Journal: Journal of Biomedical Science

Article Title: CXCL5 neutralization mitigates cancer cachexia by disrupting CAF-cancer cell crosstalk

doi: 10.1186/s12929-025-01192-0

Figure Lengend Snippet: A Schematic of the protocol to compare the effects of conditioned media (CM) from CCD-18Co human normal colon tissue fibroblasts (NF CM), primary human colon cancer-associated fibroblasts (CAF CM), and human HCT 116 colon cancer cell-stimulated CAF CM (CAF CCM) on C2C12 myotube wasting. B Myosin heavy chain 2 (MYH2) immunostaining of C2C12 myotubes cultured in normal myotube differentiation media (DM) and treated with NF CM, CAF CM, and CAF CCM for 72 h (scale bar = 150 μm). C Calculation of mean myotube diameter. D Western blot analysis of MYH2 and atrogin-1 expression in the treated myotubes. E Densitometry of MYH2 and atrogin-1 expression relative to GAPDH. F Cytokine array analysis CAF CM, CAF CCM, and HCT 116 cancer cell CM. Red boxes and numbers indicate upregulated cytokines in the CAF CCM compared to CAF CM. G Quantification of the fold-change for the upregulated cytokines. H ELISA-based detection of CXCL5 in NF CM, CAF CM, CAF CCM, and HCT 116 cancer cell CM. The CAF CM and CCM values are the mean obtained from three sources of CAF: two derived from patients and one provided commercially. All experiments were conducted 3 times independently and the values are indicated as the mean ± SD. For C and E : * = p < 0.05 and *** = p < 0.001 compared to DM. For H : ** = p < 0.01 compared to CAF CM

Article Snippet: To inhibit CXCL5, 120 μg/kg of neutralizing antibody (R&D Systems, MAB-254, clone 33160, MN, USA) and IgG1 monoclonal antibody (R&D Systems, MAB-002, clone 11711, MN, USA) were treated for 3 d prior to transplantation via intraperitoneal (IP) injection.

Techniques: Immunostaining, Cell Culture, Western Blot, Expressing, Enzyme-linked Immunosorbent Assay, Derivative Assay

Journal: Journal of Biomedical Science

Article Title: CXCL5 neutralization mitigates cancer cachexia by disrupting CAF-cancer cell crosstalk

doi: 10.1186/s12929-025-01192-0

Figure Lengend Snippet: A Myosin heavy chain 2 (MYH2) immunostaining of C2C12 myotubes cultured in normal differentiation media (DM), DM plus 10 ng/mL CXCL5, or DM plus 20 ng/mL CXCL5 for 72 h. (scale bar = 150 μm) B Calculation of mean myotube diameter. C Western blot analysis of atrogin-1 and MuRF-1 expression in the treated myotubes. D Densitometry of atrogin-1 and MuRF-1 expression relative to GAPDH. E Western blot analysis of total extracellular signal-regulated kinase 1/2 (ERK1/2) and phosphorylated ERK1/2 in myotubes treated with CAF CCM or 20 ng/mL CXCL5 for 0.5 h and 2 h. F Densitometry of phosphorylated ERK1/2 relative to ERK1/2. G MHY2 immunostaining of C2C12 myotubes cultured for 72 h as follows: (1) Normal differentiation media (DM), (2) DM:CAF CCM (1:1) plus vehicle (0.1% DMSO), (3) DM:CAF CCM (1:1) plus 0.5 μg/mL CXCL5 neutralizing antibody, and (4) DM:CAF CCM (1:1) plus 22 nM SB225005 for 96 h (scale bar = 150 μm). H Calculation of mean myotube diameter. All experiments were conducted 3 times independently and the values are indicated as the mean ± SD. For B , D , F and H : * = p < 0.05, ** = p < 0.01 and *** = p < 0.001 indicate significantly increased compared to untreated. For H : # = p < 0.05 and ## = p < 0.01 indicates significantly increased compared to the DM:CCM (1:1) group

Article Snippet: To inhibit CXCL5, 120 μg/kg of neutralizing antibody (R&D Systems, MAB-254, clone 33160, MN, USA) and IgG1 monoclonal antibody (R&D Systems, MAB-002, clone 11711, MN, USA) were treated for 3 d prior to transplantation via intraperitoneal (IP) injection.

Techniques: Immunostaining, Cell Culture, Western Blot, Expressing

Journal: Journal of Biomedical Science

Article Title: CXCL5 neutralization mitigates cancer cachexia by disrupting CAF-cancer cell crosstalk

doi: 10.1186/s12929-025-01192-0

Figure Lengend Snippet: A IVIS imaging of NOD-SCID mice 3 weeks post-xenograft with 1 × 10 6 human HCT 116 luc2 colon cancer cells, or 1 × 10 6 HCT 116 luc2 cancer cells plus 2 × 10 6 human colon CAF. B Mean total flux detected from the tumor at the end point (ns = not significant). C Dissected tumors and tumor mass at the 3 week endpoint. D Tumor free body weight (B.W) at the 3 week end point. E Representative images of H&E stained gastrocnemius muscle (scale bar = 50 μm). F Calculation of the myofiber cross sectional area. G qPCR analysis of CXCL1, 2, 3, 5, 6, 7, and 8 (known CXCR2 ligands) and IL-6 in the dissected tumor. H IVIS imaging of NOD-SCID mice at 3 weeks post-xenograft with human HCT 116 luc2 cancer cells plus human colon CAF, with or without CXCL5 neutralization (CXCL5 Neu Ab). I Mean total flux detected from the tumor at the 3 week end point. J Dissected tumors and tumor mass. K Tumor free body weight (B.W) at the 3 week end point. L Hanging tolerance in the treated mice. M Tibialis anterior (TA) muscle mass. N Representative H&E staining of the TA muscle (scale bar = 100 µm). O Calculation of the myofiber cross sectional area. P CXCR2 immunostaining of the TA muscle (scale bar = 100 µm). Q Proportion of CXCR2 positive myofibers. 5 mice per group were used for the experiments and the values are indicated as the mean ± SEM. For D and F * = p < 0.05 and ** = p < 0.01 indicate significantly decreased compared to PBS treated mice. For G , * = p < 0.05 indicates significantly increased compared to HCT 116 injected mice. For I – Q 5–7 mice per group were used for the experiments and the values are indicated as the mean ± SEM. * = p < 0.05 and ** = p < 0.01 indicate significantly decreased compared to vehicle alone. # = p < 0.05 and ## = p < 0.01 indicate significantly increased compared to HCT 116 plus CAF

Article Snippet: To inhibit CXCL5, 120 μg/kg of neutralizing antibody (R&D Systems, MAB-254, clone 33160, MN, USA) and IgG1 monoclonal antibody (R&D Systems, MAB-002, clone 11711, MN, USA) were treated for 3 d prior to transplantation via intraperitoneal (IP) injection.

Techniques: Imaging, Staining, Neutralization, Immunostaining, Injection

Journal: Journal of Biomedical Science

Article Title: CXCL5 neutralization mitigates cancer cachexia by disrupting CAF-cancer cell crosstalk

doi: 10.1186/s12929-025-01192-0

Figure Lengend Snippet: A Heat map of genes showing differential expression between the tibialis anterior (TA) muscles of NOD-SCID mice treated as follows: (1) No xenograft (Normal); (2) Xenograft with HCT 116 human cancer cells plus CAF (HCT 116 + CAF); (3) Xenograft with HCT 116 human cancer cells plus CAF, followed by treatment with a CXCL5 neutralizing antibody (Neu Ab). B KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway analysis for the HCT-116 + CAF compared with Neu Ab treatment groups. C Gene ontology (GO) functional analysis for the HCT-116 + CAF group compared with Neu Ab treatment group. D Western blot analysis of PI3K-AKT and ERK1/2 phosphorylation in the dissected TA muscle from vehicle alone, HCT 116 + CAF, and Neu Ab-treated groups. E Densitometry of PI3K-AKT and ERK1/2 phosphorylation relative to PI3K-AKT and ERK1/2. F Heat map for genes showing differential expression between (1) HCT 116 + CAF xenograft compared to vehicle alone (HCT-116 + CAF/Normal), and (2) HCT 116 + CAF xenograft treated with a CXCL5 neutralizing antibody compared to HCT 116 + CAF xenograft alone (Neu Ab/HCT 116 + CAF). G qPCR analysis of the differentially expressed genes, in addition to atrogin-1 and MuRF-1 in the TA muscles. H Western blot analysis of PI3K-AKT phosphorylation in CAF CCM treated myotubes. I Densitometry of PI3K-AKT phosphorylation relative to PI3K-AKT. J Protein synthesis rate as determined by western blot analysis of puromycin incorporation (SUnSET assay). K Densitometry of puromycin incorporation normalized by GAPDH expression. For E and G : 4–5 mice per group were used for the experiments and the values are indicated as the mean ± SEM. * = p < 0.05, ** = p < 0.01 and *** = p < 0.001 indicate significantly increased or decreased compared to vehicle alone-injected mice (Normal). # = p < 0.05, ## = p < 0.01 and ### = p < 0.001 indicate significantly decreased compared to HCT 116 + CAF. For I and K : All values are indicated as the mean ± SD. * = p < 0.05 and *** = p < 0.001 indicate significantly decreased compared to vehicle alone. # = p < 0.05 indicates significantly increased compared to CAF CCM treatment

Article Snippet: To inhibit CXCL5, 120 μg/kg of neutralizing antibody (R&D Systems, MAB-254, clone 33160, MN, USA) and IgG1 monoclonal antibody (R&D Systems, MAB-002, clone 11711, MN, USA) were treated for 3 d prior to transplantation via intraperitoneal (IP) injection.

Techniques: Quantitative Proteomics, Muscles, Functional Assay, Western Blot, Phospho-proteomics, Expressing, Injection

Journal: Journal of Biomedical Science

Article Title: CXCL5 neutralization mitigates cancer cachexia by disrupting CAF-cancer cell crosstalk

doi: 10.1186/s12929-025-01192-0

Figure Lengend Snippet: A Schematic of the protocol to investigate CXCL5 neutralization in a model of cytokine-induced skeletal muscle wasting. C57BL6/J mice were treated with 40 ng/kg CXCL5 and 80 ng/kg IL-6 for 4 weeks with or without 120 μg/kg CXCL5 neutralizing antibody. 120 μg/kg IgG1 was used as control. B Body weight at the end point of experiment. C Quadriceps muscle mass. D Gastrocnemius muscle mass. E Tibialis anterior (TA) muscle mass. F Laminin staining of the TA muscle (scale bar = 150 µm). G TA myofiber cross sectional area. H CXCR2 staining of the TA muscle (scale bar = 150 µm). I The proportion of CXCR2 positive fibers in TA muscle. J Western blot analysis of atrogin-1, MuRF-1 and CXCR2 expression in the TA muscle. K Densitometry of atrogin-1, MuRF-1 and CXCR2 expression normalized by the expression of GAPDH. For B – I, 7 mice per group were used for the experiments and for J – K, 4 mice per group were used for the experiments and the analysis was carried out two times. The values were indicated as the mean ± SEM. * = p < 0.05 and ** = p < 0.01 indicate significantly increased or decreased compared to IgG1 control. # = p < 0.05 and ## = p < 0.01 indicate significantly decreased compared to CXCL5 + IL-6 + IgG1

Article Snippet: To inhibit CXCL5, 120 μg/kg of neutralizing antibody (R&D Systems, MAB-254, clone 33160, MN, USA) and IgG1 monoclonal antibody (R&D Systems, MAB-002, clone 11711, MN, USA) were treated for 3 d prior to transplantation via intraperitoneal (IP) injection.

Techniques: Neutralization, Control, Staining, Western Blot, Expressing

Journal: Journal of Biomedical Science

Article Title: CXCL5 neutralization mitigates cancer cachexia by disrupting CAF-cancer cell crosstalk

doi: 10.1186/s12929-025-01192-0

Figure Lengend Snippet: A Representative MYH2-stained images of human donor myotubes cultured as follows: (1) Differentiation media (DM) for 72 h; (2) Treatment with human HCT 116 colon cancer cell-stimulated CAF CM (CAF CCM) for 72 h; (3) Treatment with CAF CCM and CXCL5 neutralizing antibody (CAF CCM + Neu Ab) for 72 h (scale bar = 100 μm). B Calculation of mean myotube diameter. C Western blot analysis of MYH2 and atrogin-1 expression. D Densitometry of MYH2 and atrogin-1 expression relative to α–tubulin. E Immunohistochemical analysis of CXCL5 and vimentin expression in tumor-stromal and normal tissues obtained from a colon carcinoma patient. White arrows indicate overlapping CXCL5 and vimentin immunostaining. Quantification of CXCL5 fluorescence in the tumor-stromal tissues is also shown (** = p < 0.01 compared to normal). All experiments were conducted 3 times independently and the values were indicated as the mean ± SD. For B and D : * = p < 0.05 indicate significantly increased or decreased compared to DM alone. # = p < 0.05 and ## = p < 0.01 indicate significantly increased or decreased compared to CAF CCM. G Working model of the role of CAF in cancer cachexia progression. Molecular crosstalk between cancer cells and CAF in the tumor microenvironment induces the secretion of chemokine CXCL5 by CAF. CXCL5 activates muscle atrophy signaling, as shown by decreased PI3K-AKT and ERK phosphorylation and upregulation of the key atrogenes, atrogin-1 and MuRF-1, causing the skeletal muscle loss observed in cancer cachexia

Article Snippet: To inhibit CXCL5, 120 μg/kg of neutralizing antibody (R&D Systems, MAB-254, clone 33160, MN, USA) and IgG1 monoclonal antibody (R&D Systems, MAB-002, clone 11711, MN, USA) were treated for 3 d prior to transplantation via intraperitoneal (IP) injection.

Techniques: Staining, Cell Culture, Western Blot, Expressing, Immunohistochemical staining, Immunostaining, Fluorescence, Phospho-proteomics

Journal: Scientific reports

Article Title: ADSCs stimulated by resistin promote breast cancer cell malignancy via CXCL5 in a breast cancer coculture model.

doi: 10.1038/s41598-022-19290-6

Figure Lengend Snippet: Figure 3. Secreted CXCL5 in the conditioned medium of the co-culture model with resistin-stimulated ADSCs promoted malignant behaviors of breast cancer cells. The isolated ADSCs were treated with resistin at 0 and 100 ng/ml (R0 and R100, respectively) for 48 h, followed by co-culture with MDA-MB-231 cells in the transwell model for another 72 h before the analyses in (A,B,F,G). (A) The conditioned medium from the co-culture of R-ADSCs (R100) or control ADSCs (R0) with MDA-MB-231 cells was collected and analyzed by cytokine/ chemokine proteome array. A total of 102 proteins were detected with duplicates for each protein. The position of CXCL5 on the array was highlighted. (B) Secreted protein level of CXCL5 in the conditioned medium from the co-culture of R-ADSCs (R100) or control ADSCs (R0) with MDA-MB-231 cells was analyzed by ELISA. (C) MDA-MB-231 cells treated with recombinant CXCL5 (0, 20, and 40 ng/ml) for 48 h were collected and evaluated by cell migration assay. (D) MDA-MB-231 cells treated with recombinant CXCL5 (0, 20, and 40 ng/ml) for 48 h were collected and evaluated by cell invasion assay. (E) MDA-MB-231 cells treated with recombinant CXCL5 (0, 20, and 40 ng/ml) for 48 h were collected and analyzed for the protein expression of mesenchymal marker Slug and cancer stemness marker Oct4 by Western blot. The original blot images in (A) and (E) were available in Supplementary Information. (F,G) CXCL5 neutralizing antibody was added ( +) or omitted (–) during the co-culture of R-ADSCs (R100) or control ADSCs (R0) with MDA-MB-231 cells. After the co-culture, MDA-MB-231 cells were collected and evaluated by cell migration assay in (F) and cell invasion assay in (G). Data were obtained from three independent experiments and presented as mean ± SEM. Statistical difference was determined by t-test comparing R100 group versus their corresponding R0 group as control, or comparing recombinant CXCL5 treatment group (20 and 40 ng/ml) versus recombinant CXCL5 control group (0 ng/ml). *p < 0.05; **p < 0.01; ***p < 0.001.

Article Snippet: The primary antibodies used in this study for IHC are listed as follows: rabbit polyclonal antibody against human Slug (GeneTex, Hsinchu, Taiwan); goat polyclonal antibody against human CXCL5 (R&D Systems, Minneapolis, MN, USA); mouse monoclonal antibody against human resistin (clone C-10; Santa Cruz Biotechnology, Dallas, TX, USA); rabbit monoclonal antibody against human phosphorylated ERK1/2 at Thr202/Tyr204 (clone D13.14.4E; Cell Signaling Technology, Danvers, MA, USA).

Techniques: Co-Culture Assay, Isolation, Control, Enzyme-linked Immunosorbent Assay, Recombinant, Cell Migration Assay, Invasion Assay, Expressing, Marker, Western Blot

Journal: Scientific reports

Article Title: ADSCs stimulated by resistin promote breast cancer cell malignancy via CXCL5 in a breast cancer coculture model.

doi: 10.1038/s41598-022-19290-6

Figure Lengend Snippet: Figure 5. Enhanced breast tumor growth and protein expression of CXCL5, Slug, and ERK phosphorylation in mice via xenograft of breast cancer cells after co-culture with resistin-stimulated ADSCs. The isolated ADSCs were treated with resistin at 0 and 50 ng/ml (denoted as R0 and R50, respectively) for 48 h, followed by co-culture with MDA-MB-231 cells in the transwell model for another 72 h. After the co-culture, MDA-MB-231 cells were collected and injected into the fourth mammary fat pads of female NOD/SCID mice for the following analyses. (A) The tumor volume, calculated by (width2 × length)/2, was measured weekly after palpable tumor mass was formed. (B) Upon sacrifice of the mice on week eight, the weight of individual tumor mass was measured. (C) The body weight of the mice was measured weekly. (D) The tumor mass was collected after sacrifice of the mice on week eight, and analyzed for CXCL5, Slug, and phospho-ERK1/2 protein expression by immunohistochemistry (IHC). The quantitative IHC scores were manually evaluated and calculated by multiplying the categorized percentage of stained cells (0, 0–24%; 1, 25‑49%; 2, 50‑74%; 3, 75‑100%) by the categorized intensity of staining (0, negative; 1, weak; 2, moderate; 3, strong). Data were obtained from three to six mice in each group and presented as mean ± SEM or box plots. Statistical difference was determined by t-test comparing R50 group versus R0 group as control. *p < 0.05; **p < 0.01; ***p < 0.001.

Article Snippet: The primary antibodies used in this study for IHC are listed as follows: rabbit polyclonal antibody against human Slug (GeneTex, Hsinchu, Taiwan); goat polyclonal antibody against human CXCL5 (R&D Systems, Minneapolis, MN, USA); mouse monoclonal antibody against human resistin (clone C-10; Santa Cruz Biotechnology, Dallas, TX, USA); rabbit monoclonal antibody against human phosphorylated ERK1/2 at Thr202/Tyr204 (clone D13.14.4E; Cell Signaling Technology, Danvers, MA, USA).

Techniques: Expressing, Phospho-proteomics, Co-Culture Assay, Isolation, Injection, Immunohistochemistry, Staining, Control

Journal: Scientific reports

Article Title: ADSCs stimulated by resistin promote breast cancer cell malignancy via CXCL5 in a breast cancer coculture model.

doi: 10.1038/s41598-022-19290-6

Figure Lengend Snippet: Figure 6. Correlation analysis for the protein expression of resistin, CXCL5, and ERK phosphorylation in the tumor and serum specimens from breast cancer patients. (A) Representative images of immunohistochemistry (IHC) staining for resistin, CXCL5, and phospho-ERK1/2 expression in breast tumor sections from breast cancer patients. (B–D) The quantitative IHC scores were evaluated by HistoQuest software, followed by Pearson correlation (r) analysis between resistin and CXCL5 in (B) (n = 96), resistin and phospho-ERK1/2 in (C) (n = 45), and CXCL5 and phospho-ERK1/2 in (D) (n = 45). (E) The serum levels of resistin and CXCL5 in breast cancer patients were determined by ELISA, followed by Pearson correlation (r) analysis (n = 120). The mean ± SD of resistin and CXCL5 was 31.4 ± 15.4 ng/ml and 707.9 ± 293.4 pg/ml, respectively. (F) Schematic summary of the current study. Our preclinical and clinical data together suggest that CXCL5 may be secreted by resistin-stimulated ADSCs in the breast tumor microenvironment, promoting breast cancer cell malignancy via the participation of ERK pathway and epithelial-to-mesenchymal transition. The schematic summary in (F) was produced using the illustration elements from Servier Medical Art (https://smart.servier.com), which is in compliance with the terms of the Creative Commons Attribution 3.0 Unported License (https://creativeco mmons.org/licenses/by/3.0/).

Article Snippet: The primary antibodies used in this study for IHC are listed as follows: rabbit polyclonal antibody against human Slug (GeneTex, Hsinchu, Taiwan); goat polyclonal antibody against human CXCL5 (R&D Systems, Minneapolis, MN, USA); mouse monoclonal antibody against human resistin (clone C-10; Santa Cruz Biotechnology, Dallas, TX, USA); rabbit monoclonal antibody against human phosphorylated ERK1/2 at Thr202/Tyr204 (clone D13.14.4E; Cell Signaling Technology, Danvers, MA, USA).

Techniques: Expressing, Phospho-proteomics, Immunohistochemistry, Software, Enzyme-linked Immunosorbent Assay, Produced

Journal: Journal for Immunotherapy of Cancer

Article Title: METTL3 promotes an immunosuppressive microenvironment in bladder cancer via m6A-dependent CXCL5/CCL5 regulation

doi: 10.1136/jitc-2024-011108

Figure Lengend Snippet: METTL3 regulates the expression and secretion of CXCL5 and CCL5 in bladder cancer. (A) Schematic of the transcriptome sequencing workflow following METTL3 knockdown in MB49 cells. (B) Heatmap of differentially expressed genes after METTL3 knockdown (criteria for differential genes: p<0.05, fold change >1.5 or <0.67. (C) Volcano plot of differentially expressed genes after METTL3 knockdown. (D) Network diagram of GO enrichment analysis of differentially expressed genes. (E) Chemokines with differential expression following METTL3 silencing. (F–G) Reverse Transcription Quantitative Polymerase Chain Reaction (RT-qPCR) analysis of CXCL5 and CCL5 mRNA expression levels following METTL3 overexpression or knockdown in MB49 cells. (H) RT-qPCR analysis of CXCL5 and CCL5 mRNA expression levels in MB49 cells treated with DMSO or STM2457 for 72 hours. (I) ELISA of CXCL5 and CCL5 secretion levels in the culture supernatant of MB49 cell lines; ELISA of CXCL5 and CCL5 levels in (J) mouse tumor tissues and (K) in peripheral blood serum. RT-qPCR analysis of CXCL5 and CCL5 mRNA expression levels in (L) 5637 cells and (M) T24 cells treated with DMSO or STM2457 for 72 hours. *p<0.05; **p<0.01; ***p<0.001.

Article Snippet: Secretion levels of CXCL5 and CCL5 were measured using the Mouse CXCL5 ELISA Kit (Elabscience, #E-EL-M0471) and the Mouse RANTES ELISA Kit (Elabscience, #E-EL-M0009), respectively.

Techniques: Expressing, Sequencing, Knockdown, Quantitative Proteomics, Reverse Transcription, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Over Expression, Enzyme-linked Immunosorbent Assay

Journal: Journal for Immunotherapy of Cancer

Article Title: METTL3 promotes an immunosuppressive microenvironment in bladder cancer via m6A-dependent CXCL5/CCL5 regulation

doi: 10.1136/jitc-2024-011108

Figure Lengend Snippet: METTL3 regulates CXCL5 expression through N6-methyladenosine (m6A) modification, thereby promoting myeloid-derived suppressor cell (MDSC) chemotaxis. (A) Schematic diagram of the workflow used for Methylated RNA Immunoprecipitation (MeRIP) sequencing and data analysis in METTL3-knockdown MB49 stable cell lines. (B) Bar chart showing the number of m6A modification sites identified in MeRIP sequencing results. (C) Venn diagram of downstream target gene screening for METTL3, intersecting chemokines significantly altered in RNA sequencing with those showing significant downregulation in m6A modification levels in m6A sequencing. (D) m6A peak map of CXCL5 mRNA modification sites. (E) Bar chart of MeRIP-qPCR results showing the m6A modification level of CXCL5 mRNA in MB49 cells after METTL3 knockdown. (F) RNA Immunoprecipitation (RIP) assay detecting the interaction between METTL3 and CXCL5 mRNA. (G) RNA degradation assay showing CXCL5 mRNA stability after silencing METTL3. (H) RNA degradation assay showing CXCL5 mRNA stability after treatment with METTL3 inhibitor STM2457 (2 µg/mL, 72 hours) in MB49 cells. (I) Reverse Transcription Quantitative Polymerase Chain Reaction (RT-qPCR) analysis of IGF2BP1 and CXCL5 mRNA expression levels in MB49 cells after silencing IGF2BP1. (J) RT-qPCR analysis of IGF2BP2 and CXCL5 mRNA expression levels in MB49 cells after silencing IGF2BP2. (K) RT-qPCR analysis of METTL3, IGF2BP1, and CXCL5 mRNA expression levels in MB49 cells after overexpression of METTL3 and/or silencing of IGF2BP1. (L) Schematic of the animal experiment. (M) Images of bladder cancer tumors in mice. (N) Growth curves of bladder cancer tumors in mice. (O) Tumor weights of bladder cancer tumors in mice. ns, no significance. *p<0.05; **p<0.01; ***p<0.001.

Article Snippet: Secretion levels of CXCL5 and CCL5 were measured using the Mouse CXCL5 ELISA Kit (Elabscience, #E-EL-M0471) and the Mouse RANTES ELISA Kit (Elabscience, #E-EL-M0009), respectively.

Techniques: Expressing, Modification, Derivative Assay, Chemotaxis Assay, Methylation, RNA Immunoprecipitation, Sequencing, Knockdown, Stable Transfection, RNA Sequencing, Degradation Assay, Reverse Transcription, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Over Expression