Journal: Redox Biology

Article Title: Oxidative stress-driven m 5 C methylation by NSUN5 confers cisplatin resistance in lung adenocarcinoma through promoting glycolysis

doi: 10.1016/j.redox.2026.104193

Figure Lengend Snippet: NSUN5 regulates the m5C modification and expression of its downstream target gene GLUT1. (A) Dot blot assay illustrating global m 5 C modification levels of total RNA in shNC or shNSUN5 A549/DDP cells. (B) Distribution profile of m 5 C modifications across diverse RNA regions (CDS, downstream, exon, intron, upstream, 3′UTR, and 5′UTR) from RNA Bis-seq in shNC- and shNSUN5-transfected A549/DDP cells. (C) Line chart depicting m 5 C site distribution by methylation level after NSUN5 knockdown. (D) Expression of differentially expressed genes (DEGs) from RNA-seq analysis of shNC- vs. shNSUN5-transfected A549/DDP cells. (E) Enriched pathways of those DEGs (D) in the RNA-seq. (F) Venn diagram of significantly m 5 C-modified genes (BiS-seq) and DEGs (RNA-seq). (G) Integrated volcano plot showing methylation (BiS-seq) and expression (RNA-seq) changes for 149 overlapping genes. GLUT1 exhibited the most pronounced methylation decrease in hypo-down group. (H) Correlation between NSUN5 and GLUT1 mRNA expression in TCGA-LUAD cohort. (I) IHC of NSUN5 and GLUT1 in serial sections from the same LUAD tumor tissue sample (left). Frequency of GLUT1 overexpression stratified by high/low NSUN5 expression. Scale bars (the upper panel), 200 μm. Scale bars (the lower panel), 50 μm. (J) Representative immunofluorescence staining showing the subcellular localization of GLUT1 (red) in shNC or shNSUN5 A549/DDP cells. Nuclei were stained with DAPI (blue). Scale bars, 15 μm. (K) Protein expression of GLUT1 in shNC and NSUN5-knockdown cells was assessed by Western blot assays. (L) m 5 C-MeRIP-qPCR analysis showing m 5 C modification of GLUT1 mRNA in shNC- or shNSUN5-transfected A549/DDP cells. (M) GLUT1 mRNA stability after actinomycin D (4 μg/mL) treatment. Half-life calculated from decay curves. (N) Western blot assays evaluating relative GLUT1 protein expression in NSUN5-overexpressing vs. control cells. (O) m 5 C-MeRIP-qPCR quantifying m 5 C modification levels of GLUT1 mRNA in NSUN5-overexpressing vs. control cells. (P) Actinomycin D assay determining GLUT1 mRNA half-life in NSUN5-overexpressing vs. control cells. Rep: Repeat. Data were representative of at least three independent experiments and presented as mean (SD). Statistical significance was determined using Student's t-test (L, O), Pearson correlation test (H) or Chi-square test (I). ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001. n.s, not significant.

Article Snippet: Short hairpin RNA (shRNA) oligonucleotides targeting NSUN5, YBX1, and GLUT1, as well as lentiviruses encoding NSUN5 and YBX1 overexpression constructs, were purchased from GeneChem (Shanghai, China).

Techniques: Modification, Expressing, Dot Blot, Transfection, Methylation, Knockdown, RNA Sequencing, Over Expression, Immunofluorescence, Staining, Western Blot, Control

Journal: Redox Biology

Article Title: Oxidative stress-driven m 5 C methylation by NSUN5 confers cisplatin resistance in lung adenocarcinoma through promoting glycolysis

doi: 10.1016/j.redox.2026.104193

Figure Lengend Snippet: NSUN5 confers cisplatin resistance in a GLUT1-dependent manner in vitro and in vivo . (A) Effect of GLUT1 knockdown on cisplatin sensitivity in NSUN5-overexpressing cells. Cellular viability and cisplatin IC 50 values were determined by CCK-8 assay in NSUN5-overexpressing A549 cells following GLUT1 knockdown. (B) Effect of GLUT1 knockdown on cisplatin-induced apoptosis in NSUN5-overexpressing cells. Apoptosis was assessed by flow cytometry in NSUN5-upregulated A549 cells after GLUT1 knockdown and cisplatin exposure. (C) Western blot analysis of indicated proteins in NSUN5-overexpressing A549 (left panel) and PC9 (right panel) cells, with or without cisplatin exposure and with or without GLUT1 knockdown. (D) Representative comet assay images (left panel) and quantitative tail moment analysis (right panel) in NSUN5-overexpressing A549 cells following GLUT1 knockdown. (E) Immunofluorescence showing nuclear γ-H2AX foci density in designated treatment groups. Scale bars, 10 μm. (F) Bioluminescence images of xenograft tumors across groups. (G) Tumor volume measurements in nude mice under indicated conditions. (H) Terminal tumor weights across groups. (I) H&E staining and IHC for NSUN5, GLUT1, p -RPA2, γ-H2AX, and Cleaved Caspase 3 (Cleaved C3) in mice tumor sections. Scale bars (the upper panel), 200 μm. Scale bars (the lower panel), 50 μm. Data were representative of at least three independent experiments and presented as mean (SD). Statistical significance was determined using Student's t-test (A, D, E, H). ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001. n.s, not significant.

Article Snippet: Short hairpin RNA (shRNA) oligonucleotides targeting NSUN5, YBX1, and GLUT1, as well as lentiviruses encoding NSUN5 and YBX1 overexpression constructs, were purchased from GeneChem (Shanghai, China).

Techniques: In Vitro, In Vivo, Knockdown, CCK-8 Assay, Flow Cytometry, Western Blot, Single Cell Gel Electrophoresis, Immunofluorescence, Staining

Journal: Redox Biology

Article Title: Oxidative stress-driven m 5 C methylation by NSUN5 confers cisplatin resistance in lung adenocarcinoma through promoting glycolysis

doi: 10.1016/j.redox.2026.104193

Figure Lengend Snippet: Cisplatin-induced ROS enhances methyltransferases activity of NSUN5 to promote m 5 C modification of GLUT1 mRNA. (A, B) NSUN5-bound m 5 C RNA detection by Co-IP. Western blot revealed m 5 C-modified RNA bound by HA-NSUN5 treated with cisplatin or Tempol. (C) Three-step catalytic mechanism of NSUN5-mediated m 5 C methylation. First, deprotonated Cys359 (motif VI, purple) initiated nucleophilic attack on cytosine C6, forming a covalent S-thioester intermediate (II) that polarizes C5. Second, Cys308 (motif IV, orange) abstractd the C5 proton, enabling methyl transfer from SAM to generate methylated intermediate (III). Finally, general base-catalyzed β-elimination released m 5 C-modified RNA and regenerates the enzyme. Top: Amino acid sequence alignment of regions forming the active sites of m 5 C methyltransferases NSUN5; The conserved motifs of NSUN5 (IV and VI) were boxed. Bottom: Reaction pathway of m 5 C formation. (D) Schematic of single-site (NSUN5 C308A , NSUN5 C359A ) and double mutant (NSUN5 DM ) constructs. Domains: N-terminal globular (green), RNA methyltransferase (blue), C-terminal (grey). Catalytic cysteines (C308/C359, orange) and SAM binding site (pink) were shown. Amino acid positions were numbered from the N-terminus. (E) Western blot revealed m 5 C-modified RNA bound by wild-type or mutant HA-NSUN5 treated with cisplatin or Tempol. (F) RNA pull-down assay coupled with Western blot validated NSUN5 as a binding protein for GLUT1 mRNA in resistant cells. (G) RNA immunoprecipitation (left panel) and agarose gel electrophoresis (right panel) assays confirmed direct binding between NSUN5 protein and GLUT1 mRNA in A549/DDP cells. (H) Western blot of GLUT1 expression after overexpression of NSUN5 WT , NSUN5 C308A , or NSUN5 C359A in A549 cells under cisplatin treatment. (I) RIP assay comparing the binding ability of NSUN5 with GLUT1 mRNA in overexpressed NSUN5 WT , NSUN5 C308A or NSUN5 C359A cells when treated with cisplatin or Tempol. (J) m 5 C-MeRIP-qPCR analysis of GLUT1 mRNA m 5 C modification levels in cells transfected with wild-type or single-point mutation constructs, following cisplatin or Tempol treatment. (K) GLUT1 mRNA half-life measured by actinomycin D assay after NSUN5 WT versus NSUN5 DM overexpression in A549 cells after cisplatin exposure. (L) Luciferase activity of wild-type and m 5 C-site-mutated GLUT1 reporters in A549 cells overexpressing NSUN5 WT or NSUN5 DM . Data were representative of at least three independent experiments and presented as mean (SD). Statistical significance was determined using Student's t-test (G, I, J, L). ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001. n.s, not significant.

Article Snippet: Short hairpin RNA (shRNA) oligonucleotides targeting NSUN5, YBX1, and GLUT1, as well as lentiviruses encoding NSUN5 and YBX1 overexpression constructs, were purchased from GeneChem (Shanghai, China).

Techniques: Activity Assay, Modification, RNA Detection, Co-Immunoprecipitation Assay, Western Blot, Methylation, Sequencing, Mutagenesis, Construct, Binding Assay, Pull Down Assay, RNA Immunoprecipitation, Agarose Gel Electrophoresis, Expressing, Over Expression, Transfection, Luciferase

Journal: Redox Biology

Article Title: Oxidative stress-driven m 5 C methylation by NSUN5 confers cisplatin resistance in lung adenocarcinoma through promoting glycolysis

doi: 10.1016/j.redox.2026.104193

Figure Lengend Snippet: NSUN5-catalyzed m 5 C modification of GLUT1 mRNA maintains its YBX1-mediated stability. (A) Silver staining of whole-cell extract, biotin-NC pull-down (Bio-NC), and biotin-GLUT1 mRNA (Bio-GLUT1) pull-down proteins from A549/DDP cells (left panel). HPLC-MS/MS results showing the sequence HT score and relative abundance of YBX1 (right panel). (B) Correlation between YBX1 and GLUT1 mRNA expression in TCGA-LUAD cohort. (C) IHC staining of serial sections from the same LUAD patients showing co-expression of YBX1 and GLUT1. Scale bars (the upper panel), 200 μm. Scale bars (the lower panel), 50 μm. (D, E) GLUT1 expression at mRNA and protein levels following YBX1 depletion (shRNA #1/#2) in cisplatin resistant cells. (F) GLUT1 mRNA half-life determined by actinomycin D chase assay after YBX1 knockdown in A549/DDP cells. (G, H) GLUT1 mRNA (G, qPCR) and protein (H, Western blot) expression upon YBX1 overexpression in cisplatin sensitive LUAD cells. (I) GLUT1 mRNA half-life was measured by actinomycin D assay after YBX1 overexpression. (J) RIP assay showing enrichment of GLUT1 mRNA by the YBX1 antibody compared with the negative control IgG. (K) RNA-pulldown assay demonstrating direct binding between GLUT1 mRNA and YBX1. (L) Western blotting showed that YBX1 depletion reversed the increase in GLUT1 protein levels induced by NSUN5 overexpression upon cisplatin exposure. (M) RIP analysis evaluating YBX1 binding to GLUT1 mRNA in A549 cells overexpressing NSUN5 WT or NSUN5 DM with cisplatin treatment. (N) Dual-luciferase reporter assay measuring YBX1-mediated activity of GLUT1-WT and GLUT1-MUT reporters. Data were representative of at least three independent experiments and presented as mean (SD). Statistical significance was determined using Student's t-test (D, G, J, M, N), Pearson correlation test (B) or Chi-square test (C). ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001. n.s, not significant.

Article Snippet: Short hairpin RNA (shRNA) oligonucleotides targeting NSUN5, YBX1, and GLUT1, as well as lentiviruses encoding NSUN5 and YBX1 overexpression constructs, were purchased from GeneChem (Shanghai, China).

Techniques: Modification, Silver Staining, Tandem Mass Spectroscopy, Sequencing, Expressing, Immunohistochemistry, shRNA, Knockdown, Western Blot, Over Expression, Negative Control, Binding Assay, Luciferase, Reporter Assay, Activity Assay

Journal: Redox Biology

Article Title: Oxidative stress-driven m 5 C methylation by NSUN5 confers cisplatin resistance in lung adenocarcinoma through promoting glycolysis

doi: 10.1016/j.redox.2026.104193

Figure Lengend Snippet: NSUN5 promotes glycolysis and HR through GLUT1. (A) The glucose uptake was measured in NSUN5-overexpressing A549 cells with shNC or shGLUT1 transfection by fluorescently labeled glucose analogue 2-NBDG. The nucleus (blue) was stained with Hoechst. Scale bars, 100 μm. (B) Glycolytic flux analysis by extracellular acidification rate (ECAR). Real-time ECAR tracing in A549 cells sequentially treated with glucose, oligomycin (oligo), and 2-DG across experimental groups (left panel). Quantification of glycolytic parameters, including the basal glycolytic rate, maximal glycolytic capacity, and spare glycolytic capacity (right panel). (C) Mitochondrial respiration analysis by oxygen consumption rate (OCR). Real-time OCR tracing in A549 cells sequentially treated with oligomycin, FCCP, and rotenone/antimycin A across groups (left panel). Quantification of mitochondrial parameters, including basal respiration, ATP production, maximal respiration, and spare respiratory capacity (right panel). (D) Relative lactate production in designated A549 cell groups. (E) Schematic representation of the HR reporter. (F) The HR levels of the indicated HEK293T cells were detected by flow cytometry. (G-J) Representative immunofluorescence images of MRE11 (G), p -RPA2 (H), BrdU (I), and RAD51 (J) foci in A549 cells under indicated treatments. Scale bars, 10 μm. Data were representative of at least three independent experiments and presented as mean (SD). Statistical significance was determined using Student's t-test (B-D, F-J), ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, n.s, not significant.

Article Snippet: Short hairpin RNA (shRNA) oligonucleotides targeting NSUN5, YBX1, and GLUT1, as well as lentiviruses encoding NSUN5 and YBX1 overexpression constructs, were purchased from GeneChem (Shanghai, China).

Techniques: Transfection, Labeling, Staining, Flow Cytometry, Immunofluorescence

Journal: iScience

Article Title: Decoding neuron-specific lineage to identify diagnostic biomarkers and therapeutic targets for ischemic stroke

doi: 10.1016/j.isci.2026.116257

Figure Lengend Snippet: Cherp serving an effective treatment target for IS (A) The neuron counts of cluster 3 and 5. (B) The Cherp expression in each cluster. (C) The WB image of Cherp expression after OGD/R process. (D) The IF images of Cherp expression after OGD/R process. Scale bars = 100 μm. (E) The efficiency validation of Cherp knocking down using shRNA. (F) The cell viability assay after Cherp knocking down. (G) LDH leakage assay after Cherp knocking down. (H) TUNEL assay after Cherp knocking down. Scale bars = 100 μm. (I) WB image of apoptosis-related markers after Cherp knocking down. (J) Morphology of cerebral edema in sham, MCAO and MCAO-shRNA intervention groups at 72 h post-surgery. (K) TTC staining of cerebral infarction in sham, MCAO and MCAO-shRNA intervention groups at 72 h post-surgery (Dashed area represents the infarcted brain region). (L) Statistics analysis of brain water content ( n = 5 mice/group). (M) Quantification of infarct area ( n = 5 mice/group). (N) The functional enrichment of Cherp high cells related pathways. (O) The intracellular calcium imaging after Cherp knocking down. Scale bars = 100 μm (P) Quantitative analysis of intracellular Ca 2+ fluorescence intensity in primary neurons under indicated conditions ( n = 3 independent experiments). Data are presented as mean ± SD. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001 and ∗∗∗∗ p < 0.0001 as determined by Student’s t test.

Article Snippet: The shRNA sequences targeting Cherp were generated at Sangon Biotech.

Techniques: Expressing, Biomarker Discovery, shRNA, Viability Assay, TUNEL Assay, Staining, Functional Assay, Imaging, Fluorescence

Journal: iScience

Article Title: Stromal peroxidasin drives early tumor growth in breast cancer

doi: 10.1016/j.isci.2026.116078

Figure Lengend Snippet: Significantly differentially expressed ECM molecules in early, mid and late-stage mammary tumor samples from the PyMT mouse model of breast cancer in comparison to healthy fat pad tissue (A) PCA of individual samples ( n = 4 or 5 mice per group). Principal components 1 and 2 account for 81.6% of the variability in the dataset. (B) Heatmap of significantly differentially deposited matrisomal elements between groups within the dataset (left). Missing values are shaded in gray. Four major clusters were identified based on Euclidian hierarchical clustering of proteins (middle) and the matrisome categorical annotation for these clustered matrisomal elements based on the matrisome project is shown (right). Expression of PXDN in each sample (cluster 3) is highlighted in red.

Article Snippet: Lentiviral PXDN shRNA knockdown constructs and a Scrambled control construct containing GFP sequences were obtained from OriGene (TL513178V).

Techniques: Comparison, Expressing

Journal: iScience

Article Title: Stromal peroxidasin drives early tumor growth in breast cancer

doi: 10.1016/j.isci.2026.116078

Figure Lengend Snippet: Association of PXDN expression with patient overall survival Significance between Kaplan-Meier survival curves were calculated with the log-rank test. (A) Overall survival of 1,082 patients from the TCGA breast cancer cohort stratified by median mRNA expression of PXDN. (B) Overall survival of 1,082 patients from the TCGA breast cancer cohort separated by disease stage, and stratified by median mRNA expression of PXDN (dotted line = low PXDN expression, solid line = high PXDN expression) and stage of disease (stage I, red; II, gold; III, green; IV, blue; or unknown, purple). (C) Extraction of data from (B) showing patients with stage II breast cancer at the time of diagnosis stratified by median mRNA expression of PXDN (dotted line = low PXDN expression, solid line = high PXDN expression) ( n = 615). (D) Examples of PXDN IHC staining intensity and corresponding intensity scores for the stromal and epithelial compartments of tumors ( n = 334). Scale bars, 50 μm. (E) Overall survival of 311 invasive ductal carcinoma patients from the CREA tumor microarray cohort stratified by high vs. low Allred-scores for PXDN IHC staining of epithelial compartments of tumors. (F) Overall survival of 309 invasive ductal carcinoma patients from the CREA tumor microarray cohort stratified by high vs. low Allred-scores for PXDN IHC staining of stromal compartments of tumors. (G) Overall survival of invasive ductal carcinoma patients from the CREA tumor microarray cohort stratified by combined stromal and epithelial Allred-scores for PXDN IHC staining of cores. Log-rank p values between each curve of (G) are listed in .

Article Snippet: Lentiviral PXDN shRNA knockdown constructs and a Scrambled control construct containing GFP sequences were obtained from OriGene (TL513178V).

Techniques: Expressing, Extraction, Biomarker Discovery, Immunohistochemistry, Microarray

Journal: iScience

Article Title: Stromal peroxidasin drives early tumor growth in breast cancer

doi: 10.1016/j.isci.2026.116078

Figure Lengend Snippet: Single cell analysis of breast tumor cells expressing PXDN (A–C) Murine PyMT tumor single cell data (11,490 cells) obtained from Valdés-Mora et al. (A) UMAP of the major cell types identified in the murine single cell data. (B) UMAP of PXDN expression across cells. (C) Violin plots of fold change in PXDN expression according to cell type in the murine dataset. (D and E) Human breast cancer single cell and spatial data containing 130,246 cells from 26 invasive breast cancer patients. (D) UMAP of the major cell types identified in the human single cell data. (E) UMAP of PXDN expression across human cells. (F) Boxplots of fold change in PXDN expression according to cell type in the human dataset.

Article Snippet: Lentiviral PXDN shRNA knockdown constructs and a Scrambled control construct containing GFP sequences were obtained from OriGene (TL513178V).

Techniques: Single-cell Analysis, Expressing, Single Cell

Journal: iScience

Article Title: Stromal peroxidasin drives early tumor growth in breast cancer

doi: 10.1016/j.isci.2026.116078

Figure Lengend Snippet: PXDN affects cell proliferation and spheroid formation dynamics in CAFs Differences between shScr CAFs and sh#1 CAFs, or between WT CAFs and OE CAFs, were calculated using Student’s t tests and p values are shown on each graph. p values <0.05 were considered significant. ns = not significant. (A and C) RNA expression of PXDN in shScr and sh#1 (A), or WT and OE (C) CAFs. Error bars show the standard deviation of six independent experiments. (B and D) Representative western blots showing PXDN levels in conditioned media (top) from shScr and sh#1 CAFs (B) or WT and OE CAFs (D), with densitometry quantification of three biological replicate western blots (bottom). Densitometry measurements were normalized to total protein loading (measured by Ponceau S stain) and calculated relative to control (shScr or WT) protein expression. (E and F) 2D proliferation of shScr and sh#1 CAFs (E) or WT and OE CAFs (F) as measured by Alamar blue assay five days of culture (left). Differences between CAF lines were directly compared on day 5 (right). Error bars indicate the standard error of the mean for three biological replicates. (G–L) 3D growth of shScr (blue), sh#1 (red), WT (purple), and OE (gold) CAFs as spheroids over the course of 16 days. (G and I) Spheroid area and (K and L) representative images over the course of 16 days. Scale bars, 400 μm. Dotted lines indicate media changes on days 4, 8, and 12. Error bars indicate the standard error of the mean for three biological replicates. (H and J) The day of spheroid growth at which spheroids reached their smallest size before expanding. Error bars indicate the standard error of 24 spheroids across three biological replicates.

Article Snippet: Lentiviral PXDN shRNA knockdown constructs and a Scrambled control construct containing GFP sequences were obtained from OriGene (TL513178V).

Techniques: RNA Expression, Standard Deviation, Western Blot, Staining, Control, Expressing, Alamar Blue Assay

Journal: iScience

Article Title: Stromal peroxidasin drives early tumor growth in breast cancer

doi: 10.1016/j.isci.2026.116078

Figure Lengend Snippet: PXDN inhibits the ability of CAFs to remodel collagen matrices (A and C) Representative images of collagen matrices contraction over the course of 12 days when seeded with shScr or sh#1 CAFs (A) or WT or OE CAFs (C). Scale bars, 1 cm. (B and D) Area of matrices during contraction over the course of 12 days (left), with differences in final area of matrices compared at day 12 (right). Error bars indicate the standard error of the mean for three biological replicates. p values were calculated using Student’s t tests. p values <0.05 were considered significant.

Article Snippet: Lentiviral PXDN shRNA knockdown constructs and a Scrambled control construct containing GFP sequences were obtained from OriGene (TL513178V).

Techniques:

Journal: iScience

Article Title: Stromal peroxidasin drives early tumor growth in breast cancer

doi: 10.1016/j.isci.2026.116078

Figure Lengend Snippet: Impact of CAF-produced PXDN on cancer cell proliferation and motility Differences between shScr CAFs and sh#1 CAFs, or between WT CAFs and OE CAFs, were calculated using Student’s t tests and p values are indicated on each graph. p values <0.05 were considered significant. ns = not significant. (A) Schematic of generation of CAF-generated CDMs, onto which cancer cells were seeded. (B and C) Proliferation rates of cancer cells seeded on CDMs produced by sh#1 or shScr CAFs (B), or by WT and OE CAFs (C) as measured by Alamar blue at day 7. Error bars represent the standard deviation of eight replicates. (D and F) Mean velocity of cancer cells when seeded onto CDMs produced by shScr or sh#1 CAFs (D) or by WT or OE CAFs (F). (E and G) Distance traveled by cancer cells when seeded onto CDMs produced by shScr or sh#1 CAFs (E) or by WT or OE CAFs (G). A minimum of ten cells were tracked per biological replicate, across three biological replicates. Error bars indicate the standard deviation.

Article Snippet: Lentiviral PXDN shRNA knockdown constructs and a Scrambled control construct containing GFP sequences were obtained from OriGene (TL513178V).

Techniques: Produced, Generated, Standard Deviation

Journal: iScience

Article Title: Stromal peroxidasin drives early tumor growth in breast cancer

doi: 10.1016/j.isci.2026.116078

Figure Lengend Snippet: Effect of CAF PXDN expression on breast tumor progression in vivo Differences between shScr CAFs and sh#1 CAFs, or between WT CAFs and OE CAFs, were calculated using Student’s t tests and p values are indicated on each graph. p values <0.05 were considered significant; ns = not significant. (A and B) Latency of tumor formation from the time cells were implanted into mice along with shScr or sh#1 CAFs (A) or with WT or OE CAFs (B) until tumors reached 50 mm 3 in size. (C and D) Final weights of tumors upon collection from mice implanted with cancer cells and shScr or sh#1 CAFs (C) or with WT or OE CAFs (D). (E) Representative images of α-SMA staining in tumors from shScr or sh#1 CAF tumors. Scale bars, 100 μm. (F) Quantification of α-SMA staining in shScr and sh#1 CAF tumors. Error bars indicate the standard deviation. (G) Representative images of α-SMA staining in tumors from WT or OE CAF tumors. Scale bars, 100 μm. (H) Quantification of α-SMA staining in WT and OE CAF tumors. Error bars indicate the standard deviation.

Article Snippet: Lentiviral PXDN shRNA knockdown constructs and a Scrambled control construct containing GFP sequences were obtained from OriGene (TL513178V).

Techniques: Expressing, In Vivo, Staining, Standard Deviation

Journal: iScience

Article Title: Stromal peroxidasin drives early tumor growth in breast cancer

doi: 10.1016/j.isci.2026.116078

Figure Lengend Snippet: Effect of PXDN inhibition on breast tumor progression using AZD5904 (A) Inhibition of PXDN by a range of concentrations of AZD5904 measured on live cells using a modified Amplex red activity assay. Resorufin fluorescence was monitored continuously for 1 h. Error bars indicate standard error of the mean across three biological replicates, each consisting of eight technical replicates. (B) Final resorufin fluorescence readings at 60 min were used to determine to IC50 inhibitory concentration of AZD5904. (C) Kaplan-Meier curve of mouse survival measured from the start of treatment with AZD5904 or vehicle (when tumors reached a detectable size of 50 mm 3 ) until tumors reached a maximum ethical size of 1 cm by 1 cm and mice were sacrificed. Survival curves were significantly different, with a log-rank p value of 0.0088. (D) Final weights of tumors upon collection from mice in the AZD5904 in vivo study. Error bars indicate the standard deviation. Student’s t test showed no statistical significance between vehicle and AZD5904 treatment groups. (E) Time taken for tumors to reach the detectable size of 50 mm 3 at which point treatment started. Error bars indicate the standard deviation. Student’s t test showed no statistical significance between vehicle and AZD5904 treatment groups.

Article Snippet: Lentiviral PXDN shRNA knockdown constructs and a Scrambled control construct containing GFP sequences were obtained from OriGene (TL513178V).

Techniques: Inhibition, Modification, Activity Assay, Fluorescence, Concentration Assay, In Vivo, Standard Deviation

Journal: Molecular Therapy Oncology

Article Title: CBX6 and CA9 as predictive indicators and therapeutic targets in GBM

doi: 10.1016/j.omton.2026.201159

Figure Lengend Snippet: CBX6 expression is downregulated in multiple cancer types (A) Statistical analysis of The Cancer Genome Atlas (TCGA) Pan-Cancer RNA sequencing (RNA-seq) database shows CBX6 expression is downregulated in various cancer types. (B) CBX6 expression is significantly suppressed in brain cancer, breast cancer, lung adenocarcinoma (abbreviated as LUAD), and prostate cancer, based on analysis of TCGA tumor microarray data using the BRowse All Variants Online (BRAVO) database method. (C and D) Comparison of CBX6 expression in patient-derived GBM tissues and primary tumor cells relative to respective normal controls using qRT-PCR. GAPDH was used as a loading control. (D) Reduced CBX6 expression is significantly associated with poor prognosis in patients with glioma, as shown in Kaplan-Meier survival curves derived from a public patient-derived microarray database, p = 1.94∗ e−7. PBT, primary brain tumor; RBT, recurrent brain tumor.

Article Snippet: A pCMV6-entry plasmid containing Myc-DDK (same as Flag)-tagged human CBX6 cDNA (Cat#: RC204166) and a CBX6 Human shRNA Plasmid Kit (Cat#: TF314170) were purchased from OriGene.

Techniques: Expressing, RNA Sequencing, Microarray, Comparison, Derivative Assay, Quantitative RT-PCR, Control

Journal: Molecular Therapy Oncology

Article Title: CBX6 and CA9 as predictive indicators and therapeutic targets in GBM

doi: 10.1016/j.omton.2026.201159

Figure Lengend Snippet: CBX6 modulation affects tumor cell morphology and proliferation (A) CBX6 modulation affects the morphology of U-251 MG cells in two-dimensional culture. (B) Downregulation of CBX6 increases growth and invasion of U-251 MG cells in 3D culture. (C) Overexpression of CBX6 significantly suppresses U-251 MG proliferation on days 3 and 5 post-plating, as measured by MTT assay. (D) MTT assay of KLuc cells stably overexpressing human CBX6 or transfected with control plasmid on days 3 and 5 post-plating. (E) Silencing CBX6 increases U-251 MG proliferation compared to shRNA controls. Images were captured randomly from different fields under 20× magnification. VC, vector control; OE, CBX6 overexpression; KD, CBX6 knockdown. “∗” indicated p < 0.05.

Article Snippet: A pCMV6-entry plasmid containing Myc-DDK (same as Flag)-tagged human CBX6 cDNA (Cat#: RC204166) and a CBX6 Human shRNA Plasmid Kit (Cat#: TF314170) were purchased from OriGene.

Techniques: Over Expression, MTT Assay, Stable Transfection, Transfection, Control, Plasmid Preparation, shRNA, Knockdown

Journal: Molecular Therapy Oncology

Article Title: CBX6 and CA9 as predictive indicators and therapeutic targets in GBM

doi: 10.1016/j.omton.2026.201159

Figure Lengend Snippet: Effects of CBX6 modulation on invasion and migration of U-251 MG cells (A and B) Representative images and cell quantification from transwell migration assays of U-251 MG cells with modulated CBX6 expression compared to corresponding controls. (C and D) Representative images and cell quantification from transwell invasion assays of U-251 MG cells with modulated CBX6 expression compared to respective controls. VC, vector control; OE, CBX6 overexpression; KD, CBX6 knockdown. “∗” Indicates p < 0.05.

Article Snippet: A pCMV6-entry plasmid containing Myc-DDK (same as Flag)-tagged human CBX6 cDNA (Cat#: RC204166) and a CBX6 Human shRNA Plasmid Kit (Cat#: TF314170) were purchased from OriGene.

Techniques: Migration, Expressing, Plasmid Preparation, Control, Over Expression, Knockdown

Journal: Molecular Therapy Oncology

Article Title: CBX6 and CA9 as predictive indicators and therapeutic targets in GBM

doi: 10.1016/j.omton.2026.201159

Figure Lengend Snippet: Effect of human CBX6 overexpression on glioma tumor growth (A and B) Bioluminescent imaging of NSG mice (A) and quantification of bioluminescence signal intensity as fold change (B) on days 4 and 8 ( n = 5). (C) Bioluminescent imaging at week 2 showing reduced Kluc tumor size in mice with human CBX6 overexpression compared to controls ( n = 10). (D and E) Kaplan-Meier survival analysis demonstrating that overexpression of human CBX6 in U-251 MG cells (D) ( n = 5) and Kluc cells (E) ( n = 10) improved mouse survival. (F) Histological analysis of tumor invasion and microsatellite metastasis in CBX6-overexpressing tumors. “∗” Indicates p < 0.05.

Article Snippet: A pCMV6-entry plasmid containing Myc-DDK (same as Flag)-tagged human CBX6 cDNA (Cat#: RC204166) and a CBX6 Human shRNA Plasmid Kit (Cat#: TF314170) were purchased from OriGene.

Techniques: Over Expression, Imaging

Journal: Molecular Therapy Oncology

Article Title: CBX6 and CA9 as predictive indicators and therapeutic targets in GBM

doi: 10.1016/j.omton.2026.201159

Figure Lengend Snippet: CBX6 binds to the CA9 promoter (A) Genes related to tumor cell invasion, proliferation, or migration were selected based on RNA sequencing data from U-251 MG cells with CBX6 knockdown compared to negative controls. (B) qRT-PCR results show that CA9 expression is affected by CBX6 dysregulation in U-251 MG cells, with overexpression or shRNA-mediated knockdown of CBX6, using GAPDH as a reference gene. (C) Gene expression correlation analysis from CGGA reveals an inverse relationship between CBX6 and CA9 expression patterns ( http://www.cgga.org.cn ). (D) Western blot and qRT-PCR data demonstrate changes in CBX6 and CA9 expression in U-251 MG and PBT030 cells under normoxic (N) and hypoxic (H) conditions for 24 and 48 h, using 28S as a reference gene. (E) ChIP assay results from CBX6-overexpressing U-251 MG cells show detection of the CA9 promoter sequence using two primer sets in CBX6/Flag pull-down products compared to a negative IgG control via qRT-PCR. VC, vector control; OE, CBX6 overexpression; KD, CBX6 knockdown. “∗” Indicates p < 0.05.

Article Snippet: A pCMV6-entry plasmid containing Myc-DDK (same as Flag)-tagged human CBX6 cDNA (Cat#: RC204166) and a CBX6 Human shRNA Plasmid Kit (Cat#: TF314170) were purchased from OriGene.

Techniques: Migration, RNA Sequencing, Knockdown, Quantitative RT-PCR, Expressing, Over Expression, shRNA, Gene Expression, Western Blot, Sequencing, Control, Plasmid Preparation