Journal: Nucleic Acids Research

Article Title: Repeat-rich RNA guides repetitive genomic elements into biomolecular condensates for heterochromatin organization and muscle integrity

doi: 10.1093/nar/gkag168

Figure Lengend Snippet: CU-rich RNA promotes heterochromatin condensate organization during differentiation. ( A ) Nuclei of C2C12 myotubes (MT) treated with 1.5% 1,6-hexanediol (1,6-HD) or 1.5% 2,5-hexanediol (2,5-HD). Left: Representative images of 4,6-diamidino-2-phenylindole (DAPI)-stained nuclei. Scale bar, 5 μm. Middle: Time-course quantification of the number of heterochromatin foci per nucleus. Right: Boxplots show foci area (μm 2 , y -axis) at 5, 10, and 15 min posttreatment ( x -axis). n = 55 nuclei, three biological replicates. ( B ) Representative live-cell images of Hoechst 33342-stained MT nuclei before and after 1,6-HD treatment (0 and 15 min, respectively), taken from . Arrows indicate changes in heterochromatin foci intensity (pink, increased; blue, decreased), and the red arrow highlights an alteration in chromocenter integrity. ( C ) Number of heterochromatin foci per nucleus in MT following recovery from 1.5% 1,6-hexanediol (1,6-HD) treatment for 15 min, measured at indicated time points. n = 68 nuclei, three biological replicates. ( D ) Representative images of nuclei of myoblast (MB) and MT with or without 1,6-HD treatment (1.5%, 15 min). Right: Quantification of the number of heterochromatin foci per nucleus. n = 40 (MB), n = 60 (MT), three biological replicates. ( E ) Quantification of colocalization between indicated proteins and DAPI foci in MT with or without 1,6-HD treatment (1.5%, 15 min), by Pearson’s correlation coefficients. n = 60, three biological replicates. ( F ) Boxplot showing the distribution of Z -score of the interchromosomal interaction frequencies in MB and MT. ( G ) RNA FISH using ChRO1 and LacZ biotinylated probes. Biotin signal was detected by Fluorescein-conjugated Avidin DCS and amplified with biotinylated anti-Avidin and additional Fluorescein Avidin DCS. Right: Quantification of colocalization between biotin signal and DAPI foci. n = 50 nuclei. ( H ) Number of heterochromatin foci per nucleus of mouse fibroblast cells (NIH3T3) with or without doxycyline (Dox)-induced ChRO1a expression and/or 1,6-HD treatment (1.5%, 15 min). (EV; empty vector). n = 50, three biological replicates. ( I ) Number of heterochromatin foci per nucleus in MB with or without Dox-induced ChRO1a fragment (1–413, CUR) expression and/or 1,6-HD treatment (1.5%, 15 min). n = 75, three biological replicates. Statistical analyses and data presentation details are described in the “Materials and methods” section.

Article Snippet: C2C12 murine myoblast cells and NIH3T3 mouse fibroblast cells were obtained from the American-type culture collection and grown in a growth medium (GM) consisting of Dulbecco’s modified Eagle medium (DMEM) with 10% (v/v) fetal bovine serum at 37°C and 5% CO 2 .

Techniques: Staining, Avidin-Biotin Assay, Amplification, Expressing, Plasmid Preparation

Journal: Scientific Reports

Article Title: Freeform micropatterning of living cells into cell culture medium using direct inkjet printing

doi: 10.1038/s41598-017-14726-w

Figure Lengend Snippet: Demonstration of various cell patterns that were directly printed into culture medium. ( a ) Printed cell migration assay model with 1-mm and 0.5-mm gaps. Images were captured at 2 hours and 48 hours after printing. Scale bar: 500 µm. ( b–e ) Fluorescent images of red-, green-, and blue-labeled cell patterns. Cells were pre-labeled and printed in patterns. Scale bar: 1 mm. ( f ) Printed zigzag pattern using a heterotypic co-culture model of NIH3T3 and HEK293A cells. Scale bar: 1 mm.

Article Snippet: Mouse NIH3T3 fibroblasts and HEK293A human embryonic kidney cells were provided by Prof. Kyungtae Kim, POSTECH, Korea.

Techniques: Cell Migration Assay, Labeling, Co-Culture Assay

Journal: Scientific Reports

Article Title: Murine neonatal cardiac regeneration depends on Insulin-like growth factor 1 receptor signaling

doi: 10.1038/s41598-024-72783-4

Figure Lengend Snippet: Efficient IGF1R knockdown in the neonatal heart. A Time course of Igf1r mRNA expression in vivo in whole hearts during postnatal (P) days 1 to 7, analyzed by qPCR. qPCR was performed in biological triplicates. B In vitro KD efficiency of the Renilla control, and the three indicated Igf1r KD hairpins used for in vivo and in vitro experiments. Mouse embryonic fibroblasts (MEFs) expressing a dTomato reporter tagged with target sites of the probed shRNAmir were transduced with the indicated shRNAs. Note that “expression” refers to the expression of the dTomato reporter tagged with the target site for the responding shRNAmirs. Therefore, the Ren.713 hairpin reduced expression of the reporter tagged with the Ren target site, not with the Igf1r target sites. d = day. C Immunoblotting of in vitro IGF1R KD efficiency using the indicated hairpins, the Renilla control hairpin delivered by retroviruses, and the empty retrovirus control, tested in NIH3T3 cells. Cells were harvested on day 4 after 1 st infection. GAPDH protein expression is shown as a loading control. D In vitro Igf1r knockdown efficiency of the respective siRNAs and controls in neonatal mouse cardiomyocytes determined by qPCR. The target gene expression levels were normalized to the house keeping gene TATA-binding protein (TBP). E Representative Western blot of in vitro Igf1r knockdown efficiency of the respective siRNA and controls in neonatal mouse cardiomyocytes. For Western blotting, two runs of neonatal mouse cardiomyocyte cell culture with four cell culture wells per siRNA were performed; four cell culture wells were pooled per Western blot sample. Graph indicates mean relative signaling intensity of IGF1R relative to GAPDH ± SD, normalized to Ren.713. F In vivo luciferase imaging to confirm recombinant adeno-associated virus (rAAV) infection of the heart. The upper panel shows the plasmid sequence of the rAAV used, containing a luciferase as a reporter gene under the control of a cardiomyocyte specific troponin T promoter. The lower panel shows two mice after injection of the rAAVs on the left and a control mouse on the right into which no rAAVs were injected. Color represents the light emitted by the luciferase. While there is some leakiness of the reporter in the liver, constructs are expressed exclusively in the hearts of the mice. ITR = Inverted terminal repeat. TropT = Cardiomyocyte specific troponin T promoter. Luc2 = Firefly luciferase. MCS = Multiple cloning site. PolA = Poly-A tail. G Representative images of immunohistochemical staining of hearts to confirm in vivo plasmid expression. The upper panel shows the plasmid sequence containing GFP as a reporter gene, under the control of a cardiomyocyte specific troponin T promoter. The lower panels show the sections stained for GFP expression. Hairpins were subcloned into the multiple cloning site (MCS). The panels on the left show a negative control heart, the panels on the right represent a heart 5 days after injection of the rAAVs constructs. Brown marks GFP. Blue represents counterstaining with Hematoxylin. Areas chosen for magnification are marked with a black box. The red triangle marks the aortic valve, the orange triangle the aorta. Both do not express GFP, implicating the construct is not expressed in these tissues, i.e. fibroblasts, myofibroblasts, and smooth muscle cells, in contrast to cardiomyocytes (cyan triangle and bottom magnification panel). ITR = Inverted terminal repeat. TropT = Cardiomyocyte specific troponin T promoter. GFP = Green fluorescent protein. PolA = Poly-A tail. H Representative Western blots of in vivo rAAV KD efficiency of the three Igf1r KD hairpins compared to the Ren.713 control hairpin. Hearts were harvested on P5 after rAAV injections on day one after birth and blotted for IGF1R and GAPDH protein levels. Bar graphs indicate mean relative signaling intensity of IGF1R relative to GAPDH ± SD, normalized to Ren.713. Values are normalized to the Ren.713 group. One-way ANOVA with Dunnett’s correction for multiple comparisons was used for statistical analysis in panels A, D, E. Unpaired two-tailed Student’s t-Test were used for statistical analysis in panel H. Full blots are supplied in Supplementary Fig. 3.

Article Snippet: NIH3T3 mouse fibroblasts (Merck KGaA, Darmstadt, Germany) were transduced with the retroviral particles.

Techniques: Knockdown, Expressing, In Vivo, In Vitro, Control, Transduction, Western Blot, Infection, Targeted Gene Expression, Binding Assay, Cell Culture, Luciferase, Imaging, Recombinant, Virus, Plasmid Preparation, Sequencing, Injection, Construct, Cloning, Immunohistochemical staining, Staining, Negative Control, Two Tailed Test

Journal: Cell reports

Article Title: Sarcosine Is Uniquely Modulated by Aging and Dietary Restriction in Rodents and Humans

doi: 10.1016/j.celrep.2018.09.065

Figure Lengend Snippet: (A) Mouse fibroblasts in culture (NIH 3T3 cells) expressing the tandem reporter mCherry-GFP-LC3 were exposed to the indicated concentrations of sarcosine for16 hr in the absence or presence of other autophagy inducers. Representative images of both controls and cells treated with 500 μM sarcosine in the presence or absence of serum are shown. (B) Quantification of autophagic flux (number of autophagosomes matured into autolysosomes) at the indicated sarcosine concentrations shows a dose responseto sarcosine (n > 2,500 cells). (C–E) Number of autophagic vacuoles (AVs; C), autophagosomes (APGs; D) and autolysosomes (AUTs; E) in cells treated with sarcosine (500 μM) show increased induction (vesicle count) and efficient clearance (AUT/APGs) of APGs (n > 2,500 cells). (F) Degradation of the autophagic cargo p62 in cells treated with increasing concentrations of sarcosine. Top: representative immunoblot. Bottom: quantification of the changes in p62 upon addition of lysosomal inhibitors ammonium chloride and leupeptin ammonium chloride and leupeptin (N/L) (n > 4 cells per condition). (G) A comparative analysis of several well-known inducers of autophagy with sarcosine. Sarcosine is more effective than metformin at inducing autophagy but less effective than rapamycin and spermidine (n > 2,500 cells). Quantification was done using high-content microscopy. Differences with untreated (“none”) are indicated. (H) Effect of the indicated treatments alone or in combination with 500 μM sarcosine on autophagic flux in cultured mouse fibroblasts. Several methods of autophagic induction were investigated, including oxidative damage (paraquat [PQ]), ER stress (thapsigargin [TG]) and lipotoxicity (oleic) in addition to serum-starved induction. Sarcosine showed an additive effect to serum starvation and paraquat, suggesting alternate mechanisms of activation, but not to thapsigargin or lipotoxicity (n > 2,500 cells). (I and J) Representative immunoblots (I) and densitometry analysis (J) in 3T3 cells demonstrate that sarcosine activates the mTOR signaling pathway in cells, but that this occurs in concert with Ulk and LAMP1 activation, suggesting that AMPK activity is also increased, thereby permitting increased autophagy in spite of mTOR activation. All results were obtained from a minimum of 3 independent experiments unless otherwise stated. Bars and lines indicate means ± SEMs (n = 3–4 per treatment). Significantly different from control: *p < 0.05, **p < 0.01, and ***p < 0.001.

Article Snippet: Human: NIH 3T3 mouse fibroblasts , Coriell Institute , N/A.

Techniques: Expressing, Western Blot, Microscopy, Cell Culture, Activation Assay, Activity Assay, Control

Journal: Cell reports

Article Title: Sarcosine Is Uniquely Modulated by Aging and Dietary Restriction in Rodents and Humans

doi: 10.1016/j.celrep.2018.09.065

Figure Lengend Snippet: (A) Autophagic flux increases in rat liver after short-term (10 days) dietary sarcosine feeding. Treatment with the inhibitors of lysosomal proteolysis (20 mM ammonium chloride and 100 μm leupeptin; N/L) revealed that degradation of LC3 and p62 in lysosomes was accelerated in the sarcosine-treated group. Representative immunoblot (left) and quantification of the levels and flux of LC3 and p62 (right) are both shown. n = 3 per group. (B) Effect of a short-term (10 days) dietary sarcosine treatment on the autophagic compartments in aged rat liver. Low-magnification images (left) and examples of the autophagic compartments more abundant in each of the groups (APGs in untreated and autolysosomes [AUTs] in sarcosine treated). Red arrows indicate AUTs, and yellow arrows indicate APGs. Quantification of the number of AVs, APGs, AUTs and lysosomes (LYSs) per section (left) and the percentage of AVs that display characteristics of APGs or AUTs (right) are shown. Data reveal improved maturation of APGs into AUTs after sarcosine treatment, which is indicative of increased autophagic flux (n = 20 sections from 3 different animals). More examples of each compartment are shown in Figure S7E. (C) Effect of old control and sarcosine-treated rat serum on autophagy. Heat-inactivated serum collected from old control or sarcosine-treated rats at 9 a.m. was added to the culture media of NIH 3T3 cells stably expressing the autophagy reporter mCherry-GFP-LC3. Cells were imaged, and the number per cell of AVs (mCherry+ vesicles), APGs (mCherry+ and GFP+ vesicles), and AUTs (ALs, mCherry+ GFP– vesicles) were quantified using high-content microscopy (n > 2,500 cells). Differences with controls (supplemented with serum from untreated rats) were significant at most serum concentrations tested. (D and E) Evaluation of signaling pathway activation in old rat liver following a short-term (10 days) dietary sarcosine treatment, as demonstrated by representative western blots (D) and the corresponding densitometry measurements (E), revealed that sarcosine increased pS6 in vivo, while a tendency toward increased activation of Akt and AMPK was observed (n = 8 per group). All results were obtained from a minimum of 3 independent experiments unless otherwise stated. Lines and bars indicate means ± SEMs. Significantly different from controls: *p < 0.05, **p < 0.01, and ***p < 0.001.

Article Snippet: Human: NIH 3T3 mouse fibroblasts , Coriell Institute , N/A.

Techniques: Western Blot, Control, Stable Transfection, Expressing, Microscopy, Activation Assay, In Vivo

Journal: Cell reports

Article Title: Sarcosine Is Uniquely Modulated by Aging and Dietary Restriction in Rodents and Humans

doi: 10.1016/j.celrep.2018.09.065

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Human: NIH 3T3 mouse fibroblasts , Coriell Institute , N/A.

Techniques: Virus, Recombinant, Protease Inhibitor, Staining, Western Blot, Fluorescence, Software, Modification, Microscopy, Imaging, Transmission Assay