Journal: Nature Communications

Article Title: ACE2 localizes to the respiratory cilia and is not increased by ACE inhibitors or ARBs

doi: 10.1038/s41467-020-19145-6

Figure Lengend Snippet: a Representative double immunofluorescence staining of ACE2 and endothelial cell marker CD31 in the blood vessels of human nasal turbinates using six different anti-ACE2 antibodies and anti-CD31. b Double immunofluorescence staining of ACE2 and type II pneumocyte marker mucin 1 (MUC1) in the human lung using six different anti-ACE2 antibodies and anti-MUC1. Abcam ab15348 clone yielded the most robust staining of pneumocytes, while the other clones showed negligible or less specific membrane staining. Scale bars: 20 μm (top) and 5 μm (bottom).

Article Snippet: Amplification of the ISH probes was performed the next day according to manufacturer’s protocol (323100, Bio-Techne), with the final deposition of Cyanine 3 for ACE2 mRNA probe targets (NEL744001KT, Akoya Biosciences).

Techniques: Double Immunofluorescence Staining, Marker, Staining, Clone Assay, Membrane

Journal: Nature Communications

Article Title: ACE2 localizes to the respiratory cilia and is not increased by ACE inhibitors or ARBs

doi: 10.1038/s41467-020-19145-6

Figure Lengend Snippet: Representative images of human tissues on a tissue microarray (TMA) stained by chromogenic immunohistochemistry using antibodies targeting the ACE2 protein (brown) and counterstained with hematoxylin (blue). Highest ACE2 expression was observed in the villi of the intestinal tract (jejunum), renal tubules, testis, and glandular cells in the seminal vesicle. Minimal to no/non-specific staining can be seen in the heart, stomach, spleen, skin, and liver. Staining of lung pneumocytes was observed using Abcam ab15348, and less specifically with Sigma HPA000288 (Fig. 2b; Supplementary Table 1 ). Scale bars: 100 μm.

Article Snippet: Amplification of the ISH probes was performed the next day according to manufacturer’s protocol (323100, Bio-Techne), with the final deposition of Cyanine 3 for ACE2 mRNA probe targets (NEL744001KT, Akoya Biosciences).

Techniques: Microarray, Staining, Immunohistochemistry, Expressing

Journal: Nature Communications

Article Title: ACE2 localizes to the respiratory cilia and is not increased by ACE inhibitors or ARBs

doi: 10.1038/s41467-020-19145-6

Figure Lengend Snippet: a Representative double immunofluorescence staining of ACE2 and acetylated α-tubulin (ACTUB) on normal human nasal turbinate, ethmoid sinus, uncinate process (sinus), trachea, and bronchus, using anti-ACE2 and anti-ACTUB antibodies, respectively. b Representative double immunofluorescence staining of ACE2 and ACTUB on normal C57BL/6J mouse nasal turbinate and trachea. c Immunofluorescent staining of (top panel) ACE2, cilia marker ADP-ribosylation factor-like protein 13B (ARL13B), and cilia centrosome marker FGFR1 oncogene partner (FOP); (bottom panel) ACE2, and cilia markers ACTUB and ARL13B in a ciliated mouse cell line, IMCD3. d Immunofluorescent staining of ACE2 in the primary cilia of IMCD3 cells transiently transfected with human ACE2 (yellow outline) compared to endogenous mouse ACE2 (blue outline). e Quantified percentages of endogenous ACE2-positive cilia (34.67 ± 13.58%; control (Ctrl)) versus cilia with overexpressed human ACE2 (82.67 ± 4.73%). Ciliated cells were identified by staining of ARL13B. Error bars represent mean ± SD. ( n = 100 cells examined per experiment over three independent experiments). (Two-tailed Student’s t test, ** p = 0.004). f Representative multiplexed images of in situ hybridization against the SARS-CoV-2 Spike mRNA, in combination with immunofluorescence staining of ACE2 and the differentiated epithelial cell marker cytokeratin 8 (KRT8). SARS-CoV-2 Spike mRNA expression (red) was detected within ciliated epithelial cells containing motile cilia positive for ACE2 (green). The nuclei were stained using DAPI (blue) as a counterstain. Scale bars: 20 μm ( a , b top panels; f large panels); 5 μm ( a , b bottom panels; f small panels); 2 μm ( c , d ).

Article Snippet: Amplification of the ISH probes was performed the next day according to manufacturer’s protocol (323100, Bio-Techne), with the final deposition of Cyanine 3 for ACE2 mRNA probe targets (NEL744001KT, Akoya Biosciences).

Techniques: Double Immunofluorescence Staining, Staining, Marker, Transfection, Control, Two Tailed Test, In Situ Hybridization, Immunofluorescence, Expressing

Journal: Nature Communications

Article Title: ACE2 localizes to the respiratory cilia and is not increased by ACE inhibitors or ARBs

doi: 10.1038/s41467-020-19145-6

Figure Lengend Snippet: a Representative immunofluorescence double staining of ACE2 and mucin 5AC (MUC5AC) reveals absence of co-localization of ACE2 within secretory goblet cells in the human nasal turbinate, uncinate process, and bronchus. b Representative in situ hybridization using an ACE2 probe in combination with an anti-MUC5AC antibody. ACE2 mRNA expression (red dots) was not detected within goblet cells marked by MUC5AC in the nasal turbinate, uncinate process, and trachea. Nuclei were stained using DAPI. Scale bars: 20 μm (top) and 5 μm (bottom).

Article Snippet: Amplification of the ISH probes was performed the next day according to manufacturer’s protocol (323100, Bio-Techne), with the final deposition of Cyanine 3 for ACE2 mRNA probe targets (NEL744001KT, Akoya Biosciences).

Techniques: Immunofluorescence, Double Staining, In Situ Hybridization, Expressing, Staining

Journal: Nature Communications

Article Title: ACE2 localizes to the respiratory cilia and is not increased by ACE inhibitors or ARBs

doi: 10.1038/s41467-020-19145-6

Figure Lengend Snippet: a No statistically significant changes in ACE2 expression was detected among patients less than or greater than 65 years of age, males versus females, and patients with varying smoking history. (Two-tailed Mann–Whitney test or Kruskal–Wallis test, p > 0.05). b No statistically significant difference in ACE2 expression was observed between healthy controls and patients with chronic rhinosinusitis with polyps (CRSwNP) or without polyps (CRSsNP). (Kruskal–Wallis test, p > 0.05). c No statistically significant difference in ACE2 expression was noted between distinct human nasal tissue sites/regions. (Two-tailed Mann–Whitney test or Kruskal–Wallis test, p > 0.05). UNC uncinate process, Turb nasal turbinates, Eth ethmoid sinus, NP benign nasal polyps. The bottom and top of the box plots represent the 25th and 75th percentiles, respectively. The bands within the box show the median value, and the whiskers extending from both ends of the boxes are minimum and maximum values. Each dot represents one patient.

Article Snippet: Amplification of the ISH probes was performed the next day according to manufacturer’s protocol (323100, Bio-Techne), with the final deposition of Cyanine 3 for ACE2 mRNA probe targets (NEL744001KT, Akoya Biosciences).

Techniques: Expressing, Two Tailed Test, MANN-WHITNEY

Journal: Nature Communications

Article Title: ACE2 localizes to the respiratory cilia and is not increased by ACE inhibitors or ARBs

doi: 10.1038/s41467-020-19145-6

Figure Lengend Snippet: a Quantification of ACE2 in controls and patients taking ARBs and ACEI. In the Stanford cohort, ACE2 is slightly but statistically significantly lower in patients taking ACEI (0.19 ± 0.02) compared to controls (0.26 ± 0.06). (Kruskal–Wallis test p = 0.021; Dunn’s multiple comparison post-hoc test, *adjusted p = 0.043). There were no statistically significant differences in ACE2 expression between patients taking ARBs and controls in the Stanford, National Taiwan University Hospital (NTUH), and China Medical University Hospital (CMUH) cohorts. b In the Stanford cohort, when including only controls with hypertension (HTN) on other medications (“HTN w/o ARBs/ACEI”), ACE2 expression was statistically different between the groups (Kruskal–Wallis test, p = 0.044) but Dunn’s multiple comparison post-hoc test did not reveal any statistical significance between the three groups. No statistically significant differences were seen among patients taking ARBs compared to controls. c When cohorts from all three institutions were normalized by Z -score and integrated, patients taking ACEI (−0.72 ± 0.42) had a lower ACE2 expression compared to controls with hypertension (0.41 ± 1.07). (Kruskal–Wallis test, p = 0.032; Dunn’s multiple comparison post-hoc test, *adjusted p = 0.043). Patients taking ARBs (−0.15 ± 0.95) showed a trend towards lower ACE2 compared to controls with hypertension, but this was not statistically significant. d ACE2 expression among patients of older (≥65 years) and younger (<65 years) age taking ARBs or ACEI was not statistically divergent from control patients of the same age group. (Kruskal–Wallis test, p > 0.05). e ACE2 expression among male and female patients on ARBs or ACEI trended comparably or lower than same-sex controls except for males taking ARBs in the CMUH group who showed a trend towards higher ACE2 expression. No statistically significant differences were observed. (Kruskal–Wallis test, p > 0.05). f Among non-smokers, there was a statistically significant trend towards lower ACE2 expression in patients taking ACEI compared to controls in the Stanford group (Kruskal–Wallis test, p = 0.021; Dunn’s multiple comparison post-hoc test, *adjusted p = 0.035). No statistical significance was observed with the non-smokers on ARBs. All data are noted as mean ± SD. Kruskal–Wallis test was used for three group comparisons and two-tailed Mann–Whitney test was used for two-group comparisons. Box plots are similar in format to Fig. .

Article Snippet: Amplification of the ISH probes was performed the next day according to manufacturer’s protocol (323100, Bio-Techne), with the final deposition of Cyanine 3 for ACE2 mRNA probe targets (NEL744001KT, Akoya Biosciences).

Techniques: Comparison, Expressing, Medications, Control, Two Tailed Test, MANN-WHITNEY

Journal: Nature Communications

Article Title: ACE2 localizes to the respiratory cilia and is not increased by ACE inhibitors or ARBs

doi: 10.1038/s41467-020-19145-6

Figure Lengend Snippet: The luminal differentiated airway epithelial cells consist of ciliated columnar cells (~80%) and secretory goblet cells (~20%). Club cells are infrequently found in the human upper airway. The basal cell layer, which faces the lamina propria, is comprised of both basal and suprabasal cells, which are considered multipotent progenitors capable of renewing the airway epithelium. This schematic depicts how SARS-CoV-2 may bind to ACE2 expressed on the cilia of the nasal cavity following exposure to respiratory droplets or airborne particles.

Article Snippet: Amplification of the ISH probes was performed the next day according to manufacturer’s protocol (323100, Bio-Techne), with the final deposition of Cyanine 3 for ACE2 mRNA probe targets (NEL744001KT, Akoya Biosciences).

Techniques:

Journal: eLife

Article Title: Stem cell regionalization during olfactory bulb neurogenesis depends on regulatory interactions between Vax1 and Pax6

doi: 10.7554/eLife.58215

Figure Lengend Snippet: ( A ) Representation of the strategy used for transcriptomic analysis in time and space in the dorsal and lateral OB lineages. pCX-GFP plasmid was introduced into neural stem cells (NSCs) residing within the dorsal or lateral V-SVZ and GFP-positive cells were isolated by FACS at different time points after electroporation (Elpo). The mRNA content was analyzed by micro-array . ( B ) Quantification of Vax1 mRNA expression detected by micro-array analysis in dorsal (brown) and lateral (purple) progenies during neurogenesis. ( C–H ) In situ hybridization revealing Vax1 mRNA (in blue) combined with immuno-histochemistry using antibodies detecting (in brown) PAX6 ( C, C’, G ), ASCL1 ( D, D’ ), DLX2 ( E, E’, H ) or KI67 ( F, F’ ) proteins in the V-SVZ ( C–F ) or RMS ( G, H ) at postnatal day 3 ( P3 ). ( C’–F’ ) High magnification of cellular staining in the V-SVZ (area indicated by the yellow bracket in C-F). Arrows ( C’ ): examples of strong PAX6 only positive cell in the dorso-lateral SVZ; blue staining underneath labels cells from a distinct plane. Arrow heads ( E’, F’ ): double positive cells for DLX2 and KI67, respectively. High magnification of the RMS highlights the differential expression of Vax1 and Pax6 along the dorso-ventral axis ( G’,G” ) and the co-localization with Dlx2 ( H’,H” ). ( I ) Schematic representation of gene expression profile in different cell types of the neurogenic sequence. Circular arrow indicates proliferating cells. LV: lateral ventricle, RG: radial glia, TAP: transit amplified precursor, VZ: ventricular zone, SVZ: sub-ventricular zone. D: dorsal, L: lateral, S: septal, V: ventral. Scale bars: 100 µm ( C–F ), 20 µm ( C’–F’ ), 50 µm ( G–H ).

Article Snippet: Sequence-based reagent , Cre mRNA , Miltenyi Biotec , 130-101-113 , .

Techniques: Plasmid Preparation, Isolation, Electroporation, Microarray, Expressing, In Situ Hybridization, Immunohistochemistry, Staining, Quantitative Proteomics, Gene Expression, Sequencing, Amplification

Journal: eLife

Article Title: Stem cell regionalization during olfactory bulb neurogenesis depends on regulatory interactions between Vax1 and Pax6

doi: 10.7554/eLife.58215

Figure Lengend Snippet: Cells were analyzed 15 days after electroporation of lateral V-SVZ progenitors by Cre mRNA in WT or Vax1cKO mice. Data are shown as means ± SD, dots represent individual animals. WT: n = 12, Vax1cKO: n = 17. Figure 2—figure supplement 2—source data 1. Quantification of tdTomato+ PGC in Vax1 mutant.

Article Snippet: Sequence-based reagent , Cre mRNA , Miltenyi Biotec , 130-101-113 , .

Techniques: Electroporation, Mutagenesis

Journal: eLife

Article Title: Stem cell regionalization during olfactory bulb neurogenesis depends on regulatory interactions between Vax1 and Pax6

doi: 10.7554/eLife.58215

Figure Lengend Snippet: ( A ) Strategy used to determine the expression of microRNAs in Vax1-overexpressing progenitors. PCAG-Vax1 and pCX-GFP were simultaneously introduced into NSCs by electroporation of the lateral wall of postnatal P1 brains. Lateral V-SVZ was dissected out 2 days after electroporation and GFP+ cells were isolated by flow cytometry (FACS) to perform quantitative RT-PCR analysis. ( B ) Quantification of Vax1 mRNA level by qRT-PCR in control and Vax1OE conditions, normalized to beta-actin and reported in Vax1 condition as relative level to control, validating the overexpression of Vax1 after electroporation. ( C ) Quantification of miR-7 expression in both conditions. Expression level of miR-7 was normalized by invariant expression of microRNA let-7a and reported in Vax1 condition as relative level to control. Experiments in B and C were performed in triplicate, and data were obtained from ( B ) two independent biological replications or ( C ) three technical replications. ( D ) Genome browser images representing the chromosomal portions encoding the three MiR-7 loci (depicted in pink). Mir-7–1 lies within an intronic sequence of the Hnrnpk gene whereas MiR-7–2 and MiR-7b reside within intergenic sequences. Vax1-binding sites found in the upstream regulatory region of the three MiR-7 are represented by red boxes. ( E ) Model of cross-regulatory interaction between Vax1 , miR-7, and Pax6 in the lateral V-SVZ to control the number of dopaminergic neurons generated by the neural stem cells regionalized in this aspect. This model is supported by our present data and previous work where it was shown that miR-7 was required to inhibit PAX6 expression in lateral NSCs to produce the correct number of dopaminergic neurons in the postnatal OB. Here, we propose that Vax1 acts upstream of miR-7 by positively regulating its expression and consequently inhibiting PAX6. However, it is also possible that Vax1 directly represses the expression of Pax6 mRNA (dashed line) by acting on its promoter . Additionally, Vax1 is required to generate Calbindin neurons from the ventral aspect of the lateral V-SVZ. Figure 5—source data 1. Quantification of expression level of Vax1 and miR-7 in V-SVZ cells.

Article Snippet: Sequence-based reagent , Cre mRNA , Miltenyi Biotec , 130-101-113 , .

Techniques: Expressing, Electroporation, Isolation, Flow Cytometry, Quantitative RT-PCR, Control, Over Expression, Sequencing, Binding Assay, Generated

Journal: eLife

Article Title: Stem cell regionalization during olfactory bulb neurogenesis depends on regulatory interactions between Vax1 and Pax6

doi: 10.7554/eLife.58215

Figure Lengend Snippet: ( A ) Strategy used to target lateral V-SVZ NSCs with Cre -mRNA. Tomato+ recombined cells were isolated 2 days after electroporation. ( B ) Vax1 mRNA level quantified by RT-PCR was normalized to beta-actin and reported in Vax1cKO condition as relative level to control (WT).

Article Snippet: Sequence-based reagent , Cre mRNA , Miltenyi Biotec , 130-101-113 , .

Techniques: Isolation, Electroporation, Reverse Transcription Polymerase Chain Reaction, Control

Journal: eLife

Article Title: Stem cell regionalization during olfactory bulb neurogenesis depends on regulatory interactions between Vax1 and Pax6

doi: 10.7554/eLife.58215

Figure Lengend Snippet:

Article Snippet: Sequence-based reagent , Cre mRNA , Miltenyi Biotec , 130-101-113 , .

Techniques: Sequencing, Recombinant, Plasmid Preparation, SYBR Green Assay, Software, Imaging

Journal: Cell Death & Disease

Article Title: Long noncoding RNA related to periodontitis interacts with miR-182 to upregulate osteogenic differentiation in periodontal mesenchymal stem cells of periodontitis patients

doi: 10.1038/cddis.2016.125

Figure Lengend Snippet: LncRNA-POIR expression, which is significantly inhibited in inflammatory microenvironments, correlates with the osteogenic differentiation of pPDLSCs. ( a ) Heat map of differentially expressed lncRNAs (the top 10 in upregulated lncRNAs and the top 10 in downregulated lncRNAs) between hPDLSCs and pPDLSCs. ( b ) The results were confirmed using qPCR. ( c ) The expression levels of lncRNAs with high fold changes (fold change >5.0, P -value <0.05) were determined by qPCR at 7 days after osteogenic induction. ( d ) The expression levels of lncRNA-POIR were determined by qPCR at 0, 1, 7 and 14 days. ( e ) Correlation analysis between lncRNA-POIR levels and Runx2 and Col1 mRNA levels in pPDLSCs 0, 1, 7 and 14 days after osteogenic induction. All experiments were repeated three times. A total of six samples (three pPDLSCs and three hPDLSCs from six individuals) are tested in lncRNA profiling. Relative expressions of lncRNAs expression were normalized by β -actin in qPCR. All data are the mean±S.D. * P <0.05, ** P <0.01 and NS, not significant. C, hPDLSCs; Con, control; lncRNA-POIR, pPDLSCs osteogenesis impaired-related lncRNA, ENST00000446358; Osteo, osteogenic induction; T, pPDLSCs; Undiff, without osteogenic induction

Article Snippet: All labeled lncRNAs and mRNAs were hybridized onto an Arraystar Human LncRNA Microarray V3.0 (Arraystar, Kangchen, Shanghai, China), which included 30 586 lncRNAs and 26 109 coding transcripts.

Techniques: Expressing, Control

Journal: Cell Death & Disease

Article Title: Long noncoding RNA related to periodontitis interacts with miR-182 to upregulate osteogenic differentiation in periodontal mesenchymal stem cells of periodontitis patients

doi: 10.1038/cddis.2016.125

Figure Lengend Snippet: LncRNA-POIR promotes osteogenesis of pPDLSCs. ( a and b ) Transfection effects of shlncRNA-POIR plasmids and lncRNA-POIR overexpression lentiviruses were determined by qPCR. ( c and d ) Runx2, ALP and Col1 expressions were measured by qPCR at 0 and 7 days after osteogenic induction. ( e – g ) Osteogenic differentiations of pPDLSCs were determined by Alizarin Red S, ALP staining and ALP activity assay 7 or 14 days after osteogenic induction. ( h and i ) LncRNA-POIR promotes osteogenesis of pPDLSCs in vivo. pPDLSCs were mixed with HA-TCP and transplanted into the dorsal region of nude mice for 4 weeks. Then, the results were measured by H&E staining and Masson's trichrome staining. Quantitative analysis of the new bone area determined by Image-Pro Plus 6.0 software (Media Cybernetics, Washington, USA). At least three fields were randomly selected from each transplant. Six implants were engrafted into three mice per treatment. All experiments were repeated three times. Relative expressions of mRNAs and lncRNA-POIR were normalized by β -actin in qPCR. All data are the mean±S.D. * P <0.05, ** P <0.01 and NS, not significant. The scale bar in the micrographs represents 200 nm. Con, Control; diff, osteogenic induction; lncRNA-POIR, lentivirus for upregulating lncRNA-POIR; NB, new bone; NC, lentivirus negative control; OD, optical density; shNC, plasmids negative control, shlncRNA-POIR, plamids for downregulating lncRNA-POIR

Article Snippet: All labeled lncRNAs and mRNAs were hybridized onto an Arraystar Human LncRNA Microarray V3.0 (Arraystar, Kangchen, Shanghai, China), which included 30 586 lncRNAs and 26 109 coding transcripts.

Techniques: Transfection, Over Expression, Staining, ALP Activity Assay, In Vivo, Software, Control, Negative Control

Journal: Cell Death & Disease

Article Title: Long noncoding RNA related to periodontitis interacts with miR-182 to upregulate osteogenic differentiation in periodontal mesenchymal stem cells of periodontitis patients

doi: 10.1038/cddis.2016.125

Figure Lengend Snippet: LncRNA-POIR knockdown decreases osteogenic differentiation of hPDLSCs ( a ) Runx2, ALP and Col1 expressions were measured by qPCR at 0 and 7 days after osteogenic induction. ( b – d ) Osteogenic differentiations of pPDLSCs were determined by Alizarin Red S, ALP staining and ALP activity assay 7 or 14 days after osteogenic induction. ( e and f ) shLncRNA-POIR inhibits osteogenesis of hPDLSCs in vivo. hPDLSCs were mixed with HA-TCP and transplanted into the dorsal region of nude mice for 4 weeks. Then, the results were measured by H&E staining and Masson's trichrome staining. Quantitative analysis of the new bone area determined by Image-Pro Plus 6.0 software. At least three fields were randomly selected from each transplant. Six implants were engrafted into three mice per treatment. All experiments were repeated three times. Relative expressions of mRNAs and lncRNA-POIR were normalized by β -actin in qPCR. Data represent mean±S.D. * P <0.05, ** P <0.01 and NS, not significant. The scale bar in the micrographs represents 200 nm. Con, control; diff, osteogenic induction; shlncRNA-POIR, plamids for downregulating lncRNA-POIR; shNC, plasmids negative control; NB, new bone; OD, optical density

Article Snippet: All labeled lncRNAs and mRNAs were hybridized onto an Arraystar Human LncRNA Microarray V3.0 (Arraystar, Kangchen, Shanghai, China), which included 30 586 lncRNAs and 26 109 coding transcripts.

Techniques: Knockdown, Staining, ALP Activity Assay, In Vivo, Software, Control, Negative Control

Journal: Cell Death & Disease

Article Title: Long noncoding RNA related to periodontitis interacts with miR-182 to upregulate osteogenic differentiation in periodontal mesenchymal stem cells of periodontitis patients

doi: 10.1038/cddis.2016.125

Figure Lengend Snippet: LncRNA-POIR acts as a sponge of miR-182. Besides, lncRNA-POIR and miR-182 could negatively regulate each other. ( a ) Transfection effects of miR-182 inhibitor (anti-miR-182) were determined by qPCR. ( b ) Schematic of the miR-182 putative target site in the lncRNA-POIR. ( c ) After transfection of shlncRNA-POIR in pPDLSCs, the expression of miR-182 was determined by qPCR. ( d ) LncRNA-POIR expression was measured by qPCR in pPDLSCs transfected with anti-miR-182. ( e ) Correlation analysis between lncRNA-POIR levels and miR-182 levels in pPDLSCs 0, 1, 7 and 14 days after osteogenic induction. ( f ) The luciferase reporter assay for the lncRNA-POIR in the presence of miR-182. pPDLSCs were co-transfected with miR-Control or miR-182 and wild-type lncRNA-POIR or mutant lncRNA-POIR. Luciferase constructs values are reported as firefly luciferase activity to Renilla luciferase activity. ( g and h ) RIP assays were performed using input from cell lysate, normal mouse IgG or anti-Ago2. Relative expression levels of lncRNA-POIR and miR-182 in pPDLSCs were detected by qPCR. All experiments were repeated three times. Relative expressions of lncRNA-POIR and miR-182 were normalized by β -actin and U6 in qPCR, respectively. Data represent mean±S.D. * P <0.05, ** P <0.01 and NS, not significant. Anti-miR-NC, siPORT reagent alone; anti-miR-182, miR-182 inhibitor; Con, control; lncRNA-POIR wt, lncRNA-POIR wild-type; lncRNA-POIR mut, lncRNA-POIR-mutated type; shlncRNA-POIR, plamids for downregulating lncRNA-POIR; shNC, plasmids negative control

Article Snippet: All labeled lncRNAs and mRNAs were hybridized onto an Arraystar Human LncRNA Microarray V3.0 (Arraystar, Kangchen, Shanghai, China), which included 30 586 lncRNAs and 26 109 coding transcripts.

Techniques: Transfection, Expressing, Luciferase, Reporter Assay, Control, Mutagenesis, Construct, Activity Assay, Negative Control

Journal: Cell Death & Disease

Article Title: Long noncoding RNA related to periodontitis interacts with miR-182 to upregulate osteogenic differentiation in periodontal mesenchymal stem cells of periodontitis patients

doi: 10.1038/cddis.2016.125

Figure Lengend Snippet: lncRNA-POIR modulated FoxO1 by regulating miR-182 ( a and b ) After transfection of anti-miR-182, FoxO1 expression in pPDLSCs was measured by qPCR and western blot. ( c ) Schematic of the miR-182 putative target site in the FoxO1 3′-UTR. ( d ) The luciferase reporter assay for the lncRNA-POIR in the presence of miR-182. PPDLSCs were co-transfected with miR-Control or miR-182 and wild-type FoxO1 3′-UTR or mutant FoxO1 3′-UTR luciferase constructs. Values are reported as firefly luciferase activity to Renilla luciferase activity. ( e – h ) After transfection of lncRNA-POIR and shlncRNA-POIR in pPDLSCs, FoxO1 expression was measured by qPCR and western blot. All experiments were repeated three times. Relative expressions of FoxO1 were normalized by β -actin in qPCR. Data represent mean±S.D. * P <0.05, ** P <0.01 and NS, not significant. Anti-miR-NC, siPORT reagent alone; anti-miR-182, miR-182 inhibitor; FoxO1 3′-UTR wt, FoxO1 3′-UTR wild-type; FoxO1 3′-UTR mut, FoxO1 3′-UTR-mutated type, Con, Control; lncRNA-POIR, lentivirus for upregulating lncRNA-POIR; NC, lentivirus negative control; shlncRNA-POIR, plamids for downregulating lncRNA-POIR; shNC, plasmids negative control; UTR, untranslated regions

Article Snippet: All labeled lncRNAs and mRNAs were hybridized onto an Arraystar Human LncRNA Microarray V3.0 (Arraystar, Kangchen, Shanghai, China), which included 30 586 lncRNAs and 26 109 coding transcripts.

Techniques: Transfection, Expressing, Western Blot, Luciferase, Reporter Assay, Control, Mutagenesis, Construct, Activity Assay, Negative Control

Journal: Cell Death & Disease

Article Title: Long noncoding RNA related to periodontitis interacts with miR-182 to upregulate osteogenic differentiation in periodontal mesenchymal stem cells of periodontitis patients

doi: 10.1038/cddis.2016.125

Figure Lengend Snippet: The opposite effects of miR-182 and FoxO1 on osteogenic differentiation. ( a and b ) Transfection effects of siFoxO1 was determined by qPCR and western blot. ( c – f ) The functions of siFoxO1 on the expression of osteogenic markers of pPDLSCs were measured by Alizarin red staining, qPCR and western blot 7 or 14 days after osteogenic induction. ( g and h ) Runx2 and Col1 mRNA levels and Runx2 protein level in pPDLSCs were measured after being transfected with siFoxO1 under the treatment of overexpression of lncRNA-POIR or controls at 7 or 14 days after osteogenic induction. ( i and j ) siFoxO1 inhibits osteogenesis of hPDLSCs in vivo. hPDLSCs were mixed with HA-TCP and transplanted into the dorsal region of nude mice for 4 weeks. Then, the results were measured by H&E staining and Masson's trichrome staining. Quantitative analysis of the new bone area determined by Image-Pro Plus 6.0 software. At least three fields were randomly selected from each transplant. Six implants were engrafted into three mice per treatment. All experiments were repeated three times. Relative expressions of mRNAs were normalized by β -actin in qPCR. Data represent mean±S.D. * P <0.05, ** P <0.01 and NS, not significant. The scale bar in the micrographs represents 200 nm. Con, control; lncRNA-POIR, lentivirus for upregulating lncRNA-POIR; NB, new bone; NC, lentivirus negative control; OD, optical density; siFoxO1, FoxO1 oligo; siNC, negative control

Article Snippet: All labeled lncRNAs and mRNAs were hybridized onto an Arraystar Human LncRNA Microarray V3.0 (Arraystar, Kangchen, Shanghai, China), which included 30 586 lncRNAs and 26 109 coding transcripts.

Techniques: Transfection, Western Blot, Expressing, Staining, Over Expression, In Vivo, Software, Control, Negative Control

Journal: Cell Death & Disease

Article Title: Long noncoding RNA related to periodontitis interacts with miR-182 to upregulate osteogenic differentiation in periodontal mesenchymal stem cells of periodontitis patients

doi: 10.1038/cddis.2016.125

Figure Lengend Snippet: Overactivation of the NF- κ B pathway in inflammation is the main cause of dysregulation of the lncRNA-POIR and miR-182 regulatory network. ( a ) After transfection of anti-miR-182, lncRNA-POIR levels in hPDLSCs and pPDLSCs were measured by qPCR. ( b ) After transfection of shlncRNA-POIR, miR-182 levels in hPDLSCs and pPDLSCs were measured by qPCR. ( c ) Western blot was performed to detect the level of P65 in the cytoplasm and nucleus of hPDLSCs and pPDLSCs. β -Actin was used as the control for cytoplasmic P65 and HDAC1 was used as the control for P65 in the nucleus. ( d ) Schematic representation of the human pri-miR-182 promoter region in 2000 bp upstream of the transcription start site (TSS). ChIP assays were performed using input from cell lysate, normal mouse IgG, anti-P65 or anti-c-Rel. Relative expression levels of control and binding regions in pPDLSCs were detected by qPCR. Control: regions without binding sites of P65/c-Rel. Binding regions: regions with several binding sites of P65/c-Rel. ( e ) Transfection effect of siIKK α was determined by qPCR. ( f and g ) The pPDLSCs were transfected with IKK α SiRNA for 48 h and qPCR was performed. ( h ) Working model of lncRNA-POIR-miR-182 network in regulating osteogenesis of pPDLSCs. LncRNA-POIR and miR-182 could form a negative regulatory network and lead to a reduction of miR-182 target gene, FoxO1 , which in turn inhibits canonical Wnt pathway. Besides, inflammation can increase miR-182 expression through the NF- κ B pathway and the overexpressed miR-182 in the inflammatory microenvironment resulted in an imbalance in the lncRNA-POIR-miR-182 regulatory network. All experiments were repeated three times. Relative expressions of mRNAs and lncRNA-POIR were normalized by β -actin and relative expression of miR-182 was normalized by U6 in qPCR, respectively. Data represent mean±S.D. * P <0.05, ** P <0.01 and NS, not significant. Anti-miR-NC, siPORT reagent alone; anti-miR-182, miR-182 inhibitor; Con, Control; shlncRNA-POIR, plamids for downregulating lncRNA-POIR; shNC, plasmids negative control; siNC, negative control; siIKK α , IKK α oligo

Article Snippet: All labeled lncRNAs and mRNAs were hybridized onto an Arraystar Human LncRNA Microarray V3.0 (Arraystar, Kangchen, Shanghai, China), which included 30 586 lncRNAs and 26 109 coding transcripts.

Techniques: Transfection, Western Blot, Control, Expressing, Binding Assay, Negative Control

Journal: Stem Cell Reports

Article Title: Long Noncoding RNA ADINR Regulates Adipogenesis by Transcriptionally Activating C/EBPα

doi: 10.1016/j.stemcr.2015.09.007

Figure Lengend Snippet: lncRNA ADINR Is Upregulated during Adipogenic Differentiation (A) Mean-centered, hierarchical clustering of 1,423 differentially (≥2-fold) expressed (two-tailed, paired Student’s t test, FDR < 0.2), previously annotated noncoding RNAs on days 0, 3, and 6 of adipogenic differentiation. The microarray data are from three independent biological replicates. NC, negative control. (B) ChIP-seq analysis of H3K4me3 and H3K27me3 at the C/EBPα and ADINR loci in adipose-derived hMSCs on day 20 of adipogenic differentiation relative to the undifferentiated cells (day 0). The data were obtained from the Roadmap Epigenomics Project. (C) qRT-PCR analysis of C/EBPα and ADINR expression across three time points (days 0, 3, and 6) of adipogenic differentiation. The relative expression levels after normalizing to the amount of GAPDH signal in each sample are shown. qPCR data are presented as the mean ± SD in three independent experiments. (D) 5′ and 3′ RACE and RT-PCR assays detecting full-length ADINR RNA in undifferentiated (0d) and 3-day adipogenic-differentiated (3d) hMSCs. The longest bands (arrows) for ADINR RNA in the RACE assays were indicated. Through sequencing the PCR product of 5′ RACE, we found that the two shorter bands are non-specific PCR products. +, RT-PCR using DNase-treated 3d total RNA; -, PCR using DNase-treated 3d total RNA (no RT; negative control). (E) Single-molecule RNA fluorescence in situ hybridization shows greatly increased abundance of ADINR molecules during adipogenic differentiation, and ADINR RNA is exclusively localized in the nucleus of hMSCs and day-3 differentiated cells. Scale bars, 50 μm. See also Figure S1 .

Article Snippet: Total RNAs were hybridized using mRNA-lncRNA-combined microarray (CapitalBio).

Techniques: Two Tailed Test, Microarray, Negative Control, ChIP-sequencing, Derivative Assay, Quantitative RT-PCR, Expressing, Reverse Transcription Polymerase Chain Reaction, Sequencing, Fluorescence, In Situ Hybridization

Journal: Frontiers in Immunology

Article Title: IFIH1 Contributes to M1 Macrophage Polarization in ARDS

doi: 10.3389/fimmu.2020.580838

Figure Lengend Snippet: IFIH1 was identified as a hub gene involved in ARDS. (A) The network plot represents interactions among 36 genes according to protein interaction analyses. (B) The histogram illustrates the hub genes. The numbers in the histogram represent the numbers of interactions among the 36 genes based on protein interaction analyses. There is an inflection point between IRF1 and CXCL10 . Therefore, the top 5 genes ( STAT1 , IFIH1 , GBP1 , IFIT3 , and IRF1 ) were selected as the hub genes since they have the most interactions. (C) The mRNA levels of IFIH1 , IRF1 , IFIT3 , GBP1 , and STAT1 in bronchoalveolar lavage fluid (BALF) from patients with ARDS (n=6) and patients without ARDS (postoperative patients, n=6) were measured by qRT-PCR. The mRNA expression was calculated based on 2 -ΔΔCt of each sample /2 -ΔΔCt of Ctrl . Student’s t test indicated that the mRNA levels of IFIH1 , GBP1 , and STAT1 were obviously upregulated in the BALF of the ARDS patients ( P <0.05). All error bars represent the SDs. (D) The mRNA levels of Ifih1 , Irf1 , Ifit3 , Gbp1 and Stat1 in lung tissue homogenates from ARDS model mice (n=6) and control mice (n=6) were measured by qRT-PCR. Student’s t test indicated that the mRNA levels of Ifih1 , Irf1 , Ifit3 , Gbp1 , and Stat1 were obviously upregulated in the ARDS mice ( P < 0.05). All error bars represent the SDs. (E) The plots show the associations of IFIH1 , IRF1 , IFIT3 , GBP1 , and STAT1 mRNA expression profiles in 26 ARDS patients’ peripheral blood with the severity of ARDS. Plot length represents the correlation coefficient between the mRNA expression profiles of each gene and the severity of ARDS. Plot color depth represents the P value from Spearman’s correlation analysis of the expression profiles of each gene and the severity of ARDS. (F) The heatmap represents the P values from panels (C–E) and the interrelated P values. The RobustRankAggreg algorithm helped us find the gene with the strongest ARDS-related correlation (minimum P value and maximum Spearman correlation coefficient). The interrelated P value was calculated via the RobustRankAggreg algorithm based on the P values from human and animal experiments.

Article Snippet: Furthermore, these labeled cRNAs were hybridized onto the Human mRNA Microarray V4.0 (Arraystar) chip, including 20,730 genes.

Techniques: Quantitative RT-PCR, Expressing, Control