Expression, Expressing

Journal: bioRxiv

Article Title: Nuclear m6A Reader YTHDC1 Promotes Muscle Stem Cell Activation/Proliferation by Regulating mRNA Splicing and Nuclear Export

doi: 10.1101/2022.08.07.503064

Figure Lengend Snippet: YTHDC1 is induced upon SC activation and proliferation to promote SC activation and proliferation upon acute injury induced muscle regeneration. Mechanistically, it functions through both facilitating mRNA splicing synergistically with hnRNPG and promoting mRNA export possibly by binding with the THO nuclear export complex.

Article Snippet: Two donor plasmids were generated by overlap PCR ∼500bp Ythdc1 genomic sequence flanking BSD/HygR-P2A-mAID-mCherry2 sequence (Addgene plasmid #121180, #121183) and cloning into pMD20-T vectors by T-A ligation.

Techniques: Activation Assay, Binding Assay

Journal: The EMBO Journal

Article Title: Metabolic priming by multiple enzyme systems supports glycolysis, HIF1α stabilisation, and human cancer cell survival in early hypoxia

doi: 10.1038/s44318-024-00065-w

Figure Lengend Snippet: Reagents and tools table

Article Snippet: Plasmids pLenti-HA-IRES-BSD (PS100104) and pLenti-GFP-P2A-BSD (PS100103) were from Origene. pUC57- Lb NOX was a gift from Vamsi Mootha (Addgene plasmid # 75285) and used to subclone Lb NOX into the MluI and XhoI sites of pLenti-HA-IRES-BSD.

Techniques: Subcloning, Recombinant, Bicinchoninic Acid Protein Assay, Cell Viability Assay, RNA Sequencing, Amplification, CRISPR, Knock-Out, Biomarker Discovery, Software