Journal: Biochimica et biophysica acta. Molecular cell research

Article Title: Overcoming MET-targeted drug resistance in MET-amplified lung cancer by aurora kinase B inhibition.

doi: 10.1016/j.bbamcr.2025.120001

Figure Lengend Snippet: Fig. 2. Stimulation of STAT3-BCL2 axis by AURKB. (A) Gene set enrichment (GSEA) data of upregulated STAT3 target genes in the H1993 PR-S2 cells. (B) Protein expressions of p-STAT3, STAT3, and GAPDH. (C) Protein expressions of p-STAT3, STAT3, p-AURKB, AURKB, and GAPDH after barasertib single or PHA665752 combination treatment of indicated drug concentration for 48 h. (D) Immunoblot analysis of the H1993 and H1993 PR-S2 cells transfected with scramble and siAURKB for 48 h. (E) and (F) The indicated protein ex pressions of the GFP and AURKB-GFP virus-infected H1993 stable cell lines and MG132 treatment for the indicated treatment time. The line graph represents the protein stability of p-STAT3. (G) and (H) RT-qPCR and immunoblotting measured the expressions of STAT3 downstream targets. (I) mRNA expression of BCL2 after treatment of 10, 100, and 1000 nM concentrations of barasertib. (J) mRNA expression levels of BCL2 and STAT3 in the scramble or siSTAT3 transfected H1993 and H1993 PR-S2 cells. (K) Protein expression of cleaved-caspase3 and BCL2 after treatment with barasertib for 48 h. The error bars represent the mean ± SEM (n = 3). Statistical analysis was performed using a two-sample t-test. ns, not significant; *p < 0.05; **p < 0.01; ***p < 0.001.

Article Snippet: PHA665752, barasertib, VX680, MLN8237, AT9283, cycloheximide, and MG132 were purchased from Selleckchem Korea (Seoul, Korea).

Techniques: Concentration Assay, Western Blot, Transfection, Virus, Infection, Stable Transfection, Quantitative RT-PCR, Expressing

Journal: Biochimica et biophysica acta. Molecular cell research

Article Title: Overcoming MET-targeted drug resistance in MET-amplified lung cancer by aurora kinase B inhibition.

doi: 10.1016/j.bbamcr.2025.120001

Figure Lengend Snippet: Fig. 3. Induced apoptotic cell death by AURKB inhibition. (A) RT-qPCR measured the mRNA expression of apoptosis-related genes in the H1993 and H1993 PR-S2 cells. (B) Western blot analysis after treatment PHA665752 or barasertib (each 1 uM) for 48 h. The bar graph represents the band intensity of BIMEL. (C) The activated form expression of BIM protein (S69 and S87) in the H1993 and H1993 PR-S2 cells. (D) Histogram indicated cell cycles of the H1993 and H1993 PR-S2 cells after AURKB inhibitor dose-dependent treatment for 24 h. The bar graph indicates the cell cycle proportion of G1, S, and G2/M. (E) Annexin-V-FITC/PI flow cytometry analysis of the H1993 and H1993 PR-S2 cells treated with 100, 250, and 500 nM of barasertib for 48 h. The bar graph represents the early and late apoptotic cell population of the barasertib-treated H1993 and H1993 PR-S2 cells. (F) Immunofluorescence analysis of nucleus (blue) and cleaved-caspase3 (green) after treatment with barasertib. Scale bars, 100 μm. The error bars represent the mean ± SEM (n = 3). Statistical analysis was performed using a two-sample t-test. ns, not significant; *p < 0.05; **p < 0.01; ***p < 0.001, Abbre viation: Bara, Barasertib. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: PHA665752, barasertib, VX680, MLN8237, AT9283, cycloheximide, and MG132 were purchased from Selleckchem Korea (Seoul, Korea).

Techniques: Inhibition, Quantitative RT-PCR, Expressing, Western Blot, Flow Cytometry, Immunofluorescence

Journal: Biochimica et biophysica acta. Molecular cell research

Article Title: Overcoming MET-targeted drug resistance in MET-amplified lung cancer by aurora kinase B inhibition.

doi: 10.1016/j.bbamcr.2025.120001

Figure Lengend Snippet: Fig. 4. AURKB inhibition effect in xenograft model. (A) Diagram summarizing xenograft experiment strategy in NOD-2/Shi-scid IL2rgamma(null) (N2G) mice. (B) and (C) The measurement of tumor volume (mm3) following treatment with vehicle, PHA665752 (25 mg/kg), Barasertib (50 mg/kg) and their combination for 21 days in N2G mice bearing the H1993 and H1993 PR- S2 cell xenografts. Tumor volumes were measured twice weekly by caliper (mean ± SEM, n = 6–7 for each group). (D) The fold change of the tumor volume during the period of drug injection, representing 0, 6, 14, 16, 18, and 20 days. (E) Representative hematoxylin and eosin (H&E) and Ki-67 staining images of xenograft tumors. Scale bars, 100 μm. The bar graph indicates the relative percentage of the Ki-67-positive cells. The error bars represent the mean ± SEM (n = 3). Statistical analysis was performed using a two-sample t-test. ns, not significant; *p < 0.05; **p < 0.01; ***p < 0.001, Abbreviation: PHA, PHA665752; Bara, Barasertib.

Article Snippet: PHA665752, barasertib, VX680, MLN8237, AT9283, cycloheximide, and MG132 were purchased from Selleckchem Korea (Seoul, Korea).

Techniques: Inhibition, Injection, Staining

Journal: bioRxiv

Article Title: Hypoxia-induced histone methylation and NF-κB activation in pancreas cancer fibroblasts promote EMT-supportive growth factor secretion

doi: 10.1101/2025.01.30.635486

Figure Lengend Snippet: A) 0082T CAF cells were cultured in 21% or 1% O 2 for 72 hr, and qRT-PCR was performed for the indicated transcripts and normalized to CASC3 . n=3, t-test. B) 0082T CAFs were cultured in 21% or 1% O 2 for 120 hr, and immunofluorescence microscopy was performed for IGF-2. n=3, t-test. C) 430 inhibitors were screened for their ability to counteract hypoxic CAF-induced EMT in HPAF-II cells. HPAF-II cells were treated with hypoxic 0082T CM and inhibitors (1 μM for all compounds) for 120 hr. The ability of each inhibitor to reduce vimentin expression was calculated using positive and negative controls contained on each plate (see Methods). Data are displayed as a plot of the kernel density function (displayed as density) for the percent vimentin inhibition. Inhibitors with >50% reduction in vimentin-positive cells were designated as effective (orange region of the density plot of the inhibitor screen). D) Fraction of vimentin-positive HPAF-II cells treated with CM and MET inhibitors (blue) or MEK inhibitors (orange) compared to untreated and CM-only controls. The percentage of effective inhibitors was calculated for each inhibitor class in the inhibitor library. Representative images are shown of effective (i.e., >50% vimentin reduction) MEKi- or METi-treated samples and controls. E) 0082T cells were cultured in 21% or 1% O 2 for 120 hr and assessed for HGF expression by immunofluorescence microscopy. n=3, t-test. F) Immunofluorescence microscopy was performed on HPAF-II cells treated with hypoxic 0082T CM with linsitinib (IGF1Ri, 2.5 μM), PHA665752 (METi, 2.5 μM), both, or DMSO. G) Immunofluorescence microscopy was performed on KPCY tumor samples to determine fraction of HGF-positive CAFs (PDPN+) in oxygenated (HYP-) or hypoxic (HYP+) tumor regions. n=3, paired t-test. Data are presented as mean ± SEM for bar plots (A, B, E, F) or as a box-and- whisker plot that represent the first quartile (lower box bound), median (center bound), or third quartile (upper box bound) (G). * p <0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001

Article Snippet: PHA665752 (Santa Cruz Biotechnology) and linsitinib (Selleck) were used at 2.5 μM ( , ).

Techniques: Cell Culture, Quantitative RT-PCR, Immunofluorescence, Microscopy, Expressing, Inhibition, Whisker Assay

Journal: bioRxiv

Article Title: Hypoxia-induced histone methylation and NF-κB activation in pancreas cancer fibroblasts promote EMT-supportive growth factor secretion

doi: 10.1101/2025.01.30.635486

Figure Lengend Snippet: A) 0082T CAFs were cultured in 21% or 1% O 2 for 120 hr, and qRT-PCR was performed on extracted mRNA for HGF transcripts. CASC3 was used for normalization. n=3. B) Conditioned medium was collected from 0082T cells cultured in 21% or 1% O 2 and analyzed for HGF by ELISA. n=3, t-test. C) HPAF-II cells were untreated or treated with conditioned medium (CM) from PSCs grown at 1% O 2 +/- PHA665752 (METi, 2.5 μM) for 120 hr. Immunofluorescence microscopy was performed for the indicated proteins. n=3, one-way ANOVA with Tukey’s multiple comparison test. D) HPAF-II cells were treated with IGF-2 (100 ng/mL), HGF (5 ng/mL), or IGF-2+HGF for 120 hr. Immunofluorescence microscopy was performed for the indicated proteins. n=3, one-way ANOVA with Tukey’s multiple comparison test. E) Fluorescence-based immunohistochemistry was performed on sections of KPCY tumors for podoplanin (PDPN) and Hypoxyprobe (HYP) to calculate the percentage of CAFs within hypoxic regions. n=3. F) In sections of subcutaneous tumors formed using KPCY-derived 7160.c2 cells, HGF expression was assessed in Hypoxyprobe-positive and -negative CAFs (PDPN+ cells). n=3, t-test. Data are presented as mean ± SEM for bar plots (A, B, C, D, F) or as a mosaic plot indicating the percentage of PDPN+ cells that are positive or negative for Hypoxyprobe (HYP) across biological replicates. * p <0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001

Article Snippet: PHA665752 (Santa Cruz Biotechnology) and linsitinib (Selleck) were used at 2.5 μM ( , ).

Techniques: Cell Culture, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Immunofluorescence, Microscopy, Comparison, Fluorescence, Immunohistochemistry, Derivative Assay, Expressing