Journal: Molecular Oncology

Article Title: Single‐cell RNA‐seq reveals the invasive trajectory and molecular cascades underlying glioblastoma progression

doi: 10.1002/1878-0261.12569

Figure Lengend Snippet: TFs and lncRNAs identified in data from Patel et al . . List of upregulated (A) and downregulated (B) TFs as well as their Spearman correlation coefficient with pseudotime. (C) Venn diagrams showed the significant overlaps of TFs between both GBM data sets. P values were calculated by hypergeometric test. (D) Expression profiles of the most positively correlated TF EPAS1 and the most negatively correlated TF OLIG1. Data points are fitted with local polynomial regression fitting (red lines) with 95% confidence interval (gray area). Cells are colored based on their states. List of the top 100 upregulated (E) and downregulated (F) lncRNAs as well as their Spearman correlation coefficient with pseudotime in GBM1.

Article Snippet: Short‐interfering RNAs (siRNAs) specifically against EPAS1 were purchased from RiboBio (Guangzhou, China) and then transfected into GBM cells using Lipofectamine 2000 reagent (Invitrogen, Shanghai, China) according to the manufacturer’s protocol.

Techniques: Expressing

Journal: Molecular Oncology

Article Title: Single‐cell RNA‐seq reveals the invasive trajectory and molecular cascades underlying glioblastoma progression

doi: 10.1002/1878-0261.12569

Figure Lengend Snippet: Knockdown of EPAS1 inhibited GBM cell migration and invasion in vitro . (A) Endogenous EPAS1 expression status in three GBM cell lines. (B) EPAS1 expression was efficiently knocked down by two targeted siRNAs (siRNA1 and siRNA2) in U251 cells and LN229 cells as detected by Western blotting. Silencing EPAS1 expression suppressed cell migration and invasion capacity of U251 (C) and LN229 (D) cells in the Transwell migration and invasion assay (magnification 100×). Scale bars = 500 μm. Results were summarized as mean ± SD of three independent experiments (** P < 0.01; *** P < 0.001, independent Student’s t test).

Article Snippet: Short‐interfering RNAs (siRNAs) specifically against EPAS1 were purchased from RiboBio (Guangzhou, China) and then transfected into GBM cells using Lipofectamine 2000 reagent (Invitrogen, Shanghai, China) according to the manufacturer’s protocol.

Techniques: Knockdown, Migration, In Vitro, Expressing, Western Blot, Invasion Assay

Journal: Scientific Reports

Article Title: Inhibition of GPR68 induces ferroptosis and radiosensitivity in diverse cancer cell types

doi: 10.1038/s41598-025-88357-x

Figure Lengend Snippet: Knock down of GPR68 reduces survival of A549 and Panc02 cells 72-hour cell survival assays. ( A ) Cotransfection of dCas9 with GPR68 targeting sgRNAs results in decreased A549 survival, transfection of dCas9 alone or sgRNA alone had no impact on A549 survival ( B ). ( C ) Cotransfection of dCas9 with GPR68 targeting sgRNAs results in decreased GPR68 expression in A549, transfection of dCas9 or sgRNA alone had no impact on GPR68 expression ( D ). ( E ) Cotransfection of dCas9 with GPR68 targeting sgRNAs results in decreased A549 survival, transfection of dCas9 alone or sgRNA alone had no impact on A549 survival ( F ). ( G ) Cotransfection of dCas9 with GPR68 targeting sgRNAs results in decreased GPR68 expression in A549, transfection of dCas9 or sgRNA alone had no impact on GPR68 expression ( H ). ( I ) siRNA targeting GPR68 results in decreased survival of A549, while non-targeting siRNA had no effect. ( J ) siRNA knockdown of GPR68 in A549 was verified in qRT-PCR. ( K ) siRNA targeting GPR68 results in decreased survival of Panc02, while non-targeting siRNA had no effect. ( L ) siRNA knockdown of GPR68 in Panc02 was verified in qRT-PCR. ( C ), ( D ), ( J ), ( L ) were normalized to GAPDH. ( A )–( L ) n = 3 biological repeats with n = 3 technical repeats; mean +/- SD with significance determined by multiple two-tailed, equal variance with Bonferroni Correction. ( A )–( H ) α-level of 0.001 is *** p < 0.00033. ( I )–( L ) α-level of 0.01 is ** p < 0.0025, 0.001 is *** p < 0.00025.

Article Snippet: For GPR68 overexpressions control (VB230630-1409kyc) and hGPR68 (VB221110-1429uwp) plasmids were obtained from Vectorbuilder.

Techniques: Knockdown, Cotransfection, Transfection, Expressing, Quantitative RT-PCR, Two Tailed Test

Journal: Scientific Reports

Article Title: Inhibition of GPR68 induces ferroptosis and radiosensitivity in diverse cancer cell types

doi: 10.1038/s41598-025-88357-x

Figure Lengend Snippet: GPR68 inhibition reduces cellular survival. ( A ) and ( B ) OGM inhibits clonogenic growth at a dose of 1 μm and higher in a 6-day clonogenic assay for both A549 and Panc02 cells. ( C ) and ( D ) Quantification of colony size in the clonogenic assay using low doses of OGM show decreased colony sizes for both A549 and Panc02 cells. ( A )–( D ) mean +/- SD; n ≥ 3 biological repeats with Bonferroni Correction and an α-level of 0.001 is *** p < 0.0002.

Article Snippet: For GPR68 overexpressions control (VB230630-1409kyc) and hGPR68 (VB221110-1429uwp) plasmids were obtained from Vectorbuilder.

Techniques: Inhibition, Clonogenic Assay

Journal: Scientific Reports

Article Title: Inhibition of GPR68 induces ferroptosis and radiosensitivity in diverse cancer cell types

doi: 10.1038/s41598-025-88357-x

Figure Lengend Snippet: Inhibition of GPR68 increases adenocarcinoma radiosensitivity. Clonogenicity assays of cells irradiated with 2 or 4 Gy and treated with OGM 100 min later. ( A ) A549 cells showed a significant decrease in colony size when treated with 0.7 μm OGM after irradiation with 2 Gy. ( B ) At 4 Gy treatment with both 0.7 μm and 0.8 μm OGM significantly decreased colony size in A549 cells. ( C ) and ( D ) At both 2 and 4 Gy, the 0.7 μm OGM treatment significantly decreased colony size in Panc02 cells. OGM treatment groups were compared to their corresponding unirradiated controls. ( A )–( D ) mean +/- SD; n ≥ 3 biological repeats and an α-level of 0.001 is *** p < 0.001.

Article Snippet: For GPR68 overexpressions control (VB230630-1409kyc) and hGPR68 (VB221110-1429uwp) plasmids were obtained from Vectorbuilder.

Techniques: Inhibition, Irradiation

Journal: Scientific Reports

Article Title: Inhibition of GPR68 induces ferroptosis and radiosensitivity in diverse cancer cell types

doi: 10.1038/s41598-025-88357-x

Figure Lengend Snippet: GPR68 inhibition synergizes with radiation ( A ) and ( B ) flow-cytometry of A549 shows cells exposed to OGM and radiation have elevated lipid peroxidation compared to OGM or radiation alone. ( C ) statistical analysis of curves in ( A ) and ( B ). ( D )–( F ) flow-cytometry of Panc02 shows cells exposed to OGM and radiation have elevated lipid peroxidation compared to OGM or radiation alone, with statistics. ( C ) and ( D ) CDI < < 1 indicates the combinatorial effect is synergistic, not additive. ( G ) Cell cycle analysis of OGM treatment and Irradiated Panc02 cells. ( H ) Quantification of % change in G2 phase fraction from (G) shows synergy between OGM and 3 Gy radiation. ( A )–( H ) n = 3 biological repeats with n = 10,000 events. ( C ) and ( F ) α-level of 0.001 is *** p < 0.00017.

Article Snippet: For GPR68 overexpressions control (VB230630-1409kyc) and hGPR68 (VB221110-1429uwp) plasmids were obtained from Vectorbuilder.

Techniques: Inhibition, Flow Cytometry, Cell Cycle Assay, Irradiation

Journal: Scientific Reports

Article Title: Inhibition of GPR68 induces ferroptosis and radiosensitivity in diverse cancer cell types

doi: 10.1038/s41598-025-88357-x

Figure Lengend Snippet: Inhibition of GPR68 promotes intracellular Fe 2+ levels. ( A ) FerroOrange staining of A549 and Panc02 cells treated with DMSO or 2 µM OGM for 24 h. ( B ) Quantification of FerroOrange staining in A549 and Panc02 cells demonstrating an increase in intracellular Fe 2+ levels. ( B ) n = 3 biological repeats with n = 6 technical repeats. ( B ) mean +/- SD with significance determined by multiple two-tailed, equal variance with an α-level of 0.001 is *** p < 0.001.

Article Snippet: For GPR68 overexpressions control (VB230630-1409kyc) and hGPR68 (VB221110-1429uwp) plasmids were obtained from Vectorbuilder.

Techniques: Inhibition, Staining, Two Tailed Test

Journal: Clinical and Translational Medicine

Article Title: Single‐cell RNA‐sequencing and spatial transcriptomic analysis reveal a distinct population of APOE − cells yielding pathological lymph node metastasis in papillary thyroid cancer

doi: 10.1002/ctm2.70172

Figure Lengend Snippet: APOE gene expression is associated with tumour cell metastasis. (A) Distribution of APOE expression in cells from samples with different metastasis characters (left: centre metastasis; right: centre plus neck metastasis). (B) APOE expression in lymph node and thyroid tumour cells from samples with different metastasis characters. Lymph tumour: metastatic tumour cells in LN; Thyroid tumour: Thyroid primary tumours. (C) TCGA survival analysis of PTC patients regarding different APOE expression levels. N = 256. (D) Pseudo‐time trajectory for tumour cells. The colour of cells represents the inferred pseudo‐time. (E) APOE expression along the pseudo‐time. Each data point represents the APOE expression in a cell. The colour of cells represents the inferred states of tumour cells along the trajectory. (F) Pathways (GO Biological Process) enriched in differentially expressed gene sets between APOE + and APOE − cells (left two panels for thyroid tumour location; right two panels for lymph node location). (G, K) Pathology annotation of two lymph node histology slides from two patients with metastatic PTC. Yellow: Tumour region. (H, L) Tumour region identified by TESLA using four indicated marker expression levels. (I, M) Tumour Edge detected by TESLA. The red colour indicates the tumour edge, while purple denotes the tumour core. (J−N) APOE expression marked by TESLA DE program, red highlights the region where APOE is significantly overexpressed.

Article Snippet: APOE overexpression control and lentiviral vectors were purchased from GenePharma.

Techniques: Gene Expression, Expressing, Marker

Journal: Clinical and Translational Medicine

Article Title: Single‐cell RNA‐sequencing and spatial transcriptomic analysis reveal a distinct population of APOE − cells yielding pathological lymph node metastasis in papillary thyroid cancer

doi: 10.1002/ctm2.70172

Figure Lengend Snippet: Overexpression of APOE inhibits tumour cell proliferation and invasion in vitro. (A) Hth‐7 and TPC‐1 cells were transfected with control and APOE ‐overexpression lentivirus (OE) for 48 h. Cell lines that stably overexpressed APOE were obtained through puromycin selection. Total cellular RNA was extracted for RT‐PCR analysis. Overexpression efficiencies were verified via RT‐PCR. * p < .05; ** p < .01; *** p < .001. (B, C) The proliferation of indicated cells was analysed with a Cell Counting Kit‐8 (CCK8) assay (B) and colony formation (C). Control and OE cells were cultured and evaluated at 0, 24, 48, 72 and 96 h. n = 5 biologically independent experiments. Colony formation was assessed after 2 weeks. The results from three independent experiments are indicated as means ± standard deviations. (D) Transwell invasion analysis revealed the invasive ability of cells. (E) Each bar represents the mean ± standard deviation of three independent experiments. (F) GSEA enrichment analysis reveals that ‘TNFA_signaling_via_NFKB’ is the most enriched hallmark pathway in both cell lines. (G) The bar plot displays the RNA‐seq raw counts (raw expression levels) of ABCA1 in both cell lines, with and without APOE overexpression.

Article Snippet: APOE overexpression control and lentiviral vectors were purchased from GenePharma.

Techniques: Over Expression, In Vitro, Transfection, Control, Stable Transfection, Selection, Reverse Transcription Polymerase Chain Reaction, Cell Counting, CCK-8 Assay, Cell Culture, Standard Deviation, RNA Sequencing, Expressing

Journal: Clinical and Translational Medicine

Article Title: Single‐cell RNA‐sequencing and spatial transcriptomic analysis reveal a distinct population of APOE − cells yielding pathological lymph node metastasis in papillary thyroid cancer

doi: 10.1002/ctm2.70172

Figure Lengend Snippet: In vivo validation of APOE function in tumour inhibition and its large‐scale human validation. (A–C) APOE overexpression inhibited xenograft tumours. 1×10 7 APOE ‐overexpression Hth‐7 and TPC‐1 cells and control cells were subcutaneously grafted in athymic, female nude mice. Tumour volume (A, B) and body weight (C) were measured every 3−4 days (volume = width 2 × length × 1/2) (* p < .05; ** p < .01; *** p < .001). (D) Tumour tissues were removed from mice after 2 weeks and slides were immunohistochemically stained with APOE and ABCA1 . (E) Quantification results for the APOE and ABCA1 expression of the immunohistochemistry analysis. (F) Tumour sections were haematoxylin and eosin (HE)‐stained and observed via dual colour fluorescence. Fluorescent images of thyroid tumours in which CK19 ‐positive cells are stained red and APOE ‐positive cells are stained green. Nuclei were stained blue (DAPI). (G) Quantification of the colocalization of APOE and CK19 immunofluorescence data. (H) Representative are images of Gr‐1 immunofluorescence staining in cells treated with saline (top) or RGX‐104 (bottom). Green staining indicates Gr‐1‐positive cells, while blue staining marks nuclei. (I) Quantification results for the Gr‐1‐positive cells of the immunofluorescence analysis. (J) Representative images of APOE expression after treatment with the saline (top) or RGX‐104 (bottom). (K) Quantification results for the APOE expression of the immunohistochemistry analysis.

Article Snippet: APOE overexpression control and lentiviral vectors were purchased from GenePharma.

Techniques: In Vivo, Biomarker Discovery, Inhibition, Over Expression, Control, Staining, Expressing, Immunohistochemistry, Fluorescence, Immunofluorescence, Saline

Journal: Clinical and Translational Medicine

Article Title: Single‐cell RNA‐sequencing and spatial transcriptomic analysis reveal a distinct population of APOE − cells yielding pathological lymph node metastasis in papillary thyroid cancer

doi: 10.1002/ctm2.70172

Figure Lengend Snippet: Differential immune−tumour interactions associated with APOE expression profiles. (A) Visualization of re‐clustered immune cell type annotation in 2D UMAP. (B) Violin plots demonstrating marker genes used to annotate immune cell types. (C) Summary of overall interaction between immune cells with APOE + tumour cells and immune cells with APOE − tumour cells (left total numbers of interaction events; right: normalized interaction strength). The figure illustrates the total number of interactions and interaction strength within the inferred cell−cell communication networks between the ‘APOE neg’ and ‘APOE pos’ groups, as determined using CellChat. (D) Overview of ligand‐receptor pairs showing differential interaction profiles between the APOE + and APOE − cells (with immune cells). (E, F) Demonstration of signalling pathway network of CD6 (E) and JAM (F). The width of the strings represents the interaction strength of the interaction. Neg: tumour cells were APOE ‐negative cells; POS: tumour cells were APOE ‐positive cells; the figure showcases a representative differentially expressed ligand‐receptor pathway, CD6 , obtained from the CellChat package. This pathway demonstrates the variations in cell communication between the ‘ APOE neg’ and ‘ APOE pos’ groups. (G, I) CellTrek analysis mapped the specific cell type back to the histology slides from different tissues (G: primary tumour, I: Lymph node). The figure represents the co‐embedding analysis of spatial transcriptomic (ST) and single‐cell RNA‐seq (scRNA‐seq) datasets utilizing the CellTrek ‘traint’ function. The objective of this step is to assess the overlap between these two data modalities. Following the co‐embedding process, single cells can be mapped to their spatial positions. (H, J) Cell−cell colocalization/interaction analysis of different cell types inferred by CellTrek analysis.

Article Snippet: APOE overexpression control and lentiviral vectors were purchased from GenePharma.

Techniques: Expressing, Marker, RNA Sequencing