Journal: The Prostate

Article Title: The protein kinase C super-family member PKN is regulated by mTOR and influences differentiation during prostate cancer progression.

doi: 10.1002/pros.23400

Figure Lengend Snippet: FIGURE 1 Regulation of PKN kinase activity. IP-kinase assays with WT and TM mutants of PKN1 (S916A) and PKN2 (T958A). Torin inhibited the PKN kinase activity to about the same extent as mutating the TM in both PKN isoforms. B, The PKN1 TM mutant S916A has reduced kinase activity toward multiple substrates. C, Deletion of the PKN N-terminus results in constitutive histone H3 phosphorylation in vitro and in cells. D and E, The PKN1 TM mutant S916A dramatically reduces autophosphorylation as well as Histone H3 and MARCKS phosphorylation

Article Snippet: PKN1 (Homo sapiens transcript variant 2, Origene, Rockville, MD, TC118456) and PKN2 (Addgene, Cambridge, MA, #20587) were cloned into pcDNA3 (Thermo Fisher Scientific, Grand Island, NY) along with anN-terminal Flag tag.

Techniques: Activity Assay, Mutagenesis, Phospho-proteomics, In Vitro

Journal: The Prostate

Article Title: The protein kinase C super-family member PKN is regulated by mTOR and influences differentiation during prostate cancer progression.

doi: 10.1002/pros.23400

Figure Lengend Snippet: FIGURE 3 Torin and rapamycin sensitivity of PKN, AKT, and PKCα. A, Cells stably transduced with WT PKN1 were treated with a range of torin and rapamycin concentrations for 24 h, and analyzed by using pan- and phosphosite-specific antibodies. B, Cells were treated with torin and rapamycin during a time course up to 24 h and subsequently analyzed by using pan- and phosphosite- specific antibodies

Article Snippet: PKN1 (Homo sapiens transcript variant 2, Origene, Rockville, MD, TC118456) and PKN2 (Addgene, Cambridge, MA, #20587) were cloned into pcDNA3 (Thermo Fisher Scientific, Grand Island, NY) along with anN-terminal Flag tag.

Techniques: Stable Transfection, Transduction, Phospho-proteomics

Journal: The Prostate

Article Title: The protein kinase C super-family member PKN is regulated by mTOR and influences differentiation during prostate cancer progression.

doi: 10.1002/pros.23400

Figure Lengend Snippet: FIGURE 2 PKN contains a TM phosphorylated by a torin- sensitive kinase. A, Alignment of TM sequences with the predicted phosphorylated residues indicated (bold). B, Transfection of PKN1 bearing mutations in the TM (S916A), activation loop (T774E) and ATP binding pocket (K644E) probed with antibodies specific for phos-S916 and phos-T774. Including nonphospho-TM peptide during the antibody incubation reduces the detection of non- phosphorylated PKN. C, IP-blot of WT PKN1 expressed in cells treated with torin and rapamycin

Article Snippet: PKN1 (Homo sapiens transcript variant 2, Origene, Rockville, MD, TC118456) and PKN2 (Addgene, Cambridge, MA, #20587) were cloned into pcDNA3 (Thermo Fisher Scientific, Grand Island, NY) along with anN-terminal Flag tag.

Techniques: Transfection, Activation Assay, Binding Assay, Incubation

Journal: The Prostate

Article Title: The protein kinase C super-family member PKN is regulated by mTOR and influences differentiation during prostate cancer progression.

doi: 10.1002/pros.23400

Figure Lengend Snippet: FIGURE 4 Cell motility functions of PKN. A, Localization of Flag-tagged PKN1 (green) at the cleavage furrow during mitosis, imaged by confocal microscopy. B, Examples of binucleate cells generated in response to depletion of PKN1, PKN2, and Ect2 (positive control), indicative of cytokinesis failure. C, Quantification of cytokinesis failure data as a consequence of PKN1 and PKN2 depletion. D, Expression levels (immunoblotting) of PKN1 and PKN2 after siRNA depletion. E, Stable C4-2b cell lines showing that (E) ectopic expression and (F) knockdown increase and decrease, respectively, cell migration in a Boyden chamber assay (****P < 0.0001). G, Transient depletion of PKN1, PKN2, and the TORC2 subunit Rictor reduces cell invasion of PC-3 cells to a similar extent as torin treatment

Article Snippet: PKN1 (Homo sapiens transcript variant 2, Origene, Rockville, MD, TC118456) and PKN2 (Addgene, Cambridge, MA, #20587) were cloned into pcDNA3 (Thermo Fisher Scientific, Grand Island, NY) along with anN-terminal Flag tag.

Techniques: Confocal Microscopy, Generated, Positive Control, Expressing, Western Blot, Knockdown, Migration, Boyden Chamber Assay

Journal: The Prostate

Article Title: The protein kinase C super-family member PKN is regulated by mTOR and influences differentiation during prostate cancer progression.

doi: 10.1002/pros.23400

Figure Lengend Snippet: FIGURE 5 Analysis of PKN isoform expression in human prostate cancer. A, Representative IHC showing PKN1 protein levels in normal, primary tumor, and lymph node metastasis. B, PKN1 and PKN2 expression (using microarray data from reference 47) in normal prostate, primary tumor, and metastases. C, RNA expression (using RNAseq data from TCGA) of PKN1-3 isoforms, PTEN, PKCα, AKT, and select mTOR components. **P < 0.01, ***P < 0.001, ****P < 0.0001

Article Snippet: PKN1 (Homo sapiens transcript variant 2, Origene, Rockville, MD, TC118456) and PKN2 (Addgene, Cambridge, MA, #20587) were cloned into pcDNA3 (Thermo Fisher Scientific, Grand Island, NY) along with anN-terminal Flag tag.

Techniques: Expressing, Microarray, RNA Expression

Journal: The Prostate

Article Title: The protein kinase C super-family member PKN is regulated by mTOR and influences differentiation during prostate cancer progression.

doi: 10.1002/pros.23400

Figure Lengend Snippet: FIGURE 6 Pkn2 is required for embryonic development. A, Embryos from Pkn1 and Pkn2 lacZ reporter mice were stained for β- galactosidase activity, and are shown as whole mount images. Upper row: E10.5, E11.5, E11.5. Scale bars: 1.0 mm. Bottom row: E6.5, E8.5 (side and dorsal view), E9.5, E9.5. Scale bars: 0.2 mm, 0.5 mm, 1.0 mm. B, Whole mount images of Pkn2 heterozygotes and homozygous null embryos at E7.0, E7.75, and E9.5. Scale bars 0.2 mm (upper four panels) 1.0 mm. C, Whole mount images of wild-type and Pkn2 null embryos analyzed by whole mount in situ hybridization for Otx2 (E7.5) and Bra (E7.25) are shown. Scale bars: 0.2 mm

Article Snippet: PKN1 (Homo sapiens transcript variant 2, Origene, Rockville, MD, TC118456) and PKN2 (Addgene, Cambridge, MA, #20587) were cloned into pcDNA3 (Thermo Fisher Scientific, Grand Island, NY) along with anN-terminal Flag tag.

Techniques: Staining, Activity Assay, In Situ Hybridization

Journal: The Prostate

Article Title: The protein kinase C super-family member PKN is regulated by mTOR and influences differentiation during prostate cancer progression.

doi: 10.1002/pros.23400

Figure Lengend Snippet: FIGURE 7 Analysis of PKN1 overexpression in prostate. A, Immunoblots showing transgenic expression of full-length (Tg-PKN1) and constitutively active (Tg-PKN1ΔN) proteins in anterior, dorsal, lateral, and ventral lobes (AP, DP, LP, VP). B-E, H&E stained images of sections through the ventral prostates from mice of the indicated genotypes are shown. The ages of the mice are as follows: WT, 53 weeks; Tg-PKN1, 58 weeks; Tg-PKN1ΔN, 58 weeks; Tg-AKT1, 52 weeks; Tg-AKT1;Tg-PKN1, 41 weeks; Tg-AKT1;Tg-PKN1ΔN, 52 weeks; TRAMP and TRAMP;Tg-PKN1, 16 weeks (showing HGPIN); TRAMP and TRAMP;Tg-PKN1, 17 weeks (showing small cell carcinoma). All images were captured at 200× magnification. Lower magnification views of the same samples are also provided (Supplemental Figure S3)

Article Snippet: PKN1 (Homo sapiens transcript variant 2, Origene, Rockville, MD, TC118456) and PKN2 (Addgene, Cambridge, MA, #20587) were cloned into pcDNA3 (Thermo Fisher Scientific, Grand Island, NY) along with anN-terminal Flag tag.

Techniques: Over Expression, Western Blot, Transgenic Assay, Expressing, Staining

Journal: The Prostate

Article Title: The protein kinase C super-family member PKN is regulated by mTOR and influences differentiation during prostate cancer progression.

doi: 10.1002/pros.23400

Figure Lengend Snippet: FIGURE 8 Analysis of PKNs in Pten null prostate tumors. H&E stained images of sections through the prostates from mice of the indicated genotypes are shown. All images were captured at 200× magnification and are of the ventral prostate, except for the right-most image in panel D, which shows squamous differentiation from the anterior prostate. The ages of the mice (panels A–C) are as follows: Ptenr/r, 12 and 45 weeks; Ptenr/r;Tg-PKN1, 12 and 43 weeks; Ptenr/r;Pkn1r/r;Pkn2r/r, 26 and 45 weeks. D, The images of invasive cancer (left and middle) are from 53-week ventral prostates, the squamous differentiation shown to the right is from the anterior prostate of a 53-week animal. Lower magnification views of the same samples are also provided (Supplemental Figure S4)

Article Snippet: PKN1 (Homo sapiens transcript variant 2, Origene, Rockville, MD, TC118456) and PKN2 (Addgene, Cambridge, MA, #20587) were cloned into pcDNA3 (Thermo Fisher Scientific, Grand Island, NY) along with anN-terminal Flag tag.

Techniques: 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 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:

Article Title: ZBED4, a BED-Type Zinc-Finger Protein in the Cones of the Human Retina

doi: 10.1167/iovs.08-2751

Figure Lengend Snippet: PCR Primers

Article Snippet: The human ZBED4 gene is 36.2 kb long, has two exons, and maps to chromosome 22. table ft1 table-wrap mode="anchored" t5 caption a7 cDNAs Number of Isolated cDNA Clones Cone opsin 3 ATPase, H+ transporting, lysosomal, subunit 1 4 Cone transducin α 2 2 Cone transducin β 3 1 Cone transducin γ 8 1 CRX 2 PDE α ′ 3 Dog Na/Cl dependent taurine 4 Dog glycoprotein 80 5 Dog mitochondrial genome (different regions) 32 Hydroxyl methylbilane synthase 1 Kinesin 6 Phosphatidyl serine receptor (PTDSR) 2 Ribosomal protein 2 Vascular protein sorting 35 (VPS 35) 4 Unknown cDNAs 8 Open in a separate window List of the Isolated cDNAs Resulting from Microarray Analysis of Subtracted Adult Retinal cd Dog mRNAs from mRNAs of Normal Dog Retina ZBED4 mRNA in Human and Mouse Retinas The HZB22 probe ( ) from the ZBED4 3′ UTR was hybridized to blots containing mRNAs from multiple human tissues (OriGene Technologies, Inc., Rockville, MD), retina and cone-derived Y79 cells.

Techniques:

Journal:

Article Title: ZBED4, a BED-Type Zinc-Finger Protein in the Cones of the Human Retina

doi: 10.1167/iovs.08-2751

Figure Lengend Snippet: Distribution of expressed ZBED4 in HEK293. (A) ZBED4 expression construct. (B) Immunocytochemical detection of the ZBED4 protein after transient transfection of the expression vector into HEK293 cells. Cells were double stained with antibodies against ZBED4 and Xpress and the resultant images were merged. Nuclear localization of expressed ZBED4 was detected by both antibodies and confirmed by staining with DAPI. (C) Western blot of nuclear and cytoplasmic extracts of HEK293 cells transfected for 24 or 72 hours. The blot was hybridized with anti-Xpress antibody. The expressed protein was present in the nuclear extract, confirming its immunocytochemical localization. Lanes 1 and 3: nuclear extracts; lanes 2 and 4: cytosolic extracts; lanes 1 and 2: extracts obtained after transfecting cells for 24 hours; lanes 3 and 4: extracts obtained after transfecting cells for 72 hours. (D) Localization of ZBED4 protein in Y79 cells. Note the granular pattern of ZBED4 staining in the nuclei.

Article Snippet: The human ZBED4 gene is 36.2 kb long, has two exons, and maps to chromosome 22. table ft1 table-wrap mode="anchored" t5 caption a7 cDNAs Number of Isolated cDNA Clones Cone opsin 3 ATPase, H+ transporting, lysosomal, subunit 1 4 Cone transducin α 2 2 Cone transducin β 3 1 Cone transducin γ 8 1 CRX 2 PDE α ′ 3 Dog Na/Cl dependent taurine 4 Dog glycoprotein 80 5 Dog mitochondrial genome (different regions) 32 Hydroxyl methylbilane synthase 1 Kinesin 6 Phosphatidyl serine receptor (PTDSR) 2 Ribosomal protein 2 Vascular protein sorting 35 (VPS 35) 4 Unknown cDNAs 8 Open in a separate window List of the Isolated cDNAs Resulting from Microarray Analysis of Subtracted Adult Retinal cd Dog mRNAs from mRNAs of Normal Dog Retina ZBED4 mRNA in Human and Mouse Retinas The HZB22 probe ( ) from the ZBED4 3′ UTR was hybridized to blots containing mRNAs from multiple human tissues (OriGene Technologies, Inc., Rockville, MD), retina and cone-derived Y79 cells.

Techniques: Expressing, Construct, Transfection, Plasmid Preparation, Staining, Western Blot

Journal:

Article Title: ZBED4, a BED-Type Zinc-Finger Protein in the Cones of the Human Retina

doi: 10.1167/iovs.08-2751

Figure Lengend Snippet: Northern blot analyses show ZBED4 mRNA in various human tissues and Y79 cells. (A) A human multitissue blot, and a separate blot containing human retina and Y79 cells mRNAs were probed with a 32P-labeled ZBED4 cDNA. Two transcripts were detected when using a 3′ UTR probe (HZB22). (B) An additional 4-kb transcript was detected in retina and Y79 cells when using a 5′ UTR probe (HZB4). Lanes: 1, brain; 2, colon; 3, heart; 4, kidney; 5, liver; 6, lung; 7, muscle; 8, placenta; 9, small intestine; 10, spleen; 11, stomach; 12, testis; 13, retina; and 14, Y79 cells.

Article Snippet: The human ZBED4 gene is 36.2 kb long, has two exons, and maps to chromosome 22. table ft1 table-wrap mode="anchored" t5 caption a7 cDNAs Number of Isolated cDNA Clones Cone opsin 3 ATPase, H+ transporting, lysosomal, subunit 1 4 Cone transducin α 2 2 Cone transducin β 3 1 Cone transducin γ 8 1 CRX 2 PDE α ′ 3 Dog Na/Cl dependent taurine 4 Dog glycoprotein 80 5 Dog mitochondrial genome (different regions) 32 Hydroxyl methylbilane synthase 1 Kinesin 6 Phosphatidyl serine receptor (PTDSR) 2 Ribosomal protein 2 Vascular protein sorting 35 (VPS 35) 4 Unknown cDNAs 8 Open in a separate window List of the Isolated cDNAs Resulting from Microarray Analysis of Subtracted Adult Retinal cd Dog mRNAs from mRNAs of Normal Dog Retina ZBED4 mRNA in Human and Mouse Retinas The HZB22 probe ( ) from the ZBED4 3′ UTR was hybridized to blots containing mRNAs from multiple human tissues (OriGene Technologies, Inc., Rockville, MD), retina and cone-derived Y79 cells.

Techniques: Northern Blot, Labeling

Journal:

Article Title: ZBED4, a BED-Type Zinc-Finger Protein in the Cones of the Human Retina

doi: 10.1167/iovs.08-2751

Figure Lengend Snippet: Expression level of ZBED4 and cell-specific marker mRNAs in normal and rd mouse retinas and in cone-sorted cells. (A) RT-PCR amplification of 80-day-old normal and rd mouse retinal mRNAs using primer sets, PDEα (for PDEα) and PDEα′-1 (for PDEα′) and ZB27 (for ZBED4). The resulting RT-PCR products were separated on a 1.2% agarose gel and stained with ethidium bromide. ZBED4 is expressed in rd retina but at a lower level than in normal retina, similar to the expression of PDEα′, suggesting the presence of ZBED4 mRNA in cones but not ruling out its expression in the inner retina. (B) Quantitative real-time RT-PCR. Levels of ZBED4 mRNA in normal and rd mouse retinas at different times of postnatal development relative to those of 40-day-old normal retina were compared to the relative levels of cell-specific markers expressed in the same samples (normalized to β-actin mRNA level using primer set mA1; Table 1). Rod-specific marker: PDEα; cone-specific marker: PDEα′. (C) Relative expression of rod PDEα, cone PDEα′, and ZBED4 mRNA between flow cytometry–sorted mouse cones and dissociated retinal cells measured by QPCR using β-actin cDNA as normalizer. The primer sets are described in Table 1.

Article Snippet: The human ZBED4 gene is 36.2 kb long, has two exons, and maps to chromosome 22. table ft1 table-wrap mode="anchored" t5 caption a7 cDNAs Number of Isolated cDNA Clones Cone opsin 3 ATPase, H+ transporting, lysosomal, subunit 1 4 Cone transducin α 2 2 Cone transducin β 3 1 Cone transducin γ 8 1 CRX 2 PDE α ′ 3 Dog Na/Cl dependent taurine 4 Dog glycoprotein 80 5 Dog mitochondrial genome (different regions) 32 Hydroxyl methylbilane synthase 1 Kinesin 6 Phosphatidyl serine receptor (PTDSR) 2 Ribosomal protein 2 Vascular protein sorting 35 (VPS 35) 4 Unknown cDNAs 8 Open in a separate window List of the Isolated cDNAs Resulting from Microarray Analysis of Subtracted Adult Retinal cd Dog mRNAs from mRNAs of Normal Dog Retina ZBED4 mRNA in Human and Mouse Retinas The HZB22 probe ( ) from the ZBED4 3′ UTR was hybridized to blots containing mRNAs from multiple human tissues (OriGene Technologies, Inc., Rockville, MD), retina and cone-derived Y79 cells.

Techniques: Expressing, Marker, Reverse Transcription Polymerase Chain Reaction, Amplification, Agarose Gel Electrophoresis, Staining, Quantitative RT-PCR, Flow Cytometry

Journal:

Article Title: ZBED4, a BED-Type Zinc-Finger Protein in the Cones of the Human Retina

doi: 10.1167/iovs.08-2751

Figure Lengend Snippet: In situ hybridization shows ZBED4 mRNA localized to inner segments of human photoreceptor cells in human retina. (A) Sections (8 μm thick) of fixed and cryoprotected human retina were hybridized with either antisense or sense ZBED4 digoxigenin (DIG)–labeled riboprobes. ZBED4 message localized to the inner segment of presumptive cone cells; no signal was detected with the sense probe. (B) Higher magnification of a labeled retinal section showing patchy expression of ZBED4, suggesting its expression in cone cells. OS, outer segments; IS, inner segments; ONL, outer nuclear layer; INL, inner nuclear layer; GCL, ganglion cell layer. Magnification: (A) ×200; (B) ×400.

Article Snippet: The human ZBED4 gene is 36.2 kb long, has two exons, and maps to chromosome 22. table ft1 table-wrap mode="anchored" t5 caption a7 cDNAs Number of Isolated cDNA Clones Cone opsin 3 ATPase, H+ transporting, lysosomal, subunit 1 4 Cone transducin α 2 2 Cone transducin β 3 1 Cone transducin γ 8 1 CRX 2 PDE α ′ 3 Dog Na/Cl dependent taurine 4 Dog glycoprotein 80 5 Dog mitochondrial genome (different regions) 32 Hydroxyl methylbilane synthase 1 Kinesin 6 Phosphatidyl serine receptor (PTDSR) 2 Ribosomal protein 2 Vascular protein sorting 35 (VPS 35) 4 Unknown cDNAs 8 Open in a separate window List of the Isolated cDNAs Resulting from Microarray Analysis of Subtracted Adult Retinal cd Dog mRNAs from mRNAs of Normal Dog Retina ZBED4 mRNA in Human and Mouse Retinas The HZB22 probe ( ) from the ZBED4 3′ UTR was hybridized to blots containing mRNAs from multiple human tissues (OriGene Technologies, Inc., Rockville, MD), retina and cone-derived Y79 cells.

Techniques: In Situ Hybridization, Labeling, Expressing

Journal:

Article Title: ZBED4, a BED-Type Zinc-Finger Protein in the Cones of the Human Retina

doi: 10.1167/iovs.08-2751

Figure Lengend Snippet: Specificity of anti-ZBED4 antibodies. (A) Protein extracts from both thymus and retina were separated on a Tris/Tricine-buffered 6% acrylamide/3% cross-linking gel and transferred onto PVDF membranes. Blots were incubated with the N terminus ZBED4 antibody (left) at a 1:3000 dilution. For the competition reaction (right), the antibody was incubated overnight with a 3000 molar excess of the peptide used to generate it. The labeled band that corresponds to ZBED4, apparent molecular mass of 135 kDa, is absent in the anti-ZBED+peptide blot. Similar results were obtained with the C terminus ZBED4 antibody. (B, C) Y79 cells were transiently transfected with siRNA duplexes targeted to the 5′ or 3′ regions of ZBED4 mRNA. (B) Ninety-six hours after transfection, total RNA was extracted, reverse transcribed, and subjected to QPCR. The level of mRNA expression in all samples is relative to that in control cells (C) and shows the silencing of ZBED4 in HEK 293 cells after transiently transfecting all three siRNAs. ZBED4 antibodies barely detected or did not detect at all the expression of ZBED4 in the silenced samples. C, control cells; TC, cells with transfection reagent only; NC, negative control (totally unrelated to ZBED4 siRNA); S1, S2, S3, double-stranded ZBED4 RNA oligomers (siRNAs). (C) Protein extracts from transfected HEK 293 cell lysates were assayed for ZBED4 gene silencing by Western blot analysis.

Article Snippet: The human ZBED4 gene is 36.2 kb long, has two exons, and maps to chromosome 22. table ft1 table-wrap mode="anchored" t5 caption a7 cDNAs Number of Isolated cDNA Clones Cone opsin 3 ATPase, H+ transporting, lysosomal, subunit 1 4 Cone transducin α 2 2 Cone transducin β 3 1 Cone transducin γ 8 1 CRX 2 PDE α ′ 3 Dog Na/Cl dependent taurine 4 Dog glycoprotein 80 5 Dog mitochondrial genome (different regions) 32 Hydroxyl methylbilane synthase 1 Kinesin 6 Phosphatidyl serine receptor (PTDSR) 2 Ribosomal protein 2 Vascular protein sorting 35 (VPS 35) 4 Unknown cDNAs 8 Open in a separate window List of the Isolated cDNAs Resulting from Microarray Analysis of Subtracted Adult Retinal cd Dog mRNAs from mRNAs of Normal Dog Retina ZBED4 mRNA in Human and Mouse Retinas The HZB22 probe ( ) from the ZBED4 3′ UTR was hybridized to blots containing mRNAs from multiple human tissues (OriGene Technologies, Inc., Rockville, MD), retina and cone-derived Y79 cells.

Techniques: Incubation, Labeling, Transfection, Reverse Transcription, Expressing, Control, Negative Control, Western Blot

Journal:

Article Title: ZBED4, a BED-Type Zinc-Finger Protein in the Cones of the Human Retina

doi: 10.1167/iovs.08-2751

Figure Lengend Snippet: Localization of ZBED4 in human retina. (A, C) Human retinal sections were double-stained with N terminus ZBED4 antibody followed by FITC-conjugated secondary antibody (green), rhodamine-conjugated PNA (red), and DAPI (blue). (A) Cone nuclei (arrows) and inner segments (arrowheads) are stained green, and the cone matrix is stained red. Note also the anti-ZBED4 staining of the innermost layer of the retina and of the cone pedicles (open arrowhead). (B) Magnified images individually stained with anti-ZBED4, PNA, and DAPI with the use of appropriate filters, and the merging of the three images. (C) An obliquely cut retinal section shows the cone inner segment localization of ZBED4 surrounded by PNA-stained cone extracellular matrix. OS, outer segments; IS, inner segments; ONL, outer nuclear layer; INL, inner nuclear layer; GCL, ganglion cell layer. (D) Top: section incubated with rabbit preimmune serum, rhodamine-conjugated PNA and DAPI. Bottom: section incubated with ZBED4 antibody that had been absorbed with the ZBED4 peptide used to generate it, rhodamine-conjugated PNA and DAPI. The absence of ZBED4 staining validates the ZBED4 antibody specificity. (E) Western blot of proteins from the nuclear extract of mouse thymus (lane 1) and from the nuclear (lane 2) and cytosolic (lane 3) fractions of human retina. ZBED4 (arrow) is very abundant in mouse thymus nuclei; therefore, it was used as a positive control in all Western blot analyses. (F) An obliquely cut human retinal section double-labeled with rabbit polyclonal anti-ZBED4 (green) and anti-human vimentin (red); nuclei were stained with DAPI (blue). Arrows: colocalization of vimentin and ZBED4 in Müller cell endfeet. Magnification: (A, D, F) ×400; (C) ×600.

Article Snippet: The human ZBED4 gene is 36.2 kb long, has two exons, and maps to chromosome 22. table ft1 table-wrap mode="anchored" t5 caption a7 cDNAs Number of Isolated cDNA Clones Cone opsin 3 ATPase, H+ transporting, lysosomal, subunit 1 4 Cone transducin α 2 2 Cone transducin β 3 1 Cone transducin γ 8 1 CRX 2 PDE α ′ 3 Dog Na/Cl dependent taurine 4 Dog glycoprotein 80 5 Dog mitochondrial genome (different regions) 32 Hydroxyl methylbilane synthase 1 Kinesin 6 Phosphatidyl serine receptor (PTDSR) 2 Ribosomal protein 2 Vascular protein sorting 35 (VPS 35) 4 Unknown cDNAs 8 Open in a separate window List of the Isolated cDNAs Resulting from Microarray Analysis of Subtracted Adult Retinal cd Dog mRNAs from mRNAs of Normal Dog Retina ZBED4 mRNA in Human and Mouse Retinas The HZB22 probe ( ) from the ZBED4 3′ UTR was hybridized to blots containing mRNAs from multiple human tissues (OriGene Technologies, Inc., Rockville, MD), retina and cone-derived Y79 cells.

Techniques: Staining, Incubation, Western Blot, Positive Control, Labeling

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: The Prostate

Article Title: The Protein Kinase C Super-family Member PKN is Regulated by mTOR and Influences Differentiation During Prostate Cancer Progression

doi: 10.1002/pros.23400

Figure Lengend Snippet: (A) Alignment of TM sequences with the predicted phosphorylated residues indicated (bold). (B) Transfection of PKN1 bearing mutations in the TM (S916A), activation loop (T774E) and ATP binding pocket (K644E) probed with antibodies specific for phos-S916 and phos-T774. Including non-phospho-TM peptide during the antibody incubation reduces the detection of non-phosphorylated PKN. (C) IP-blot of WT PKN1 expressed in cells treated with torin and rapamycin.

Article Snippet: Plasmids and siRNAs PKN1 (Homo sapiens transcript variant 2, Origene, Rockville, MD, TC118456) and PKN2 (Addgene, Cambridge, MA, #20587) were cloned into pcDNA3 (Thermo Fisher Scientific, Grand Island, NY) along with an N-terminal Flag tag.

Techniques: Transfection, Activation Assay, Binding Assay, Incubation

Journal: The Prostate

Article Title: The Protein Kinase C Super-family Member PKN is Regulated by mTOR and Influences Differentiation During Prostate Cancer Progression

doi: 10.1002/pros.23400

Figure Lengend Snippet: IP-kinase assays with WT and TM mutants of PKN1 (S916A) and PKN2 (T958A). Torin inhibited the PKN kinase activity to about the same extent as mutating the TM in both PKN isoforms. (B) The PKN1 TM mutant S916A has reduced kinase activity towards multiple substrates. (C) Deletion of the PKN N-terminus results in constitutive histone H3 phosphorylation in vitro and in cells. (D, E) The PKN1 TM mutant S916A dramatically reduces autophosphorylation as well as Histone H3 and MARCKS phosphorylation.

Article Snippet: Plasmids and siRNAs PKN1 (Homo sapiens transcript variant 2, Origene, Rockville, MD, TC118456) and PKN2 (Addgene, Cambridge, MA, #20587) were cloned into pcDNA3 (Thermo Fisher Scientific, Grand Island, NY) along with an N-terminal Flag tag.

Techniques: Activity Assay, Mutagenesis, Phospho-proteomics, In Vitro

Journal: The Prostate

Article Title: The Protein Kinase C Super-family Member PKN is Regulated by mTOR and Influences Differentiation During Prostate Cancer Progression

doi: 10.1002/pros.23400

Figure Lengend Snippet: (A) Cells stably transduced with WT PKN1 were treated with a range of torin and rapamycin concentrations for 24 hrs, and analyzed by using pan- and phosphosite-specific antibodies. (B) Cells were treated with torin and rapamycin during a time course up to 24 hrs and subsequently analyzed by using pan- and phosphosite-specific antibodies.

Article Snippet: Plasmids and siRNAs PKN1 (Homo sapiens transcript variant 2, Origene, Rockville, MD, TC118456) and PKN2 (Addgene, Cambridge, MA, #20587) were cloned into pcDNA3 (Thermo Fisher Scientific, Grand Island, NY) along with an N-terminal Flag tag.

Techniques: Stable Transfection, Transduction, Phospho-proteomics

Journal: The Prostate

Article Title: The Protein Kinase C Super-family Member PKN is Regulated by mTOR and Influences Differentiation During Prostate Cancer Progression

doi: 10.1002/pros.23400

Figure Lengend Snippet: (A) Localization of Flag-tagged PKN1 (green) at the cleavage furrow during mitosis, imaged by confocal microscopy. (B) Examples of binucleate cells generated in response to depletion of PKN1, PKN2, and Ect2 (positive control), indicative of cytokinesis failure. (C) Quantification of cytokinesis failure data as a consequence of PKN1 and PKN2 depletion. (D) Expression levels (immunoblotting) of PKN1 and PKN2 after siRNA depletion. (E) Stable C4-2b cell lines showing that (E) ectopic expression and (F) knockdown increase and decrease, respectively, cell migration in a Boyden chamber assay (**** p =< 0.0001). (G) Transient depletion of PKN1, PKN2, and the TORC2 subunit Rictor reduces cell invasion of PC-3 cells to a similar extent as torin treatment.

Article Snippet: Plasmids and siRNAs PKN1 (Homo sapiens transcript variant 2, Origene, Rockville, MD, TC118456) and PKN2 (Addgene, Cambridge, MA, #20587) were cloned into pcDNA3 (Thermo Fisher Scientific, Grand Island, NY) along with an N-terminal Flag tag.

Techniques: Confocal Microscopy, Generated, Positive Control, Expressing, Western Blot, Knockdown, Migration, Boyden Chamber Assay

Journal: The Prostate

Article Title: The Protein Kinase C Super-family Member PKN is Regulated by mTOR and Influences Differentiation During Prostate Cancer Progression

doi: 10.1002/pros.23400

Figure Lengend Snippet: (A) Representative IHC showing PKN1 protein levels in normal, primary tumor, and lymph node metastasis. (B) PKN1 and PKN2 expression (using microarray data from [47]) in normal prostate, primary tumor, and metastases. (C) RNA expression (using RNAseq data from TCGA) of PKN1-3 isoforms, PTEN, PKCα, AKT and select mTOR components. ** p =< 0.01 *** p =< 0.001 **** p =< 0.0001

Article Snippet: Plasmids and siRNAs PKN1 (Homo sapiens transcript variant 2, Origene, Rockville, MD, TC118456) and PKN2 (Addgene, Cambridge, MA, #20587) were cloned into pcDNA3 (Thermo Fisher Scientific, Grand Island, NY) along with an N-terminal Flag tag.

Techniques: Expressing, Microarray, RNA Expression

Journal: The Prostate

Article Title: The Protein Kinase C Super-family Member PKN is Regulated by mTOR and Influences Differentiation During Prostate Cancer Progression

doi: 10.1002/pros.23400

Figure Lengend Snippet: Genotypes of weaned mice.

Article Snippet: Plasmids and siRNAs PKN1 (Homo sapiens transcript variant 2, Origene, Rockville, MD, TC118456) and PKN2 (Addgene, Cambridge, MA, #20587) were cloned into pcDNA3 (Thermo Fisher Scientific, Grand Island, NY) along with an N-terminal Flag tag.

Techniques:

Journal: The Prostate

Article Title: The Protein Kinase C Super-family Member PKN is Regulated by mTOR and Influences Differentiation During Prostate Cancer Progression

doi: 10.1002/pros.23400

Figure Lengend Snippet: Genotypes of weaned mice from double heterozygous intercrosses.

Article Snippet: Plasmids and siRNAs PKN1 (Homo sapiens transcript variant 2, Origene, Rockville, MD, TC118456) and PKN2 (Addgene, Cambridge, MA, #20587) were cloned into pcDNA3 (Thermo Fisher Scientific, Grand Island, NY) along with an N-terminal Flag tag.

Techniques:

Journal: The Prostate

Article Title: The Protein Kinase C Super-family Member PKN is Regulated by mTOR and Influences Differentiation During Prostate Cancer Progression

doi: 10.1002/pros.23400

Figure Lengend Snippet: (A) Embryos from Pkn1 and Pkn2 lacZ reporter mice were stained for β-galactosidase activity, and are shown as whole mount images. Upper row: E10.5, E11.5, E11.5. Scale bars: 1.0mm. Bottom row: E6.5, E8.5 (side and dorsal view), E9.5, E9.5. Scale bars: 0.2mm, 0.5mm, 1.0mm. B) Whole mount images of Pkn2 heterozygotes and homozygous null embryos at E7.0, E7.75 and E9.5. Scale bars 0.2mm (upper four panels), 1.0mm. (C) Whole mount images of wild type and Pkn2 null embryos analyzed by whole mount in situ hybridization for Otx2 (E7.5) and Bra (E7.25) are shown. Scale bars: 0.2mm.

Article Snippet: Plasmids and siRNAs PKN1 (Homo sapiens transcript variant 2, Origene, Rockville, MD, TC118456) and PKN2 (Addgene, Cambridge, MA, #20587) were cloned into pcDNA3 (Thermo Fisher Scientific, Grand Island, NY) along with an N-terminal Flag tag.

Techniques: Staining, Activity Assay, In Situ Hybridization

Journal: The Prostate

Article Title: The Protein Kinase C Super-family Member PKN is Regulated by mTOR and Influences Differentiation During Prostate Cancer Progression

doi: 10.1002/pros.23400

Figure Lengend Snippet: (A) Immunoblots showing transgenic expression of full-length (Tg-PKN1) and constitutively active (Tg-PKN1ΔN) proteins in anterior, dorsal, lateral, and ventral lobes (AP, DP, LP, VP). (B–E) H&E stained images of sections through the ventral prostates from mice of the indicated genotypes are shown. The ages of the mice are as follows: WT, 53 weeks; Tg-PKN1, 58 weeks; Tg-PKN1ΔN, 58 weeks; Tg-AKT1, 52 weeks; Tg-AKT1; Tg-PKN1, 41 weeks; Tg-AKT1; Tg-PKN1ΔN, 52 weeks; TRAMP and TRAMP; Tg-PKN1, 16 weeks (showing HGPIN); TRAMP and TRAMP; Tg-PKN1, 17 weeks (showing small cell carcinoma). All images were captured at 200× magnification. Lower magnification views of the same samples are also provided (Supplemental Fig. 3).

Article Snippet: Plasmids and siRNAs PKN1 (Homo sapiens transcript variant 2, Origene, Rockville, MD, TC118456) and PKN2 (Addgene, Cambridge, MA, #20587) were cloned into pcDNA3 (Thermo Fisher Scientific, Grand Island, NY) along with an N-terminal Flag tag.

Techniques: Western Blot, Transgenic Assay, Expressing, Staining

Journal: The Prostate

Article Title: The Protein Kinase C Super-family Member PKN is Regulated by mTOR and Influences Differentiation During Prostate Cancer Progression

doi: 10.1002/pros.23400

Figure Lengend Snippet: Prostate phenotypes in the ventral lobe

Article Snippet: Plasmids and siRNAs PKN1 (Homo sapiens transcript variant 2, Origene, Rockville, MD, TC118456) and PKN2 (Addgene, Cambridge, MA, #20587) were cloned into pcDNA3 (Thermo Fisher Scientific, Grand Island, NY) along with an N-terminal Flag tag.

Techniques:

Journal: The Prostate

Article Title: The Protein Kinase C Super-family Member PKN is Regulated by mTOR and Influences Differentiation During Prostate Cancer Progression

doi: 10.1002/pros.23400

Figure Lengend Snippet: H&E stained images of sections through the prostates from mice of the indicated genotypes are shown. All images were captured at 200x magnification and are of the ventral prostate, except for the right-most image in panel D, which shows squamous differentiation from the anterior prostate. The ages of the mice (panels A–C) are as follows: Ptenr/r, 12 and 45 weeks; Ptenr/r ;Tg-PKN1, 12 and 43 weeks; Ptenr/r;Pkn1r/r ;Pkn2r/r, 26 and 45 weeks. (D) The images of invasive cancer (left and middle) are from 53 week ventral prostates, the squamous differentiation shown to the right is from the anterior prostate of a 53 week animal. Lower magnification views of the same samples are also provided (Supplemental Fig. 4).

Article Snippet: Plasmids and siRNAs PKN1 (Homo sapiens transcript variant 2, Origene, Rockville, MD, TC118456) and PKN2 (Addgene, Cambridge, MA, #20587) were cloned into pcDNA3 (Thermo Fisher Scientific, Grand Island, NY) along with an N-terminal Flag tag.

Techniques: Staining