anti lig3  (R&D Systems)

Journal: bioRxiv

Article Title: A Novel miR-4745 -KLC2 Axis Regulates Cancer Stem Cell Traits in Colorectal Cancer

doi: 10.64898/2026.01.07.697660

Figure Lengend Snippet: (a) Quantification of the CD44v6-APC-positive population in CPP1 (n=7) and CTC44 (n=5) cells transfected with miR-4745-3p ( miR4745 ) or miRVana microRNA Mimic Negative Control #1 (miCTRL) mimics (right panel). Percentage of CD44v6-APC positive CPP1 cells is indicated in inset boxes for a representative experiment (left panel). (b) Representative images of tumorspheres (upper panel) and the percentage (lower panel) of sphere-forming cells in patient-derived CPP1 and circulating tumor (CTC44) colon cancer cells transfected with miR-4745-3p ( miR4745 ) or miCTRL mimics. Data from individual replicates (n=15 per experiment) are presented, along with the mean ± SEM from six independent experiments. (c) Percentage of sphere-forming cells in patient-derived CPP1 colon cancer cells transfected with miR4745 or negative control (miCTRL) mimics and, maintained as first-generation colonospheres (S1) or after several passages (S2, S3). Data from individual replicates (n=12 per experiment) are presented, along with the mean ± SEM from three independent experiments. (d) Percentage of surviving cancer stem cells (Aldefluor-positive) measured 48 hours following treatment with specified concentrations of FIRI (1X = 50 µM 5-FU + 500 nM SN38) in vitro . Sorted CPP1 Aldefluor-positive cells were first transfected with 50 nM miR-4745-3p ( miR-4745 ) or miRVana microRNA Mimic Negative Control #1 (miCTRL) mimics, 24 hours prior to FIRI treatment. Data are expressed as mean ± SEM (n = 3). (e) Percentage of Aldefluor-positive cells (% ALDH + cells) in CPP1 cells transfected with miR-4745 or miCTRL mimics and then exposed for 72 hours to chemotherapy (Firi=5µM 5-FU + 50nM SN38). Data are expressed as mean ± SEM (n=6). *, p<0.05; **, p<0.005, ***, p < 0.001, Mann Whitney test.

Article Snippet: CD44v6-APC (clone REA706, 130-111-238; Macs Miltenyi) antibody was incubated with 100 000 cells in PBS containing 5% FBS for 20 min at 4°C.

Techniques: Transfection, Negative Control, Derivative Assay, In Vitro, MANN-WHITNEY

Journal: Cancer Research

Article Title: MEN1 Promotes Ferroptosis by Disrupting CD44 Alternative Splicing to Suppress Lung Cancer

doi: 10.1158/0008-5472.CAN-25-0021

Figure Lengend Snippet: MEN1 regulation of ferroptosis in lung cancer cells depends on CD44 variant isoforms. A, Heatmap of the hyperactivated genes in KMS mice vs. KS mice. B, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis of hyperactivated genes in KMS vs. KS mice. C, Gene set enrichment analysis of gene ontology terms comparing KS and KMS mice. ES, enrichment score; NES, normalized enrichment score. D, Kaplan–Meier survival curves of patients with LUAD (The Cancer Genome Atlas) with high/low CD44 PSI or mRNA expression. E and F, H&E staining and fluorescence images ( E ), and quantification ( F ) of CD44v3/CD44v6 in lung cancer tissues (metastatic, n = 41; nonmetastatic, n = 74). Scale bar, 50 μm. G and H, Representative fluorescence images ( G ) and quantification ( H ) for CD44v3/CD44v6 in M-NSG mice xenografted with WT/KO NCI-H460 cells. Scale bar, 50 μm. I, Flow cytometry analysis of CD44v6 expression in WT/KO NCI-H460 cells (40 ng/mL PMA, 12 hours). J, Representative transwell images and quantification of invaded cells in WT/KO NCI-H460 cells treated with anti-CD44v6 or control IgG antibody. K, Immunoblotting analysis of indicated proteins in WT/KO NCI-H460 cells transfected with siV6#1/#2 or siNC for 48 hours. L, Cell viability of WT/KO NCI-H460 cells transfected with siV6 or siNC, followed by 48-hour treatment with erastin. OD, optical density. M and N, Lipid peroxidation and ROS levels in WT/KO NCI-H460 cells transfected with siV6 or siNC, followed by 24-hour treatment with erastin. *, P < 0.05; **, P < 0.01; ***, P < 0.001. a.u, arbitrary unit.

Article Snippet: siRNAs directed against CD44v6 , PAF1 , and negative control RNA (siNC) were constructed by Sangon Biotech.

Techniques: Variant Assay, Expressing, Staining, Fluorescence, Flow Cytometry, Control, Western Blot, Transfection

Journal: Cancer Research

Article Title: MEN1 Promotes Ferroptosis by Disrupting CD44 Alternative Splicing to Suppress Lung Cancer

doi: 10.1158/0008-5472.CAN-25-0021

Figure Lengend Snippet: MEN1 prevents CD44 variant generation by repressing RNA alternative splicing. A, RT-PCR and gel electrophoresis of CD44 variant exons (V) in lung tissues from WT, KS, and KMS mice. B–D, Representative fluorescence (CD44v3/CD44v6) and H&E staining images of lung tissues from WT ( B ), KS ( C ), and KMS ( D ) mice. E, Schematic of WT mouse normal lungs, KS/KMS lung tumors (T), and adjacent nontumor lungs (N). F and G, qPCR analysis of CD44 variant expression in N and T tissues from KS ( F ) and KMS ( G ) mice presented in E . H, Lung injury scoring of WT, KS, and KMS mice. I, qPCR analysis of CD44 pre-mRNAs and isoforms containing variable exon v6v7 in WT/KO NCI-H460 cells (100 μmol/L DRB). J, Schematic of primer pairs for detecting unspliced pre-mRNA and total mRNA. Arrows, primer positions. K and L, qPCR analysis of total (spliced) CD44 mRNA ( K ) and unspliced/total mRNA ( L ) ratio in PMA-treated WT/KO NCI-H460 cells. M, Schematics of luciferase splice reporter pETCatEBLucv6 and control pETLuc. Skipping of v6 exon creates a reading frame terminating before luciferase (stop). Inclusion of v6 exon products a luciferase fusion protein reading frame terminating after luciferase. Black arrows, RSV LTR promoter; black boxes, insulin exon 2 and 3 sequences; gray boxes, SV40 poly(A) signal. N and O, Luciferase activity in WT, KO, and r MEN1 NCI-H460 cells transfected with pETCatEBLucv6 ( N ) or pETLuc ( O ), followed by 6-hour treatment with PMA or DMSO. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ns, not significant.

Article Snippet: siRNAs directed against CD44v6 , PAF1 , and negative control RNA (siNC) were constructed by Sangon Biotech.

Techniques: Variant Assay, Alternative Splicing, Reverse Transcription Polymerase Chain Reaction, Nucleic Acid Electrophoresis, Fluorescence, Staining, Expressing, Luciferase, Control, Activity Assay, Transfection

Journal: Cancer Research

Article Title: MEN1 Promotes Ferroptosis by Disrupting CD44 Alternative Splicing to Suppress Lung Cancer

doi: 10.1158/0008-5472.CAN-25-0021

Figure Lengend Snippet: CD44 variant–GPX4 signaling is upregulated in lung cancers and associated with poor prognosis. A, qPCR analysis of CD44 variants and GPX4 expression in adjacent and tumor tissues from patients with lung cancer. B, Representative H&E and CD44v6 IF staining images of adjacent and tumor tissues from patients with lung cancer. Scale bar, 50 μm. C, Quantification of CD44v6 IF staining in adjacent and tumor tissues from patients with lung cancer presented in B ( n = 82 paired tumor and adjacent nontumor counterparts). D and E, Representative images ( D ) and quantification ( E ) of GPX4 fluorescence in normal nontumor lung tissues and stage I–IV lung cancer tissues; n = 36 (normal), 56 (I), 25 (II), 29 (III), and 5 (IV). F, Correlation analysis of CD44v6 or CD44v9 mRNA expression and GPX4 mRNA expression in lung cancer specimens. G, GPX4 expression in LUAD vs. adjacent nontumor samples from three Gene Expression Omnibus databases ( GSE46539 : 92 pairs; GSE40791 : 100 normal and 94 lung cancer). H, OS, disease-free survival (DFS), and relapse-free survival (RFS) analyses of patients with LUAD with high/low GPX4 expression from three PrognoScan datasets. I, Representative images and quantification of menin IHC staining in adjacent and tumor tissues from patients with lung cancer. J, OS analysis of patients with LUAD stratified by MEN1 expression and CD44 PSI. *, P < 0.05; **, P < 0.01; ***, P < 0.001. a.u, arbitrary unit.

Article Snippet: siRNAs directed against CD44v6 , PAF1 , and negative control RNA (siNC) were constructed by Sangon Biotech.

Techniques: Variant Assay, Expressing, Staining, Fluorescence, Gene Expression, Immunohistochemistry

Journal: Cancer Research

Article Title: MEN1 Promotes Ferroptosis by Disrupting CD44 Alternative Splicing to Suppress Lung Cancer

doi: 10.1158/0008-5472.CAN-25-0021

Figure Lengend Snippet: CD44v6 peptides suppress the growth and metastasis of preexisting lung cancers by activating ferroptosis. A, Representative lung images from KS and KMS mice treated with Ctrlpep, mv6pep, or erastin ( n = 8, 9, and 8 for Ctrlpep-, mv6pep-, and erastin-treated KS mice; n = 7 per treatment in KMS mice). B–E, Lung weight ( B ), tumor number ( C ), burden ( D ), and size ( E ) of KS and KMS mice. F, Representative images of H&E and Ki67 IHC staining of lung tissue sections from KS and KMS mice. Scale bars, 1 mm or 100 μm. G, Quantification of Ki67 IHC staining in lung tissues presented in F . H and I, Representative images ( H ) and quantification ( I ) for 4HNE fluorescence in lung tissue sections from KS and KMS mice. Scale bar, 50 μm. J and K, Iron ( J ) and LPO ( K ) levels in lungs from KS and KMS mice. L and M, Representative RNA FISH images ( L ) and quantification ( M ) of GPX4 probe numbers in lung tissues from KS and KMS mice. Scale bars, 500 or 20 μm. N, Kaplan–Meier survival analysis was performed on KS mice (2 months after tamoxifen) and KMS mice (1 week after tamoxifen), treated with Ctrlpep or mv6pep three times weekly. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ns, not significant.

Article Snippet: siRNAs directed against CD44v6 , PAF1 , and negative control RNA (siNC) were constructed by Sangon Biotech.

Techniques: Immunohistochemistry, Fluorescence

Journal: bioRxiv

Article Title: Suppression of Glucosylceramide Synthase Reverses Drug Resistance in Cancer Cells Harbor Homozygous p53 Mutants

doi: 10.1101/2025.11.02.686136

Figure Lengend Snippet: Cells were planted in 5% FBS EMEM medium containing 6 μM IRI alone or combined 4 μM Genz-161 for 6 days. A, Imaging flow cytometry analysis for colon CSCs (CD44v6 + /CD133 + ). BF, bright field. B, GCS inhibition decreased CSC population. *, p <0.001 compared to WiDr cells treated with vehicle; **, p <0.001 compared to WiDr cells treated with IRI alone. C, Representative CSC plots of cancer cells with various treatments.

Article Snippet: After treatments, suspended cells (10 6 cells/ml) were incubated with human CD44v6 Alexa Fluor ® 488-conjugated antibody (2F10; mouse IgG1; from R&D Systems, Minneapolis, MN, USA) and human CD133 APC-conjugated antibody (170411; mouse IgG2b; from R&D Systems) in 1% BSA-containing PBS at 4°C for 45 min. After washing, cells were resuspended in 1% BSA PBS (5 x 10 5 cells/150 μL) and analysed using an Amnis Imagestream Mark II Imagestream software, and the data were further analysed using the IDEAS v6.2 program.

Techniques: Imaging, Flow Cytometry, Inhibition

Journal: bioRxiv

Article Title: Suppression of Glucosylceramide Synthase Reverses Drug Resistance in Cancer Cells Harbor Homozygous p53 Mutants

doi: 10.1101/2025.11.02.686136

Figure Lengend Snippet: A, Tumor growth of mice treated with oxaliplatin. Mice-bearing tumors generated from WiDr or WiDr/UGCG - cells were treated with vehicle, oxaliplatin (Oxa 2 mg/kg, i.p , once every 6 days) and combination (Oxa 2 mg/kg, i.p, once every 6 days and Genz-161 4 mg/kg, i.p, once every 3 days) for 37 days. *, p <0.01 compared with WiDr tumors treated with vehicle or Oxa. B, Tumor growth of mice treated with irenotecan. Mice-bearing tumors generated from WiDr cells were treated with vehicle, irenotecan (IRI 6 mg/kg, i.p , once every 6 days) and combination (IRI 6 mg/kg i.p , once every 6 days and Genz-161 4 mg/kg, i.p, once every 3 days) for 30 days. *, p <0.01 compared with tumors treated with vehicle; **, p<0.01 compared with tumors treated with IRI. C, H&E and immunofluorescence staining of tumors. Tumor sections were stained with H&E or fluorescent antibodies for CSC markers (CD44v6/CD133). Green, Alexa Fluor 448−CD44v6; red, APC-CD133; blue, DAPI nuclear counterstain. Images were magnified 200x, scale bar represents to 50 μm. D, GCS mRNA levels of tumors. *, p <0.01 compared with WiDr-tumors treated with vehicle or oxaliplatin. E, Tumor stem cell clusters in snRNA. Tumor-bearing mice were treated with Oxa (2 mg/kg, i.p, once every 6 days) for 37 days. *, p <0.001 compared with WiDr tumors treated with Oxa.

Article Snippet: After treatments, suspended cells (10 6 cells/ml) were incubated with human CD44v6 Alexa Fluor ® 488-conjugated antibody (2F10; mouse IgG1; from R&D Systems, Minneapolis, MN, USA) and human CD133 APC-conjugated antibody (170411; mouse IgG2b; from R&D Systems) in 1% BSA-containing PBS at 4°C for 45 min. After washing, cells were resuspended in 1% BSA PBS (5 x 10 5 cells/150 μL) and analysed using an Amnis Imagestream Mark II Imagestream software, and the data were further analysed using the IDEAS v6.2 program.

Techniques: Generated, Immunofluorescence, Staining