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Journal: bioRxiv
Article Title: Long-chain Polyunsaturated Fatty Acids Mitigate In Vitro Skeletal Muscle Wasting Induced by Colorectal Carcinoma Cells via a 15-LOX-dependent Pathway
doi: 10.64898/2026.05.22.726995
Figure Lengend Snippet: (A) : Day 3 post-differentiation C2C12 myotubes were treated with fresh DM, CT26 CM, DM containing 50 µM NDGA, or 10 µM BLX-3887 for 72 hours. C2C12 myotubes were fixed and stained for sarcomeric myosin (MF20c, green) and myogenin (F5Dc, red). Nuclei was counter stained by DAPI (blue). (B) : Myotube diameter for C2C12 myotubes was quantified as described in Methods. Groups labeled with different letters are significantly different from one another, while groups sharing a common letter are not significantly different.
Article Snippet: The murine skeletal muscle cell line C2C12 (ATCC, CRL-1772) and
Techniques: Staining, Labeling
Journal: bioRxiv
Article Title: Long-chain Polyunsaturated Fatty Acids Mitigate In Vitro Skeletal Muscle Wasting Induced by Colorectal Carcinoma Cells via a 15-LOX-dependent Pathway
doi: 10.64898/2026.05.22.726995
Figure Lengend Snippet: Day 3 post-differentiation C2C12 myotubes were treated with CT26 conditioned media (CM) with or without the pan-LOX inhibitor nordihydroguaiaretic acid (NDGA) (50 µM) or the 15-LOX specific inhibitor BLX3887 (10 µM). Then, individual LC-PUFAs including ARA, EPA, DHA, and DPA were spiked into the wells to reach a final concentration of 25 µM. After 72 hours, C2C12 myotubes were fixed in 4% paraformaldehyde (PFA) in preparation for immunocytochemistry analysis. (A) C2C12 myotubes were stained for sarcomeric myosin (MF20c, green) and myogenin (F5Dc, red). Nuclei was counter stained by DAPI (blue). (B-C) Myotube diameter for C2C12 myotubes with or without NDGA (B) or BLX-3887 (C) was quantified as described in Methods. Scale bar is 200 μm. *P < 0.05 for difference of PUFA treatments vs. vehicle, and #P < 0.05 for difference between respective LC-PUFA treatments with or without LOX inhibitors. (D-E) C2C12 myoblasts were differentiated for 3 days and then exposed to CT26 CM and individual PUFAs including ARA, EPA, DPA, and DHA for 3 hours. RNA was extracted from mature myotubes. mRNA expression fold change of Il6 (D) and Ccl2 (E) in response to CT26 CM and PUFA treatments was shown. *P < 0.05 for effects of PUFA treatments, and #P < 0.05 for difference between DM and CT26 CM.
Article Snippet: The murine skeletal muscle cell line C2C12 (ATCC, CRL-1772) and
Techniques: Concentration Assay, Immunocytochemistry, Staining, Expressing
Journal: bioRxiv
Article Title: Long-chain Polyunsaturated Fatty Acids Mitigate In Vitro Skeletal Muscle Wasting Induced by Colorectal Carcinoma Cells via a 15-LOX-dependent Pathway
doi: 10.64898/2026.05.22.726995
Figure Lengend Snippet: Co-cultures of mature C2C12 myotubes and CT26 carcinoma cells were treated with individual LC-PUFAs including ARA, EPA, DPA, and DHA (25 µM). After 72 hours, conditioned media from the lower compartments was collected and C2C12 myotubes were fixed and stained for sarcomeric myosin (MF20c, green) and myogenin (F5Dc, red). Nuclei was counter stained by DAPI (blue) (A) . Myotube diameter for C2C12 myotubes in the base plates was quantified as described in Methods (B) . Scale bar is 200 μm. Groups labeled with different letters are significantly different from one another, while groups sharing a common letter are not significantly different. (C- G) Conditioned media from C2C12-CT26 co-culture was analyzed by targeted Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS) to profile lipid metabolites in response to PUFA treatments. (C) : A heatmap of the top 50 most differentially regulated lipid mediators detected in conditioned culture media samples from C2C12 myotubes. (D-G) : Concentration of representative lipid mediator metabolites downstream of arachidonic acid (ARA) (e.g., PGE 2 , 15-HETE, and LXA 4 ) (D) , EPA (e.g., 15-HEPE, RvE1) (E), DPA (e.g., RvD5 n-3 DPA ) (F) , and DHA (e.g., 14-HDoHE, MaR2, 17-HDoHE, and RvD1) (G) . Bars show the mean ± SEM of media from 3 wells (biological replicates). P-values were determined by two-tailed unpaired t-tests. ∗p < 0.05, **p<0.01, ***p<0.001, and ****p<0.0001 vs. C2C12 myotubes without PUFAs or CT26 inserts.
Article Snippet: The murine skeletal muscle cell line C2C12 (ATCC, CRL-1772) and
Techniques: Staining, Labeling, Co-Culture Assay, Liquid Chromatography, Mass Spectrometry, Liquid Chromatography with Mass Spectroscopy, Concentration Assay, Two Tailed Test
Journal: bioRxiv
Article Title: Long-chain Polyunsaturated Fatty Acids Mitigate In Vitro Skeletal Muscle Wasting Induced by Colorectal Carcinoma Cells via a 15-LOX-dependent Pathway
doi: 10.64898/2026.05.22.726995
Figure Lengend Snippet: Concentration of lipid mediator metabolites downstream of arachidonic acid (ARA) (e.g., 5-HETE, 6-keto-PGF 1α , PGD 2 , and PGF 2α ) (A) , EPA (e.g., 18-HEPE, RvE2) (B), DPA (e.g., MaR1 n-3 DPA , PD1 n- 3 DPA ) (C) , and DHA (e.g., 4-HDoHE, 7-HDoHE, PD1, PDX, RvD2, RvD3, RvD5, RvD6, AT-RvD3, AT-RvD6) (D) Bars show the mean ± SEM of media from 3 wells (biological replicates). P-values were determined by two- tailed unpaired t-tests. ∗p < 0.05, **p<0.01, ***p<0.001, and ****p<0.0001 vs. C2C12 myotubes without PUFAs or CT26 inserts.
Article Snippet: The murine skeletal muscle cell line C2C12 (ATCC, CRL-1772) and
Techniques: Concentration Assay, Two Tailed Test
Journal: bioRxiv
Article Title: Long-chain Polyunsaturated Fatty Acids Mitigate In Vitro Skeletal Muscle Wasting Induced by Colorectal Carcinoma Cells via a 15-LOX-dependent Pathway
doi: 10.64898/2026.05.22.726995
Figure Lengend Snippet: (A) : Day 3 post-differentiation C2C12 myotubes were treated with 100 ng/mL TNFα (top) or CT26 CM (bottom) in the presence or absence of 100 nM of individual mature SPMs including RvD1, RvD5, MaR1, PD1, RvE1, LXA 4 , and RvD2 n-3 DPA for 72 hours. Resulting C2C12 myotubes were fixed and stained for sarcomeric myosin (MF20c, green) and myogenin (F5Dc, red). Nuclei was counter stained by DAPI (blue). (B- C) Myotube diameter for C2C12 myotubes receiving TNFα (B) or CT26 CM (C) was quantified as described in Methods. Scale bar is 200 μm. *P < 0.05 for difference compared to C2C12 myotubes receiving TNFα or CT26 CM alone. (D-E) C2C12 myoblasts were differentiated for 3 days and exposed to TNFα (100 ng/mL) with or without individual SPMs including RvD1, RvD5, MaR1, PD1, RvE1, LXA 4 , and RvD2 n-3 DPA (100nM) for 3 hours, after which RNA was extracted from mature myotubes. mRNA expression was determined by RT-qPCR and fold change of Il6 (D) and Fbxo32 (E) in response to TNFα exposure and SPM treatments was shown. *P < 0.05 for difference compared to C2C12 myotubes receiving TNFα.
Article Snippet: The murine skeletal muscle cell line C2C12 (ATCC, CRL-1772) and
Techniques: Staining, Expressing, Quantitative RT-PCR
Journal: Frontiers in Immunology
Article Title: STAT6 inhibition of M2 macrophages suppresses tumor growth by modulating the tumor microenvironment in colon cancer model
doi: 10.3389/fimmu.2026.1733991
Figure Lengend Snippet: TAMpep-IP suppresses tumor growth in the colon cancer model. (A) BALB/c mice were subcutaneously inoculated with CT26 colon carcinoma cells (3 × 10 5 cells per mouse). Starting on day 7 post-inoculation, TAMpep-IP (400 nmol/kg) was administered subcutaneously every three days for a total of seven doses. (B) Representative images of tumors excised at the experimental endpoint (day 25) showed visibly reduced tumor size in the TAMpep-IP–treated group compared to control. (C) Tumor volumes were measured every 3 days following tumor implantation. Mice treated with TAMpep-IP exhibited significantly reduced tumor growth relative to the control group (control: n = 6; TAMpep-IP: n = 6). (D) Tumor proliferation was evaluated by immunohistochemical staining of Ki-67 in tumor sections. Quantitative analysis showed a significantly lower proportion of Ki-67 + proliferating cells in TAMpep-IP–treated tumors. Representative immunohistochemistry images were acquired at ×100 magnification. Scale bar = 1000 μm. All data are presented as mean ± SEM. *p<0.05, ***p<0.001.
Article Snippet: The
Techniques: Control, Tumor Implantation, Immunohistochemical staining, Staining, Immunohistochemistry
Journal: Frontiers in Immunology
Article Title: STAT6 inhibition of M2 macrophages suppresses tumor growth by modulating the tumor microenvironment in colon cancer model
doi: 10.3389/fimmu.2026.1733991
Figure Lengend Snippet: TAMpep-IP reduces M2 macrophages in tumor tissues of colon cancer model. (A) Tumor-infiltrating immune cells were isolated from CT26 tumors in control and TAMpep-IP–treated mice. Flow cytometry was used to identify CD206 + F4/80 + macrophages within the CD45 + CD11b + population. TAMpep-IP significantly decreased the proportion of M2-like tumor-associated macrophages. (B) Quantitative RT-PCR analysis of tumor tissues revealed that TGF-β mRNA expression, a key M2-associated cytokine, was significantly reduced in TAMpep-IP–treated tumors compared to controls. (C) Western blot analysis of tumor showed a marked decrease in CD206 protein levels following TAMpep-IP, indicating effective suppression of M2 macrophage markers. (D) CD206 + macrophages were further visualized by immunohistochemical staining of tumor sections. ImageJ-based quantification confirmed a significant reduction in CD206 + area in TAMpep-IP–treated tumors. Representative immunohistochemistry images were acquired at ×100 magnification. Scale bar = 1000 μm. All data are presented as mean ± SEM. *p<0.05, **p<0.01, ***p<0.001.
Article Snippet: The
Techniques: Isolation, Control, Flow Cytometry, Quantitative RT-PCR, Expressing, Western Blot, Immunohistochemical staining, Staining, Immunohistochemistry
Journal: Frontiers in Immunology
Article Title: STAT6 inhibition of M2 macrophages suppresses tumor growth by modulating the tumor microenvironment in colon cancer model
doi: 10.3389/fimmu.2026.1733991
Figure Lengend Snippet: TAMpep-IP enhances inflammatory cytokine expression and activated CD8 + T cells in tumor tissues of colon cancer model. (A) Flow cytometry was used to evaluate CD8 + T cell function in CT26 tumor tissues from control and TAMpep-IP-treated mice. TAMpep-IP significantly increased the proportion of activated Granzyme B + CD8 + T cells, while reducing the frequency of exhausted Tim-3 + CD8 + T cells, indicating enhanced cytotoxic T cell activity. (B, C) Confocal immunofluorescence analysis was performed on tumor sections stained with DAPI (nuclei), anti-CD8 (green), anti-Granzyme B (red), and anti-PD-1 (red). Activated CD8 + T cells were identified by co-localization of CD8 and Granzyme B, whereas exhausted CD8 + T cells were identified by co-localization of CD8 and PD-1. Quantification revealed a significant increase in intertumoral CD8 + Granzyme B + T cells and a concomitant decrease in CD8 + PD-1 + exhausted T cells following TAMpep-IP. Representative confocal images were acquired using a 40× objective lens. Scale bar = 20 μm. (D) Quantitative RT-PCR analysis of CT26 tumor tissues showed significantly elevated mRNA levels of pro-inflammatory cytokines TNF-α, IL-1β, and IL-12 in TAMpep-IP–treated mice compared to controls, indicating induction of a pro-inflammatory tumor microenvironment. All data are presented as mean ± SEM. *p<0.05, **p<0.01, ***p<0.001, ****p < 0.0001.
Article Snippet: The
Techniques: Expressing, Flow Cytometry, Cell Function Assay, Control, Activity Assay, Immunofluorescence, Staining, Quantitative RT-PCR
Journal: Applied Microbiology and Biotechnology
Article Title: Characterization and evaluation of a novel polylactic acid-made bioreactor for large-scale adherent cell expansion
doi: 10.1007/s00253-026-13880-4
Figure Lengend Snippet: Attachment kinetics of CT-26 WT cells to CellScrew® mini surface. CT-26 WT cells were seeded into CellScrew® mini at a cell density of 1 × 10E4 cells/cm 2 and attachment was monitored by taking supernatant at indicated time points. ( A ) shows attached cells over time at 2 rpm rotational speed. ( B ) shows glucose (black square) and lactate concentration (black triangle) of supernatant over time. Values represent the mean ± SD of n = 3 biological replicates; in some cases, error bars are smaller than the symbols and therefore not visible
Article Snippet: The murine melanoma cell line B16-F10 (CRL-6475, RRID: CVCL_0159, C57BL/6 background, ATCC, Manassas, VA, USA) and the
Techniques: Concentration Assay
Journal: Applied Microbiology and Biotechnology
Article Title: Characterization and evaluation of a novel polylactic acid-made bioreactor for large-scale adherent cell expansion
doi: 10.1007/s00253-026-13880-4
Figure Lengend Snippet: Growth kinetics of CT-26 WT cells. CT-26 WT cells were cultivated in CellScrew® mini at 2 rpm rotational speed for 96 h. The seeding concentration for CT-26 WT cells was 1 × 10E4 cells/cm 2 . ( A ) shows the cell density [x 10E4] harvested per cm 2 over time. ( B ) shows glucose (black square) and lactate concentration (black triangle) in supernatant over time. Data are presented as mean (± SD) of n = 3 per time point; in some cases, error bars are smaller than the symbols and therefore not visible
Article Snippet: The murine melanoma cell line B16-F10 (CRL-6475, RRID: CVCL_0159, C57BL/6 background, ATCC, Manassas, VA, USA) and the
Techniques: Concentration Assay
Journal: Applied Microbiology and Biotechnology
Article Title: Characterization and evaluation of a novel polylactic acid-made bioreactor for large-scale adherent cell expansion
doi: 10.1007/s00253-026-13880-4
Figure Lengend Snippet: Reduction and replacement of dissociation reagent on PLA surface. Harvest efficiency [%] and cell viability [%] of B16-F10 ( A ) and CT-26 WT ( B ) cell lines after 96 h of culture on PLA surface in the CellScrew® mini. Cells were detached using 100% Accutase, 50% Accutase + 50% DPBS, or 100% Envzyme™ Super under identical conditions ( n = 1)
Article Snippet: The murine melanoma cell line B16-F10 (CRL-6475, RRID: CVCL_0159, C57BL/6 background, ATCC, Manassas, VA, USA) and the
Techniques:
Journal: Communications Biology
Article Title: The MYB-related transcription factor MYPOP acts as a selective regulator of cancer cell growth
doi: 10.1038/s42003-026-10272-2
Figure Lengend Snippet: a Protein expression of untreated, control mRNA and MYPOP mRNA-transfected CT-26 cells and B16-F10 cells at the indicated time points was analyzed by western blotting using anti-MYPOP and anti-GAPDH antibodies. b Cell counts of untreated, control mRNA and MYPOP mRNA-transfected CT-26 cells and B16-F10 cells at the indicated time points. Mean values ( n = 3) are shown as a line diagram + individual data points. Statistical significance was determined comparing Control and MYPOP cell counts at 54 h p.t. with p = 0.0650 for CT- 26 and with p = 0.0313 for B16-F10.
Article Snippet: The
Techniques: Expressing, Control, Transfection, Western Blot
Journal: bioRxiv
Article Title: Lung-targeted cytokine-coding RNA-lipoplexes induce T and NK cell-mediated anti-tumor immune response
doi: 10.64898/2026.05.06.723126
Figure Lengend Snippet: ( A-D ) Luc–encoding RNA formulated as RNA-LPX was administered i.v. to non-tumor bearing (A, B; n=3) or CT26 metastases-bearing (C, D) BALB/c mice (n=3-9). ( A ) BLI of total body, ( B ) normalized organ signal from explanted organs. Data were analyzed by ordinary one-way ANOVA and Tukey’s test for multiple comparisons. ( C ) Kinetics of BLI of the lung signal in metastases-bearing mice, significance was determined by mixed-effects analysis with Geisser-Greenhouse correction and Tukey’s test for multiple comparisons. ( D ) Luc RNA, luc protein and CD31 (PECAM-1) were detected via RNAscope and immunohistochemistry on consecutive sections 1, 6 or 24 hours post injection, scale bar: 100 µm, tumor tissue is indicated with dashed line. ( E ) Cytokine quantification in lung after the indicated time points. ( F ) Cytokine fold increase in lung normalized to spleen at 6 hours after injection. ( G ) FDG positron emission tomography (PET) imaging 24 h after cytokine RNA mix injection into naïve mice, ( H ) ex vivo measurement of FDG uptake. Significance was determined using a two-tailed t-test for unpaired samples.
Article Snippet:
Techniques: RNAscope, Immunohistochemistry, Injection, Positron Emission Tomography, Imaging, Ex Vivo, Two Tailed Test
Journal: bioRxiv
Article Title: Lung-targeted cytokine-coding RNA-lipoplexes induce T and NK cell-mediated anti-tumor immune response
doi: 10.64898/2026.05.06.723126
Figure Lengend Snippet: ( A ) Experimental design: BALB/c mice (n=15 per group) were injected i.v. with CT26 tumor cells; subsequently, cytokine RNA mix or irrelevant RNA was administered i.v. as RNA-LPX twice per week for a total of 8 injections from d3 to d27. Survivor mice from the cytokine RNA mix-treated group (n=5) compared to naïve BALB/c mice (n=10) were re-challenged with CT26 tumor cells. ( B ) Survival after the initial i.v. CT26 tumor cell inoculation. ( C ) Survival after re-challenge. Significance was determined via Mantel-Cox logrank. ( D-F ) BALB/c mice (n=5 per group) were injected i.v. with CT26 tumor cells, RNA-LPX was administered i.v. at d3, d6 and d10 p.t.i., mice were sacrificed at d11, lungs were collected and analyzed via flow cytometry. Significance for pairwise comparisons was determined by unpaired two-tailed t-test. Flow cytometric analysis of ( D ) tumor burden, ( E ) CD8 + T and NK cells and ( F ) CD4 + Foxp3 + CD25 + T reg . ( G ) Functional analysis: after tumor cell inoculation, mice were treated with RNA-LPX at d3, d6, d10 and d13 p.t.i., mice were sacrificed at d14 for an ex vivo stimulation assay with PMA/Ionomycin ( H ) qRT-PCR was done to determine fold-change expression of cytokine RNA mix-treated normalized to irrelevant RNA LPX-treated lung samples (n=5 per group indicated in rows) for the indicated cytokines and chemokines.
Article Snippet:
Techniques: Injection, Flow Cytometry, Two Tailed Test, Functional Assay, Ex Vivo, Quantitative RT-PCR, Expressing
Journal: bioRxiv
Article Title: Lung-targeted cytokine-coding RNA-lipoplexes induce T and NK cell-mediated anti-tumor immune response
doi: 10.64898/2026.05.06.723126
Figure Lengend Snippet: Experimental design: BALB/c mice (n=5 per group) were injected i.v. with CT26 tumor cells; cytokine RNA mix or irrelevant RNA was administered i.v. at d3, d6 and d10 post tumor cell injection, mice were sacrificed at d11, lungs were collected and pooled at same ratios before sorting of CD45 + cells. CD45 + cells were subjected to scRNAseq. ( A,B ) Uniform manifold approximation and projection (UMAP) of 22 assigned clusters ( A ) and cell type frequencies ( B ) of CD45 + cells isolated from the lungs of cytokine RNA mix-treated versus irrelevant RNA-treated mice. ( C ) Effector function visualized as bubble plot. ( D ) Selected differentially expressed genes in cytokine RNA mix-treated versus irrelevant RNA-treated samples are shown as average log2 fold change. Note only significantly expressed values (p ≤ 0.05) are shown in colouring (blue = downregulated, red = upregulated), non-significant values are set to “0”/white.
Article Snippet:
Techniques: Injection, Isolation
Journal: bioRxiv
Article Title: Lung-targeted cytokine-coding RNA-lipoplexes induce T and NK cell-mediated anti-tumor immune response
doi: 10.64898/2026.05.06.723126
Figure Lengend Snippet: ( A ) Experimental design for ( B-C; CT26 tumor model) and ( E; CT26 B2M k.o. tumor model); n=15 BALB/c mice per group. Depletion or blocking antibody treatment was started 2 days prior to RNA-LPX treatment to ensure depletion before treatment start. ( B, C ) Survival according to termination criteria. ( D ) Experimental design and survival in CT26B2M k.o. or CT26gp70 k.o. tumor cells i.v. tumor model, n=15 mice per group. ( E ) BALB/c mice injected i.v. with CT26B2M k.o and depletion/neutralization antibodies were applied as shown in ( A ). Note that data in ( E ) were generated within the same experiment, irrelevant RNA + isotype mix as well as cytokine mix RNA + isotype mix refers to the same groups in all 3 plots. Survival was analyzed via Mantel-Cox logrank test. For ( B ) and ( C ), groups 1 and 2 were furthermore compared via logrank test with emphasis on early and late differences, ( B ) ## Logrank test with emphasis on late differences ((rho=0, lambda=1): group 1 vs 2: p=0,00235; ( C ) # Logrank test with emphasis on early differences (rho=1, lambda=0): group 1 vs 2: p=0,0378.
Article Snippet:
Techniques: Blocking Assay, Injection, Neutralization, Generated