Journal: Frontiers in immunology

Article Title: Signal peptide exchange alters HIV-1 envelope antigenicity and immunogenicity.

doi: 10.3389/fimmu.2024.1476924

Figure Lengend Snippet: FIGURE 3 Changes in antigenicity of signal peptide (SP) DNA immunogens. (A) Schematic representation of WT and SP swapped gp160 Env. Dashes (-) indicate missing residues. Differences between the SP are shown in bold and are underlined. (B) Mouse muscle cell line C2C12 were transfected with gp160 expressing plasmids. Cells were probed with mAbs, 24 hours post-transfection followed by detecting the ligand binding by flow cytometry. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001 by 2-way ANOVA, p>0.05 was left unmarked. (C) Summary of the changes in antigenicity of gp160 proteins upon SP swapping. Fold changes (MFI) of SP swapped/WT were calculated for each mAb tested from (B). Significant increases or decreases are marked with blue (fold change of >1 with p<0.05) or red (fold change of <1 with p<0.05), respectively.

Article Snippet: Mouse myoblast cell line C2C12 was obtained from ATCC and was maintained in the ATCC-formulated Frontiers in Immunology 03 Dulbecco’s Modified Eagle’s Medium supplemented with 10% FBS.

Techniques: Transfection, Expressing, Ligand Binding Assay, Cytometry

Journal: Frontiers in immunology

Article Title: Signal peptide exchange alters HIV-1 envelope antigenicity and immunogenicity.

doi: 10.3389/fimmu.2024.1476924

Figure Lengend Snippet: FIGURE 4 Changes in glycosylation of Env upon signal peptide (SP) swapping. (A) Relative amounts of unoccupied, complex and oligomannose/hybrid glycans at each glycosites on SP-swapped vs WT gp120 proteins as determined by LC-MS/MS. ND: glycosites not detected. (B) WT and SP swapped gp120 proteins were coated onto ELISA plates (2 mg/ml) and probed with serially diluted biotinylated lectins. Representative data are shown depicting the binding of biotinylated lectins specific to mannose, fucose, and sialic acid sugars on the N-linked Env glycans. (C) Mouse muscle cell line C2C12 were transfected with gp160 expressing plasmids. Cells were probed with lectins, 24 hours post-transfection followed by detecting the ligand binding by flow cytometry. Comparisons were made between AA05 versus AC02, AA05 versus AA05-02 and AC02 versus AC02-05. *p < 0.05; ***p < 0.001; ****p < 0.0001 by 2-way ANOVA, p>0.05 was left unmarked. nd, not detected.

Article Snippet: Mouse myoblast cell line C2C12 was obtained from ATCC and was maintained in the ATCC-formulated Frontiers in Immunology 03 Dulbecco’s Modified Eagle’s Medium supplemented with 10% FBS.

Techniques: Glycoproteomics, Liquid Chromatography with Mass Spectroscopy, Enzyme-linked Immunosorbent Assay, Binding Assay, Transfection, Expressing, Ligand Binding Assay, Cytometry

Journal: Cell Reports Medicine

Article Title: Molecular Profiling Reveals a Common Metabolic Signature of Tissue Fibrosis

doi: 10.1016/j.xcrm.2020.100056

Figure Lengend Snippet:

Article Snippet: Rabbit anti-αSMA monoclonal antibody (EPR5368) IHC , Abcam , Cat#Ab124964; RRID: AB_11129103.

Techniques: Transduction, Virus, Ligation, Recombinant, Saline, Injection, Protease Inhibitor, Mass Spectrometry, Staining, cDNA Synthesis, Extraction, RNA Library Preparation, Enzyme-linked Immunosorbent Assay, Software, Simple Western

Journal: Cell reports

Article Title: Integrated genomic and proteomic analyses identify stimulus-dependent molecular changes associated with distinct modes of skeletal muscle atrophy

doi: 10.1016/j.celrep.2021.109971

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Rabbit anti-alpha- tubulin (11H10) , Cell Signaling Technologies , 2125S; RRID:AB_2619646.

Techniques: Recombinant, SYBR Green Assay, Membrane, Electron Microscopy, Bicinchoninic Acid Protein Assay, Cell Based Assay, Reverse Transcription, Muscles, Gene Expression, Control, Mass Spectrometry, Software

Journal: bioRxiv

Article Title: ACTN3 genotype influences androgen response in skeletal muscle

doi: 10.1101/2024.04.25.591034

Figure Lengend Snippet: Orchidectomy decreased a) body mass, b) seminal vesicle, c) levator ani bulbocavernosus (LABC), and d) gastrocnemius mass of WT and Actn3 KO mice; KO showed greater atrophy for the gastrocnemius muscle compared to WT. e) The size of fast 2B, 2X and 2A fibres is reduced in KO-ORX but not WT- ORX gastrocnemius muscles compared to controls. f) Muscles of orchidectomised WT but not KO show significant reductions in fibre count. g) Western blot analyses quantify the effect of orchidectomy in WT and KO gastrocnemius muscles on the expression of h) ACTN3, i) ACTN2, j) AR, k) p-4ebp1 Thr /46 , l) total 4ebp1, m) ratio of p-4ebp1 Thr37/46 /4ebp1, n) p- S6RP Ser235/236 , o) total S6RP, p) ratio of p-S6RP Ser235/236 /S6RP, and q) RCAN1-4. Data are represented as mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 by Mann- Whitney U test; # p < 0.05 by two-way ANOVA (d-f).

Article Snippet: These were blocked with 5% BSA in 1× TBST, probed overnight at 4°C with primary antibodies against α-actinin-3, α-actinin-2 (ab68204, ab68167, abcam), Akt1 (#C73H10, CST), p-Akt (Ser463) (#4060, CST), mTOR (#2983, CST), p-mTOR (#5536, CST), p-4ebp1 (#2855, CST), 4ebp1 (#9452, CST), S6RP (#2217, CST), p-S6RP (#4856, CST), SQSTM1/p62 (#5114, CST), LC3B (#3868, CST), p-AR (ab45089, abcam).

Techniques: Muscles, Western Blot, Expressing, MANN-WHITNEY

Journal: bioRxiv

Article Title: ACTN3 genotype influences androgen response in skeletal muscle

doi: 10.1101/2024.04.25.591034

Figure Lengend Snippet: A) Liquid chromatography-mass spectrometry confirms increases in serum DHT in male (Sham/ORX) and female WT and Actn3 KO mice that were implanted with silastic tubing containing 10 mg of crystalline DHT for 6 weeks, relative to mice that were implanted with empty tubing. b) DHT treatment prevented atrophy of spinalis muscles in orchidectomised WT and KO mice c) Female WT-DHT but not KO-DHT mice show increases in spinalis mass relative to empty controls. D) Western blot analysis quantify the effect of DHT in female WT and KO spinalis muscles on the expression of e) ACTN3, f) ACTN2, g) AR, h) p-4ebp1 Thr37/46 , i) total 4ebp1, j) ratio of p-4ebp1 Thr37/46 /4ebp1, k) p-S6RP Ser235/236 , l) total S6RP, m) ratio of p- S6RP Ser235/236 /S6RP, n) p-Akt Ser473 , o) total Akt, p) ratio of p-Akt Ser473 /Akt and q) RCAN1-4. Data are represented as mean ± SEM; * p < 0.05, ** p < 0.01 by Mann-Whitney U test; # p < 0.05, ## p < 0.0001 by two-way ANOVA (c, h, j, k, m, n, p, q).

Article Snippet: These were blocked with 5% BSA in 1× TBST, probed overnight at 4°C with primary antibodies against α-actinin-3, α-actinin-2 (ab68204, ab68167, abcam), Akt1 (#C73H10, CST), p-Akt (Ser463) (#4060, CST), mTOR (#2983, CST), p-mTOR (#5536, CST), p-4ebp1 (#2855, CST), 4ebp1 (#9452, CST), S6RP (#2217, CST), p-S6RP (#4856, CST), SQSTM1/p62 (#5114, CST), LC3B (#3868, CST), p-AR (ab45089, abcam).

Techniques: Liquid Chromatography, Mass Spectrometry, Muscles, Western Blot, Expressing, MANN-WHITNEY

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Inhibition of COX-2, mPGES-1 and CYP4A by isoliquiritigenin blocks the angiogenic Akt signaling in glioma through ceRNA effect of miR-194-5p and lncRNA NEAT1

doi: 10.1186/s13046-019-1361-2

Figure Lengend Snippet: Isoliquiritigenin (ISL) prolongs survival, delays growth and induces vessel normalization in C6 gliomas with the decreased intratumoral level of 20-HETE and PGE 2 . a In the C6 intracranial glioma model, the survival time of the rats ( n = 10) injected intraperitoneally with ISL (10 and 20 mg/kg), Sunitinib (80 mg/kg) or vehicle was measured, and 20-HETE and PGE 2 in the tumor tissues from each group were determined at day 12 by LC-MS/MS or ELISA. b In the C6 subcutaneous glioma model, rat C6 glioma cells (5 × 10 6 ) were injected subcutaneously into the right flank of Wistar rats. When tumors reached a size of about 100 mm 3 , the rats ( n = 8) received ISL (10 and 20 mg/kg), Sunitinib (80 mg/kg) or vehicle by intraperitoneal injection once daily for a week. Tumor weight was measured, and 20-HETE and PGE 2 were determined by LC-MS/MS or ELISA. The values are presented as the mean ± SEM, * P < 0.05, ** P < 0.01 vs. control, # P < 0.05, ## P < 0.01 vs. sunitinib (80 mg/kg)-treated group. c - h Rat C6 glioma cells (5 × 10 6 ) were injected subcutaneously into the right flank of Wistar rats. When tumors reached a size of about 100 mm 3 , the rats ( n = 8) received ISL (10 and 20 mg/kg) or vehicle by intraperitoneal injection once daily for a week. Tumor perfusion at day 0, 2, 4, 6 and 8 was measured using a laser Doppler analyzer. Scale bars, 2 mm ( c ). The quantitative analysis showed the relative level of tumor perfusion ( d ). After sacrificing the rats at day 8, hypoxia induced factor (HIF)-1α in the tumor tissues was measured by Western blot ( e ). Double staining for CD31 (green) and α-SMA (red) in the tumor tissues was shown. Scale bars, 50 μm ( f - h ). i In the C6 intracranial glioma model, the survival time of the rats ( n = 10) injected intraperitoneally with ISL (20 mg/kg), temozolomide (TMZ, 20 mg/kg), ISL (20 mg/kg) plus TMZ (20 mg/kg) or vehicle was measured, and TMZ uptake into tumor tissues was determined at day 12 by high performance liquid chromatography (HPLC). j In another experiment, rat C6 glioma cells (5 × 10 6 ) were injected subcutaneously into the right flank of Wistar rats. When tumors reached a size of about 100 mm 3 , the rats ( n = 8) received ISL (20 mg/kg), TMZ (20 mg/kg), ISL (20 mg/kg) plus TMZ (20 mg/kg) or vehicle by intraperitoneal injection once daily for a week. Tumor weight was measured, and TMZ uptake into tumor tissues was determined by HPLC. The values are presented as the mean ± SEM, * P < 0.05, ** P < 0.01 vs. control, # P < 0.05, ## P < 0.01 vs. TMZ (20 mg/kg)-treated group

Article Snippet: The antibodies against rat CD31, α-smooth muscle actin (SMA) and hypoxia-inducible factor (HIF)-1α were purchased from Santa Cruz Biotechnology, Inc. (Texas, USA).

Techniques: Injection, Liquid Chromatography with Mass Spectroscopy, Enzyme-linked Immunosorbent Assay, Control, Western Blot, Double Staining, High Performance Liquid Chromatography

Journal: Cell Death Discovery

Article Title: Limiting serine availability during tumor progression promotes muscle wasting in cancer cachexia

doi: 10.1038/s41420-024-02271-1

Figure Lengend Snippet: A Metascape functional enrichment analysis. Common and coherent metabolic DEGs, extracted from GSE24112 and GSE48363, were analyzed for ontology purposes. B PHGDH, PSAT1, PSPH protein levels in CRC cell lines. Cell lysates from HCT-116, HT29, HCT8, CACO2, RKO, and LS174T cells were analyzed by western blotting with the anti-PHGDH antibody. An anti-actin antibody was used to ensure equal protein loading. The image is representative of three independent experiments. C AA composition of cancer conditioned media (CM). Amino acids content in CM was analyzed by GC-MS on media collected following 48 h of incubation with muscle cells. Data are reported as normalized to serum-free DMEM abundance and are average of three independent experiments. Black label indicates relative value as 1. D Ser media content analyzed by GC-MS on media collected following 48 h of incubation with CRC cells. Data are expressed as relative to serum-free DMEM. One-way ANOVA with Sidak’s post hoc test ( n = 3). Each dot represents a single experiment. E PHGDH protein levels-CM Ser content correlation analysis. Pearson correlation between PHGDH protein levels in CRC cell lines and Ser content from corresponding CM. F Schematic representation of CM-culturing experiments. G Alterations in myotubes width following CM incubation. Relative C2C12 myotubes fibers width following 96 h of incubation with boiled CMs derived from CRC cell lines cultures. CRC cell lines were incubated in serum-free DMEM medium for 48 h before collecting conditioned media (CM) and boiling them for 20 min to remove all protein components. Data are expressed as relative to serum-free DMEM. One-way ANOVA with Sidak’s post hoc test ( n = 3). Each dot represents a single experiment. H Ser media content-myotubes width correlation analysis. Pearson correlation between Ser content in CM from CRC cell lines and C2C12 myotubes width following 96 h of incubation with CM. Data are normalized on values from C2C12 myotubes incubated in normal serum-free DMEM. I PHGDH protein levels-myotubes width correlation analysis. Pearson correlation between PHGDH protein levels in CRC cell lines and C2C12 myotubes width following 96 h of incubation with corresponding CM. J Alterations in myotubes width following incubation with CM from HT29-high and HT29-low cells. Relative C2C12 myotubes fibers width following 96 h of incubation with boiled CM derived from high PHGDH HT29 and low PHGDH HT29-expressing cells. Data are normalized to width values from C2C12 cells incubated in serum-free DMEM. One-way ANOVA with Dunnett’s post hoc test ( n = 3). Each dot represents a single experiment. K Ser supplementation rescues C2C12 myotubes width reduction under CM incubation. Relative C2C12 myotubes fibers width following 96 h of incubation with boiled CM derived from CACO2 cells supplemented or not with exogenous Ser (0.4 mM). Data are normalized to width values from C2C12 cells incubated in serum-free DMEM. One-way ANOVA with Dunnett’s post hoc test ( n = 3). Each dot represents a single experiment. L Representative microscopic pictures of C2C12 myotubes. C2C12 myotubes were incubated with boiled CM derived from CACO2 cells supplemented or not with exogenous Ser (0.4 mM). Pictures were taken following 96 h of incubation. The image is representative of three independent experiments. Data are represented as mean ± standard error of the mean of at least three independent biological replicates. ns, not significant. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Mouse C2C12 myoblasts were obtained from ATCC and cultured in DMEM with 10% fetal bovine serum (FBS).

Techniques: Functional Assay, Western Blot, Gas Chromatography-Mass Spectrometry, Incubation, Derivative Assay, Expressing

Journal: Cell Death Discovery

Article Title: Limiting serine availability during tumor progression promotes muscle wasting in cancer cachexia

doi: 10.1038/s41420-024-02271-1

Figure Lengend Snippet: A , B Total tumor mass. Mass quantification of tumors obtained from HT29 low-PHGDH ( A ) and CT26 ( B ) tumor-bearing mice treated as in Fig. . Mass values were calculated based on tumor weight measured at the endpoint of the experiment. Each dot represents a plasma sample derived from a single mouse. Data are represented as mean ± SEM, Student’s t test. C , D Intra-tumor Ser levels. Relative Ser levels quantification in tumor tissue-derived samples from HT29 low-PHGDH ( C ) and CT26 ( D ) tumor-bearing mice treated as in Fig. . Metabolites from tissue samples were extracted as described in the “Materials and methods” section and tissue Ser content was quantified by GC-MS analysis. Each dot represents a plasma sample derived from a single mouse. Data are represented as mean ± SEM, Student’s t test. E PHGDH protein levels in CRC cell lines under Ser starvation. HCT-116, CACO2, and RKO cells were incubated in a medium containing (+Ser +Gly) or lacking (-Ser -Gly) Ser and Gly for 24 h before protein extraction. Cell lysates were analyzed by western blotting with the anti-PHGDH antibody. An anti-actin antibody was used to ensure equal protein loading. Data are expressed as relative to +Ser+Gly condition. Student’s t-test ( n = 3). Each dot represents a single experiment. F Representative images of western blotting analysis evaluating PHGDH expression in HCT-116, CACO2, and RKO cells incubated in +Ser +Gly or -Ser -Gly medium for 24 h. G PHGDH protein levels in C2C12 myotubes under Ser starvation. C2C12 myotubes were incubated in a medium containing (+Ser +Gly) or lacking (-Ser -Gly) Ser and Gly for 6, 12, and 24 h before protein extraction. Cell lysates were analyzed by western blotting with the anti-PHGDH antibody. An anti-actin antibody was used to ensure equal protein loading. Data are expressed as relative to +Ser+Gly condition. Student’s t-test ( n = 3). Each dot represents a single experiment. H Representative images of western blotting analysis evaluating PHGDH expression in C2C12 myotubes incubated in +Ser +Gly or -Ser -Gly medium for 24 h. I Ser synthesis pathway (SSP) activity analysis by [U- 13 C]-glucose labeling assay. Relative incorporation of [U- 13 C]-glucose-derived carbons in Ser. C2C12, CACO2, RKO, and HCT116 cells were incubated in a (-Ser -Gly) medium containing [U- 13 C]-Glucose for 3 h. Metabolite abundance and labeling enrichment were evaluated by GC-MS analysis. One-way ANOVA with Dunnett post hoc test ( n = 3). Each dot represents a single experiment. J Competition for Ser availability between tumor and muscle cells. C2C12 myotubes were pre-incubated with a medium containing 13 C 1 -Ser for 24 h before replacing growth medium with standard serum-starved-DMEM and adding tumor cells in the upper compartment of a Boyden chamber (pores diameter 0.4 µm). The fate of C2C12-derived 13 C 1 -Ser was assessed by GC-MS analysis 24 h after tumor cell’s introduction. K Workflow for the 13 C 1 -Ser labeling experiment described in J . One-way ANOVA with Tukey’s post hoc test ( n = 3). Each dot represents a single experiment. Data are represented as mean ± SEM of n independent experiments, ns, not significant. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Mouse C2C12 myoblasts were obtained from ATCC and cultured in DMEM with 10% fetal bovine serum (FBS).

Techniques: Clinical Proteomics, Derivative Assay, Gas Chromatography-Mass Spectrometry, Incubation, Protein Extraction, Western Blot, Expressing, Activity Assay, Labeling

Journal: Cell Death Discovery

Article Title: Limiting serine availability during tumor progression promotes muscle wasting in cancer cachexia

doi: 10.1038/s41420-024-02271-1

Figure Lengend Snippet: A Representative microscopic pictures of C2C12 myotubes. C2C12 myotubes were incubated in a medium containing (+Ser +Gly) or lacking (-Ser -Gly) Ser and Gly. Pictures were taken following 96 h of incubation. The image is representative of three independent experiments. B Alterations in myotubes width following Ser/Gly starvation. Relative C2C12 myotubes fibers width following 96 h of incubation with a medium containing (+Ser +Gly) or lacking (-Ser -Gly) Ser and/or Gly. 10 nM TNFα was used as wasting-inducer positive control. Data are expressed as relative to width values of myotubes grown in normal serum-free DMEM. One-way ANOVA with Tukey’s post hoc test ( n = 3). Each dot represents a single experiment. C Alterations in total protein content in myotubes width following Ser/Gly starvation. C2C12 myotubes fibers were incubated for 96 h of incubation with a medium containing (+Ser +Gly) or lacking (-Ser -Gly) Ser and/or Gly before lysing cells for protein quantification analysis. Data are expressed as relative to protein content of myotubes grown in normal serum-free DMEM. One-way ANOVA with Tukey’s post hoc test ( n = 3). Each dot represents a single experiment. D Myotubes AAs content following Ser/Gly starvation. C2C12 myotubes were incubated for 48 h with a medium containing (+Ser +Gly) or lacking (-Ser -Gly) Ser and/or Gly before metabolomic analysis. Metabolites were extracted as described in the “Materials and methods” section and intracellular AAs content was quantified by GC-MS. Data are expressed as relative to AAs levels measured in samples from cells incubated in serum-free DMEM. Data are reported as average of three independent experiments. E , F Atrogin-1 and MURF-1 expression levels. mRNA levels of Atrogin-1 ( E ) and MuRF-1 ( F ) were analyzed by quantitative RT-PCR in C2C12 myotubes following 48 h of incubation with +Ser +Gly/-Ser -Gly media. +Ser +Gly condition was used as comparator. Student’s t-test ( n > 3). Each dot represents a single experiment. G Total protein synthesis in C2C12 myotubes under Ser/Gly starvation. Protein synthesis was evaluated on the incorporation of puromycin into newly synthesized proteins. C2C12 myotubes were incubated with +Ser +Gly/-Ser -Gly media for 24 h, starved in Hank’s balanced salt solution (HBSS) for 1 h, and subsequently reactivated (30 min) in a medium containing puromycin (10 mg/mL). Cell lysates were analyzed by western blotting with the anti-puromycin antibody. An anti-actin antibody was used to ensure equal protein loading. The image is representative of three independent experiments. H Phosphorylation of AKT and mTORC1 targets S6 and S6K1 in C2C12 myotubes under Ser/Gly starvation. C2C12 myotubes were incubated with +Ser +Gly/-Ser -Gly media for 6, 16, and 24 h before analyzing cell lysates by western blotting. An anti-actin antibody was used to ensure equal protein loading. The image is representative of three independent experiments. I Phosphorylation of AKT and S6K1 in C2C12 myotubes incubated in low Ser containing CM. C2C12 myotubes were incubated with boiled CM derived from CACO2 cells supplemented or not with exogenous Ser (0.4 mM) for 24 h before analyzing cell lysates by western blotting. An anti-actin antibody was used to ensure equal protein loading. The image is representative of three independent experiments. J Proliferation curves of C2C12 cells cultured in presence or absence of Ser/Gly. Proliferation curves of C2C12 cell lines grown in medium with Ser/Gly (+ser+gly, gray dots) or without Serine/Gly (-ser-gly, blue dots). Cell number was assessed every 24 h ( n = 3). K Proliferation curves of C2C12 cells following 24 h of Ser/Gly starvation. C2C12 cells were incubated for 24 h in presence (+ser+gly, gray dots) or absence (-ser-gly, blue dots) of Ser/Gly before assessing cell proliferation in normal medium by counting cell number every 24 h ( n = 3). L Cell Cycle distribution of C2C12 cells cultured in presence or absence of Ser/Gly. Quantitative analysis of relative cell number in G1, S, and G2/M of C2C12 cells incubated in grown in medium with or without Serine/Gly for 24. C2C12 cells were stained with Propidium Iodide (PI) to analyze the cell cycle distribution by flow cytometry. Quantitation was determined by measuring the area corresponding to the indicated phase. M Time-dependent changes in MHC and MYOD protein levels during differentiation under Ser/Gly starvation. C2C12 myotubes were incubated with differentiation media (dialyzed FBS 2%) in presence or not of Ser/Gly for 1, 2, 3, 6 days before analyzing cell lysates by western blotting with the anti-MHC and anti-MYOD antibodies. An anti-Vinculin antibody was used to ensure equal protein loading. The image is representative of three independent experiments. Data are represented as mean ± SEM of n independent experiments, ns, not significant. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Mouse C2C12 myoblasts were obtained from ATCC and cultured in DMEM with 10% fetal bovine serum (FBS).

Techniques: Incubation, Positive Control, Gas Chromatography-Mass Spectrometry, Expressing, Quantitative RT-PCR, Synthesized, Western Blot, Phospho-proteomics, Derivative Assay, Cell Culture, Staining, Flow Cytometry, Quantitation Assay

Journal: Cell Death Discovery

Article Title: Limiting serine availability during tumor progression promotes muscle wasting in cancer cachexia

doi: 10.1038/s41420-024-02271-1

Figure Lengend Snippet: A Total ROS levels in C2C12 myotubes under Ser/Gly starvation. Intracellular ROS were quantified in C2C12 myotubes incubated with +Ser +Gly/-Ser -Gly media for 24 h by staining with DCFDA probe and analyzing them by FACS. Data are expressed as relative to +Ser+Gly condition. Student’s t-test ( n = 3). Each dot represents a single experiment. B Mitochondrial ROS levels in C2C12 myotubes under Ser/Gly starvation. Mitochondrial ROS were quantified in C2C12 myotubes incubated with +Ser +Gly/-Ser -Gly media for 24 h by staining with MitoSOX probe and analyzing by FACS. Data are expressed as relative to +Ser+Gly condition. Student’s t-test ( n = 3). Each dot represents a single experiment. C Intracellular ATP levels in C2C12 myotubes under Ser/Gly starvation. C2C12 myotubes were incubated with +Ser +Gly/-Ser -Gly media for 24 h before analyzing ATP content. Data are expressed as relative to +Ser+Gly condition. Student’s t-test ( n = 3). Each dot represents a single experiment. D Phosphorylation of AMPK in C2C12 myotubes under Ser/Gly starvation. C2C12 myotubes were incubated with +Ser +Gly/-Ser -Gly media 24 h before analyzing cell lysates by western blotting with the anti-phospho-AMPK and anti-AMPK antibody. AMPK phosphorylation levels are reported as relative to total AMPK levels. An anti-actin antibody was used to ensure equal protein loading. Student’s t-test ( n = 3). Each dot represents a single experiment. E – G Oxidative metabolism in myotubes under Ser/Gly starvation. Oxygen consumption rate (OCR) was measured in C2C12 myotubes previously incubated with +Ser +Gly/-Ser -Gly media for 24 and 48 h. E Basal OCR was calculated as the OCR values before Oligomycin injection. F ATP-linked respiration was calculated by subtracting OCR values obtained following Oligomycin injection to basal OCR. G Non-mitochondrial respiration was calculated as the OCR values after Rotenone/Antimycin A injection. Data are normalized to those from C2C12 cells incubated with +Ser +Gly medium. One-way ANOVA with Dunnett’s post hoc test ( n = 3). Each dot represents a single experiment. H Mitochondrial respiration complexes in C2C12 myotubes under Ser/Gly starvation. C2C12 myotubes were incubated with +Ser +Gly/-Ser -Gly media for 24 h before analyzing cell lysates by western blotting with the anti-OXPHOS complexes antibody. An anti-actin antibody was used to ensure equal protein loading. The image is representative of three independent experiments. I Mitochondrial ROS accumulation in Ser starved C2C12 myotubes upon mitoTEMPO treatment. C2C12 cells were incubated with +Ser +Gly/-Ser -Gly media 24 h in presence or not of 2.5 µM mitoTEMPO before quantifying mitochondrial ROS by staining them with MitoSOX probe and analyzing by FACS. Data are normalized to those from C2C12 cells incubated with +Ser +Gly medium. Two-way ANOVA with Tukey’s post hoc test ( n = 3). Each dot represents a single experiment. J Quantification of Ser starved C2C12 myotubes upon MitoTEMPO treatment. C2C12 cells were incubated with +Ser +Gly/-Ser -Gly media 72 h in presence or not of 2.5 µM mitoTEMPO before quantifying fiber width. Data are normalized to those from C2C12 cells incubated with +Ser +Gly medium. Two-way ANOVA with Tukey’s post hoc test. Data are represented as mean ± SEM of n independent experiments, ns, not significant. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Mouse C2C12 myoblasts were obtained from ATCC and cultured in DMEM with 10% fetal bovine serum (FBS).

Techniques: Incubation, Staining, Phospho-proteomics, Western Blot, Injection

Journal: International Journal of Bioprinting

Article Title: Blood-derived biomaterials for tissue graft biofabrication by solvent-based extrusion bioprinting

doi:

Figure Lengend Snippet: 3D bioprinting of specific tissue constructs using bioinks functionalized with blood-derived biomaterials

Article Snippet: Kumar et al. (2019); ACS Biomater Sci Eng [ ] , 1 mL of the bioink: 990 μL of fibrinogen solution (60 mg/mL), 155 mg of gelatin- furfuryl at 25°C Heat it at 37 °C for 1 h with stirring, add Rose Bengal (10 μL) Polymerization: 50 units/mL thrombin, and 80 mM CaCl 2 , Human-induced pluripotent stem cell (iPSC)-derived cardiomyocytes (CM) Human CM AC 16 cell lines (ATCC) , Fibrinogen Thrombin (#) , SEM, swelling assay, rheology, Fourier transform infrared spectroscopy (FTIR), biocompatibility Cardiac cell-laden constructs To validate the applicability of this scaffold toward mimicking cardiac tissue.

Techniques: Construct, Formulation, Migration, In Vivo, Clinical Proteomics, In Vitro, Micro-CT, Concentration Assay, Gene Expression, Transplantation Assay, Activation Assay, Immunohistochemistry, Immunohistochemical staining, Fourier Transform Infrared Spectroscopy, Spectroscopy, Functional Assay, In Situ, Modification, Activity Assay, Expressing, Marker, Comparison

Journal: iScience

Article Title: Exercise-acclimated microbiota improves skeletal muscle metabolism via circulating bile acid deconjugation

doi: 10.1016/j.isci.2023.106251

Figure Lengend Snippet: Different forms of BA modulate metabolic and inflammatory responses in cultured myotubes and muscle tissues Glucose uptake in C2C12 myotubes incubated in the absence or presence of αMCA and tauro α-muricholic acid (TαMCA) (10 μM) with palmitic acid (200 μM) for 24 h or incubated with or without sotrastaurin (AEB071) (2 nM) (n = 5–8, A and J). The membrane content of glucose transporter 4 (GLUT4) (n = 4, B) and AMPKα Thr172 phosphorylation (n = 7, C) in C2C12 myotubes incubated in the absence or presence of MCA and TMCA. mRNA levels of CCL-2 , CXCL-1 , and Tlr-4 (n = 5–6, F–H, K) with or without AEB071in myotubes, and levels of CCL-2 in the media (n = 8, I) in the absence or presence of αMCA and TαMCA. mRNA levels of TNF-α , IL1-β , Tlr-4 , CXCL-1 , and CCL-2 , and F4/80 (n = 6, D) and CCL-2 protein level (n = 6, E) in gastrocnemius muscles from recipient mice at week-1. Phosphorylation levels were correlated with the total content of each target in immunoblotting. RS; recipient from sedentary donor, RT; recipient from trained donor. # p < 0.1 and ∗p < 0.05 between groups. Results are presented as the mean ± SE.

Article Snippet: Then, experiments using BAs (αMCA and tauro-αMCA; Cayman Chemical, Ann Arbor, MI, USA; Toronto Research Chemical, ON, Canada), palmitic acid (TCI Chemicals, Tokyo, Japan), and sotrastaurin, a PKCθ inhibitor (AEB071; Selleck Chemicals, Houston, TX, USA), were performed following incubation for 4 h in non-supplemented medium.

Techniques: Cell Culture, Incubation, Membrane, Phospho-proteomics, Muscles, Western Blot

Journal: iScience

Article Title: Exercise-acclimated microbiota improves skeletal muscle metabolism via circulating bile acid deconjugation

doi: 10.1016/j.isci.2023.106251

Figure Lengend Snippet:

Article Snippet: Then, experiments using BAs (αMCA and tauro-αMCA; Cayman Chemical, Ann Arbor, MI, USA; Toronto Research Chemical, ON, Canada), palmitic acid (TCI Chemicals, Tokyo, Japan), and sotrastaurin, a PKCθ inhibitor (AEB071; Selleck Chemicals, Houston, TX, USA), were performed following incubation for 4 h in non-supplemented medium.

Techniques: Recombinant, Enzyme-linked Immunosorbent Assay, Protein Extraction, Gene Expression, Software, Mass Spectrometry, Real-time Polymerase Chain Reaction