Journal: Cancer cell

Article Title: Treatment-induced tumor dormancy through YAP-mediated transcriptional reprogramming of the apoptotic pathway

doi: 10.1016/j.ccell.2019.12.006

Figure Lengend Snippet: (A) Proliferation of PC-9 cells treated with DMSO, 100 nM osimertinib (O) alone or in combination with 30 nM trametinib (T). (B) Images of control cells (at 1 week) or dormant PC-9 cells (at 15 weeks). Scale bar, 200 μm. (C) Cells were treated as in (A) for 6 weeks followed by drug washout. D) Western blot analysis of EGFR downstream signaling following treatment with OT for indicated times or 21 days followed by drug washout (rebound). E) Fraction of barcodes shared among replicates following indicated treatments in barcoded PC-9 cells F) Relative abundance of individual barcodes. Shared and unique indicate barcodes shared by >2 or ≤2 replicates, respectively. (G) GSEA of Hallmark gene sets comparing dormant cells vs. DMSO-treated control cells. Normalized Enrichment Scores (NES) for gene sets with FDR<0.1 in at least two cell lines are shown. (H) Senescence-associated β-galactosidase (SA-β-gal) staining of cells treated as indicated for 10 days. Scale bar, 100 μm. (I) Quantification of (H). (J) GSEA of senescence signature comparing dormant, OT-treated PC-9 cells vs. control cells. (K) Immunofluorescence (IF) staining for H3K9Me3 in control cells or dormant cells treated with OT for 10 days. Scale bar, 20 μm. Mean ± SEM are shown in all plots except (I) where mean ± SD are shown. ANOVA (I) or t-test (K) were used for statistical analyses. ***, P<0.001; **, P<0.01. See also Figure S1, S2, and S3.

Article Snippet: Osimertinib ( in vivo ) , MedChemExpress , Cat#HY-15772A.

Techniques: Control, Western Blot, Staining, Immunofluorescence

Journal: Cancer cell

Article Title: Treatment-induced tumor dormancy through YAP-mediated transcriptional reprogramming of the apoptotic pathway

doi: 10.1016/j.ccell.2019.12.006

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Osimertinib ( in vivo ) , MedChemExpress , Cat#HY-15772A.

Techniques: Recombinant, In Vivo, In Vitro, Synthesized, Control, Reverse Transcription, Expressing, Staining, Cell Viability Assay, TaqMan Assay, Plasmid Preparation, Software

Journal: Oncogene

Article Title: RBM15 facilitates osimertinib resistance of lung adenocarcinoma through m6A-dependent epigenetic silencing of SPOCK1

doi: 10.1038/s41388-024-03220-z

Figure Lengend Snippet: A , B Representative image of IHC staining and IHC scores of RBM15 from EGFR wild-type (WT) or mutant patients. Mann–Whitney test. C , D Immunofluorescence of RBM15 protein expression in LUAD PDO with or without EGFR mutations (scale bar: 50 μm). T test. E OS in TCGA LUAD cases with EGFR mutation according to RBM15 expression. F PFS after initiation of osimertinib treatment in patients with EGFR mutations expressing high vs. low RBM15 mRNA levels. G , H Osimertinib IC 50 value of HCC827 and H1975 cells transfected with NC and RBM15-OE. T test. Error bars represent the means ± SDs. * P < 0.05, **** P < 0.0001.

Article Snippet: When the tumors reached a longitudinal diameter of 5 mm, the mice were randomly assigned to each group and subsequently treated orally with an equal volume of osimertinib mesylate (Selleck, S5078, TX, 20 mg/kg/day) or corn oil by gavage every 2 days.

Techniques: Immunohistochemistry, Mutagenesis, MANN-WHITNEY, Immunofluorescence, Expressing, Transfection

Journal: Oncogene

Article Title: RBM15 facilitates osimertinib resistance of lung adenocarcinoma through m6A-dependent epigenetic silencing of SPOCK1

doi: 10.1038/s41388-024-03220-z

Figure Lengend Snippet: A H1975 cells were exposed to increasing concentrations of osimertinib and cultured for 24 weeks to select H1975-OR cells (top panel). H1975 and H1975-OR cells were incubated with osimertinib for 48 h, cell viability was measured using the CCK-8 assay, and the IC 50 was calculated (bottom panel). T test. B Western blot investigation of EGFR, ERK, and AKT protein levels and their phosphorylation. C , D RBM15 protein and mRNA expression levels in H1975 and H1975-OR were assessed using western blot and qRT-PCR. T test. E Osimertinib IC 50 of H1975-OR cells transfected with si-NC, si-RBM15#1, or si-RBM15#2. T test. F A schematic diagram for the construction of drug-resistant organoids. G PDO1-OR and osimertinib-sensitive organoids were incubated with osimertinib for 48 h, cell viability was measured using the CCK-8 assay, and the IC 50 was calculated. T test. H Osimertinib IC 50 value of PDO1-OR transfected with sh-NC or sh-RBM15. T test. I , J Effects of RBM15 knockdown on H1975-OR and PDO1-OR apoptosis assessed using flow cytometry and TUNEL staining. T test. K Western blot analysis of EGFR and AKT proteins and their phosphorylation levels after knockdown of RBM15 and 48-h osimertinib treatment. L Representative images of xenograft tumors from sh-NC, sh-NC + Osi, and sh-RBM15 + Osi groups. M , N Tumor growth curve and tumor weights. T test. O Representative images of Ki-67 positive staining in xenografted tumors. T test. Error bars represent the means ± SDs. * P < 0.05, ** P < 0.01, **** P < 0.0001.

Article Snippet: When the tumors reached a longitudinal diameter of 5 mm, the mice were randomly assigned to each group and subsequently treated orally with an equal volume of osimertinib mesylate (Selleck, S5078, TX, 20 mg/kg/day) or corn oil by gavage every 2 days.

Techniques: Cell Culture, Incubation, CCK-8 Assay, Western Blot, Phospho-proteomics, Expressing, Quantitative RT-PCR, Transfection, Knockdown, Flow Cytometry, TUNEL Assay, Staining

Journal: Oncogene

Article Title: RBM15 facilitates osimertinib resistance of lung adenocarcinoma through m6A-dependent epigenetic silencing of SPOCK1

doi: 10.1038/s41388-024-03220-z

Figure Lengend Snippet: A Venn diagrams were generated from the set of genes enriched for transcripts substantially altered after RBM15 silencing (RNA-seq) and the set of genes enriched for m6A-modified transcripts (m6A-seq). Sixteen genes were chosen based on overlap. B Top motif identified by HOMER with m6A-seq peaks. C Peak regions of m6A modifications detected by MeRIP-seq. D , E qRT-PCR was conducted to assess the differences in mRNA expression of the above 16 genes after RBM15 knockdown in H1975 and HCC827 cells. T test. F , G Western blot assessment of the correlation between RBM15 and SPOCK1 protein expression. H , I RIP-PCR validated RBM15 and m6A binding to SPOCK1 mRNA. T test. J Changes in total RNA m6A modification levels after 48-h DAA treatment. T test. K Alteration of SPOCK1 mRNA levels in H1975 cells and HCC827 cells after DAA treatment. T test. L , M Western blot and qRT-PCR demonstrated reduced SPOCK1 expression in H1975-OR cells and PDO1-OR. T test. N Osimertinib IC 50 value of H1975-OR cells transfected with si-NC, si-RBM15, or si-SPOCK1. T test. O Osimertinib IC 50 value of PDO1-OR transfected with sh-NC, sh-RBM15, or sh-SPOCK1. T test. P , Q Effects of RBM15 and SPOCK1 knockdown on H1975-OR and PDO1-OR apoptosis assessed using flow cytometry and TUNEL staining. T test. Error bars represent the means ± SDs. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: When the tumors reached a longitudinal diameter of 5 mm, the mice were randomly assigned to each group and subsequently treated orally with an equal volume of osimertinib mesylate (Selleck, S5078, TX, 20 mg/kg/day) or corn oil by gavage every 2 days.

Techniques: Generated, RNA Sequencing, Modification, Quantitative RT-PCR, Expressing, Knockdown, Western Blot, Binding Assay, Transfection, Flow Cytometry, TUNEL Assay, Staining

Journal: Oncogene

Article Title: RBM15 facilitates osimertinib resistance of lung adenocarcinoma through m6A-dependent epigenetic silencing of SPOCK1

doi: 10.1038/s41388-024-03220-z

Figure Lengend Snippet: A , B GSEA pathway enrichment was performed using DEGs. C Volcano plot of RNA-seq results displaying DEGs in H1975 and H1975-OR. D , E Western blot and immunofluorescence detection of protein expression of EMT-related indicators (scale bar: 50 μm). T test. F Immunofluorescence detection of protein expression of EMT-related indicators in PDO-OS and PDO1-OR (scale bar: 25 μm). T test. G Change in CDH1, ZEB1, AXL, and RBM15 expression in osimertinib refractory tissues and matched pre-treatment tissues. H – J Spearman’s correlation of change in CDH1, ZEB1, AXL, and RBM15 expression in eight patients with matched pre- and post-osimertinib treatment. Pearson test. K , L Western blot and immunofluorescence assays reveal changes in protein expression of EMT indicators in H1975-OR and PDO1-OR after RBM15 knockdown. M Immunofluorescence assays reveal changes in protein expression of EMT indicators in PDO1-OR after RBM15 and SPOCK1 knockdown (scale bar: 50 μm). T test. Error bars represent the means ± SDs. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: When the tumors reached a longitudinal diameter of 5 mm, the mice were randomly assigned to each group and subsequently treated orally with an equal volume of osimertinib mesylate (Selleck, S5078, TX, 20 mg/kg/day) or corn oil by gavage every 2 days.

Techniques: RNA Sequencing, Western Blot, Immunofluorescence, Expressing, Knockdown

Journal: Oncogene

Article Title: RBM15 facilitates osimertinib resistance of lung adenocarcinoma through m6A-dependent epigenetic silencing of SPOCK1

doi: 10.1038/s41388-024-03220-z

Figure Lengend Snippet: A H1975 cells were exposed to 1 μm osimertinib and cultured for 20 days to select H1975-DTP cells (top panel). H1975 and H1975-DTP cells were incubated with osimertinib for 48 h, cell viability was measured using the CCK-8 assay, and the IC 50 was calculated (bottom panel). T test. B Western blot investigation of EGFR, ERK, and AKT protein levels and their phosphorylation. C Western blot assessment of RBM15 protein expression levels in H1975 and H1975-DTPCs. D , E Western blot and immunofluorescence assessment of EMT marker proteins in H1975 and H1975-DTP cells (scale bar: 50 μm). T test. F A model diagram demonstrating that RBM15 suppresses SPOCK1 mRNA expression through m6A modification, enhancing EMT-mediated osimertinib resistance in refractory tumors. This figure was created using the BioRender website ( https://biorender.com ). Error bars represent the means ± SDs. *** P < 0.001, **** P < 0.0001.

Article Snippet: When the tumors reached a longitudinal diameter of 5 mm, the mice were randomly assigned to each group and subsequently treated orally with an equal volume of osimertinib mesylate (Selleck, S5078, TX, 20 mg/kg/day) or corn oil by gavage every 2 days.

Techniques: Cell Culture, Incubation, CCK-8 Assay, Western Blot, Phospho-proteomics, Expressing, Immunofluorescence, Marker, Modification