Journal: Biochemical and biophysical research communications

Article Title: Sortilin facilitates VLDL-B100 secretion by insulin sensitive McArdle RH7777 cells

doi: 10.1016/j.bbrc.2016.07.096

Figure Lengend Snippet: (A) VLDL-apo B and VLDL-B100 secretion were measured following 18 h incubation of McA cells in cDMEM (black bars) and in 1% BSA/DMEM (gray bars) by immuno slot blotting. (B) Representative standard curve generated from an immuno slot blot (duplicate standards) where VLDL protein is plotted against average chemiluminescence signal in arbitrary units (AU) generated using anti-rat B100 monoclonal antibody and HRP-anti-mouse IgG. (C) Immunoblots of McA cell lysates from four independent experiments comparing McA cells incubated in cDMEM with 1% BSA/DMEM. Anti-GAPDH blotting display equal protein loading. (D) Relative expression of Apob, Sort1 and Atf3 mRNA in McA cells incubated in 1% BSA/DMEM as a percentage of mRNA present in McA cells incubated in cDMEM. Results are averages from 4 independent experiments.* Indicates means are significantly different.

Article Snippet: McA cells were transfected using Fugene6 according to manufacturer’s protocol (Promega Corp., Madison WI) using three different pGIPZ based vectors expressing shRNAi targeting rat Sort1 mRNA (V2LMM_58553, V3LMM_450660, V3LMM_450662), and one scrambled, non-silencing control (GE Healthcare Dharmacon, Lafayette, CO).

Techniques: Incubation, Generated, Dot Blot, Western Blot, Expressing

Journal: Biochemical and biophysical research communications

Article Title: Sortilin facilitates VLDL-B100 secretion by insulin sensitive McArdle RH7777 cells

doi: 10.1016/j.bbrc.2016.07.096

Figure Lengend Snippet: (A) Following siRNA-mediated KD of sortilin, three clones of McA cells were selected representing high (H, McA60), medium (M, McA62) and low (L, McA53) sortilin expression evaluated by immunoblotting compared with wild type (WT) McA cells. The effect of increasing sortilin KD on VLDL-B100 secretion by McA cells incubated in cDMEM (B) or incubated in 1% BSA/DMEM (C). Results in (B) and (C) are averages of triplicate plates for each condition (n = 2 studies). * Indicates means are significantly different from the SCR McA cell line.

Article Snippet: McA cells were transfected using Fugene6 according to manufacturer’s protocol (Promega Corp., Madison WI) using three different pGIPZ based vectors expressing shRNAi targeting rat Sort1 mRNA (V2LMM_58553, V3LMM_450660, V3LMM_450662), and one scrambled, non-silencing control (GE Healthcare Dharmacon, Lafayette, CO).

Techniques: Clone Assay, Expressing, Western Blot, Incubation

Journal: Biochemical and biophysical research communications

Article Title: Sortilin facilitates VLDL-B100 secretion by insulin sensitive McArdle RH7777 cells

doi: 10.1016/j.bbrc.2016.07.096

Figure Lengend Snippet: (A) McA cell lines with variable sortilin KD were incubated with vehicle (black bars) or with 10 μM cpd984 (gray bars) for 18 h (3-100 mm plates per condition). Viability of McA cells was not compromised by incubations with cpd984 as there was no significant release of LDH into the medium compared with control incubations. VLDL was isolated from media of each plate and VLDL-B100 was quantified by immuno slot blotting. Results presented are averages of the 3 plates ± S.D. (B) McA cells were incubated for 18 h in 1% BSA/DMEM ± 10 μM cpd984. McA cells were then stimulated with a time course of 250 nM insulin (0, 5, 10 and 15 min). IRβ and AKT, pY-IRβ, and p-AKT (S473) were evaluated by immunoblotting using protein and phosphospecific antibodies.

Article Snippet: McA cells were transfected using Fugene6 according to manufacturer’s protocol (Promega Corp., Madison WI) using three different pGIPZ based vectors expressing shRNAi targeting rat Sort1 mRNA (V2LMM_58553, V3LMM_450660, V3LMM_450662), and one scrambled, non-silencing control (GE Healthcare Dharmacon, Lafayette, CO).

Techniques: Incubation, Control, Isolation, Western Blot

Journal: G3: Genes|Genomes|Genetics

Article Title: RNA sequencing-based screen for reactivation of silenced alleles of autosomal genes

doi: 10.1093/g3journal/jkab428

Figure Lengend Snippet: Hit validation for 5-aza-dC. (A and B) Confirmation of Screen-seq results for 5-aza-dC-treated cells using an orthogonal method to measure AI. cDNA samples from day 7 of screening were assessed using ddPCR with allele-specific fluorescent probes. (A) Scatterplots for 20,000 droplets targeting the readout gene, Col6a5 . 5-aza-dC concentration is shown in the plots. Black : empty droplets; blue : droplets with the Cast paternal allele amplified (labeled by FAM fluorophore); red : droplets with the 129 maternal allele amplified (labeled by HEX fluorophore). Ratio of red: blue droplets are shown. AI value written in red is the maternal AI. Note that the double-positive droplets (orange) contained both maternal and paternal templates; a small number of such double-positives is expected with higher concentrations of biallelic template. These droplets are ignored in the quantitative analysis. (B) left— summary of AI measurements shown in (A) for Col6a5 on day 7; right— summary of AI measurements for Dnajc12 on day 7. (C and D) Biological replicate of Abl.1 cells were treated with 5-aza-dC and AI was measured using ddPCR. (C) Scatterplots representation as shown in (A) after 2 days of exposure. (D) Summary of AI measurement for Col6a5 (left) and Dnajc12 (right) after 2, 5, and 7 days of exposure (denoted by color). Gray vertical dashed lines for Col6a5 dose-response were used to determine “low,” “medium,” and “high” 5-aza-dC concentrations for the future experiments. Results for readout gene Adnp2 are in . (E–G) Analysis of Dnmt1 knock-down (KD) in Abl.1 cells. (E) Real-time quantitative PCR (RT-qPCR) analysis of Dnmt1 relative expression (expression in the empty vector control, normalized to Nono , taken as 1.0). Abl.1 cells were transduced with an empty plKO vector (control) or with two separate Dnmt1 shRNA knockdown constructs ( Dnmt1 KD construct 1 or 2) and grown for 2 days. Transduced cells were then selected by growing in the presence of a selection antibiotic for an additional 17 days. RT-qPCR quantification was performed on cells collected 19 days after transduction. Mean and SEM for three technical replicates are shown. (F) Representative scatterplots show AI measurement for Col6a5 in the transduced Abl.1 cells. AI was measured using ddPCR. (G) Summary of the AI measurement for Col6a5 (left) and Dnajc12 (right) after Dnmt1 KD.

Article Snippet: Two shRNA vectors targeting Dnmt1 (SHR000038801.1_TRC001.1 and SHR000373188.1_TRC005.1) and a control empty vector (NUL003.3_TRC021.1) packaged in lentiviral vectors obtained from the Genetic Perturbation Platform at the Broad Institute were tested.

Techniques: Biomarker Discovery, Concentration Assay, Amplification, Labeling, Knockdown, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Expressing, Plasmid Preparation, Control, Transduction, shRNA, Construct, Selection

Journal: Aging (Albany NY)

Article Title: NF-κB inducible miR-30b-5p aggravates joint pain and loss of articular cartilage via targeting SIRT1-FoxO3a-mediated NLRP3 inflammasome

doi: 10.18632/aging.203466

Figure Lengend Snippet: Primer sequences of each gene.

Article Snippet: SIRT1 overexpression plasmid (SIRT1) and its corresponding negative control (vector), small inference RNA (si-RNA) SIRT1 (si-SIRT1), si-FoxO3a, miR-30b-5p mimics, miR-30b-5p inhibitors (miR-30b-5p-in), and the negative controls (si-NC and miR-NC) were constructed and synthesized by Guangzhou FulenGen Co., Ltd., China The NF-κB inhibitor BAY 11-7082 (Article No. HY-13453) was bought from MedChemExpress (Monmouth Junction, NJ, USA).

Techniques: Sequencing

Journal: Aging (Albany NY)

Article Title: NF-κB inducible miR-30b-5p aggravates joint pain and loss of articular cartilage via targeting SIRT1-FoxO3a-mediated NLRP3 inflammasome

doi: 10.18632/aging.203466

Figure Lengend Snippet: The miR-30b-5p profile was heightened in the joint tissues of OA patients and correlated with pro-inflammatory responses. The joint tissues of 15 non-OA patients and 40 OA patients were collected. ( A – G ) The levels of miR-30b-5p, IL-1β, IL-6, TNF-α, IL-18, SIRT1 and FoxO3a in joint tissues were compared by RT-qPCR. ( H – M ) Pearson analysis determined the correlation between miR-30b-5p, inflammatory cytokines and SIRT1/FoxO3a in OA patients’ joint tissues. *** P <0.001 (vs.Normal group).

Article Snippet: SIRT1 overexpression plasmid (SIRT1) and its corresponding negative control (vector), small inference RNA (si-RNA) SIRT1 (si-SIRT1), si-FoxO3a, miR-30b-5p mimics, miR-30b-5p inhibitors (miR-30b-5p-in), and the negative controls (si-NC and miR-NC) were constructed and synthesized by Guangzhou FulenGen Co., Ltd., China The NF-κB inhibitor BAY 11-7082 (Article No. HY-13453) was bought from MedChemExpress (Monmouth Junction, NJ, USA).

Techniques: Quantitative RT-PCR

Journal: Aging (Albany NY)

Article Title: NF-κB inducible miR-30b-5p aggravates joint pain and loss of articular cartilage via targeting SIRT1-FoxO3a-mediated NLRP3 inflammasome

doi: 10.18632/aging.203466

Figure Lengend Snippet: The influence of inhibiting miR-30b-5p on OA rats. miR-30b-5p inhibitors were added to the knee joint cavity of OA rats. ( A ) The miR-30b-5p profile was measured by RT-qPCR. ( B ) HE and Safranin O staining were used to observe the morphological differences in cartilage tissue, and the number of injured chondrocytes was counted. ( C ) The levels of IL-1β and TNF-α were compared by RT-qPCR. ( D , E ) WB was implemented to test the profiles of Bax, Cleaved-Caspase3, MMP3 and MMP13. ( F ) The level of NF-κB in the cartilage tissue was gauged by IHC. ( G ) Expression of NF-κB, SIRT1/FoxO3a in the cartilage tissue was compared by WB. ** P <0.01, *** P <0.001. N=5.

Article Snippet: SIRT1 overexpression plasmid (SIRT1) and its corresponding negative control (vector), small inference RNA (si-RNA) SIRT1 (si-SIRT1), si-FoxO3a, miR-30b-5p mimics, miR-30b-5p inhibitors (miR-30b-5p-in), and the negative controls (si-NC and miR-NC) were constructed and synthesized by Guangzhou FulenGen Co., Ltd., China The NF-κB inhibitor BAY 11-7082 (Article No. HY-13453) was bought from MedChemExpress (Monmouth Junction, NJ, USA).

Techniques: Quantitative RT-PCR, Staining, Expressing

Journal: Aging (Albany NY)

Article Title: NF-κB inducible miR-30b-5p aggravates joint pain and loss of articular cartilage via targeting SIRT1-FoxO3a-mediated NLRP3 inflammasome

doi: 10.18632/aging.203466

Figure Lengend Snippet: SIRT1 targeted miR-30b-5p. ( A, B ) The target association between miR-30b-5p and SIRT1 was predicted in the ENCORI database. ( C , D ) The targeted affinity between miR-30b-5p and SIRT1 was validated by the dual-luciferase reporter assay and RIP experiment, respectively. ( E , F ) The profiles of SIRT1 and FoxO3a were further evaluated by RT-qPCR and WB. ns P >0.05, *** P <0.001. N=3.

Article Snippet: SIRT1 overexpression plasmid (SIRT1) and its corresponding negative control (vector), small inference RNA (si-RNA) SIRT1 (si-SIRT1), si-FoxO3a, miR-30b-5p mimics, miR-30b-5p inhibitors (miR-30b-5p-in), and the negative controls (si-NC and miR-NC) were constructed and synthesized by Guangzhou FulenGen Co., Ltd., China The NF-κB inhibitor BAY 11-7082 (Article No. HY-13453) was bought from MedChemExpress (Monmouth Junction, NJ, USA).

Techniques: Luciferase, Reporter Assay, Quantitative RT-PCR

Journal: Aging (Albany NY)

Article Title: NF-κB inducible miR-30b-5p aggravates joint pain and loss of articular cartilage via targeting SIRT1-FoxO3a-mediated NLRP3 inflammasome

doi: 10.18632/aging.203466

Figure Lengend Snippet: Overexpressing SIRT1 weakened the miR-30b-5p-mediated effect. ( A , B ) SIRT1 mimics were transfected into IL-1β-treated HC-A cells, and the transfection validity was verified by RT-qPCR and WB. ( C , D ) The expression of SIRT1 /FoxO3a in IL-1β-treated HC-A cells was assessed by RT-qPCR and WB. ( E , F ) CCK8 and flow cytometry monitored cell viability and apoptosis, respectively. ( G ) Profiles of Bax and Cleaved-Caspase3 were measured by WB. ( H ) RT-qPCR monitored the expression of IL-1β and TNF-α. ( I , J ) The profiles of MMP3, MMP13 and NLRP3-ASC- cleaved Caspase1 were verified by WB. NS P >0.05, * P <0.05, ** P <0.01, *** P <0.001. N=3.

Article Snippet: SIRT1 overexpression plasmid (SIRT1) and its corresponding negative control (vector), small inference RNA (si-RNA) SIRT1 (si-SIRT1), si-FoxO3a, miR-30b-5p mimics, miR-30b-5p inhibitors (miR-30b-5p-in), and the negative controls (si-NC and miR-NC) were constructed and synthesized by Guangzhou FulenGen Co., Ltd., China The NF-κB inhibitor BAY 11-7082 (Article No. HY-13453) was bought from MedChemExpress (Monmouth Junction, NJ, USA).

Techniques: Transfection, Quantitative RT-PCR, Expressing, Flow Cytometry

Journal: Aging (Albany NY)

Article Title: NF-κB inducible miR-30b-5p aggravates joint pain and loss of articular cartilage via targeting SIRT1-FoxO3a-mediated NLRP3 inflammasome

doi: 10.18632/aging.203466

Figure Lengend Snippet: Inhibiting SIRT1/FoxO3a curbed the protection of miR-30b-5p knockdown on IL-1β-mediated HC-A cells. ( A – D ) si-SIRT1 and si-FoxO3a were transfected into IL-1β-treated HC-A cells and their transfection effects were verified by RT-qPCR and WB. ( E , F ) The SIRT1/FoxO3a expression in IL-1β-treated HC-A cells was checked by RT-qPCR and WB, respectively. ( G , H ) The CCK8 method and flow cytometry were utilized to gauge cell viability and apoptosis. ( I ) The levels of IL-1β and TNFα were monitored by RT-qPCR. ( J – L ) The expression of Bax, cleaved Caspase3, MMP3, MMP13 and NLRP3-ASC-cleaved Caspase1 was examined by WB. nsP>0.05, **P<0.01, ***P<0.001. N=3.

Article Snippet: SIRT1 overexpression plasmid (SIRT1) and its corresponding negative control (vector), small inference RNA (si-RNA) SIRT1 (si-SIRT1), si-FoxO3a, miR-30b-5p mimics, miR-30b-5p inhibitors (miR-30b-5p-in), and the negative controls (si-NC and miR-NC) were constructed and synthesized by Guangzhou FulenGen Co., Ltd., China The NF-κB inhibitor BAY 11-7082 (Article No. HY-13453) was bought from MedChemExpress (Monmouth Junction, NJ, USA).

Techniques: Knockdown, Transfection, Quantitative RT-PCR, Expressing, Flow Cytometry

Journal: Aging (Albany NY)

Article Title: NF-κB inducible miR-30b-5p aggravates joint pain and loss of articular cartilage via targeting SIRT1-FoxO3a-mediated NLRP3 inflammasome

doi: 10.18632/aging.203466

Figure Lengend Snippet: Inhibition of NF-κB reduced miR-30b-5p levels and IL-1β-mediated HC-A cell injury. BAY 11-7082 was added to IL-1β-mediated HC-A cells and miR-30b-5p mimics were given. ( A , B ) NF-κB and miR-30b-5p expression was monitored by WB and RT-qPCR, respectively. ( C , D ) Cell proliferation and apoptosis were determined by CCK8 and flow cytometry, respectively. ( E ) Expression of Bax and Cleaved-Caspase3 was determined by WB. ( F , G ) RT-qPCR and WB were conducted to compare the levels of IL-1β, TNF-α, MMP3 and MMP13. ( H , I ) The expression of SIRT1 and FoxO3a was assessed by RT-qPCR. ( J ) WB detected the expression of NLRP3-ASC-cleaved Caspase1 inflammasomes. ** P <0.01, *** P <0.001. N=3.

Article Snippet: SIRT1 overexpression plasmid (SIRT1) and its corresponding negative control (vector), small inference RNA (si-RNA) SIRT1 (si-SIRT1), si-FoxO3a, miR-30b-5p mimics, miR-30b-5p inhibitors (miR-30b-5p-in), and the negative controls (si-NC and miR-NC) were constructed and synthesized by Guangzhou FulenGen Co., Ltd., China The NF-κB inhibitor BAY 11-7082 (Article No. HY-13453) was bought from MedChemExpress (Monmouth Junction, NJ, USA).

Techniques: Inhibition, Expressing, Quantitative RT-PCR, Flow Cytometry

Journal: Aging (Albany NY)

Article Title: NF-κB inducible miR-30b-5p aggravates joint pain and loss of articular cartilage via targeting SIRT1-FoxO3a-mediated NLRP3 inflammasome

doi: 10.18632/aging.203466

Figure Lengend Snippet: Graphical abstract. In OA chondrocytes, NF-κB-mediated miR-30b-5p activates NLRP3 inflammasomes by targeting and abating the SIRT1/FoxO3a expression, thereby aggravating joint pain and articular cartilage damage in OA patients.

Article Snippet: SIRT1 overexpression plasmid (SIRT1) and its corresponding negative control (vector), small inference RNA (si-RNA) SIRT1 (si-SIRT1), si-FoxO3a, miR-30b-5p mimics, miR-30b-5p inhibitors (miR-30b-5p-in), and the negative controls (si-NC and miR-NC) were constructed and synthesized by Guangzhou FulenGen Co., Ltd., China The NF-κB inhibitor BAY 11-7082 (Article No. HY-13453) was bought from MedChemExpress (Monmouth Junction, NJ, USA).

Techniques: Expressing

Journal: Cancers

Article Title: The Cx43-Mediated Autophagy Mechanism Influences Triple-Negative Breast Cancer Through the Regulation of Rab31

doi: 10.3390/cancers17243923

Figure Lengend Snippet: Interaction between Cx43 and Rab31 and its functional role in autophagy. ( A ) The GeneMANIA and STRING databases identify Rab31 as a potential Cx43-interacting protein. ( B ) Confocal microscopy shows co-localization of Cx43 and Rab31 in BT-549 (scale bar = 10 μm). ( C ) The interaction between Cx43 and Rab31 was confirmed using co-immunoprecipitation (Co-IP). ( D ) WB analysis of Rab31 expression after modulation of Cx43 levels. ( E , F ) WB evaluates the impact of autophagy by Interference and overexpression of Cx43 (n = 3 independent experiments (biological replicates). Statistical significance was determined using unpaired t -test. Mean ± SD, * p < 0.05, ** p < 0.01). Original western blots are presented in .

Article Snippet: Three siRNAs targeting Rab31 (si1-Rab31, si2-Rab31, si3-Rab31, NO. RX088541) and negative control (siRNA-NC), overexpression plasmid for Rab31 (OE-Rab31), and its empty vector control (Vector) were synthesized from KeyGEN Biotechnology Co., Ltd. (Nanjing, China) All transfections were performed using Lipo8000 reagent (Beyotime, Shanghai, China) following the manufacturer’s protocol.

Techniques: Functional Assay, Confocal Microscopy, Immunoprecipitation, Co-Immunoprecipitation Assay, Expressing, Over Expression, Western Blot

Journal: Cancers

Article Title: The Cx43-Mediated Autophagy Mechanism Influences Triple-Negative Breast Cancer Through the Regulation of Rab31

doi: 10.3390/cancers17243923

Figure Lengend Snippet: Functional characterization of Rab31 in TNBC. ( A ) WB and ( B ) qRT-PCR were utilized to evaluate the differential expression of Rab31 at the protein and mRNA levels in normal breast epithelial cells (MCF-10A) and triple-negative breast cancer cells (MDA-MB-231, BT-549). ( C ) WB analysis confirming Rab31 overexpression efficiency. ( D ) qRT-PCR and ( E ) WB were used to assess the interference efficiency of Rab31. ( F ) A CCK8 assay was utilized to measure cell proliferation following Rab31 modulation (statistical significance was determined using two-way ANOVA). ( G ) A wound healing assay assesses cell migration. ( H ) A transwell assay was used to evaluate cell migration and invasion ability. ( I ) The influence of Rab31 on autophagy-related protein expression was examined using Western blotting (n = 3 independent experiments (biological replicates). Two-group comparisons used unpaired t -tests, while multi-group comparisons used one-way ANOVA with Tukey’s post hoc test. Mean ± SD, * p < 0.05, ** p < 0.01). Original western blots are presented in .

Article Snippet: Three siRNAs targeting Rab31 (si1-Rab31, si2-Rab31, si3-Rab31, NO. RX088541) and negative control (siRNA-NC), overexpression plasmid for Rab31 (OE-Rab31), and its empty vector control (Vector) were synthesized from KeyGEN Biotechnology Co., Ltd. (Nanjing, China) All transfections were performed using Lipo8000 reagent (Beyotime, Shanghai, China) following the manufacturer’s protocol.

Techniques: Functional Assay, Quantitative RT-PCR, Quantitative Proteomics, Over Expression, CCK-8 Assay, Wound Healing Assay, Migration, Transwell Assay, Expressing, Western Blot

Journal: Cancers

Article Title: The Cx43-Mediated Autophagy Mechanism Influences Triple-Negative Breast Cancer Through the Regulation of Rab31

doi: 10.3390/cancers17243923

Figure Lengend Snippet: Cx43 regulates the autophagy pathway through Rab31 in MAD-MB-231. ( A , B ) WB analysis of Cx43 and key autophagy markers (P62, LC3, ATG5, ULK1) in Cx43-overexpressing breast cancer cells (MDA-MB-231) following Rab31 modulation. ( C ) A wound healing assay was employed to assess cell migration and migration rate. ( D ) The CCK8 assay was employed to evaluate the cell proliferation (statistical significance was determined using two-way ANOVA). ( E ) Transwell assay was employed to evaluate cell migration and invasion (n = 3 independent experiments (biological replicates). Statistical significance was determined using one-way ANOVA with Tukey’s post hoc test. Mean ± SD, * p < 0.05, ** p < 0.01, ns, not significant ( p > 0.05).). Original western blots are presented in .

Article Snippet: Three siRNAs targeting Rab31 (si1-Rab31, si2-Rab31, si3-Rab31, NO. RX088541) and negative control (siRNA-NC), overexpression plasmid for Rab31 (OE-Rab31), and its empty vector control (Vector) were synthesized from KeyGEN Biotechnology Co., Ltd. (Nanjing, China) All transfections were performed using Lipo8000 reagent (Beyotime, Shanghai, China) following the manufacturer’s protocol.

Techniques: Wound Healing Assay, Migration, CCK-8 Assay, Transwell Assay, Western Blot

Journal: Cancers

Article Title: The Cx43-Mediated Autophagy Mechanism Influences Triple-Negative Breast Cancer Through the Regulation of Rab31

doi: 10.3390/cancers17243923

Figure Lengend Snippet: Overexpression of Cx43 promotes breast tumor growth in vivo. ( A ) Representative images of orthotopic transplanted tumors in the control and Cx43 overexpression groups. ( B ) Tumor growth in vivo was assessed every five days and the growth curves were plotted (statistical significance was determined using two-way ANOVA). ( C ) Animals were sacrificed to remove tumors, and the weight and volume of tumors were determined. ( D ) The expression of Rab31 and autophagy-related proteins in tumor tissues was assessed using WB (n = 6 independent tumor samples per group; statistical significance was determined using an unpaired t -test, mean ± SD, ** p < 0.01). Original western blots are presented in .

Article Snippet: Three siRNAs targeting Rab31 (si1-Rab31, si2-Rab31, si3-Rab31, NO. RX088541) and negative control (siRNA-NC), overexpression plasmid for Rab31 (OE-Rab31), and its empty vector control (Vector) were synthesized from KeyGEN Biotechnology Co., Ltd. (Nanjing, China) All transfections were performed using Lipo8000 reagent (Beyotime, Shanghai, China) following the manufacturer’s protocol.

Techniques: Over Expression, In Vivo, Control, Expressing, Western Blot

Journal: Nature

Article Title: MicroRNA sequence codes for small extracellular vesicle release and cellular retention

doi: 10.1038/s41586-021-04234-3

Figure Lengend Snippet: Cell type-specific miRNAs in sEV and cells, and selectivity of sEV versus cellular distribution of certain miRNAs. a) The experimental setup and cell lines used in this study. b) Principal Component Analysis (PCA) showing cellular (triangles) and sEV (circles) miRNA profiles for each cell type (n=3–4). c) Venn diagram showing the number of miRNAs with specific expression in cell bodies of each cell type. The number in the center refers to those miRNAs with no specific expression in the cell body of any of the five cell lines studied. d) Venn diagram for miRNAs with specific expression in the sEV of each cell type. e) Heatmap showing the abundance of the indicated miRNAs in sEV and cell bodies for each cell type studied. High expression based on normalized Ct is shown in red and low expression in blue. f) Waterfall diagram showing sEV enrichment for all expressed miRNAs in brown adipocytes as log 2 fold difference for relative abundance in sEV versus relative abundance for the same miRNA in cell body. The dashed lines indicate a four-fold difference. g) Table indicating the number of miRNAs showing significant cellular- or sEV-enrichment (FDR<0.1) or no enrichment for each cell type. The last column indicates the percentage of miRNAs with a selective distribution as the ratio between the sum of sEV and cellular enriched miRNAs divided by the total number of measured miRNAs. h) Venn diagram indicating the number of miRNAs significantly enriched in the cell bodies compared to their respective sEV for the indicated cell-types and the different combinations among them. i) Venn diagram indicating the number of miRNAs significantly enriched in the sEV compared to their respective cell body of each cell type and their combinations.

Article Snippet: A scramble non-targeting stem-loop precursor miRNA cassette into the same backbone lentiviral vector (MMIR000-PA-1, System Biosciences) was used as expression control.

Techniques: Expressing

Journal: Nature

Article Title: MicroRNA sequence codes for small extracellular vesicle release and cellular retention

doi: 10.1038/s41586-021-04234-3

Figure Lengend Snippet: Motifs over-represented in miRNAs preferentially sorted into sEV/exosomes (EXOmotifs) or cell retained (CELLmotifs) for each cell type. a) Table showing the sEV/exosomal motifs (EXOmotifs) identified for each cell type in their extended version (left) and core version (right). For each motif, the P-value, false discovery rate (FDR), the percentage of miRNAs significantly enriched in exosomes/sEV that contain the motif, the percentage of miRNAs not enriched in exosomes/sEV containing the motif (background) and the fold-enrichment as the ratio between the previous two columns are displayed. b) Table showing the cell retention-associated motifs (CELLmotifs) identified for each cell type in their extended version (left) and core version (right). Calculated are as in panel a.

Article Snippet: A scramble non-targeting stem-loop precursor miRNA cassette into the same backbone lentiviral vector (MMIR000-PA-1, System Biosciences) was used as expression control.

Techniques:

Journal: Nature

Article Title: MicroRNA sequence codes for small extracellular vesicle release and cellular retention

doi: 10.1038/s41586-021-04234-3

Figure Lengend Snippet: CELLmotifs and EXOmotifs regulate miRNA distribution. Alyref and Fus participate in sorting of miRNAs containing CNGGNG-EXOmotif. a) Experimental setup for interrogating motif functionality. b) sEV enrichment calculated as the ratio of sEV expression divided by cellular expression in AML12 hepatocytes for either miR-431-5p wild-type (miR-431-5p-WT) or its version containing the CELLmotif AGAAC (miR-431-5p-AGAAC). c) The same motif AGAAC was removed from miR-140-3p (miR-140-3p NO AGAAC) in brown adipocytes and the sEV enrichment was calculated for it as for the wild-type version (miR-140-3p-WT). d) Core-CELLmotifs CAGU and AUU[A/G] were removed from miR-677-5p (miR-677-5p-NO CELLmotifs) in AML12 hepatocytes and its sEV enrichment was calculated as for the wild-type version (miR-677-5p-WT). e) sEV enrichment for wild-type miR-34c-5p (miR-34c-WT) or the miR-34c versions containing UGUGU, CAUG or CGGGAG EXOmotifs expressed in brown adipocytes (BAT). f) sEV enrichment for wild-type miR-26a-5p and its versions containing EXOmotifs CAUG and CGGGAG expressed in BAT. g) Experimental setup for the miRNA pulldown experiments. h and i) Proteins displaying at least 8-fold better (log 2 > 3) binding to the EXOmotif-containing version relative to its wild-type counterpart. h) Binding to miR-34c-WT was set as 1, and the binding to scrambled miRNA and miR-34c-CGGGAG were calculated relative to miR-34c-WT (n=3). i) Similar to h, but relative to miR-26a-WT (n=3). *P≤0.05 Motif-containing vs WT miRNA (paired T-test with Storey’s method for group-wise correction). j) sEV enrichment for miR-34c-WT and miR-34c-CGGGAG in BAT treated with Alyref, Fus or non-targeting control siRNAs. In panels b-f and j, the dashed line separates sEV enrichment (above line) versus cellular enrichment (below line). The data are from 3–4 independent experiments, i.e., biological replicates, with each replicate being the average of duplicate/triplicate qPCR reactions. Expression was normalized to miR-138-5p. Data are expressed as mean ± SEM, *P≤0.05 (Kruskal-Wallis followed by Mann-Whitney-U test).

Article Snippet: A scramble non-targeting stem-loop precursor miRNA cassette into the same backbone lentiviral vector (MMIR000-PA-1, System Biosciences) was used as expression control.

Techniques: Expressing, Binding Assay, Control, MANN-WHITNEY

Journal: Nature

Article Title: MicroRNA sequence codes for small extracellular vesicle release and cellular retention

doi: 10.1038/s41586-021-04234-3

Figure Lengend Snippet: Incorporation of EXOmotifs enhances miRNA delivery and target inhibition. a) Diagram illustrating the transwell experiment. b) miRNA expression for endogenous basal miR-34c (grey bar), induced wild-type miR-34c over the basal (blue), miR-34c-CAUG (green) and miR-34c-CGGGAG (red) in the donor cells and expressed as the number of copies of each miRNA per 10 3 copies of miR-103-3p as an endogenous control. c) miRNA expression for basal miR-34c (grey bar), induced wild-type miR-34c over the basal (blue), miR-34c-CAUG (green) and miR-34c-CGGGAG (red) in the recipient cells after co-incubation with the donor cells overexpressing these miRNAs respectively, as indicated in the x-axis and normalized per 10 3 copies of miR-103-3p. d) Average number of copies of each miRNA induced in the donor cells above the levels measured in control Scr cells (second column) and the number of copies in the recipient cells over the levels in cells cocultured with the O/E-Scr donors (third column). Values are normalized per 10 3 copies of miR-103-3p. The last column represents the ratio between the second and third columns. e) Change in gene expression for miR-34c targets in the recipient cells induced by the coculture with miR-34c-WT, miR-34c-CAUG or miR-34c-CGGGAG donor cells as indicated in the x-axis and relative to recipient cells co-incubated with O/E-Scr donors. n=3; Data are expressed as mean ± SEM. *P≤0.05 motif-containing vs WT-miR-34c for each gene (Kruskal-Wallis followed by Mann-Whitney-U test). f) Proposed model for the sEV/exosomal sorting and cellular retention of miRNAs mediated by the cell-specific repertoire of EXO- and CELLmotifs. In this, “reader” proteins (i.e., Alyref and Fus for motif CGGGAG) recognize the miRNA motifs that promote sorting of EXOmotif-harboring miRNAs into exosomes/multivesicular bodies (MVB) or cellular retention (CELLmotif). The released exosomes then deliver their miRNA cargo to nearby or distal cells leading to changes in gene expression and cellular function.

Article Snippet: A scramble non-targeting stem-loop precursor miRNA cassette into the same backbone lentiviral vector (MMIR000-PA-1, System Biosciences) was used as expression control.

Techniques: Inhibition, Expressing, Control, Incubation, Gene Expression, MANN-WHITNEY, Cell Function Assay

Journal: Nature

Article Title: MicroRNA sequence codes for small extracellular vesicle release and cellular retention

doi: 10.1038/s41586-021-04234-3

Figure Lengend Snippet: General features of sEV secretion among the five cell types studied. a) Number of vesicles over 48 hours released by each cell type normalized by the number of cells in the tissue-culture plate (n=4). *P≤0.05 (indicated cell type versus all other cell types), § P≤0.05 (indicated cell type versus 3T3-L1, C2C12 and AML-12) (Kruskal-Wallis followed by Mann-Whitney U test). b) Average vesicle size of the sEV as determined by Nanoparticle tracking analysis (NTA) for each cell type (n=4). c) Average size distribution and number of vesicles released per cell for each of the five cell types. d) Immunoblotting for the indicated sEV (ALIX, TSG101 and CD9) and cellular (GM130, CANX) markers in sEV and cell lysates from AML12 hepatocytes and BAT brown adipocytes. e) Electron micrograph showing CD63 gold immunostaining of sEV isolated from C2C12 cells. f) RNA yield obtained from sEV isolated from each cell type and normalized by the number of cells in the tissue-culture plate (n=3). *P≤0.05 (indicated cell type versus all other cell types); § P≤0.05 (indicated cell type versus 3T3-L1 and BAT) (Kruskal-Wallis followed by Mann-Whitney U test). g) Principal component analysis showing cellular miRNA profiles for each cell type. h) Heatmap showing the top 10 representative cellular miRNAs of each cell type. i) Heatmap showing the top 10 representative sEV miRNAs of each cell type. j) Comparative miRNA profile between cell-derived sEV and non-conditioned medium (NCM). Same volume of NCM as in cell-conditioned medium was processed for sEV isolation by differential ultracentrifugation. RNA was isolated and a miRNA profiling was performed for NCM. The miRNA expressions for the 13 miRNAs found sEV-enriched in all 5 cell types were compared to the NCM average Ct by ΔΔct method and represented as fold change. Each dot is the relative average value of each of the five cell types. Data are expressed as mean ± SEM.

Article Snippet: A scramble non-targeting stem-loop precursor miRNA cassette into the same backbone lentiviral vector (MMIR000-PA-1, System Biosciences) was used as expression control.

Techniques: MANN-WHITNEY, Western Blot, Immunostaining, Isolation, Derivative Assay

Journal: Nature

Article Title: MicroRNA sequence codes for small extracellular vesicle release and cellular retention

doi: 10.1038/s41586-021-04234-3

Figure Lengend Snippet: Cell-type specific sEV enrichment of miRNAs and sEV versus cell housekeeping miRNAs. Effect of percentage of CG and Gibbs free energy (ΔG) in miRNA sorting or cellular retention. a) Normalized gene expression of representative miRNAs showing cell-type specific sEV enrichment: miR-696 (BAT), miR-770-5p (C2C12), miR-1927 (3T3-L1), miR-1931 (AML12) and miR-718 (SVEC). Average Ct from the whole miRNA profile was used for normalization for each sample (n=3–4). *P≤0.05 (Limma t-test) b) Normalized gene expression of two representative miRNAs (miR-138-5p and miR-501-5p) showing similar sEV and cellular expression for each cell type. Average Ct from the whole miRNA profile was used for normalization for each sample (n=3–4). c) Percentage of CG content in the sequence of the miRNAs: those sorted into the sEV in all cells are shown in red (n=13); those sorted into sEV in 3 or 4 of the five cell types are shown in green (n=90); those not enriched in either sEV or cells are shown in black (n=109); those retained in 3 or 4 cell types are shown in pink (n=97); and those retained in the cell bodies of all cell types are shown in blue (n=43). d) The calculated Gibbs free energy (ΔG) for each of the miRNA in each of the five groups described in panel c. For c and d lines indicate mean value, **P≤0.01, *** P≤0.001 between the indicated group and the other four groups (ANOVA followed by Bonferroni post-hoc test in c and d). Data are expressed as mean ± SEM.

Article Snippet: A scramble non-targeting stem-loop precursor miRNA cassette into the same backbone lentiviral vector (MMIR000-PA-1, System Biosciences) was used as expression control.

Techniques: Gene Expression, Expressing, Sequencing

Journal: Nature

Article Title: MicroRNA sequence codes for small extracellular vesicle release and cellular retention

doi: 10.1038/s41586-021-04234-3

Figure Lengend Snippet: Comparison of all identified Extended and Core EXOmotifs (a) and CELLmotifs (b) among the five different cell types. a) Fold enrichment (left half) and abundance (right half, as percentage of sEV-enriched miRNAs) containing the sEV-associated miRNA motifs. The first column indicates the cell type where these motifs were originally identified (see Main Figure 2a ) and here shown in its predominant form in the second column (Extended motifs) and fifth column (Core motifs). The fold enrichment is shown in a red (high sEV enrichment)-white (neutral)-blue (cell enrichment) color gradient for indicated cell types displayed below. The abundance is shown in a red (high)-white (low) color gradient. The rectangles highlight the enrichment and presence of the motifs in the cell types where they were originally identified. b) Fold enrichment (left half) and abundance (right half, as percentage of cell-enriched miRNAs) containing the Cell-associated miRNA motifs. As in a), the first column indicates the cell type where these motifs were originally identified (see Main Figure 2b ) and here shown in its predominant form in the second column (Extended motifs) and fifth column (Core motifs). The fold enrichment is shown in a blue (high cell enrichment)-white (neutral)-blue (red sEV enrichment) color gradient for indicated cell types displayed below. The abundance is shown in a blue (high)-white (low) color gradient. The rectangles highlight the enrichment and presence of the motifs in the cell types where they were originally identified.

Article Snippet: A scramble non-targeting stem-loop precursor miRNA cassette into the same backbone lentiviral vector (MMIR000-PA-1, System Biosciences) was used as expression control.

Techniques: Comparison

Journal: Nature

Article Title: MicroRNA sequence codes for small extracellular vesicle release and cellular retention

doi: 10.1038/s41586-021-04234-3

Figure Lengend Snippet: Location of EXO and CELLmotifs and comparison between small miRNAseq (smRNAseq) and qPCR-based miRNA profiling. a-b) Percentage of miRNAs showing indicated EXOmotifs (a) or CELLmotifs (b) either in the 5’ half (nucleotides 1–9, light yellow bars) or 3’ half (from nucleotide 10 to the 3’ end, orange bars) of the miRNA sequence. c) sEV and cell lysates from differentiated 3T3-L1 white adipocytes and AML12 hepatocytes were subjected to smRNAseq or qPCR-based profiling (n=4 for each cell type and compartment). Detected miRNAs by each method in each cell type is displayed in black bars. Selective distribution (either sEV-enriched or cell-enriched) was calculated for each miRNA using both methods and the number of miRNAs displaying significant selective distribution is shown in blue bars. The percentages in the blue bars refers to the ratio between the number of selectively distributed miRNA and the total number of detected miRNAs for each method and cell type. d) Venn diagrams indicating the number of miRNAs with a selective distribution in sEV or cells detected by smRNAseq (blue circles) and qPCR (green circles) in 3T3-L1 (above) or AML12 (below). The total number of miRNAs detected simultaneously by these two methods was 180. e) Table depicting the top EXOmotif found by HOMER software in sEV-enriched miRNAs from 3T3-L1 and AML12 detected by smRNAseq. Fold enrichment refers to the ratio between presence in the sEV-enriched miRNAs and presence in the rest of miRNAs (background).

Article Snippet: A scramble non-targeting stem-loop precursor miRNA cassette into the same backbone lentiviral vector (MMIR000-PA-1, System Biosciences) was used as expression control.

Techniques: Comparison, Sequencing, Software

Journal: Nature

Article Title: MicroRNA sequence codes for small extracellular vesicle release and cellular retention

doi: 10.1038/s41586-021-04234-3

Figure Lengend Snippet: Comparison of miRNA profiling of AML12 and primary hepatocytes. Primary hepatocytes were isolated from C57Bl/6J wild-type mice (n=4) and cultured for 48 h in exosome-free medium to collect sEV and cell lysates, which were later subjected to RNA isolation and miRNA profiling for comparison to AML12 hepatocytes. a) Number of miRNAs showing selective cellular retention, non-selective distribution or selective sEV sorting in AML12 and primary hepatocytes. The selective distribution column is the sum of sEV- and cell-enriched miRNAs divided by the total number of miRNAs detected as percentage. FDR < 0.1 b) Venn-diagram showing the number of sEV- (red) and cell-enriched miRNAs (blue) in AML12 and primary hepatocytes and the overlap between them. c-d) Motifs associated to sEV (c) and cell (d) enrichment in primary hepatocytes. The table shows the significance of the enrichment (P-value), false discovery rate (FDR), the percentage of miRNAs significantly enriched in sEV (in c) or cell (in d) that contain the motif, the percentage of miRNAs not enriched in the background miRNAs containing the motif and the fold-enrichment as the ratio between the previous two columns.

Article Snippet: A scramble non-targeting stem-loop precursor miRNA cassette into the same backbone lentiviral vector (MMIR000-PA-1, System Biosciences) was used as expression control.

Techniques: Comparison, Isolation, Cell Culture

Journal: Nature

Article Title: MicroRNA sequence codes for small extracellular vesicle release and cellular retention

doi: 10.1038/s41586-021-04234-3

Figure Lengend Snippet: Isolation of sEV and NV using an additional step of size exclusion chromatography. a) Diagram of the isolation method used to obtain cellular, sEV-p100 pellet, sEV-SEC and NV-SEC samples using two rounds of ultracentrifugation followed by size exclusion chromatography (SEC). (n=4). b) NTA analysis for EV concentration (top graph) of the 30 fractions obtained from SEC. These fractions were pooled in pairs in some cases and concentrated using Amicon centrifugal 3 KDa filter prior assessing protein concentration (bottom graph). c) Immunoblot for classical exosomal markers CD63 and CD9 for the concentrated fractions shown in b, bottom graph. d) PCA plot for the miRNA profile from the cells, sEV-p100, sEV-SEC and NV-SEC samples. e) Heatmap of the top differentially expressed miRNAs among sEV-p100, sEV-SEC and NV-SEC. High expression is shown in red and low expression in blue. f) Pearson correlation between averaged normalized miRNA expression levels in sEV-p100 and sEV-SEC. Expression levels were first normalized to average ct of each sample. g) Motifs found overrepresented in sEV-SEC enriched-miRNAs compared to cellular-enriched miRNAs.

Article Snippet: A scramble non-targeting stem-loop precursor miRNA cassette into the same backbone lentiviral vector (MMIR000-PA-1, System Biosciences) was used as expression control.

Techniques: Isolation, Size-exclusion Chromatography, Concentration Assay, Protein Concentration, Western Blot, Expressing

Journal: Nature

Article Title: MicroRNA sequence codes for small extracellular vesicle release and cellular retention

doi: 10.1038/s41586-021-04234-3

Figure Lengend Snippet: Additional information about mutations in miR-431-5p, miR-140-3p and miR-677-5p. a) Table depicting the name and sequence of the CELLmotif AGAAC incorporated in miR-431-5p. Bold underlying text in the sequence indicated changed nucleotides in the guide strand of the miRNA used to introduce the CELLmotif AGAAC. Nucleotides in the passenger strand were also modified to maintain miRNA structure. b) Predicted structure for the hairpin miRNA for the constructs shown in a. Red means high probability of pairing, while blue indicates low probability calculated by RNAfold WebServer software. Arrow indicates the location of the mutated nucleotides. c) Normalized gene expression for miR-431-5p wild-type in cells and sEV for each cell type. Average Ct from the whole miRNA profile was used for normalization for each sample. d) Table depicting the name and sequence of the wild-type miR-140-3p and mutated version without CELLmotif AGAAC. Bold underlying text in the sequence indicated changed nucleotides in the guide strand of the miRNA used to remove the CELLmotif AGAAC. Nucleotides in the passenger strand were also modified to maintain miRNA structure. e) Predicted structure for the hairpin miRNA for the constructs shown in d. Red means high probability of pairing, while blue indicates low probability. Arrow indicates the location of the mutated nucleotides. f) Normalized gene expression for miR-140-3p wild-type in cells and sEV for each cell type. Average Ct from the whole miRNA profile was used for normalization for each sample. g) Table showing the name and sequence of the wild-type miR-677-5p and the version in which CELLmotifs were mutated. Bold underlined text in the sequence indicated mutated nucleotides in the guide strand of the miRNA to remove CELLmotifs CAGU and AUU[A/G]. Nucleotides in the passenger strand were also modified accordingly to maintain miRNA structure. h) Predicted structure for the hairpin miRNA for the constructs shown in g. Red means high probability of pairing while blue indicates low probability. Arrows indicate the location of the mutated nucleotides. i) Normalized gene expression for miR-677-5p wild-type in cells and exosomes for each cell type. Average Ct from the whole miRNA profile was used for normalization for each sample. Data are expressed as mean ± SEM. n=3–4. *P≤0.05 (Limma t-test).

Article Snippet: A scramble non-targeting stem-loop precursor miRNA cassette into the same backbone lentiviral vector (MMIR000-PA-1, System Biosciences) was used as expression control.

Techniques: Sequencing, Introduce, Modification, Construct, Software, Gene Expression

Journal: Nature

Article Title: MicroRNA sequence codes for small extracellular vesicle release and cellular retention

doi: 10.1038/s41586-021-04234-3

Figure Lengend Snippet: Additional information about mutations in miR-34c. a) Table depicting the name and sequence of the different EXOmotifs introduced in miR-34c-5p. Bold underlying text in the sequence indicates changed nucleotides in the guide strand of the miRNA. Nucleotides in the passenger strand were also modified accordingly to maintain miRNA structure. b) Predicted structure for the hairpin miRNA for each of the constructs shown in a. Red means high probability of pairing while blue indicates low probability calculated by RNAfold WebServer software. Arrows indicate the location of the mutated nucleotides. c) Normalized expression for miR-34c-5p wild-type in cells and sEV for each cell type. Average Ct from the whole miRNA profile was used for normalization for each sample. *P≤0.05 (Limma t-test). d) To visualize the changes in the sEV and cell content of each miRNA construct before sEV enrichment calculation, normalized expression of miR-34c wild-type (WT) or its EXOmotif-containing versions miR-34-UGUGU, miR-34-CAUG and miR-34-CGGGAG are displayed for the cells overexpressing each of the miR-34c versions. miR-138-5p was used to normalize expression in sEV versus cells as we previously showed that the levels of this miRNA are equivalent in both compartments.*P≤0.05 (Mann-Whitney U test comparing sEV and cell expression for the same miRNA version, i.e. red versus blue bars). e) Absolute copy number of the miRNAs displayed in the x-axis overexpressed in brown adipocytes were quantified in sEV isolated by ultracentrifugation followed by size exclusion chromatography and normalized by the miRNA copy number in all producing cells for each sample. f) sEV enrichment calculated as the ratio of sEV expression divided by cellular expression for each of the constructs expressed in and secreted from AML12 hepatocytes. g) sEV enrichment calculated as the ratio of sEV expression divided by cellular expression for each of the constructs expressed in and secreted from SVEC endothelial cells. In both panels e and f, the dashed line separates preferential sEV enrichment (above line) versus preferential cellular enrichment (below line). Expression was normalized to the expression of miR-501-5p, which is to be equally abundant in sEV and cells for each cell type. Data are expressed as mean ± SEM. n=3, *P≤0.05, Kruskal-Wallis followed by Mann-Whitney U tests.

Article Snippet: A scramble non-targeting stem-loop precursor miRNA cassette into the same backbone lentiviral vector (MMIR000-PA-1, System Biosciences) was used as expression control.

Techniques: Sequencing, Modification, Construct, Software, Expressing, MANN-WHITNEY, Isolation, Size-exclusion Chromatography

Journal: Nature

Article Title: MicroRNA sequence codes for small extracellular vesicle release and cellular retention

doi: 10.1038/s41586-021-04234-3

Figure Lengend Snippet: Additional information about mutations in miR-26a. a) Table depicting the name and sequence of the different EXOmotifs introduced in miR-26a-5p. Bold underlying text in the sequence indicates changed nucleotide/s in the guide strand of the miRNA. Nucleotides in the passenger strand were also modified accordingly to maintain miRNA structure. b) Predicted structure for the hairpin miRNA for each of the constructs shown in a. Red means high probability of pairing while blue indicates low probability calculated by RNAfold WebServer software. Arrows indicate the location of the mutated nucleotide/s. c) Normalized expression for miR-26a-5p wild-type in cells and sEV for each cell type. Average Ct from the whole miRNA profile was used for normalization for each sample. *P≤0.05 (Limma t-test). d) sEV enrichment calculated as the ratio of sEV expression divided by cellular expression for each of the constructs expressed in and secreted from SVEC endothelial cells. The dashed line separates preferential sEV enrichment (above line) versus preferential cellular enrichment (below line). Expression was normalized to the expression of miR-501-5p. *P≤0.05 (Kruskal-Wallis followed by Mann-Whitney U test), n=3–4.

Article Snippet: A scramble non-targeting stem-loop precursor miRNA cassette into the same backbone lentiviral vector (MMIR000-PA-1, System Biosciences) was used as expression control.

Techniques: Sequencing, Modification, Construct, Software, Expressing, MANN-WHITNEY

Journal: Nature

Article Title: MicroRNA sequence codes for small extracellular vesicle release and cellular retention

doi: 10.1038/s41586-021-04234-3

Figure Lengend Snippet: Further information miRNA pulldown and direct transfection of AML12 cells. a) Analyses for Molecular Function (left) and Cellular Component Gene Ontology (right) of the 67 proteins identified in the proteomic study. b) Table showing average values for relative binding enrichment of the proteins listed in the first column to the miRNA constructs shown in the top row. The columns 2–5 refer to miR-34c and its CGGGAG-containing version, while columns 6–8 refer to miR-26a and its CGGGAG-containing version. In both cases, binding to wild-type miRNAs was set as 1, and the binding of the other miRNA constructs (scramble and CGGGAG-containing version) was normalized respect to that. Only those proteins showing a log2 Fold Enrichment of CGGGAG-containing miRNA version versus wild-type miRNA >3 (>8 fold) were included. c) Brown adipocytes overexpressing wild type miR-34c (OE-miR-34c-5p-WT) or CGGGAG-containing miR-34c (OE-miR-34c-5p-CGGGAG) were transfected with either control siRNA (grey bars), Alyref siRNA (red bars) or Fus siRNA (green bars) and analyzed for knockdown efficiency by qPCR (left) and immunoblotting (right). *P≤0.05 (Kruskal-Wallis followed by Mann-Whitney U test), n=3. d) EXOmotif-containing miR-34c versions have the same efficiency in reducing target gene expression than wild-type miR-34c. AML12 hepatocytes were directly transfected with mimic miR-34c wild-type or its mutant versions miR-34-UGUGU, miR-34-CAUG and miR-34-CGGGAG or non-targeting miRNA (control) for 24 hours and expression of predicted and experimentally-validated miR-34c target genes were analyzed. TATA-box binding protein ( Tbp ) was used as housekeeping gene. n=6. *P≤0.05, ***P≤0.001 (ANOVA followed by Bonferroni post-hoc test). Data are expressed as mean ± SEM.

Article Snippet: A scramble non-targeting stem-loop precursor miRNA cassette into the same backbone lentiviral vector (MMIR000-PA-1, System Biosciences) was used as expression control.

Techniques: Transfection, Binding Assay, Construct, Control, Knockdown, Western Blot, MANN-WHITNEY, Targeted Gene Expression, Mutagenesis, Expressing