Transposable Element RNA Dysregulation hauv Mutant KRAS (G12C) 3D Lung Cancer Spheroids

TSAB NTAWV

Mutant KRAS tswj kev hloov pauv hloov pauv (TE) RNA thiab interferon-stimulated gene (ISG) qhia, tab sis nws tseem tsis paub meej tias muaj ntau yam kev hloov pauv hauv KRAS cuam tshuam rau TE RNAs sib txawv thoob plaws hauv genome. Peb tau txheeb xyuas cov ntawv sau tseg ntawm 3D tib neeg lub ntsws qog noj ntshav spheroids uas muaj kev hloov pauv KRAS (G12C) los txiav txim siab thaj chaw ntawm TE RNAs tswj los ntawm mutant KRAS (G12C). Peb pom tias KRAS (G12C) kev taw qhia yog xav tau rau kev qhia ntawm LINE- thiab LTR-los ntawm TE RNAs uas txawv ntawm TE RNAs yav dhau los tau pom tias tau tswj hwm los ntawm mutant KRAS (G12D) lossis KRAS (G12V). Ntxiv mus, KRAS (G12C) inhibition tshwj xeeb upregulates SINE-derived TE RNAs los ntawm tus yau tshaj Alu subfamily AluY. Peb cov txiaj ntsig tau nthuav tawm tias TE RNA dysregulation hauv KRAS uas tau tsav lub ntsws qog nqaij hlav qog noj ntshav yog hloov pauv, thaum tseem qhia txog cov tub ntxhais hluas, Alu-derived TE RNAs uas tau sib koom ua ke nrog kev tiv thaiv kab mob hauv lub cev raws li KRAS (G12C) inhibition.

Cov txiaj ntsig ntawm cistanche tubulosa-Antitumor

Taw qhia

Transposable element (TE) RNAs yog recurrently dysregulated nyob rau hauv cov ntsiab lus ntawm mob qog noj ntshav 1. Nyob rau hauv mutant KRAS lub ntsws cancer hlwb, KRAB zinc-ntiv tes (KZNF) noob yog dav downregulated, ua rau lub aberrant upregulation ntawm TE RNAs muab los ntawm LINE, SINE, thiab LTR cov ntsiab lus 2,3. Ntxiv rau TE RNAs, ntev noncoding RNAs (lncRNAs) kuj tseem tswj hwm nrog RAS signaling genes 4, thiab lawv cov qauv qhia tau zoo sib xws hauv ntau cov qog nqaij hlav 5-8. Rau TE RNAs, lawv cov kev tswj hwm hauv kev mob qog noj ntshav ua rau lub xeev ntawm cov kab mob qog noj ntshav, ua rau muaj kev ua haujlwm ntawm lub cev tiv thaiv kab mob xws li interferon-stimulated genes (ISGs) 3,9,10. Tshwj xeeb, Alu tsev neeg ntawm SINEs yog ib qho tseem ceeb ntawm immunogenic TE RNAs 11, uas yog induced los ntawm epigenetic hloov los ntawm DNA methyltransferase inhibitors (DNMTi) los yog mutant KRAS-mediated KZNF inhibition 3,9,10.

Txhawm rau tshawb xyuas seb qhov kev hloov pauv hauv KRAS cuam tshuam li cas rau TE RNA toj roob hauv pes hauv cov qog nqaij hlav cancer hauv lub ntsws, peb pom cov ntawv sau tseg ntawm 3D mob ntsws cancer spheroids uas harbor KRAS (G12C) mutations nyob rau hauv lub xub ntiag los yog tsis muaj mutant KRAS (G12C) inhibitor 12. Peb qhia. hais tias KRAS (G12C) signaling yog yuav tsum tau rau kev qhia ntawm LINE thiab LTR-derived TE RNAs, thaum KRAS (G12C) inhibition tshwj xeeb upregulates SINE-derived TE RNAs thiab ib tug subset ntawm interferon (IFN) cov noob caj noob ces. Peb qhov kev tshawb pom qhia txog kev sib cuam tshuam ntawm cov mutant KRAS signaling thiab TE dysregulation nyob rau hauv lub ntsws cancer hlwb, qhov twg cov txheej txheem ntawm cov tub ntxhais hluas AluY cov ntsiab lus yog upregulated nyob rau hauv ib tug mutation-dependent yam.

Cov txiaj ntsig ntawm cistanche tubulosa-Antitumor

TSEEM CEEB

KRAS (G12C) inhibition hloov cov coding thiab noncoding transcriptome

Txhawm rau txiav txim siab li cas oncogenic KRAS (G12C) signaling tswj cov coding thiab noncoding transcriptome, peb tau ua RNA sequencing (RNA-seq) ntawm 3D mutant KRAS (G12C) ntsws cancer spheroids. Rau RNA-seq, peb tau siv cov txheej txheem ntev nrog 5 'cov cim tshwj xeeb molecular (UMIs) txhawm rau ua kom RNA suav suav suav nrog txo qis PCR amplification biases 13 (Daim duab 1A). Peb piv cov ntawv sau tseg ntawm H358 mob ntsws cancer spheroids kho nrog KRAS (G12C) inhibitor ARS-1620 (ARS) los tswj spheroids (DMSO-kho) (Daim duab S1A) thiab pom tias ARS kev kho mob txo qis qib ntawm phosphorylated ERK. (p-ERK) (Daim duab S1B), qhia tias kev kho ARS yog inhibiting downstream KRAS (G12C) signaling. Kev tawm tsam ntawm KRAS (G12C) kev taw qhia los ntawm kev kho mob ARS kuj tau ua pov thawj los ntawm kev txo qis hauv spheroid loj thiab cell viability (Daim duab S1C thiab S1D).

Nyob rau theem RNA, peb tau soj ntsuam cov txheeb ze ntawm ntau hom biotypes thiab TE superfamilies hauv ARS- lossis DMSO-kho 3D mob ntsws cancer spheroids, uas qhia txog kev hloov pauv hauv TE RNA muaj pes tsawg leeg raws li KRAS (G12C) inhibition (Daim duab 1B). Thaum peb kuaj pom tsuas yog 32% ntawm GENCODE-annotated protein-coding noob thiab 15% ntawm lncRNA noob, 92-96% ntawm TE superfamilies (92% LTR, 95% SINE, 96% LINE) tau sawv cev hauv peb UMI-tagged RNA-seq cov ntaub ntawv, nthuav tawm dav dysregulation ntawm TE RNAs nyob rau hauv KRAS (G12C)-tsav transcriptome. Txog li ib feem peb ntawm tag nrho cov kuaj pom RNA molecules nyob rau hauv ARS-kho mob ntsws cancer spheroids tau muab los ntawm TEs, qhia tias TE RNAs sawv cev ib feem loj ntawm cov transcriptional output ntawm mutant KRAS (G12C) ntsws cancer hlwb.

cistanche cov txiaj ntsig rau txiv neej-ua kom muaj zog tiv thaiv kab mob

Nyem qhov no mus saib Cistanche Enhance Immunity khoom

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Peb tom ntej no tau txiav txim siab qhov sib txawv ntawm cov noob caj noob ces ntawm ARS- thiab DMSO-kho 3D ntsws cancer spheroids los txheeb xyuas cov txheej txheem lom neeg uas tau tswj hwm los ntawm oncogenic KRAS (G12C) signaling. Mob ntsws cancer spheroids nrog qhov tsis zoo KRAS (G12C) signaling tau ua kom muaj txiaj ntsig zoo rau cov noob koom nrog hauv G2M checkpoint, E2F lub hom phiaj, MYC lub hom phiaj, thiab mitotic spindle (Daim duab 1C). Raws li KRAS (G12C) inhibition, txawm li cas los xij, mob ntsws cancer spheroids tau nthuav tawm ntau qib ntawm cov noob koom nrog oxidation phosphorylation, ntxiv, thiab IFN alpha thiab gamma cov lus teb (Daim duab 1C), muab cov pov thawj ntxiv rau kev koom tes ntawm KRAS teeb liab hauv txoj cai. ntawm IFN ntsig txog cov noob 2,3.

KRAS (G12C) inhibition ua haujlwm ua rau ISGs thiab cov tub ntxhais hluas AluY ntsiab

Txhawm rau nthuav qhia qhov kev tswj hwm qhov tseem ceeb ntawm ISGs raws li KRAS (G12C) inhibition, peb tau txheeb xyuas cov noob thiab TEs twg tau nthuav tawm ntau qhov sib txawv hauv txhua lub noob thiab TE superfamily, feem. Thoob plaws hauv ob qho tib si IFN alpha thiab IFN gamma teb cov noob, feem ntau muaj zog induced gene raws li KRAS (G12C) inhibition nyob rau hauv lub ntsws cancer spheroids yog RTP4 (Daim duab 2A), ib tug receptor transporter protein uas tsis zoo regulates TBK1 signaling 14. Tsis tas li ntawd, MHC class I complex. gene beta-2-microglobulin (B2M), uas yog recurrently inactivated nyob rau hauv lub ntsws cancer 15, kuj tseem ceeb upregulated nyob rau hauv ob qho tib si IFN-txog gene teev nyob rau hauv ARS-kho mob ntsws cancer spheroids (Daim duab 2A).

Muab lub luag haujlwm ncaj qha rau oncogenic KRAS signaling hauv TE RNA txoj cai 3, peb tau tshawb xyuas cov tsev neeg ntawm TE RNAs nyob ntawm mutant KRAS (G12C). Hauv kev mob ntsws cancer spheroids nrog cov cim KRAS (G12C) tsis zoo, peb pom tias LINE subfamilies L1M6B thiab L1PA12 tau qhia ntau heev thiab nyob ntawm KRAS (G12C), raws li pov thawj los ntawm lawv qhov kev txo qis hauv ntsws cancer spheroids kho nrog ARS (Daim duab 2B). Ntxiv mus, thaum tsuas yog ib qho DNA subfamily MER44D yog nyob ntawm KRAS (G12C) signaling, tshaj li kaum lub LTR subfamilies tau tswj hwm los ntawm mutant KRAS (G12C), suav nrog MLT1A0-int, LTR51, MER50B , thiab LTR1B0 (Daim duab 2B). Hauv qhov sib piv, SINE subfamilies tau tag nrho cov kev tswj hwm zoo raws li KRAS (G12C) inhibition thiab ua haujlwm sib koom tes nrog cov noob ISG tshwj xeeb (Daim duab 2A) hauv ntsws cancer spheroids. Cov SINE TE RNAs tag nrho tau muab los ntawm AluY subfamily (Daim duab 2B), qhia tias KRAS (G12C) inhibition dysregulates ib qho tshwj xeeb ntawm cov tub ntxhais hluas AluY ntsiab.

KRAS (G12C) inhibition downregulates ntev noncoding RNAs

Txhawm rau qhia ntxiv txog qhov cuam tshuam ntawm KRAS (G12C) inhibition ntawm daim ntawv teev lus, peb tau tshuaj xyuas txhua qhov sib txawv ntawm lncRNAs hauv ntsws cancer spheroids kho nrog ARS lossis DMSO tswj. Peb pom tias ntau tus lncRNAs tau nyob ntawm KRAS (G12C) qhov taw qhia rau lawv cov lus qhia, vim tias ntau ntawm cov lncRNAs tau raug txo qis rau KRAS (G12C) inhibition (Daim duab 3A). Peb ntawm no downregulated lncRNAs, AC114546.3, NCMAP-DT, thiab AC073575.2, tsis paub lub luag hauj lwm tab sis sib tshooj nyob rau hauv ib tug antisense orientation rau lub coding noob ZNF770, RCAN3, thiab ERP29, raws li. Raws li ib chav kawm ntawm noncoding RNA, peb tau pom dav dav downregulation ntawm lncRNAs raws li kev kho mob ARS hauv ntsws cancer spheroids (Daim duab 3B), qhia tias ntau lncRNAs nyob ntawm KRAS (G12C) signaling rau lawv qhia.

cistanche cov txiaj ntsig rau txiv neej-ua kom muaj zog tiv thaiv kab mob

Kev sib tham

Ntawm no peb qhia tau hais tias oncogenic KRAS (G12C) signaling yog yuav tsum tau rau kev qhia ntawm tshwj xeeb TE superfamilies, uas yog LINE thiab LTR ntsiab, nrog rau ib tug subset ntawm lncRNAs, ntxiv qhia tau hais tias yuav ua li cas RAS signaling tswj cov noncoding transcriptome 2-4. Peb kuj tau siv 3D mob ntsws cancer spheroid qauv rau peb cov KRAS (G12C) inhibitor thwmsim txij li cov qauv 3D tau pom tias muaj kev ntseeg ntau dua cov lus teb hauv vivo thaum piv rau 2D kab lis kev cai qauv 16. Tsis tas li ntawd, peb daim ntawv thov ntawm UMI-raws li tag nrho. -length RNA-seq cov txheej txheem 13 tau tso cai rau peb kom raug ntau dua ntes TE RNA muaj pes tsawg leeg thiab kev ua haujlwm hauv peb lub ntsws cancer spheroids los ntawm kev tshem tawm PCR duplicates hauv peb cov ntaub ntawv RNA-seq.

Peb qhov kev tshawb pom tau zoo ib yam nrog cov kev tshawb fawb yav dhau los ntawm KRAS (G12C) inhibition, qhov twg IFN alpha thiab gamma teb cov noob tau raug kho nyob rau hauv ARS-kho H358 mob qog noj ntshav hlwb 17. Raws li paub txog cov tshuaj tiv thaiv kab mob ntawm Alu-derived RNAs 11, peb cov txiaj ntsig tau qhia tias qhov tshwj xeeb upregulation ntawm cov tub ntxhais hluas AluY ntsiab raws li KRAS (G12C) inhibition yog tsawg kawg yog ib feem ntawm lub luag hauj lwm rau lub zog upregulation ntawm ISGs nyob rau hauv ARS-kho mob ntsws cancer spheroids. Qhov tseem ceeb, qhov tseem ceeb ntawm IFN-txog cov noob nyob rau hauv cov lus teb rau KRAS (G12C) inhibitor kev kho mob tsis suav nrog kev txhawb nqa ntxiv ntawm RNA sensor ISGs xws li MDA-5, RIG-I, lossis PKR, uas pib los ua kev tswj hwm hauv Lub ntsws hlwb teb rau oncogenic KRAS signaling 2,3.

Peb tau pom yav dhau los tias mutant KRAS signaling ib leeg yog txaus los ntxias TE RNA upregulation hauv tib neeg lub ntsws hlwb uas tau hloov pauv hauv vitro 2,3, thiab peb cov txiaj ntsig tau piav qhia ntawm no txuas ntxiv cov kev soj ntsuam no rau cov qog nqaij hlav ntsws nrog qhov sib txawv ua kom KRAS hloov pauv. KRAS (G12D) lossis KRAS (G12V) kev hloov pauv ob qho tib si ua rau muaj kev txhim kho tseem ceeb ntawm LTR12C subfamily hauv cov qog hlwb hloov pauv 3, tab sis peb tsis pom qhov tseem ceeb ntawm LTR12C-derived TE RNAs hauv peb KRAS (G12C) mob ntsws cancer spheroids. Hloov chaw, peb pom kev tswj hwm ntawm LTR51, LTR1B0, LTR14B, thiab LTR28B, qhia tias muaj ntau yam kev hloov pauv ntawm kev ua haujlwm hauv KRAS tswj hwm qhov sib txawv ntawm TE RNA cov ntawv sau tseg.

Peb txoj haujlwm muab kev ntsuam xyuas dav dav txog yuav ua li cas cov noncoding / TE RNA transcriptome dynamically teb rau KRAS (G12C) inhibition. Cov kev tshawb fawb yav tom ntej tuaj yeem muab kev nkag siab tshiab rau hauv lub luag haujlwm ntawm noncoding / TE RNAs hauv cov txheej txheem ntawm KRAS (G12C) inhibitor resistance. Tsis tas li ntawd, TE RNAs uas tau zais los ntawm cov qog nqaij hlav cancer thaum KRAS inhibition tuaj yeem ua haujlwm ntxiv rau RNA biomarkers 2,3,5,18,19 ntawm cov lus teb thiab / lossis tsis kam rau KRAS inhibitor kho 20.

Cov khoom siv thiab cov txheej txheem

Cell kab

H358 cov kab mob qog nqaij hlav qog nqaij hlav uas muaj KRAS (G12C) kev hloov pauv tau coj mus rau hauv RPMI 1640 nruab nrab (Invitrogen) ntxiv nrog 10% fetal bovine serum (Sigma) ntawm 37 degree , 5% CO2 hauv lub tshuab nqus tsev vaum. Txhua kab ntawm tes tau kuaj pom tsis zoo rau mycoplasma. Cov kab xov tooj ntawm tes tau yuav los ntawm American Type Culture Collection (ATCC).

Cell viability assay

Rau spheroid viability assys, 10,000 hlwb/zoo tau noob nyob rau hauv qis adhesion puag ncig hauv qab 96- zoo daim hlau thiab incubated ntawm 37 degree , 5% CO2 rau 24 teev. Tom qab ntawd serially diluted ARS - 1620 los yog DMSO tau ntxiv rau hauv lub hlwb, thiab cov phaj tau incubated nyob rau hauv tus txheej txheem kab lis kev cai tej yam kev mob rau 72 teev, nrog tshiab ARS thiab DMSO media raug hloov txhua hnub. Cell viability raug ntsuas siv Cell Titer-Glo® Luminescent Cell Viability Assay kit (Promega) raws li cov chaw tsim khoom raws tu qauv. Lub teeb liab luminescence ntawm ARS kho cov qauv yog normalized rau DMSO tswj. Luminescence tau ntsuas ntawm SpectraMax iD3 molecular ntaus ntawv.

Kev rho tawm RNA

Tag nrho cov RNA raug cais tawm ntawm kwv yees li 100 H358 spheroids (ib yam mob) siv Quick-RNA Mini-Prep cov khoom siv (Zymogen) raws li cov chaw tsim khoom raws tu qauv. RNA raug ntsuas los ntawm NanoDrop-8000 Spectrophotometer.

RNA-seq npaj tsev qiv ntawv

Kev yoog raws Smart-seq3 raws tu qauv 13 tau siv los tsim cov tsev qiv ntawv RNA-seq los ntawm tag nrho RNA los suav thiab ntsuas tag nrho-ntev RNA molecules. Luv luv, 10ng ntawm tag nrho RNA tau thim rov qab siv lub barcoded oligoDT primer (125nM) ua raws li cov qauv hloov nrog tus qauv barcoded hloov oligo (125nM). Cov oligo sequences no tau ua haujlwm ua primers rau PCR amplification. Cov khoom siv Nextera HT (Illumina) tau siv los hloov cDNA cov tsev qiv ntawv mus rau hauv cov tsev qiv ntawv sib txuas nrog qhov sib ntxiv ntawm UMI tshwj xeeb primer los nthuav cov cDNA kawg uas muaj cov kab ntawv molecular raws li tau piav qhia hauv Smart-seq3 raws tu qauv. cDNA thiab lub tsev qiv ntawv zoo tau raug soj ntsuam siv Agilent bioanalyzer DNA siab rhiab heev nti thiab ntsuas siv qhov kev ntsuas siab DNA ntawm Qubit 3.0.

Western blot

Kwv yees li 100 H358 spheroids (ib yam mob) raug cais tawm tom qab ARS lossis DMSO kho. Spheroids ces incubated rau dej khov hauv RIPA tsis ntxiv nrog protease inhibitor rau 15 feeb. Lysates tau spun ntawm 10, 000 RCF rau 10 feeb. Supernatant tau raug xa mus rau lub raj tshiab rau cov qauv SDS-PAGE tom ntej npaj hauv Laemmli tsis, boil rau 5 feeb ntawm 95 C rau qhov kawg ntawm 1mg / ml. SDS PAGE tau ua kom cais cov protein los ntawm qhov loj thiab tom qab hloov mus rau PVDF daim nyias nyias. Membranes tau incubated nrog thawj p-ERK (CST) thiab HSP90 (CST) antibodies hmo ntuj ntawm 4 C. Secondary antibodies (Abcam) ces incubated rau 3 zaug TBST ntxuav daim nyias nyias nyob rau hauv thaiv tsis pub rau cov duab tom ntej.

cistanche cov txiaj ntsig rau txiv neej-ua kom muaj zog tiv thaiv kab mob

UMI deduplication

Paired kawg illumina nyeem tau adapter trimmed siv FastP 21 nrog rau qhov teeb meem. UMIs tau muab rho tawm los ntawm kev nyeem thiab txav mus rau lub npe nyeem siv umi_cov cuab yeej_ rho tawm los ntawm UMI-cov cuab yeej pob 22 nrog tus qauv barcode teem rau "NNNNNNNN". UMI tshem tawm cov ntawv nyeem tau ua raws li HG38 siv STAR aligner nrog GENCODE v38 annotation teeb. Aligned nyeem tau deduplicated siv "UMI-cov cuab yeej dedup" nrog lub neej ntawd nqis.

RNA-seq tsom xam

Tag nrho cov ntaub ntawv fastq tau raug txiav nrog Trimmomatic 2 (0.38) 23 thiab cov ntaub ntawv trimmed tau raug soj ntsuam nrog FastQC 24 thiab tom qab ntawd ua tiav nrog cov kav dej ntsuas hauv qab no: Salmon (1.3.0): pseudoalignment ntawm RNA -seq nyeem ua nrog Salmon 25 siv cov lus sib cav hauv qab no: {{1{{20}}}}validateMappings –gcBias --seqBias --recoverOrphans --rangeFactorizationBins 4 siv ib qho Performance index tsim los ntawm GENCODE version 35 transcriptome fasta cov ntaub ntawv siv decoy sequences los pab xaiv kev sib raug zoo. Ib qho ntxiv, TE-paub qhov ntsuas tau tsim nyob rau hauv ib qho zoo sib xws tab sis ntxiv nrog cov sequences generated los ntawm UCSC Repeat Masker khiav. DESeq2 (1.32.0): Cov zis salmon tau raug xa mus rau hauv cov khoom DESeq siv tximport 26 thiab kev tshuaj xyuas qhov sib txawv ntawm cov lus sib txawv tau ua nrog cov qauv sib cav 27. Tag nrho cov txiaj ntsig tau lim kom muaj padj <0.05. Qhov twg suav cov ntaub ntawv tau siv, nws tau ua raws li cov qauv siv DESeq.

Gene teem enrichment tsom xam

Cov noob qhia txawv txawv tau raug txheeb xyuas los ntawm qhov shrunken log2FoldChange qhov tseem ceeb tsim los ntawm DESeq2. Cov noob noob tau txais los ntawm kev siv R pob msigdbr (7.4.1) thiab lim kom tsuas muaj cov noob teev nrog 'Hallmark' raws li txoj cai. R pob fgsea (1.18.

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TXOJ CAI

Peb ua tsaug rau cov tswv cuab ntawm Kim Lab rau kev sib tham muaj txiaj ntsig. Txoj haujlwm no tau txais kev txhawb nqa los ntawm cov nyiaj los ntawm Baskin School of Engineering (rau DHK). DC tau txais kev txhawb nqa los ntawm Predoctoral Fellowship Award los ntawm Kev Tshawb Fawb Txog Kev Haus Luam Yeeb (T30DT0997), RER tau txais kev txhawb nqa los ntawm F99/K00 NIDDK KUH Predoctoral rau Postdoctoral Fellow Transition Award los ntawm National Institutes of Health (1F99DK{ {7}}), thiab VP tau txais kev txhawb nqa los ntawm Predoctoral Fellowship Award los ntawm Kev Tshawb Fawb Txog Kev Haus Luam Yeeb (T32DT4904).

AUTHOR CONTRIBUTIONS

DHK lub tswv yim kev tshawb fawb, DC thiab DHK tsim kev tshawb fawb, DC, JL, thiab GM tau ua cov kev sim, RER thiab VP txheeb xyuas cov ntaub ntawv, thiab DC thiab DHK tau sau ntawv nrog cov tswv yim los ntawm cov kws sau ntawv.

Cov duab

Daim duab 1. KRAS(G12C) inhibition hloov cov coding thiab noncoding transcriptome A. Kev sim schematic. B. Distribution of suav muab rau GENCODE coding, lncRNA, thiab TE/repeat superfamilies nyob rau hauv ARS-kho (ars) los yog DMSO-kho (dmso) ntsws cancer spheroid RNA-seq tsev qiv ntawv, qhov twg txhua kem sawv cev rau ib tug biological replicate. C. Tseem ceeb Gene Set Enrichment Analysis tau pom nyob rau hauv DMSO-kho (txoj cai, zoo NES) los yog ARS-kho (sab laug, tsis zoo NES) mob ntsws cancer spheroids siv cov noob sib txawv qhia los ntawm cov qhab nia normalized enrichment (NES).

Daim duab 2. KRAS(G12C) inhibition ua haujlwm ua rau ISGs thiab cov tub ntxhais hluas AluY cov ntsiab lus

A. Volcano plots depicting tseem ceeb sib txawv qhia pom nyob rau hauv cov noob tseem ceeb ntawm DMSO-kho (txoj cai, zoo fold-hloov) los yog ARS-kho (sab laug, tsis zoo fold hloov) ntsws cancer spheroids. B. Volcano plots depicting tseem ceeb sib txawv qhia pom nyob rau hauv TE superfamilies ntawm DMSO-kho (txoj cai, zoo fold hloov) los yog ARS-kho (sab laug, tsis zoo fold-hloov) ntsws cancer spheroids.

Daim duab 3. KRAS(G12C) inhibition downregulates ntev noncoding RNAs

A. Volcano zaj duab xis ntawm qhov sib txawv tseem ceeb ntawm GENCODE protein-coding RNAs thiab lncRNAs ntawm DMSO-kho (txoj cai, zoo fold-hloov) lossis ARS-kho (sab laug, tsis zoo fold-hloov) ntsws cancer spheroids. B. Cov phiaj xwm ntawm qhov sib txawv tseem ceeb ntawm GENCODE protein-coding RNAs thiab lncRNAs ntawm DMSO-kho (sab saum toj, zoo fold-hloov) lossis ARS-kho (hauv qab, tsis zoo fold-hloov) ntsws cancer spheroids (Wilcoxon).

SUPPLEMENTARY FIGURE

Daim duab S1.

A. H358 3D mob ntsws cancer spheroids kho nrog ARS lossis DMSO. B. Western blot rau p-ERK thiab HSP90 siv H358 3D mob ntsws cancer spheroids kho nrog ARS lossis DMSO. C. Kev ntsuas qhov ntev (hauv micrometers) (sab laug zaj duab xis) thiab cell viability (Cell Titer-Glo® luminescent cell viability nyob rau hauv cov txheeb ze fluorescence units) (txoj cai) ntawm H358 3D mob ntsws cancer spheroids kho nrog ARS lossis DMSO tom qab 3 lossis 5 hnub ntawm kev kho mob (500 nM ARS-1620 lossis DMSO). D. Kev ntsuas qhov ntev (hauv micrometers) ntawm H358 3D mob ntsws cancer spheroids kho nrog ntau qhov sib txawv ntawm ARS-1620 (nM) rau 7 hnub.