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: Endocytic recycling protein EHD1 regulates primary cilia morphogenesis and SHH signaling during neural tube development

doi: 10.1038/srep20727

Figure Lengend Snippet: ( A ) WT NIH3T3 cells stably expressing SMO-GFP were starved in low serum media for 24 hours and stimulated with SAG in starvation media for another 24 hours. Under these conditions EHD1 was seen to co-localize with SMO in the primary cilia. A profile scan for SMO and EHD1 in the primary cilia merged panel is shown. Under these conditions, EHD1 was seen to traffic to the cilia of 60% cells studied. ( B ) WT NIH3T3 cells stably expressing SMO-GFP and transiently expressing EHD1-DsRed were starved in low serum media for 24 hours and stimulated with SAG immediately before starting live imaging of Smoothened and EHD1.As reported in earlier studies, Smoothened was found in preciliary vesicles under non-stimulated conditions but upon SHH pathway activations, EHD1 was seen to associate with Smoothened vesicles and co-traffic with SMO into the primary cilia.

Article Snippet: NIH3T3 SMO-GFP cells were generated by transfecting NIH3T3 cells with a SMO-GFP reporter construct (Genecopoeia #MPRM17869-LvPF02) and selecting stable clones after FACS sorting.

Techniques: Stable Transfection, Expressing, Imaging

Journal: Scientific Reports

Article Title: Endocytic recycling protein EHD1 regulates primary cilia morphogenesis and SHH signaling during neural tube development

doi: 10.1038/srep20727

Figure Lengend Snippet: ( A ) Immunoblotting was used to assess the presence of SMO in anti-EHD1 immunoprecipitates from WT NIH3T3 cells stably expressing SMO GFP treated with SAG (24 hrs). Input samples show the amount of each protein in the whole extract before the IP. Immunoblots showing the amount of SMO that co-precipitated with EHD1 from cells that stably expressed the protein and were treated with SAG for 24 hrs. A control IP was performed with non-specific rabbit IgG instead of EHD1 antibody and WT NIH3T3 cell lysates stably expressing SMO GFP treated with SAG (24 hrs). Arrows denote the molecular weight fraction of SMO that associates with EHD1. Full-length blots/gels are presented in . ( B ) WT NIH3T3 cells stably expressing SMO-GFP were starved in low serum media for 24 hours and stimulated with SAG in starvation media for another 24 hours and lysed and these lysates were incubated with GST-EHD1 or GST-EH domain deleted EHD1. The membranes were probed with antibodies to Smoothened and GST.GST-fused to the empty plasmid vector was used as a negative control. The data is representative of three separate experimental repeats. Full-length blots/gels are presented in .

Article Snippet: NIH3T3 SMO-GFP cells were generated by transfecting NIH3T3 cells with a SMO-GFP reporter construct (Genecopoeia #MPRM17869-LvPF02) and selecting stable clones after FACS sorting.

Techniques: Western Blot, Stable Transfection, Expressing, Control, Molecular Weight, Incubation, Plasmid Preparation, Negative 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) 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

Journal: Theranostics

Article Title: Open Porous Microenvironment-regulatory Microspheres Loaded with Curcumin@BSA NPs/BMSCs for Diabetic Wound Treatment

doi: 10.7150/thno.120285

Figure Lengend Snippet: Angiogenesis and migration assays In vitro . (A) Images representing scratch test of NIH-3T3 cells in a high-glucose environment. (B) Images representing the in vitro tube formation test and measurement of the total length of HUVECs exposed to BMSC@COPM in a high-glucose setting (scale bars: 100 μm). (C) Quantitative analysis of the scratch test results. (D) Under high-sugar conditions, BMSC@COPM promoted the proliferation of HUVECs, and cell viability was determined by a CCK-8 assay (n = 6). (E) Quantification of total length of HUVECs. (F) The representative WB images of HUVECs post-treatment with microspheres loaded with BMSCs. (G and H) Quantitative evaluation of HIF-1α and VEGF. (I) The expression of VEGF protein in HUVECs measured via ELISA. (J) In HUVECs, the levels of angiogenesis-related mRNAs (PDGF, FGF, and VEGF) were quantified using qRT-PCR. vs the control group, * P < 0.05, ** P < 0.01 and *** P < 0.001; vs the BMSC@COPM group, ## P < 0.01 and ### P < 0.001. The data shown in the figure are presented as the means ± SDs (n = 3, except D).

Article Snippet: The mouse embryonic fibroblast line NIH-3T3 and the macrophage-like cell line RAW 264.7 were donated by the medical research center of Mudanjiang Medical University.

Techniques: Migration, In Vitro, CCK-8 Assay, Expressing, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Control

Journal: Theranostics

Article Title: Open Porous Microenvironment-regulatory Microspheres Loaded with Curcumin@BSA NPs/BMSCs for Diabetic Wound Treatment

doi: 10.7150/thno.120285

Figure Lengend Snippet: Autophagy induction by BMSC@COPM enhances macrophage polarization and promotes migration and proliferation. (A) Transwell assays of NIH-3T3 cells were observed under a microscope, which were treated with BMSC@COPM in a high-glucose environment (Scale bar: 100 μm). (B) Representative WB blot of CD206 and CD86 in RAW 264.7 cells after were treated with LPS, rapamycin or chloroquine, respectively. (C) Quantitative analysis of CD206 and CD86 protein expression in (B). (D) Quantification of the transwell assay results. (E) Representative images of LC3-II and P62 protein blots in NIH-3T3 cells treated with HG and then treated with CQ (10 μM) or BMSC@COPM. (F) Statistical analysis for P62 and the LC3-II/LC3-I ratio in (E). (G) Representative images of LC3-I/II and P62 protein blots in RAW 264.7 cells, which were induced with LPS and then treated with 10 μM rapamycin (an autophagy inducer, RAPA), 10 μM CQ, or BMSC@COPM. (H) Statistical analysis protein expression related to autophagyin. vs the control group, ** P < 0.01 and *** P < 0.001; vs the LPS group and HG group, $ P < 0.05, $$ P < 0.01 and $$$ P < 0.001; vs the BMSC@COPM group, # P < 0.05, ## P < 0.01, ### P < 0.001. The data shown in the figure are presented as the means ± SDs (n = 3).

Article Snippet: The mouse embryonic fibroblast line NIH-3T3 and the macrophage-like cell line RAW 264.7 were donated by the medical research center of Mudanjiang Medical University.

Techniques: Migration, Microscopy, Expressing, Transwell Assay, Control

Journal: Theranostics

Article Title: Open Porous Microenvironment-regulatory Microspheres Loaded with Curcumin@BSA NPs/BMSCs for Diabetic Wound Treatment

doi: 10.7150/thno.120285

Figure Lengend Snippet: The relationship between autophagy and the PI3K/Akt/mTOR signaling pathway. (A) TEM detected autophagosomes in NIH-3T3 cells exposed to BMSC@COPM under high glucose conditions (Scale bar: 2 μm, the red arrows represent autophagosomes). (B) Representative immunohistochemical images of LC3-I/II and P62 in different animal (50 μM). (C) Representative images of CD206 immunofluorescence in RAW264.7 macrophages after coculture with BMSCs. (D) Quantification analysis of protein expression of LC3-I/II. (E) Statistical analysis for p62 protein expression. (F) Quantification of CD206 protein expression. (G) Representative immunohistochemical images of p-Akt, p-PI3K and p-mTOR in different groups of mice (50 μM). (H) Representative images of CD86 immunofluorescence in RAW264.7 macrophages after coculture with BMSCs. (I-K) Statistical analysis for protein levels of p-Akt, p-PI3K, p-mTOR in (G). (L) Quantification analysis of CD86 protein expression in (H). vs the control group, * P < 0.001, ** P < 0.01 and *** P < 0.001; vs the BMSC@COPM group , ## P < 0.01, ### P < 0.001. The data shown in the figure are presented as the means ± SDs (n = 3).

Article Snippet: The mouse embryonic fibroblast line NIH-3T3 and the macrophage-like cell line RAW 264.7 were donated by the medical research center of Mudanjiang Medical University.

Techniques: Immunohistochemical staining, Immunofluorescence, Expressing, Control