Journal: Cell Genomics

Article Title: Phenotypic heterogeneity and plasticity in colorectal cancer metastasis

doi: 10.1016/j.xgen.2025.100881

Figure Lengend Snippet: Regulation of cancer cell states (A) Left: chromatin accessibility of putative enhancers. Right: mRNA expression of genes linked to enhancers. (B) GEA of genes in k-means clusters using Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways and MSigDB Hallmarks. (C) ChromVAR motif deviation Z scores for TFs in mCRC states (Wilcoxon, false discovery rate < 0.05), annotated based on the DNA binding domain. (D) Accessibility of chromatin regions in the indicated SCENIC+ regulons. (E) Z -scored expression of genes in SCENIC+ regulons across mCRC states. AP-1 and NF-κB regulons were formed by combining the regulons of FOS and JUN family members and the NF-κB subunits. One-way ANOVA. (F) Z -scored mRNA expression of AP-1 target genes across pCRC and mCRC states. One-way ANOVA. (G) Differentially accessible chromatin regions in REC HIGH relative to REC LOW glands. (H) Expression of AP-1 regulon genes or AP-1 target genes in REC HIGH and REC LOW glands. Unpaired t test. (I) GEA of the indicated signatures in TF regulons. Numbers in brackets indicate the number of genes in the regulon. (J) RT-qPCR analysis of the indicated genes following GFP-aFOS induction by 2 μg/mL doxycycline treatment in 3D. Paired t test; no genes were significant; n = 4. (K) RT-qPCR analysis of parental CRC21LM_PDO in 3D or on collagen I-coated plates (2D). Paired t test, n = 3. (L) RT-qPCR analysis of the indicated genes following GFP-aFOS induction under 2D culture conditions. Paired t test, n = 5. (M) Time-lapse live-cell imaging showing the change in confluency of organoids in 2D following GFP-aFOS induction. 2-way ANOVA, n = 3. (N) Cell proliferation assay of CRC21LM_PDO treated with trametinib in 3D and 2D culture systems; n = 3. Data are represented as mean ± SEM. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001.

Article Snippet: Both organoids in 2D and 3D were treated with trametinib (MedChem Express, HY-10999-10mg) for 4 days.

Techniques: Expressing, Binding Assay, Quantitative RT-PCR, Live Cell Imaging, Proliferation Assay

Journal: Nature

Article Title: α-Ketoglutarate links p53 to cell fate during tumour suppression.

doi: 10.1038/s41586-019-1577-5

Figure Lengend Snippet: Fig. 1 | p53 restoration increases the ratio of cellular αKG to succinate, independently of changes in proliferation. a, Western blot (top) and quantitative PCR with reverse transcription (qRT–PCR) (bottom) of KPsh no. 1, KPsh no. 2 and KPsh no. 3 lines cultured with or without doxycycline (dox) for six days. Gene expression is represented as the fold change (expressed in log2) relative to cells cultured with doxycycline for each line. b, c, Steady-state levels of TCA-cycle metabolites (b) or αKG/succinate ratio (c) in cells cultured with or without doxycycline for eight days. d, αKG/succinate ratio in cells cultured on doxycycline with 25 nM trametinib (tram) or 3 μM etoposide (etopo) for 48 h or 96 h, as shown. Cells cultured without doxycycline for six days are included as a control. Experiments in a–d were repeated twice with similar results. Data are presented as a representative, independently treated well (a top), as individual data points (a bottom, b) or as mean ± s.d. of n = 3 independently treated wells with individual data points shown (c, d). For gel source data for a, see Supplementary Fig. 1. Significance was assessed by two-tailed Student’s t-test (c) or one-way analysis of variance (ANOVA) with Tukey’s post-test (d) in comparison with vehicle-treated cells grown with doxycycline.

Article Snippet: Where indicated, cells were treated with H2O (vehicle), DMSO (vehicle), 3 μM etoposide (Sigma), 25 nM trametinib (Selleck Chemicals), 4 mM sodium acetate (Sigma), 4 mM dimethyl-αKG (Sigma) or 4 mM diethylαKG (Sigma).

Techniques: Western Blot, Real-time Polymerase Chain Reaction, Reverse Transcription, Quantitative RT-PCR, Cell Culture, Gene Expression, Control, Two Tailed Test, Comparison

Journal: Molecular Cancer Therapeutics

Article Title: Sensitization to Ionizing Radiation by MEK Inhibition Is Dependent on SNAI2 in Fusion-Negative Rhabdomyosarcoma

doi: 10.1158/1535-7163.mct-22-0310

Figure Lengend Snippet: Figure 1. Low-dose MEK inhibitor reduces SNAI2 expression and in combination with IR induces robust myogenic differentiation and loss of cell survival in FN-RMS. A–C, Protein expression in DMSO- and trametinib-treated FN-RMS cell lines (RD, SMS-CTR, and Rh36). D, Representative confocal microscopy images of RD cells treated with DMSO or trametinib, in non-IR and IR conditions, immunostained with myogenic differentiated myosin (MF20 antibody). Scale bar ¼ 100 mm. E, Quantification of average MF20-positive cells in either non-IR or IR conditions in DMSO- and trametinib-treated RD cells. Error bars represent 1 SD. ns ¼ not significant; , P < 0.0001 by a two-way ANOVA with a posthoc Sidak multiple comparison test. F, Representative confocal microscopy images of SMS-CTR cells treated with DMSO or trametinib, in non-IR and IR conditions, immunostained with myogenic differentiated myosin (MF20 antibody). Scale bar ¼ 100 mm. G, Quantification of average MF20-positive cells in either non-IR or IR conditions in DMSO- and trametinib-treated SMS-CTR cells. Error bars represent 1 SD. ns ¼ not significant; , P < 0.001; , P < 0.0001 by a two-way ANOVA with a posthoc Sidak multiple comparison test. H, Survival fractions of DMSO- and trametinib-treated RD cells were assessed at increasing IR dose exposures. Statistical differences were observed at 2, 4, and 6 Gy. Error bars represent 1 SD. ns ¼ not significant; , P < 0.0001 by a Student t test. I, Overview of similar phenotypic characteristics of SNAI2- knockdown RMS cells and low-dose trametinib-treated FN-RMS cells.

Article Snippet: Cells were pretreated with DMSO or trametinib (10 nmol/L for 72 hours or 20 nmol/L for 24 hours) prior to being plated at 4,000 cells/well (no IR) and 10,000 cells/well (receiving IR), grown in 10% FBS DMEM or RPMI growth media, fixed at 72 hours post-IR (hpIR; 0 or 15 Gy) in 4% paraformaldehyde/PBS, permeabilized in 0.5% Triton X-100/PBS, and incubated with rabbit anti-MEF2C (Cell Signaling Technology; Catalog No. 5030) and anti-myosin heavy chain (DSHB) in 1% BSA/PBS.

Techniques: Expressing, Confocal Microscopy, Comparison, Knockdown

Journal: Molecular Cancer Therapeutics

Article Title: Sensitization to Ionizing Radiation by MEK Inhibition Is Dependent on SNAI2 in Fusion-Negative Rhabdomyosarcoma

doi: 10.1158/1535-7163.mct-22-0310

Figure Lengend Snippet: Figure 2. Combining low-dose MEK inhibition with IR results in increased apoptosis and a G1 cell-cycle arrest in FN-RMS. A, Flow cytometry plots showing propidium iodide versus Annexin V staining of DMSO- and trametinib-treated RD cells after non-IR and IR conditions. Q4 represents cells undergoing early apoptosis, whereas Q2 represents cells undergoing late apoptosis. Q3 represents live cells not undergoing apoptosis. B, Flow cytometry plots showing propidium iodide versus Annexin V staining of DMSO- and trametinib-treated SMS-CTR cells after non-IR and IR conditions. C and D, Cleaved PARP, BIM, pERK, and total ERK protein expression immunoblots in DMSO- and trametinib-treated RD (C) and SMS-CTR (D) cells 96hpIR. Increased trametinib doses were assessed in both cell lines. E, Flow cytometry plots of EdU versus DAPI staining in DMSO- and trametinib-treated RD cells in non-IR and IR conditions. F, Flow cytometry plots of EdU versus DAPI staining in DMSO- and trametinib-treated SMS-CTR cells in non-IR and IR conditions.

Article Snippet: Cells were pretreated with DMSO or trametinib (10 nmol/L for 72 hours or 20 nmol/L for 24 hours) prior to being plated at 4,000 cells/well (no IR) and 10,000 cells/well (receiving IR), grown in 10% FBS DMEM or RPMI growth media, fixed at 72 hours post-IR (hpIR; 0 or 15 Gy) in 4% paraformaldehyde/PBS, permeabilized in 0.5% Triton X-100/PBS, and incubated with rabbit anti-MEF2C (Cell Signaling Technology; Catalog No. 5030) and anti-myosin heavy chain (DSHB) in 1% BSA/PBS.

Techniques: Inhibition, Flow Cytometry, Staining, Expressing, Western Blot

Journal: Molecular Cancer Therapeutics

Article Title: Sensitization to Ionizing Radiation by MEK Inhibition Is Dependent on SNAI2 in Fusion-Negative Rhabdomyosarcoma

doi: 10.1158/1535-7163.mct-22-0310

Figure Lengend Snippet: Figure 3. SNAI2 downstream of MEK signaling protects FN-RMS from IR-induced myogenic differentiation and apoptosis. A, SNAI2 protein expression in RD pBabe and SNAI2- Flag overexpressing cell lines. B, Representative confocal microscopy images of RD pBabe and SNAI2-Flag cells treated with DMSO or trametinib after IR exposure, immunostained with myogenic differentiated myosin MF20 antibody. Scale bar ¼ 100 mm. C, Quantification of average MF20-positive cells in DMSO- and trametinib- treated RD pBabe and SNAI2-Flag cells after IR exposure. Error bars represent 1 SD. ns ¼ not significant; , P < 0.0001 by a two-way ANOVA with a posthoc Sidak multiple comparison test. D and E, Survival fractions of DMSO- and trametinib-treated RD pBabe and SNAI2-Flag cells were assessed at increasing IR dose exposures. Statistical differences were observed at 2, 4, and 6 Gy in RD pBabe, but NOT RD SNAI2-Flag. Error bars represent 1 SD. ns ¼ not significant; , P < 0.05; , P < 0.001 by a Student t test. F, Flow cytometry plots showing propidium iodide versus Annexin V staining of DMSO- and trametinib-treated RD pBabe cells after non-IR and IR conditions. G, Flow cytometry plots showing propidium iodide versus Annexin V staining of DMSO- and trametinib-treated RD SNAI2-Flag cells after non-IR and IR conditions. H, Western blots showing pERK, total ERK, and BIM protein expression of RD pBabe, RD SNAI2-Flag,SMS-CTR pBabe, and SMS-CTR SNAI2-Flag cells after DMSO and 10 nmol/L trametinib treatment.

Article Snippet: Cells were pretreated with DMSO or trametinib (10 nmol/L for 72 hours or 20 nmol/L for 24 hours) prior to being plated at 4,000 cells/well (no IR) and 10,000 cells/well (receiving IR), grown in 10% FBS DMEM or RPMI growth media, fixed at 72 hours post-IR (hpIR; 0 or 15 Gy) in 4% paraformaldehyde/PBS, permeabilized in 0.5% Triton X-100/PBS, and incubated with rabbit anti-MEF2C (Cell Signaling Technology; Catalog No. 5030) and anti-myosin heavy chain (DSHB) in 1% BSA/PBS.

Techniques: Expressing, Confocal Microscopy, Comparison, Flow Cytometry, Staining, Western Blot

Journal: Molecular Cancer Therapeutics

Article Title: Sensitization to Ionizing Radiation by MEK Inhibition Is Dependent on SNAI2 in Fusion-Negative Rhabdomyosarcoma

doi: 10.1158/1535-7163.mct-22-0310

Figure Lengend Snippet: Figure 4. MEK inhibitor treatment results in loss of SNAI2 and increases sensitivity to IR, resulting in increased event-free survival in preclinical models. A, Illustration of in vivo experimental treatment set up. B–D, Western blots showing pERK, total ERK, SNAI2, and BIM protein expression in two representative control tumors and trametinib- treated RD (B), SJRHB013_X (C), and SJRHB000026_X1 (D) tumors. E–H, RTV of RD tumors receiving control (E), trametinib (F), IR (G), or combination trametinib þ IR (H) treatment. Red line depicts 0.5 RTV; vertical dashed line represents the average EFS for each treatment arm. PD, progressive disease; PR, partial response. I–L, RTV of SJRHB013_X tumors receiving control (I), trametinib (J), IR (K), or combination trametinib þ IR (L) treatment. Red line depicts 0.5 RTV; vertical dashed line represents the average EFS for each treatment arm. PD, progressive disease; PR, partial response; CR, complete response. M–P, RTV of SJRHB000026_X1 tumors receiving control (M), trametinib (N), IR (O), or combination trametinib þ IR (P) treatment. Red line depicts 0.5 RTV; vertical dashed line represents the average EFS for each treatment arm. PD, progressive disease; PR, partial response; CR, complete response.

Article Snippet: Cells were pretreated with DMSO or trametinib (10 nmol/L for 72 hours or 20 nmol/L for 24 hours) prior to being plated at 4,000 cells/well (no IR) and 10,000 cells/well (receiving IR), grown in 10% FBS DMEM or RPMI growth media, fixed at 72 hours post-IR (hpIR; 0 or 15 Gy) in 4% paraformaldehyde/PBS, permeabilized in 0.5% Triton X-100/PBS, and incubated with rabbit anti-MEF2C (Cell Signaling Technology; Catalog No. 5030) and anti-myosin heavy chain (DSHB) in 1% BSA/PBS.

Techniques: In Vivo, Western Blot, Expressing, Control

Journal: bioRxiv

Article Title: Defining melanoma combination therapies that provide senolytic sensitivity in human melanoma cells

doi: 10.1101/2023.10.01.560354

Figure Lengend Snippet: A) Table showing the genetic background of the panel of selected melanoma cell lines (left) and treatment timeline (right). B) Representative proliferation curves of melanoma cells exposed to different treatments (Dabrafenib – Braf-i (50nM) + Trametinib – Mek-i (5 nM, Braf-i+Mek-i), Carboplatin plus Paclitaxel - CP (10 µM + 30 nM) or Radiation - XRA (10 Gray). C) Average nuclear size of melanoma cells expressing H2B-GFP (untreated or day 6 post-treatment). D ) Cell death quantification at by PI incorporation at day 2 or day 9 post-treatment through live imaging. E ) Cell cycle distribution of 9 days treated melanoma cells compared to their controls. Mean ± SD of 2-3 independent experiments is shown. Student’s t-test, * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Trametinib was from Toronto Research Chemicals (North York, ON, CA).

Techniques: Expressing, Imaging

Journal: The Journal of Clinical Investigation

Article Title: Pericyte phenotype switching alleviates immunosuppression and sensitizes vascularized tumors to immunotherapy in preclinical models

doi: 10.1172/JCI179860

Figure Lengend Snippet: ( A ) PNET from WT RIP1-Tag5 (WT), RIP1-Tag5 on a Rgs5 -knockout background ( Rgs5 KO or KO), and triple-transgenic RIP1-Tag5 × UbiCRGS5 × RGS5CreERT2 mice engineered to overexpress RGS5 ( Rgs5 hi or HI) were analyzed at 27 weeks. Images depict vascular CD31 expression (red) and infused FITC-lectin (green) as surrogate markers for tumor perfusion; arrows indicate overlay (yellow). Quantification of overlay and CD31 vessel area. n = 3–4 mice. * P = 0.02; **** P < 0.0001, 1-way ANOVA. ( B ) Extravasation of 70 kD dextran (red, arrowheads) from blood vessels into tumor parenchyma as marker for vessel leakiness. n = 3 mice. Data are represented as mean ± SEM. * P = 0.04; ** P = 0.0014, 1-way ANOVA. ( C ) Calponin (CNN1, red) expression in pericytes (NG2, green); arrows indicate overlay (yellow); quantification of overlay and frequency of NG2 + pericytes. n = 5 mice, * P = 0.0064; ** P = 0.0008; *** P < 0.0001, 1-way ANOVA. ( D ) COLI (red) deposition around pericytes (ACTA2, green); brackets indicate width of COLI deposits. n = 3–5 mice. Data are represented as mean ± SEM. * P = 0.022; *** P = 0.0006; **** P < 0.0001, 1-way ANOVA. ( E ) VE-cadherin (CDH5, red, arrowheads) coverage of CD31 (blue) vessels, n = 4–5 mice. Data are represented as mean ± SEM. ** P = 0.0023; *** P < 0.0001, 1-way ANOVA. ( F ) p-MLC (red) expression in pericytes (NG2, green); arrows indicate overlay (yellow), n = 3–4 mice. Data are represented as mean ± SEM. * P = 0.03; ** P = 0.0003; *** P < 0.0001, 1-way ANOVA. ( G ) Fasudil treatment schematic of Rgs5 KO PNET mice and assessment of tumor perfusion at endpoint. CD31 (red) overlay with infused FITC-lectin (yellow) is highlighted by arrows. Perfusion was quantified in Rgs5 KO +fasudil ( F ) group in comparison with WT and Rgs5 KO (data from A , shadowed). n = 4–5 mice. Data are represented as mean ± SEM. **** P < 0.0001, 1-way ANOVA. Scale bars: 50 μm ( A – D , F , G ); 25 μm ( E ).

Article Snippet: The following drugs, dosing, and application routes were used for in vivo treatments: BEZ235 (dactolisib, NSP-BEZ235, Selleck Chemicals, 5 or 10 mg/kg, o.g., in 10% MMP [methyl 3-([2,2-dimethylbutanoyl]thio)propanoate]/90% PEG 300), DAPT (GSI-IX, Selleck Chemicals, 5 or 10 mg/kg, o.g. in 5% DMSO/corn oil), fasudil hydrochloride (fasudil, LKT Laboratories, 30 mg/kg, i.p. 0.9% saline), trametinib (GSK1120212, Selleck Chemicals, 0.006 or 0.02 mg/kg [low dose], 1 and 2 mg/kg [high dose], o.g., in 5% DMSO and 95% PEG300).

Techniques: Knock-Out, Transgenic Assay, Expressing, Marker, Comparison