bsmbiigested lenticrisprv2 backbone  (Addgene inc)

Journal: bioRxiv

Article Title: MYC-ATF4-ASS1 axis governs intracellular arginine synthesis and dictates the immune microenvironment in melanoma

doi: 10.64898/2026.02.27.707779

Figure Lengend Snippet: MYC regulates ASS1 expression in the context of BRAF/MEK inhibition . ( a and b ) Melanoma cells were treated with Dabrafenib/Trametinib (D/T) for 5 days. Total mRNAs were isolated and subjected to qPCR to detect the mRNA expression of ASS1 and MYC ( a ). The same treatment was performed, protein lysates were collected, and ASS1 and MYC protein expression were detected by western blot ( b ). β-ACTIN was used as a loading control. ( c-f ) Non-targeting sgRNA (sgNT) or sgRNAs targeting MYC (sgMYC) were designed, cloned to lentiCRISPRv2-blasticidin vector, used to package lentivirus, and infect target melanoma cells. After blasticidin selection, total mRNAs from survived cells were isolated and subjected to qPCR to quantify mRNA expression of ASS1 ( c ), while protein lysates were collected and used for western blot to detect protein expression of ASS1 and MYC ( d ). sgAss1 was used as a positive control. β-ACTIN was used as a loading control. Meanwhile, blasticidin resistant cells were seeded onto 6-well plates, treated with PBS or ADI-PEG20 for 10 days, and crystal violet was used to stain colonies formed. Representative images were shown ( e ) and area percentage of stained colonies were quantified ( f ).

Article Snippet: Briefly, non-targeting or gene-specific targeting oligos were annealed and ligated into a lentiCRISPRv2-blasticidin backbone (Addgene, 98293) cut with BsmBI-v2 (NEB, R0739S).

Techniques: Expressing, Inhibition, Isolation, Western Blot, Control, Clone Assay, Plasmid Preparation, Selection, Positive Control, Staining

Journal: bioRxiv

Article Title: MYC-ATF4-ASS1 axis governs intracellular arginine synthesis and dictates the immune microenvironment in melanoma

doi: 10.64898/2026.02.27.707779

Figure Lengend Snippet: ATF4 correlates with and regulates ASS1 expression . ( a ) WM4380-2 cells were treated with DMSO, D/T, ADI or D/T+ADI as shown in . Total mRNA were isolated and subjected to bulk RNA-seq. Normalized reads of ATF4 were presented. ( b ) Correlative analysis of ATF4 and ASS1 was performed using our bulk RNA-seq data as indicated in . R and P value were shown. ( c ) Correlative analysis of ATF4 and ASS1 was performed exploiting public dataset of spatical single cell RNA-seq. R and P valude were provided. ( d-f ) Non-targeting sgRNA (sgNT) or sgRNAs targeting ATF4 (sgATF4) were designed, cloned to lentiCRISPRv2-blasticidin vector, used to package lentivirus, and infect target melanoma cells. After blasticidin selection, protein lysates were collected and used for western blot to detect protein expression of ASS1 and ATF4 ( d ). β-ACTIN was used as a loading control. Meanwhile, blasticidin resistant cells were seeded onto 6-well plates, treated with PBS or ADI-PEG20 for 10 days, and crystal violet was used to stain colonies formed. Representative images were shown ( e ) and area percentage of stained colonies were quantified ( f ).

Article Snippet: Briefly, non-targeting or gene-specific targeting oligos were annealed and ligated into a lentiCRISPRv2-blasticidin backbone (Addgene, 98293) cut with BsmBI-v2 (NEB, R0739S).

Techniques: Expressing, Isolation, RNA Sequencing, Single Cell, Clone Assay, Plasmid Preparation, Selection, Western Blot, Control, Staining