Journal: eLife

Article Title: LIN37-DREAM prevents DNA end resection and homologous recombination at DNA double-strand breaks in quiescent cells

doi: 10.7554/eLife.68466

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Article Snippet: The following antibodies were used for western blot analysis: 53BP1 (Bethyl Laboratories, A300-272A, 1:3000), LIN37 (Santa Cruz Biotechnology, sc-515686, 1:200), BLM (Bethyl Laboratories, A300-572A, 1:2000), BRCA1 for mouse (R and D Systems, gift from Dr. Andre Nussenzweig, NCI, 1:1000) , BRCA1 for human (Millipore Sigma, 07-434, 1:1000), RAD51 (Millipore Sigma, ABE257, 1:2000), BARD1 (Thermo Fisher Scientific, PA5-85707, 1:1000), CtIP (gift from Dr. Richard Baer, [Columbia University, New York], 1:1000), MRE11 (Novus Biologicals, NB100-142, 1:2000), RIF1 (Abcam, ab13422, 1:500), SHLD1/C20orf196 (Thermo Fisher Scientific, PA5-559280, 1:200), GAPDH (Sigma, G8795, 1:10,000), KAP1 (Genetex, GTX102226, 1:2000), FANCD2 (R and D Systems, MAB93691, 1:1000), BRCA2 for human (Proteintech, 19791-1-AP, 1:500), Rb1 (Thermo Fisher Scientific, LF-MA0173, 1:1000), Phospho-Rb (Ser780) (Cell Signaling Technology, 8180T, 1:1000), Phospho-Rb (Ser807/811) (Cell Signaling Technology, 8516T, 1:1000), PCNA (Bethyl Laboratories, A300-276A, 1:3000), CDK4: (Novus Biologicals, NBP1-31308, 1:1000), CDK4 (phosphor Thr 172) (GeneTex, GTX00778, 1:1000), and RPA (Cell Signaling Technology, 2208S, 1:1000).

Techniques: Recombinant, Plasmid Preparation, Genome Wide, CRISPR, Clone Assay, Flow Cytometry, Staining, Sequencing, Software, Microscopy

Journal: bioRxiv

Article Title: Fast-twitch myofibrils grow in proportion to Mylpf dosage in the zebrafish embryo

doi: 10.1101/2024.09.18.613721

Figure Lengend Snippet: The mylpfa gene is expressed more abundantly than mylpfb in fast-twitch muscle. (A) Illustration of a sarcomere and an image of a fast-twitch myofibril at 26 hpf, with MyHC labeled green using A4.1025 and F-actin labeled magenta using phalloidin. (B) Illustration of mylpfa and mylpfb gene structure and the location of frameshifting alleles used in this study. Shown features include the 5’ UTR (brown), coding sequence (purple), 3’ UTR (gray), and frameshift locations (green arrows). (C) Overlay of predicted protein structures generated using Robetta, showing a high degree of expected similarity between zebrafish Mylpfa, Mylpfb, and human MYLPF proteins. (D) Illustration of Mylpfa and Mylpfb proteins, with arrowheads marking frameshift locations. (E) Chromatogram showing the gRNA target in wild-type sequence (top) and the 5 bp mylpfb oz39 lesion sequenced from a homozygous mutant (bottom). (F-F’’’) HCR ISH imaged in somites over the mid-yolk tube of a 36 hpf embryo. Shown as a single channel for mylpfa (F), mylpfb (F’) or the slow muscle marker myl10 (F’’), and as a merged image (F’’’). (G) HCR ISH shows relative expression levels for mylpfa and mylpfb through embryonic development. (H) Box plot showing the brightness of mylpfa mylpfb in the HCR ISH images, with mylpfa : mylpfb ratios shown per time-point. (I) FPKM values for mylpfa and mylpfb at 27 hpf, from a previously reported RNA-seq dataset . (J) Ratio of Mylpfa to Mylpfb band intensity in western blot; points represent biological replicates of pooled animals. (K) Image of a western blot showing Mylpfb and Mylpfa protein abundance at 24, 36, 48, and 72 hpf. (L) Western blot showing Mylpfb and Mylpfa protein in the wild-type and the mylpfa -/- mutant which lacks the Mylpfa band. Significance threshold determined by Tukey-Kramer comparison after one-way ANOVA; * P<0.05, ** P<0.01. Scalebar in F is for F-F ’’’.

Article Snippet: We used primary antibodies for the myonuclei (1:500, Rbfox1l) ; α-Actinin (1:500, A7732, Sigma); Myomesin [1:30, mMac, Developmental Studies Hybridoma Bank (DSHB)] , MyHC (1:1000, A4.1025, DSHB) , and Mylpf (1:2000, DZ41336, Boster).

Techniques: Labeling, Sequencing, Generated, Mutagenesis, Marker, Expressing, RNA Sequencing, Western Blot, Quantitative Proteomics, Comparison

Journal: bioRxiv

Article Title: Fast-twitch myofibrils grow in proportion to Mylpf dosage in the zebrafish embryo

doi: 10.1101/2024.09.18.613721

Figure Lengend Snippet: Zebrafish mylpfa is necessary for fast-twitch myofibril formation. (A-B’’) Three views of somites are illustrated above (A) and imaged in a wild-type animal and mylpfa -/- mutant sibling, each imaged at 48 hpf. (C) Box plot of myofibril width, measured from sagittal confocal slices. (D) Plot of muscle cross-sectional area (CSA) measured from the orthogonal view of confocal stacks from the wild-type and mylpfa -/- mutant siblings. (E) Example of a western blot for MyHC and Mylpf, including Mylpfa and Mylpfb, in the wild-type (WT) and the mylpfa -/- mutant (-/-) samples at 72 hpf, with ( E’) quantification shown as a box plot. (F-G’’’) Fast muscle myofibers, labeled at 48 hpf, showing co-label for MyHC (A4.1025), M-line (anti-Myomesin), and F-actin (phalloidin), shown as single channel or overlays. (H, I) Actinin label on comparable samples. (J) Box plot of sarcomere lengths in the mylpfa -/- mutant and their wild-type siblings, showing no change in length. (K) Plots showing that the mylpfa -/- mutant shows reduced F-actin sarcomeric periodicity (gray bars). Lightly colored regions indicate bootstrap confidence intervals. (L) Box plot showing sarcomeric fraction for each marker, calculated on a 0-1 scale, as described in . Throughout the figures, the wild-type plots are blue and the mylpfa -/- plots are red. Points within box plots represent the individual animals or a single western blot image. Scalebar in A is for A-B’’, in F is for F-G’’’, in H is for H, I. Significance thresholds for multiple comparisons determined by Tukey-Kramer HSD comparisons after one-way ANOVA; pairwise comparisons use Student’s T-test and matching results are found with Krustal-Wallis exact test. Not significant (n.s.) is P>0.1, * P<0.05, ** P<0.01, *** P<0.001.

Article Snippet: We used primary antibodies for the myonuclei (1:500, Rbfox1l) ; α-Actinin (1:500, A7732, Sigma); Myomesin [1:30, mMac, Developmental Studies Hybridoma Bank (DSHB)] , MyHC (1:1000, A4.1025, DSHB) , and Mylpf (1:2000, DZ41336, Boster).

Techniques: Mutagenesis, Western Blot, Labeling, Marker

Journal: bioRxiv

Article Title: Fast-twitch myofibrils grow in proportion to Mylpf dosage in the zebrafish embryo

doi: 10.1101/2024.09.18.613721

Figure Lengend Snippet: Levels of myofibril formation correspond to dosages predicted by mylpfa and mylpfb loss of function. (A-D) 3D renders of confocal stacks show normal myofibril structure in slow muscle fibers across Mylpf genotype. (E-H) Medial slices show a portion of myotome rich in fast-twitch fibers, with robust myofibrils in the wild-type sibling (E) and the mylpfb -/- mutant (F), but overt myofibrillar defect in the mylpfa -/- mutant (G) and total loss of myofibrils in the mylpfa -/- ;mylpfb -/- double mutant (H). Zoomed images show myofibrillar structure within fast-twitch muscle fibers (E’-H’). (I) Box plots of myofibril widths in slow-twitch and fast-twitch muscle. Slow and fast-twitch widths plotted separately because the slow-twitch fibers were measured on 3D rendered images and the fast-twitch fibers were measured on confocal slices. (J) Scatterplot showing the same myofibril width data from fast-twitch muscle (in I) replotted as a correlate with predicted protein dosage at 24hpf, with each allele scaled 6:1 for Mylpfa:Mylpfb ratio. (K) Box plots showing the fraction of sarcomeric MyHC localization. (L) Box plots showing myofibril widths in 72 hpf phalloidin-labeled animals. (M) Scatterplot of the same data with each allele scaled 6:1 for Mylpfa:Mylpfb. (N-P) Transmission electron microscopy showing normal sarcomere structure in the wild-type sibling (N), partial sarcomeric disarray in the mylpfa -/- mutant (O) and only scattered sarcomeric components in the mylpfa -/- ;mylpfb -/- double mutant (P). Scale bars in D, H, and H’ are 10 µm, applicable to their row. Scale bar in N is 1 µm. Significance thresholds: not significant (n.s.) is P>0.1, ** P<0.01, *** P<0.001 as determined by Tukey-Kramer HSD comparisons after one-way ANOVA.

Article Snippet: We used primary antibodies for the myonuclei (1:500, Rbfox1l) ; α-Actinin (1:500, A7732, Sigma); Myomesin [1:30, mMac, Developmental Studies Hybridoma Bank (DSHB)] , MyHC (1:1000, A4.1025, DSHB) , and Mylpf (1:2000, DZ41336, Boster).

Techniques: Mutagenesis, Labeling, Transmission Assay, Electron Microscopy

Journal: Cell reports

Article Title: Integrating Gene and Protein Expression Reveals Perturbed Functional Networks in Alzheimer’s Disease

doi: 10.1016/j.celrep.2019.06.073

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Article Snippet: Normal Horse Serum , Vector Laboratories , Cat# S-2000; RRID: AB_2336617.

Techniques: Plasmid Preparation, Recombinant, Software, Microscopy