Journal: Genes & Development

Article Title: Plagl1 and Lrrc58 control mammalian body size by triggering target-directed microRNA degradation of miR-322 and miR-503

doi: 10.1101/gad.353138.125

Figure Lengend Snippet: Validation of novel TDMD triggers. ( A ) Schematic of trigger site deletion (Δts) strategy to validate putative TDMD triggers. ( B , D ) Genomic organization of TDMD trigger transcripts with conservation and predicted miRNA base-pairing architecture of the trigger sites. Nucleotides predicted to base-pair with the miRNA are shown in bold. ( C , E ) qRT-PCR analysis of indicated miRNAs, relative to miR-16-5p, in WT and Δts MEFs. Parental MEFs or two independent Δts clones for each trigger site were infected with lentivirus expressing Cas9 and a nontargeting CRISPR guide (sgNT) or Zswim8- targeting guide (sg Zswim8 ). Values were normalized to expression level in sg Zswim8 for each condition. n = 3 technical replicates per clone, with individual data points plotted (mean ± SD shown). P -values were calculated by one-tailed Student's t -test. (*) P < 0.05, (**) P < 0.01, (***) P < 0.001, (****) P < 0.0001. ( F ) qRT-PCR analysis of the indicated miRNAs, relative to U6, in NIH3T3 cells at time points after serum starvation and restimulation. NIH3T3 cells expressing dCas9-KRAB were infected with lentivirus encoding a nontargeting guide (sgNT) or guides targeting Plagl1 or Lrrc58 . Parental NIH3T3 cells were infected with lentivirus expressing Cas9 and a nontargeting guide (sgNT) or Zswim8- targeting guide (sg Zswim8 ). Values were normalized to expression at the 0 h time point for each condition. n = 3 technical replicates per condition, with mean ± SD shown. P -values were calculated by one-tailed Student's t -test comparing sg Plagl1 (dCas9-KRAB) or sg Lrrc58 (dCas9-KRAB) with sgNT (dCas9-KRAB) or comparing sg Zswim8 (Cas9) with sgNT (Cas9). (*) P < 0.05.

Article Snippet: HEK293T and NIH3T3 cells were obtained from ATCC, and immortalized MEFs were generated previously by transfecting primary MEFs with a plasmid expressing SV40 large T antigen ( ).

Techniques: Biomarker Discovery, Quantitative RT-PCR, Clone Assay, Infection, Expressing, CRISPR, One-tailed Test

Journal: ACS applied materials & interfaces

Article Title: Cellular Uptake of Nanoparticles is Regulated by Integrin-Based Adhesion to the Extracellular Matrix

doi: 10.1021/acsami.5c18030

Figure Lengend Snippet: Characterization of cell spread area and focal adhesion formation on different coatings via immunostaining of cells after 24h incubation. (A) Cell spread area for NIH3T3 cells with (B) quantification of average focal adhesion size (n=39–40 cells per group from 3 independent experiments). (C) Cell spread area for RAW264.7 cells (n=45 cells per group from 3 independent experiments) and (D) ASCs with (E) quantification of average focal adhesion size (FAAS) (n=45 cells per group from 3 independent experiments). ( F ) Representative fluorescence images of focal adhesion formation via paxillin staining of NIH3T3, RAW264.7, and ASCs cells on different coating types. ( G ) Representative fluorescence images of cytoskeletal arrangement via phalloidin staining of NIH3T3, RAW264.7, and ASCs cells on different coating types. Violin plots indicate mean, median, quartiles and distribution (width). Bars indicate mean ± SEM. Cell area analyzed by One-way ANOVA with Tukey’s multiple comparison test. Symbols for statistical comparisons between indicated groups: * p ≤ 0.05; **p ≤ 0.01; or ## p<0.01 vs. all other groups. (scale bars = 40μm).

Article Snippet: Three different cell types, NIH3T3 fibroblasts (ATCC, Manassas, VA, USA, Cat. No. CRL-1658), RAW264.7 macrophages (ATCC, Manassas, VA, USA, Cat. No. TIB-71), and primary rat adipose-derived stem cells (ASCs), isolated from female 8–14 week-old Lewis rats (Charles River Laboratories, Charleston, SC, USA) were used.

Techniques: Immunostaining, Incubation, Fluorescence, Staining, Comparison

Journal: ACS applied materials & interfaces

Article Title: Cellular Uptake of Nanoparticles is Regulated by Integrin-Based Adhesion to the Extracellular Matrix

doi: 10.1021/acsami.5c18030

Figure Lengend Snippet: Images of PLGA nanoparticle (Green) uptake and quantification for NIH3T3 cells( A, B ), RAW264.7 cells( C, D ), and ASCs ( E, F ) on different coatings characterized via fluorescence microscopy with staining for nuclei (DAPI, Blue). The average fluorescence intensity of PLGA nanoparticles inside NIH3T3 cells ( B ), RAW264.7 cells ( D ), and ASCs ( F ) measured via ImageJ analysis of fluorescence images. Quantification was performed after 12h and 24h of incubation with PLGA nanoparticles. All data presented as mean ± SEM. (n>25 cells per group from 25 fields of view and 2 independent experiments). Two-way ANOVA with Tukey’s multiple comparison test. Symbols for statistical comparisons between indicated groups: * p ≤ 0.05; **p ≤ 0.01; *** p<0.001; or ## p<0.05 vs. all other groups. (scale bars = 40μm).

Article Snippet: Three different cell types, NIH3T3 fibroblasts (ATCC, Manassas, VA, USA, Cat. No. CRL-1658), RAW264.7 macrophages (ATCC, Manassas, VA, USA, Cat. No. TIB-71), and primary rat adipose-derived stem cells (ASCs), isolated from female 8–14 week-old Lewis rats (Charles River Laboratories, Charleston, SC, USA) were used.

Techniques: Fluorescence, Microscopy, Staining, Incubation, Comparison

Journal: ACS applied materials & interfaces

Article Title: Cellular Uptake of Nanoparticles is Regulated by Integrin-Based Adhesion to the Extracellular Matrix

doi: 10.1021/acsami.5c18030

Figure Lengend Snippet: PLGA nanoparticle uptake on NIH3T3 cells characterized via live imaging of cells for 60 min with 1 frame every 5 min via fluorescence microscope. ( A ) Average fluorescence intensity of PLGA nanoparticles inside the NIH3T3 cells measured via ImageJ analysis of fluorescence images. ( B ) Representative green PLGA fluorescence and Dil fluorescence images of NIH3T3 cells taken at the indicated time points on FN, COL, and LM coatings. All data presented as mean ± SEM (n=3–6 cells / group). Two-way ANOVA analysis with Tukey’s multiple comparison test were conducted. (scale bar = 40μm)

Article Snippet: Three different cell types, NIH3T3 fibroblasts (ATCC, Manassas, VA, USA, Cat. No. CRL-1658), RAW264.7 macrophages (ATCC, Manassas, VA, USA, Cat. No. TIB-71), and primary rat adipose-derived stem cells (ASCs), isolated from female 8–14 week-old Lewis rats (Charles River Laboratories, Charleston, SC, USA) were used.

Techniques: Imaging, Fluorescence, Microscopy, Comparison

Journal: ACS applied materials & interfaces

Article Title: Cellular Uptake of Nanoparticles is Regulated by Integrin-Based Adhesion to the Extracellular Matrix

doi: 10.1021/acsami.5c18030

Figure Lengend Snippet: Inhibition with Cytochalasin D limits focal adhesion formation and cytoskeletal arrangement of NIH3T3 cells on different coatings after 24h incubation of cells seeded with cytochalasin D. Representative fluorescence images of cytoskeletal arrangement via phalloidin staining and focal adhesion formation via paxillin staining of NIH3T3 cells on different ECM and ECM-mimetic coatings ( A ). Cells seeded on FN coatings without cytochalasin D was used as a control. PLGA nanoparticle uptake in NIH3T3 cells with or without the endocytosis pathway inhibitor chytochalasin D, characterized via fluorescence microscope (B) . Average fluorescence intensity of PLGA nanoparticles inside the NIH3T3 cells ( C ), after 12h and 24h incubation of PLGA nanoparticles with the cells seeded with/without Cytochalasin D on different coating types. All data presented as mean ± SEM (n= 2 with 15 images each sample). Two-way ANOVA analysis with Tukey’s multiple comparison test were conducted. ****p ≤ 0.0001; *** p ≤ 0.001; * p ≤ 0.05

Article Snippet: Three different cell types, NIH3T3 fibroblasts (ATCC, Manassas, VA, USA, Cat. No. CRL-1658), RAW264.7 macrophages (ATCC, Manassas, VA, USA, Cat. No. TIB-71), and primary rat adipose-derived stem cells (ASCs), isolated from female 8–14 week-old Lewis rats (Charles River Laboratories, Charleston, SC, USA) were used.

Techniques: Inhibition, Incubation, Fluorescence, Staining, Control, Microscopy, Comparison