Journal: Cell reports

Article Title: Progressive development of melanoma-induced cachexia differentially impacts organ systems in mice

doi: 10.1016/j.celrep.2022.111934

Figure Lengend Snippet:

Article Snippet: Mouse IgG1 anti-Myosin heavy chain type 2a , DSHB , SC-71; RRID:AB_2147165.

Techniques: Recombinant, Electron Microscopy, Gene Expression, Muscles, Microarray, Software, Control

Journal: bioRxiv

Article Title: Myf6/MRF4 is a Myogenic Niche Regulator Required for the Maintenance of the Muscle Stem Cell Pool

doi: 10.1101/691386

Figure Lengend Snippet: (a) Colormap of Myf5, MyoD and Myf6 peaks within 100kb of the Transcription Start Sites (TSS) of myokines ranging from zero (black) to six peaks (red) occupancy. The onset of differentiation coincides with increased binding of MRFs to the regulatory domains of the myokines genes. (b) Gene expression analysis of myokines during a five day time course of myogenic differentiation going from cycling myoblasts in growth media (Ham’s F10 supplemented with 20% Fetal Bovine Serum, 1% penicillin/streptomycin, 2.5 ng/ml basic Fibroblast Growth Factor) to terminally differentiated myocytes (2 days in differentiation media, DMEM supplemented with 5% horse serum) to post mitotic multinucleated myotubes (5 days in differentiation media). Gene expression was assayed in biological triplicate by microarray , . Expression of each gene was averaged across three replicates and normalized to mean zero and standard deviation of one. Expression values were then truncated at +/− 3 for color display. Blue indicates lower than average expression, while red indicates higher than average expression. (c) Distribution of Myf6 peaks within 100kb of the TSS of the myokine genes , similar to the analysis shown in “a”. (d) Depletion of Myf6 transcript by siRNAs in differentiated primary myotubes (5DM). (e-g) Depletion of Myf6 expression by siRNAs leads to a significant reduction in the gene expression output of Epidermal Growth Factor (EGF) (e) , Vascular Endothelial Growth Factor A (VEGFA) (f) and Leukemia Inhibitory Factor (LIF) (g). (h) Quantitative Real-Time PCR (RT-qPCR) analysis of a select set of myokines genes involved in the activation of STAT3 and EGFR signaling pathways. RT-qPCR analysis was performed on the total RNA isolated from primary myoblasts and multinucleated primary myotubes derived from MuSCs isolated by Fluorescence Activated Cell Sorting (FACS, see the Extended Material and Methods). Myotubes were obtained by feeding >90% confluent primary myoblasts with differentiation media (DMEM supplemented with 5% horse serum) for five days. (i) RT-qPCR analysis of the expression of select receptors for the ligands in “h” between primary myoblasts and multinucleated myotubes. (j-k) Activation of STAT3 and EGFR signaling in myoblasts treated with recombinant LIF (j) and EGF (k) respectively. Myoblasts were treated with 2 ng/ml of each recombinant protein in growth media (Ham’s F10 supplemented with 20% FBS, 2.5 ng/ml bFGF) for 15 minutes and lysed in RIPA lysis buffer. Western Blots were performed with antibodies against EGFR, phospho-EGFR (pEGFR-Y1068), STAT3 and phospho-STAT3 (pSTAT3-Y705). (l) Activation of EGFR in MuSCs on EDL myofibers treated with EGF compared to untreated control. Myofibers were cultured for 48 hours and were treated with 40ng/ml of EGF for 10 minutes before fixing and staining with antibodies for PAX7, EGFR and phospho-EGFR. (m) Activation of STAT3 signaling in MuSCs on EDL myofibers treated with LIF compared to untreated control. Myofibers were cultured for 48 hours and were treated with 1000U/ml of recombinant LIF for 10 minutes before fixation and staining with antibodies for PAX7, STAT3 and p-STAT3.

Article Snippet: Subsequently, muscle sections were incubated with the primary antibodies (Pax7, concentrated DSHB 1:100 dilution; Laminin (LAMA1), Sigma 1:750 dilution) in goat serum with BSA blocking solution overnight in a humid chamber at 4 ° C. The next day, slides were washed twice for 10 minutes each time in PBT (0.05% Triton-X 100 in PBS, v/v), and once for 10 minutes in PBS.

Techniques: Binding Assay, Gene Expression, Microarray, Expressing, Standard Deviation, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Activation Assay, Protein-Protein interactions, Isolation, Derivative Assay, Fluorescence, FACS, Recombinant, Lysis, Western Blot, Control, Cell Culture, Staining

Journal: bioRxiv

Article Title: Myf6/MRF4 is a Myogenic Niche Regulator Required for the Maintenance of the Muscle Stem Cell Pool

doi: 10.1101/691386

Figure Lengend Snippet: (a-c) Snapshots of the UCSC genome browser showing enrichment of Myf6 ChIP-Seq reads compared to control around EGF (a) , LIF and OSM (b) , and VEGFA (c) loci superimposed on reads for Histone H3 lysine 4 mono methyl (H3K4me1), Histone H3 lysine4 tri methyl (H3K4me3) and total H3 in myoblasts (pink) and myotubes (green). (d-e) Immunofluorescence staining of WT (d) and Myf6-KO (e) EDL myofibers stained with PAX7, EGFR, phospho-EGFR, STAT3 and phospho-STAT3 antibodies. Live fibers were maintained in culture media for 44 hours, followed by 4 hours of serum starving in non-supplemented DMEM. For treated samples, 40ng/ml of recombinant EGF (left) or 1000U/ml of recombinant LIF (right) was added to the non-supplemented DMEM for 10 minutes before fixation. Activation status of EGFR and STAT3 signaling pathways was assessed using antibodies against phospho-EGFR (pEGFR-Y1068) and phospho-STAT3 (pSTAT3-Y705), respectively. (f) Quantification of the percentage of all pEGFR + /PAX7 + cells (defined as PAX7 + cells showing low or high pEGFR signal) on WT, Myf6-KO, and Myf6-KO EDL myofibers treated with EGF. (g) Quantification of the percentage of EGFR high /PAX7 + cells (defined as PAX7 + cells showing high pEGFR signal) on WT, Myf6-KO, and Myf6-KO EDL myofibers treated with EGF. (h) Quantification of the percentage of all pSTAT3 + /PAX7 + (defined as PAX7 + cells showing low or high pSTAT3 signal) cells on WT, Myf6-KO, and Myf6-KO EDL myofibers treated with LIF. (i) Quantification of the percentage of pSTAT3 high /PAX7 + cells (defined as PAX7 + cells showing high pSTAT3 signal) on WT, Myf6-KO, and Myf6-KO EDL myofibers treated with LIF.

Article Snippet: Subsequently, muscle sections were incubated with the primary antibodies (Pax7, concentrated DSHB 1:100 dilution; Laminin (LAMA1), Sigma 1:750 dilution) in goat serum with BSA blocking solution overnight in a humid chamber at 4 ° C. The next day, slides were washed twice for 10 minutes each time in PBT (0.05% Triton-X 100 in PBS, v/v), and once for 10 minutes in PBS.

Techniques: ChIP-sequencing, Control, Immunofluorescence, Staining, Recombinant, Activation Assay, Protein-Protein interactions

Journal: bioRxiv

Article Title: Myf6/MRF4 is a Myogenic Niche Regulator Required for the Maintenance of the Muscle Stem Cell Pool

doi: 10.1101/691386

Figure Lengend Snippet: (a) Immunofluorescent analysis of TA muscle cross-sections from Myf6-KO and WT mice with PAX7 and Laminin (LAMA1) antibodies at postnatal day 7. (b) Quantification of the number of PAX7 + cells per unit area (mm 2 ) in TA muscle cross-sections from Myf6-KO and WT counterparts at postnatal day 7. (n=3 animals per group). (c) Immunofluorescent analysis of TA muscle cross-sections from Myf6-KO and WT mice with PAX7 and Laminin (LAMA1) antibodies at postnatal day 21. (d) Quantification of the number of PAX7 + cells per unit area (mm 2 ) in TA muscle cross-sections from Myf6-KO and WT counterparts at postnatal day 21. (n=3 animals per group). (e) Immunofluorescent analysis of TA muscle cross-sections from Myf6-KO and WT mice with PAX7 and Laminin (LAMA1) antibodies at 3 months of age. (f) Quantification of the number of PAX7 + cells per unit area (mm 2 ) in TA muscle cross-sections from Myf6-KO and WT counterparts at 3 months of age. (n=5 animals per group). (g) Immunofluorescent analysis of TA muscle cross-sections from Myf6-KO and WT mice with PAX7 and Laminin (LAMA1) antibodies at 10 months of age. (h) Quantification of the number of PAX7 + cells per unit area (mm 2 ) in TA muscle cross-sections from Myf6-KO and WT counterparts at 10 months of age. (n=3 animals per group). (i) Immunofluorescent analysis of myofiber-associated MuSCs isolated from the EDL muscles of 3 month-old Myf6-KO and WT counterparts. Fibers were stained with PAX7 antibody and counterstained with DAPI at time T=0 hours post fiber isolation. (j) Quantification of the number of MuSCs per fiber between 3 month-old Myf6-KO and WT counterparts (n=3 animals per group, p-values are based on two tailed t-test). (k) Immunofluorescent analysis of myofiber-associated MuSCs isolated from the EDL muscles of 10 month-old Myf6-KO and WT counterparts. Fibers were stained with PAX7 antibody and counterstained with DAPI at time T=0 hours post fiber isolation. (l) Quantification of the number of MuSCs per fiber between 10 month-old Myf6-KO and WT counterparts (n=3 animals per group, p-values are based on two tailed t-test).

Article Snippet: Subsequently, muscle sections were incubated with the primary antibodies (Pax7, concentrated DSHB 1:100 dilution; Laminin (LAMA1), Sigma 1:750 dilution) in goat serum with BSA blocking solution overnight in a humid chamber at 4 ° C. The next day, slides were washed twice for 10 minutes each time in PBT (0.05% Triton-X 100 in PBS, v/v), and once for 10 minutes in PBS.

Techniques: Isolation, Muscles, Staining, Two Tailed Test

Journal: bioRxiv

Article Title: Myf6/MRF4 is a Myogenic Niche Regulator Required for the Maintenance of the Muscle Stem Cell Pool

doi: 10.1101/691386

Figure Lengend Snippet: (a) Immunofluorescent analysis of TA muscle cross-sections from Myf6-KO and WT mice with PAX7 and KI67 antibodies at postnatal day 7. (b) Quantification of the percentage of KI67 + over PAX7 + MuSCs between Myf6-KO and WT mice at postnatal day 7 (n=3 animals per group). (c) Immunofluorescent analysis of TA muscle cross-sections from Myf6-KO and WT mice with PAX7 and KI67 antibodies at postnatal day 21. (d) Quantification of the percentage of KI67 + over PAX7 + MuSCs between Myf6-KO and WT mice at postnatal day 21 (n=3 animals per group). (e) Immunofluorescent analysis of TA muscle cross-sections from Myf6-KO and WT mice with PAX7 and KI67 antibodies at 3 months of age. (f) Quantification of the percentage of KI67 + over PAX7 + MuSCs between Myf6-KO and WT mice at 3 months of age (n=5 animals per group). (g) Schematic drawing of the strategy for FACS isolation of Myf6-KO and WT satellite cells for RNA-Seq library preparation or for culture and differentiation. (h) (Left) Heat Map of up and down-regulated genes in the p38 MAPK pathway (WP400) in Myf6-KO and WT satellite cells. (Right) Gene set enrichment analysis of p38 MAPK pathway genes. In each plot, the blue curve represents the gene set enrichment score profile across MAPK pathway genes that are ranked by their differential expression. The grey curve represents the null expectation. P-values are based on permutation test (10,000 permutations). (i) Immunofluorescent analysis of WT and Myf6-KO primary myotubes cultured in differentiation media for 7 days and stained with MF20 and DAPI. (j) Quantification of the fusion index between Myf6-KO and WT 7DM myotubes. Fusion index is calculated as the percentage of myonuclei out of the total number of nuclei per field of view. (k) EDL myofibers isolated from WT and Myf6-KO mice fixed at time T=0 and stained with PAX7, MYOD and DAPI. (l) Quantification of the average percentage of MYOD + /PAX7 + satellite cells per mouse in Myf6-KO and WT myofibers (n=3). (m) Quantification of the percentage of MYOD + /PAX7 + satellite cells per fiber in Myf6-KO vs WT myofibers. (n) Staining for phospho-p38 MAPK on EDL myofibers cultured for 48 hours from Myf6-KO and WT mice. (o) Quantification of the average percentage of phospho-p38 + /PAX7 + satellite cells per mouse in Myf6-KO vs WT myofibers (n=3). (p) Quantification of the percentage of phospho-p38 + /PAX7 + satellite cells per fiber in Myf6-KO vs WT myofibers.

Article Snippet: Subsequently, muscle sections were incubated with the primary antibodies (Pax7, concentrated DSHB 1:100 dilution; Laminin (LAMA1), Sigma 1:750 dilution) in goat serum with BSA blocking solution overnight in a humid chamber at 4 ° C. The next day, slides were washed twice for 10 minutes each time in PBT (0.05% Triton-X 100 in PBS, v/v), and once for 10 minutes in PBS.

Techniques: Isolation, RNA Sequencing, Quantitative Proteomics, Cell Culture, Staining

Journal: bioRxiv

Article Title: Myf6/MRF4 is a Myogenic Niche Regulator Required for the Maintenance of the Muscle Stem Cell Pool

doi: 10.1101/691386

Figure Lengend Snippet: (a) Schematic drawing of MuSC transplantation and in vivo bioluminescence experiments. Briefly, 10 000 MuSCs isolated by FACS from CAG-Luc-eGFP mice were injected into the irradiated hindlimb of immunocompromised WT or Myf6-KO mice. Bioluminescent imaging was performed at day 1, 7, 14 and 21 post donor MuSC injections. (b) Representative images of bioluminescent signal from hindlimbs of Myf6-KO and WT counterparts at day 1, 7, 14, and 21 post transplantation. (c) Time course of average bioluminescent signal (total flux in radiance) in Myf6-KO and WT mice at 1, 7, 14 and 21 days post MuSC transplantation showing engraftment dynamics of donor MuSCs (mean across 4-5 animals per group ± SEM at each time point). (d) Bioluminescent signal (radiance) in Myf6-KO and WT mice at each time point normalized to the bioluminescent signal at day 1 post MuSC transplantation. (n=4-5 animals per group ± SD) (e-f) Immunostaining of TA cross-sections from WT (e) and Myf6-KO (f) mice stained with laminin (LAMA1) and PAX7 antibodies. Acute muscle injury was performed by injection of Cardiotoxin (CTX) into the TA muscle of Myf6-KO and WT mice (n=5 animals per group). (g) Quantification of the number of PAX7 + cells per unit area of TA muscle cross sections between Myf6-KO and WT mice (n= 5 animals per group, p-value is based on two tailed t-test). (h) Immunofluorescent analysis of Myf6-KO and WT EDL myofibers cultured in growth media for 48 hours and stained with PAX7 and MYOD antibodies. (i) Quantification of the number of PAX7 + MuSCs per fiber in Myf6-KO and WT mice after 48 hours in culture. (j) Immunofluorescence staining of WT EDL myofibers cultured in growth media for 48 hours and treated with LIF (1000U/ml) or EGF (80ng/ml) every 8 hours. Fibers were stained with antibodies against PAX7 and MyoD. (k) Quantification of the number of PAX7 + and MYOD + cells on the EDL myofiber treated with recombinant LIF, EGF or vehicle control. (l) EdU staining of cultured primary myoblasts isolated by FACS (see materials and methods) from Myf6-KO and WT mice. Staining was analyzed after 6, 12 and 24 hours of EdU incorporation. (m) Quantification of the percentage of EdU + myoblasts after 6, 12, and 24 hours of EdU incorporation. (n) Schematic drawing of the mechanism of Myf6 action in maintaining MuSC homeostasis. Briefly, myofibers expressing Myf6 secrete myokines that block the activation of the p38 MAPK pathway, which is necessary for maintenance of the MuSC pool.

Article Snippet: Subsequently, muscle sections were incubated with the primary antibodies (Pax7, concentrated DSHB 1:100 dilution; Laminin (LAMA1), Sigma 1:750 dilution) in goat serum with BSA blocking solution overnight in a humid chamber at 4 ° C. The next day, slides were washed twice for 10 minutes each time in PBT (0.05% Triton-X 100 in PBS, v/v), and once for 10 minutes in PBS.

Techniques: Transplantation Assay, In Vivo, Isolation, Injection, Irradiation, Imaging, Immunostaining, Staining, Two Tailed Test, Cell Culture, Immunofluorescence, Recombinant, Control, Expressing, Blocking Assay, Activation Assay