murine myoblast cells c2c12 human prostate adenocarcinoma cell lines  (ATCC)

Journal: bioRxiv

Article Title: Pancreatic cancer extracellular vesicles carry a time-of-day-regulated miRNA cargo that disrupts the skeletal muscle clock and bioenergetics

doi: 10.64898/2026.05.03.722338

Figure Lengend Snippet: (A) Bioluminescence recording, ( B ) period analysis, and ( C ) phase and amplitude analysis of U2OS BMAL1 :Luc reporter cells treated with PANC-1 CM at 12.5%, 25%, 50%, and 100% of the recording media. ( D ) Bioluminescence recording, ( E ) period analysis, and ( F ) phase and amplitude analysis of NIH3T3 Bmal1 :Luc reporter cells treated with PANC-1 CM at the same concentrations. For all bioluminescence experiments, at least three complete oscillations were included in the period estimation, excluding the first 24 h. Mean ± SD of relative mRNA expression of NIH3T3 core clock genes Bmal1 ( G ), Per2 ( H ), and Cry2 ( I ) measured over 36 h in response to PANC-1 CM. Relative mRNA levels of core clock genes in synchronized C2C12 myotubes over 32 h following treatment with PANC-1 CM: ( J ) Bmal1 , ( K ) Per2 , and ( L ) Cry2 . Cosine curves were fit for visualization purposes only; solid lines represent rhythmic oscillations (p<0.05) detected by MetaCycle, while dashed lines indicate loss of statistical rhythmicity (Suppl. Table 1). ( G–L ) Black: Control; ( G–I ) Red: PANC-1 CM; ( J–L ) Blue: PANC-1 CM. ( M ) Schematic representation and representative images of mature C2C12 myotube atrophy in response to NIH3T3 or PANC-1 released factors using a Transwell co-culture system; three measurements per myotube (yellow arrows) were used to quantify shortening. ( N ) Quantification of normalized myotube diameter under NIH3T3 vs PANC-1 co-culture, normalized to NIH3T3 co-culture control. One-way ANOVA: ( B ) p=0.0009, ( E ) p=0.0087. ( B, E ) Dunnett’s post-hoc test: *p<0.05; **p<0.01; ***p<0.001. (N) Student’s t-test: ***p<0.001.

Article Snippet: The human pancreatic cancer cell line PANC-1, the murine fibroblast cell line NIH3T3, and the murine myoblast cell line C2C12 were purchased from American Type Culture Collection (ATCC, Manassas, VA).

Techniques: Expressing, Control, Co-Culture Assay

Journal: bioRxiv

Article Title: Pancreatic cancer extracellular vesicles carry a time-of-day-regulated miRNA cargo that disrupts the skeletal muscle clock and bioenergetics

doi: 10.64898/2026.05.03.722338

Figure Lengend Snippet: (A) Top 35 miRNAs by mean expression in PANC-1-derived sEVs. Bar plot of mean log2 CPM across 9 time-points (4–36 h). Red bars: miRNAs selected from the top 35 to be tested in the BMAL1 :Luc reporter and atrophy assays; grey bars: remaining top-35 miRNAs. miRNAs are ranked in descending order of EV expression. ( B ) GO Biological Process enrichment of the experimentally validated targets (miRTarBase) of the 11 selected miRNAs. Terms are grouped into functional categories. Dot size represents the number of validated target genes associated with each term; dot color indicates Gene Ratio (proportion of input genes annotated to the term), from light pink (low) to dark red (high). Analysis performed with clusterProfiler. ( C , top panel) Normalized C2C12 myotube diameter at 0, 24, and 48 h post-transfection with miR-27b-3p, miR-615-3p, miR-191-5p, miR-127-3p, miR-99b-5p, or negative-transfection control (NTC); dexamethasone (Dexa) included as positive control. ( C , lower panel) Normalized C2C12 myotube diameter at the same time-points after transfection with hsa-let-7f-5p, miR-183-5p, miR-92a-3p, miR-30c-5p, miR-26a-5p, miR-10a-5p, NTC, or Dexa. ( D ) Oxygen consumption rate (OCR; top), resting-phenotype plot of basal OCR vs ECAR (middle), and metabolic-capacity plot of maximal OCR vs ECAR following FCCP (lower) for mature C2C12 myotubes 48 h post-transfection with miR-27b-3p, miR-615-3p, miR-191-5p, or NTC (Control). ( E ) Same panels for myotubes transfected with miR-127-3p, miR-99b-5p, miR-183-5p, or NTC. Sequential injections of oligomycin, FCCP, and rotenone/antimycin A were used to dissect mitochondrial respiration. Data are presented as mean ± SEM. ( C ) Measurements were taken from at least 5 random fields per well in N=3 wells; statistical analysis used 2-way ANOVA with Dunnett’s post-hoc correction: *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001.

Article Snippet: The human pancreatic cancer cell line PANC-1, the murine fibroblast cell line NIH3T3, and the murine myoblast cell line C2C12 were purchased from American Type Culture Collection (ATCC, Manassas, VA).

Techniques: Expressing, Derivative Assay, Functional Assay, Transfection, Control, Positive Control

Journal: Medicina

Article Title: AKT Signaling Regulates Agrin-Mediated Acetylcholine Receptor Surface Density

doi: 10.3390/medicina62030456

Figure Lengend Snippet: AKT inhibition suppresses Agrin-induced AChR cluster formation in C2C12 myotubes. ( A ) Immunoblot analysis showing reduced phosphorylation of AKT (pSer474) and its downstream targets S6 (pSer240/244) and PRAS40 (pThr240 following MK2206 treatment (10 μM) for 3 h or 6 h. ( B ) Western blot analysis of PAN-AKT, pSer240/244-S6, S6 and HSP90 in C2C12 myotubes treated with MK2206 (10 μM, 3 h or 6 h) following Agrin treatment for 16 h. ( C ) Representative images of AChR cluster intensity visualized by fluorescent α-bungarotoxin (α-bungarotoxin) staining in C2C12 myotubes treated with n-Agrin, with or without MK2206 pretreatment. MK2206 markedly reduced the intensity of Agrin-induced AChR clusters. ( D ) mRNA expression of AChR subunits (α, δ, ε) in C2C12 myotubes treated with neural Agrin (n-Agrin, 16 h) with or without MK2206 pretreatment. MK2206 significantly reduced Agrin-induced expression of AChRα and AChRδ and robustly suppressed AChRε expression. Data are presented as mean ± SEM; * p < 0.05, *** p < 0.001, **** p < 0.0001. Images were acquired using a 40× objective; scale bar, 20 µm.

Article Snippet: Murine C2C12 myoblast cell lines (American Type Culture Collection (ATCC); Manassas, VA, USA) were maintained under standard culture conditions in high-glucose Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 20% fetal bovine serum (FBS).

Techniques: Inhibition, Western Blot, Phospho-proteomics, Staining, Expressing

Journal: Medicina

Article Title: AKT Signaling Regulates Agrin-Mediated Acetylcholine Receptor Surface Density

doi: 10.3390/medicina62030456

Figure Lengend Snippet: AKT is required for the maintenance of Agrin-induced AChR cluster intensity. ( A ) Confocal images of AChR clusters intensity visualized with Alexa547-conjugated α-bungarotoxin in C2C12 myotubes treated with Agrin alone for 16 h or Agrin + MK2206 for 3 h or 6 h. ( B ) qPCR analysis of AChR subunit transcripts (α, δ, and ε) after 3 h or 6 h of MK2206 treatment during the final phase of Agrin stimulation for 16 h. Data are presented as mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. unstimulated control. Images were acquired using a 40× objective; scale bar, 20 µm.

Article Snippet: Murine C2C12 myoblast cell lines (American Type Culture Collection (ATCC); Manassas, VA, USA) were maintained under standard culture conditions in high-glucose Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 20% fetal bovine serum (FBS).

Techniques: Control

Journal: Medicina

Article Title: AKT Signaling Regulates Agrin-Mediated Acetylcholine Receptor Surface Density

doi: 10.3390/medicina62030456

Figure Lengend Snippet: AKT maintains Agrin-mediated AChR clusters via the proteasome degradation pathway. ( A ) Representative confocal images of AChR clusters labeled with α-bungarotoxin. MK2206 treatment (6 h) markedly reduced AChR cluster intensity, whereas pretreatment with the proteasome inhibitor MG132 (10 μM, 30 min) restored cluster abundance. ( B ) qPCR analysis of AChR transcripts in C2C12 myotubes treated with Agrin or Agrin ± MK2206 and MG132. ( C ) qPCR analysis of proteasome-related genes ( Psma1 , Psmc4 , and Psmd11 ) in C2C12 myotubes treated with Agrin ± MK2206 or Agrin + MK2206 and MG132. Data are presented as mean ± SEM. * p < 0.05 vs. unstimulated control, ** p < 0.01 vs. unstimulated Agrin, *** p < 0.001 vs. Agrin+MK2206. **** p < 0.0001. Images were acquired using a 40× objective. Images were acquired using a 40× objective; scale bar, 20 µm.

Article Snippet: Murine C2C12 myoblast cell lines (American Type Culture Collection (ATCC); Manassas, VA, USA) were maintained under standard culture conditions in high-glucose Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 20% fetal bovine serum (FBS).

Techniques: Labeling, Control