Journal: Cell reports

Article Title: linc-AAM Facilitates Gene Expression Contributing to Macrophage Activation and Adaptive Immune Responses.

doi: 10.1016/j.celrep.2020.108584

Figure Lengend Snippet: Figure 1. Identification of linc-AAM in Activated Macrophages (A) Quantitative real-time PCR verification of top six upregulated lncRNAs in RAW264.7 cells treated with medium (control [Ctrl]) or AEPS (50 mg/mL) for 1 h (n = 3). (B) Quantitative real-time PCR analysis of linc-AAM, COX-2, IL-1b, IL-6, IL-10, TNF-a, CCL2, CCL5, and CCL22 in RAW264.7 cells treated with AEPS (50 mg/mL) for different times (n = 3). (C) The RACE analysis of linc-AAM using total RNA extracted from RAW264.7 cells treated by AEPS (50 mg/mL) for 1 h. (D) Northern blotting of linc-AAM in RAW264.7 cells treated with medium (Ctrl) or AEPS (50 mg/mL) for 1 h. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA served as a loading control. The images shown are representative of three independent experiments. (E and F) Electrophoretogram (E) and quantitative real-time PCR analysis (F) of linc-AAM in cytoplasm and nuclei of RAW264.7 cells treated with medium (Ctrl) or AEPS (50 mg/mL) for 1 h (n = 3). (G) linc-AAM (red) was detected in RAW264.7 cells by RNA fluorescence in situ hybridization (FISH). Nuclei (blue) were counterstained with DAPI. Scale bars, 10 mm. (H) Identification of coding capability of linc-AAM using in vitro transcription and translation system. Full-length linc-AAM was cloned into the eukaryotic expression vector pcDNA3.1() with N-terminal start codon ATG and C-terminal FLAG tag in all three coding patterns, and these plasmids were subsequently transfected into HEK293T cells, respectively. Immunoblotting was used to detect the FLAG-tagged protein. STAT3 with FLAG tag severs as a positive control. The images shown were representative of three independent experiments. (I) Quantitative real-time PCR analysis of linc-AAM in different tissues from six C57BL/6 mice per group, male and female in half. (J) Quantitative real-time PCR analysis of linc-AAM in RAW264.7 cells, BMDMs, PMs, and BMDCs treated with medium (Ctrl) or AEPS (50 mg/mL) for 1 h (n = 3). (K) Quantitative real-time PCR analysis of linc-AAM and IL-1b in BMDMs treated with AEPS (50 mg/mL) for different times (n = 3). (L) Quantitative real-time PCR analysis of linc-AAM, IL-1b, and COX-2 in RAW264.7 cells stimulated with AEPS (50 mg/mL), LPS (100 ng/mL), poly(I:C) (50 mg/mL), Alum (200 mg/mL), or Quil A (20 mg/mL) for 1 or 4 h, respectively (n = 3). Data are presented as mean ± SEM. ***p < 0.001. See also Figure S1.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Pyrrolidinedithiocarbamate ammonium (PDTC) Beyotime Cat# S1808 Red blood cell lysis buffer Beyotime C3702 Enhanced chemiluminescence (ECL) kit Beyotime P0018AS Proteinase K Beyotime Cat# ST533 Radioimmunoprecipitation (RIPA) buffer Beyotime Cat# P0013B TRIzol reagent Ambion Cat# 15596026 Lipofectamine 3000 Invitrogen Cat# L3000015 Lipofectamine LTX reagent with PLUS reagent Invitrogen Cat# 15338100 Rela (Myc-DDK-tagged)-pCMV6 OriGene Cat# MR227671 Stat3 (Myc-DDK-tagged)-pCMV6 OriGene Cat# MR227265 pCMV6-Entry OriGene Cat# PS100001 PrimeSTAR max DNA polymerase Takara Cat# R045A pLXSN retroviral Vector Clontech Cat# 631509 pcDNA3.1(-) eukaryotic expression vector Invitrogen Cat# V79520 pGL3-Basic Vector Promega Cat# E1751 Stat3 shRNA(m) lentiviral particles Santa Cruz Cat# sc-29494-V NF-kB p65 shRNA(m) lentiviral particles Santa Cruz Cat# sc-29411-V Control shRNA(m) lentiviral particles Santa Cruz Cat# sc-108080 G418 sulfate BBI Cat# A600958-0001 Puromycin InvivoGen Cat# ANT-PR Blasticidin InvivoGen Cat# ant-bl-05 FastStart Universal SYBR Green Master (Rox) Roche Cat# 4913850001 CSPD Roche Cat#11655884001 Anti-digoxigenin AP-conjungate Roche Cat#11093274910 Ribonucleoside vanadyl complex New England BioLabs Cat# S1402S MyOne T1 streptavidin beads Invitrogen Cat# 65604D Albumin from chicken egg white Sigma-Aldrich Cat# A5503 Concanavalin A Sigma-Aldrich Cat# L6397 Critical Commercial Assays Mouse IL-10 ELISA kit Boster Cat# EK0417 Mouse TNF-a ELISA kit Boster Cat# EK0527 Mouse IL-1b ELISA kit Boster Cat# EK0394 Mouse CCL2 ELISA kit Boster Cat# EK0568 Mouse IL-6 ELISA kit Boster Cat# EK0411 Mouse tail direct PCR Kit Bimake Cat# B40013 BLOCK-iT Pol II miR RNAi Expression Vector Kits with EmGFP Invitrogen Cat# K4936-00 Maxima First Strand cDNA Synthesis Kit Thermo Fisher Cat# K1671 NorthernMax Gly Kit Ambion Cat# AM1946 PrimeScript RT reagent Kit with gDNA Eraser Takara Cat# RR047A SMARTer RACE kit Clontech Cat# 634858 Dual-Luciferase Reporter Assay System Promega Cat# E1910 MEGAscript T7 Transcription Kit Ambion Cat# AM1333 Pierce RNA 30 End Desthiobiotinylation Kit Thermo Scientific Cat# 20163 PierceMagnetic RNA-Protein Pull-Down Kit Thermo Scientific Cat# 20164 (Continued on next page) Cell Reports 34, 108584, January 5, 2021 e2

Techniques: Real-time Polymerase Chain Reaction, Control, Northern Blot, In Situ Hybridization, In Vitro, Clone Assay, Expressing, Plasmid Preparation, FLAG-tag, Transfection, Western Blot, Positive Control

Journal: Cell reports

Article Title: linc-AAM Facilitates Gene Expression Contributing to Macrophage Activation and Adaptive Immune Responses.

doi: 10.1016/j.celrep.2020.108584

Figure Lengend Snippet: Figure 2. linc-AAM Silencing or Knockout (KO) Inhibits Macrophage Activation and the Expression of IRGs in Vitro and ex Vivo (A) Heatmap of differentially expressed genes in linc-AAM-RNAi RAW264.7 cells (linc-AAM KD) relative to Ctrl-RNAi cells (Scramble) treated with AEPS for 4 h. The most downregulated genes in linc-AAM KD cells compared with Scramble cells are magnified into view. (B) Quantitative real-time PCR analysis of linc-AAM, COX-2, IL-1b, IL-10, TNF-a, CCL2, CCL3, CCL4, CCL5, and CCL22 in linc-AAM KD and Scramble RAW264.7 cells treated with AEPS (50 mg/mL) for 4 h (n = 3). (C) The production of IL-1b and IL-6 from linc-AAM KD and Scramble RAW264.7 cells treated with medium (Ctrl) or AEPS (50 mg/mL) for 24 h using ELISA (n = 3). nd, not detectable. (D) Western bolt analysis of COX-2 in linc-AAM KD and Scramble RAW264.7 cells treated with AEPS (50 mg/mL) for 12 and 24 h. (E) Fluorescence-activated cell sorting (FACS) analysis of surface molecules (Ly6G, CD40, CD80, CD86, MHC I, and MHC II) in linc-AAM KD and Scramble RAW264.7 cells treated with AEPS (50 mg/mL) for 24 h (n = 3). (F) Quantitative real-time PCR analysis of linc-AAM potential target genes IL-1b, IL-6, COX-2, TNF-a, CCL2, and CXCL10 in BMDMs from WT and linc-AAM KO mice treated with medium (untreatment) or AEPS (25 mg/mL) for 4 or 8 h (n = 3). (G) The production of IL-6 and TNF-a in BMDMs from WT and linc-AAM KO mice treated with medium (Ctrl) or AEPS (25 mg/mL) for 24 h using ELISA (n = 3). (H) FACS analysis of surface molecules (CD40, CD80, CD86, MHC I, and MHC II) of BMDMs from WT or linc-AAM KO mice treated with medium (Ctrl) or AEPS (25 mg/mL) for 24 h (n = 3). (I) Quantitative real-time PCR analysis of miR155hg in linc-AAM KD and Scramble RAW264.7 cells treated by AEPS (50 mg/mL) for 4 h. (J) Quantitative real-time PCR analysis of miR155hg in BMDMs from WT and linc-AAM KO mice after treated by AEPS (25 mg/mL) for 4 h. Data are presented as mean ± SEM. *p < 0.05, **p < 0.01, and ***p < 0.001. See also Figures S2 and S3.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Pyrrolidinedithiocarbamate ammonium (PDTC) Beyotime Cat# S1808 Red blood cell lysis buffer Beyotime C3702 Enhanced chemiluminescence (ECL) kit Beyotime P0018AS Proteinase K Beyotime Cat# ST533 Radioimmunoprecipitation (RIPA) buffer Beyotime Cat# P0013B TRIzol reagent Ambion Cat# 15596026 Lipofectamine 3000 Invitrogen Cat# L3000015 Lipofectamine LTX reagent with PLUS reagent Invitrogen Cat# 15338100 Rela (Myc-DDK-tagged)-pCMV6 OriGene Cat# MR227671 Stat3 (Myc-DDK-tagged)-pCMV6 OriGene Cat# MR227265 pCMV6-Entry OriGene Cat# PS100001 PrimeSTAR max DNA polymerase Takara Cat# R045A pLXSN retroviral Vector Clontech Cat# 631509 pcDNA3.1(-) eukaryotic expression vector Invitrogen Cat# V79520 pGL3-Basic Vector Promega Cat# E1751 Stat3 shRNA(m) lentiviral particles Santa Cruz Cat# sc-29494-V NF-kB p65 shRNA(m) lentiviral particles Santa Cruz Cat# sc-29411-V Control shRNA(m) lentiviral particles Santa Cruz Cat# sc-108080 G418 sulfate BBI Cat# A600958-0001 Puromycin InvivoGen Cat# ANT-PR Blasticidin InvivoGen Cat# ant-bl-05 FastStart Universal SYBR Green Master (Rox) Roche Cat# 4913850001 CSPD Roche Cat#11655884001 Anti-digoxigenin AP-conjungate Roche Cat#11093274910 Ribonucleoside vanadyl complex New England BioLabs Cat# S1402S MyOne T1 streptavidin beads Invitrogen Cat# 65604D Albumin from chicken egg white Sigma-Aldrich Cat# A5503 Concanavalin A Sigma-Aldrich Cat# L6397 Critical Commercial Assays Mouse IL-10 ELISA kit Boster Cat# EK0417 Mouse TNF-a ELISA kit Boster Cat# EK0527 Mouse IL-1b ELISA kit Boster Cat# EK0394 Mouse CCL2 ELISA kit Boster Cat# EK0568 Mouse IL-6 ELISA kit Boster Cat# EK0411 Mouse tail direct PCR Kit Bimake Cat# B40013 BLOCK-iT Pol II miR RNAi Expression Vector Kits with EmGFP Invitrogen Cat# K4936-00 Maxima First Strand cDNA Synthesis Kit Thermo Fisher Cat# K1671 NorthernMax Gly Kit Ambion Cat# AM1946 PrimeScript RT reagent Kit with gDNA Eraser Takara Cat# RR047A SMARTer RACE kit Clontech Cat# 634858 Dual-Luciferase Reporter Assay System Promega Cat# E1910 MEGAscript T7 Transcription Kit Ambion Cat# AM1333 Pierce RNA 30 End Desthiobiotinylation Kit Thermo Scientific Cat# 20163 PierceMagnetic RNA-Protein Pull-Down Kit Thermo Scientific Cat# 20164 (Continued on next page) Cell Reports 34, 108584, January 5, 2021 e2

Techniques: Knock-Out, Activation Assay, Expressing, In Vitro, Ex Vivo, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Western Blot, Fluorescence, FACS

Journal: Cell reports

Article Title: linc-AAM Facilitates Gene Expression Contributing to Macrophage Activation and Adaptive Immune Responses.

doi: 10.1016/j.celrep.2020.108584

Figure Lengend Snippet: Figure 6. linc-AAM Facilitates Chromatin Activation to Promote the Transcription of IRGs (A) Cross-linked RIP to detect linc-AAM association with H3 and H3K4me3. The nuclear lysates of RAW264.7 cells were IPed with control IgG, anti-histone H3, or anti-histone H3K4me3 antibody, and then the complexes were analyzed for the presence of linc-AAM by Quantitative real-time PCR (n = 3). Signals were normalized to 10% input samples. (B) Western blot analysis of histone H3 in RNA pull-down assay samples of linc-AAM and its different mutants as in Figure 5G. (C and D) coIP analysis of the interaction between hnRNPL and histone H3 or H3K4me3 in RAW264.7 cells treated by medium or AEPS for 1 h (n = 3). (E) coIP analysis of the interaction between hnRNPL and histone H3 in BMDMs from WT or linc-AAM KO mice treated by medium or AEPS for 2 h (n = 3). (F) Mouse IL-1b promoter-driven Luc activities in HEK293T cells transfected with linc-AAM overexpression plasmids or empty plasmids (Ctrl). (G) ChIRP enrichment analysis for linc-AAM and control IL-1b. LacZ antisense DNA probes are used as negative controls. (H) linc-AAM ChIRP-qPCR in AEPS-treated RAW264.7 cells. The sequences of primers used in qPCR for CCL2, IL-1b, COX-2, CCL5, TNF-a, CXCL10, IL-6, and GAPDH are within their promoter regions. GAPDH served as a negative control. (I) Integrated model depicting linc-AAM functioning to facilitate inducible expression of IRGs in macrophages. TF, transcription factor. Data are presented as mean ± SEM. **p < 0.01 and ***p < 0.001. See also Figure S6.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Pyrrolidinedithiocarbamate ammonium (PDTC) Beyotime Cat# S1808 Red blood cell lysis buffer Beyotime C3702 Enhanced chemiluminescence (ECL) kit Beyotime P0018AS Proteinase K Beyotime Cat# ST533 Radioimmunoprecipitation (RIPA) buffer Beyotime Cat# P0013B TRIzol reagent Ambion Cat# 15596026 Lipofectamine 3000 Invitrogen Cat# L3000015 Lipofectamine LTX reagent with PLUS reagent Invitrogen Cat# 15338100 Rela (Myc-DDK-tagged)-pCMV6 OriGene Cat# MR227671 Stat3 (Myc-DDK-tagged)-pCMV6 OriGene Cat# MR227265 pCMV6-Entry OriGene Cat# PS100001 PrimeSTAR max DNA polymerase Takara Cat# R045A pLXSN retroviral Vector Clontech Cat# 631509 pcDNA3.1(-) eukaryotic expression vector Invitrogen Cat# V79520 pGL3-Basic Vector Promega Cat# E1751 Stat3 shRNA(m) lentiviral particles Santa Cruz Cat# sc-29494-V NF-kB p65 shRNA(m) lentiviral particles Santa Cruz Cat# sc-29411-V Control shRNA(m) lentiviral particles Santa Cruz Cat# sc-108080 G418 sulfate BBI Cat# A600958-0001 Puromycin InvivoGen Cat# ANT-PR Blasticidin InvivoGen Cat# ant-bl-05 FastStart Universal SYBR Green Master (Rox) Roche Cat# 4913850001 CSPD Roche Cat#11655884001 Anti-digoxigenin AP-conjungate Roche Cat#11093274910 Ribonucleoside vanadyl complex New England BioLabs Cat# S1402S MyOne T1 streptavidin beads Invitrogen Cat# 65604D Albumin from chicken egg white Sigma-Aldrich Cat# A5503 Concanavalin A Sigma-Aldrich Cat# L6397 Critical Commercial Assays Mouse IL-10 ELISA kit Boster Cat# EK0417 Mouse TNF-a ELISA kit Boster Cat# EK0527 Mouse IL-1b ELISA kit Boster Cat# EK0394 Mouse CCL2 ELISA kit Boster Cat# EK0568 Mouse IL-6 ELISA kit Boster Cat# EK0411 Mouse tail direct PCR Kit Bimake Cat# B40013 BLOCK-iT Pol II miR RNAi Expression Vector Kits with EmGFP Invitrogen Cat# K4936-00 Maxima First Strand cDNA Synthesis Kit Thermo Fisher Cat# K1671 NorthernMax Gly Kit Ambion Cat# AM1946 PrimeScript RT reagent Kit with gDNA Eraser Takara Cat# RR047A SMARTer RACE kit Clontech Cat# 634858 Dual-Luciferase Reporter Assay System Promega Cat# E1910 MEGAscript T7 Transcription Kit Ambion Cat# AM1333 Pierce RNA 30 End Desthiobiotinylation Kit Thermo Scientific Cat# 20163 PierceMagnetic RNA-Protein Pull-Down Kit Thermo Scientific Cat# 20164 (Continued on next page) Cell Reports 34, 108584, January 5, 2021 e2

Techniques: Activation Assay, Control, Real-time Polymerase Chain Reaction, Western Blot, Pull Down Assay, Transfection, Over Expression, Negative Control, Expressing

Journal: RNA

Article Title: Novel small RNA expression libraries uncover hsa-miR-30b and hsa-miR-30c as important factors in anoikis resistance

doi: 10.1261/rna.039461.113

Figure Lengend Snippet: A functional screening using a novel small RNAs expression library reveals hsa-miR-30b and hsa-miR-30c as repressors of anoikis. ( A ) Immortalized RPE-1 cells were infected with a lentiviral expression library containing a small RNAs collection from the MDA-MB-231 cell line. RPE-1-infected cells were cultured for 19 d in agar dishes at low confluence avoiding any contact and surviving clones transferred to adhesive culture dishes to allow the growth of anoikis-resistant cells. DNA from these clones was isolated and sequenced. ( B ) Table showing the small RNA sequences overexpressed in anoikis-resistant clones. ( C ) Flow cytometry analysis of the levels of apoptosis in RPE-1 cells (SubG1/SubG1 control + polyHEMA) infected with lentivirus containing candidate mature miRNAs or empty vector (control). Three days after infection, cells were deprived of serum for 24 h and then cultured in polyhema (PH) plates plus methylcellulose (2%) in serum-free medium for another 24 h. ( D ) RNase protection assay of miR-30b and miR-30c transiently overexpressed in HEK-293 cells from pLENT-DUAL plasmid. Control (Ctr) cells expressed the empty vector pLENT-DUAL; Y corresponds to yeast RNA; M, Decade RNA markers (Ambion). ( E ) Levels of apoptosis in RPE-1 cells (SubG1/SubG1 control) infected with lentivirus carrying full-length miR-30b, miR-30c-1 (FL), miR-30b/c mature miRNAs (Lent-30b/c), and the corresponding empty vectors as controls in a similar assay as described in C . Results are shown as the averages ± standard errors of the means from at least three independent experiments and were analyzed by one-way ANOVA, followed by Bonferroni post-test for significance versus control cells. (*) P < 0.05, (**) P < 0.01, (***) P < 0.001.

Article Snippet: Immortalized human retinal pigment epithelial cell line hTERT RPE-1 (Clontech) was maintained in DMEM/F12 supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine, and antibiotics (50 units/mL penicillin, 50 μ/mL streptomycin).

Techniques: Functional Assay, Expressing, Infection, Cell Culture, Clone Assay, Isolation, Flow Cytometry, Plasmid Preparation, Rnase Protection Assay

Journal: RNA

Article Title: Novel small RNA expression libraries uncover hsa-miR-30b and hsa-miR-30c as important factors in anoikis resistance

doi: 10.1261/rna.039461.113

Figure Lengend Snippet: Hsa-miR-30b and hsa-miR-30c down-regulate CASP3 expression through its 3′ UTR-binding. ( A ) Western blot analysis of p53, CASP3, and BCL2-like 11 (BIM), three putative target proteins of miR-30b/c related to anoikis. RPE-1 cells were infected with lentivirus expressing full-length miR-30b, miR-30c-1 (miR-30c), or empty vector as a control. Active CASP3 was analyzed in cells cultured under anoikis conditions as described in C. Actin (ACTB) was used as the loading control. ( B ) Densitometric analysis of pro-CASP3 and active CASP3 levels. ACTB protein was used as the normalization control. Results are shown as the averages ± standard errors of the means from at least three independent experiments and were analyzed by one-way ANOVA, followed by Bonferroni post-test (* P < 0.05; ** P < 0.01). ( C ) Schematic representation of the caspase 3 3′ UTR containing two putative binding sites for miR-30b/c. The 3′ UTR of CASP3 mRNA was cloned downstream from the open reading frame of luciferase (pLuc-BS). Both broadly (1187–1193) and poorly (1222–1228) conserved putative miRNA regulatory elements of the CASP3 3′ UTR were mutated (Mut1 and Mut2, respectively) and cloned along with the wild-type 3′ UTR (WT). HEK-293T cells were transiently cotransfected with plasmids overexpressing full-length miRNAs (miR-30b, miR-30c-1, or empty vector as control), pLuc-3′ UTR CASP3 (WT, Mut1, or Mut2) and pCMV- Renilla at 100:10:1 proportions, respectively. Firefly and Renilla activities were measured 40 h after transfection. The luciferase activity normalized to Renilla is shown ( lower panel). Transfections were performed in triplicate, and results are shown as the averages ± standard errors of the means from at least three independent experiments and were analyzed by one-way and two-way ANOVA, followed by Bonferroni post-test. (*) P < 0.05, (**) P < 0.01, (***) P < 0.001, (ns) not significant.

Article Snippet: Immortalized human retinal pigment epithelial cell line hTERT RPE-1 (Clontech) was maintained in DMEM/F12 supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine, and antibiotics (50 units/mL penicillin, 50 μ/mL streptomycin).

Techniques: Expressing, Binding Assay, Western Blot, Infection, Plasmid Preparation, Cell Culture, Clone Assay, Luciferase, Transfection, Activity Assay

Journal: RNA

Article Title: Novel small RNA expression libraries uncover hsa-miR-30b and hsa-miR-30c as important factors in anoikis resistance

doi: 10.1261/rna.039461.113

Figure Lengend Snippet: Caspase 3 is the main target of miR-30b/c in the anoikis-resistance context. ( A ) RPE-1 cells were co-infected with both lentiviruses expressing full-length miR-30b/c and the ORF of caspase 3. Western blot analysis of pro-CASP3, active CASP3, and ACTB is shown for RPE-1 cells growing either in complete medium under adherent conditions (−PH) or in anoikis-inducing conditions (+PH). Flow cytometry analysis of the levels of apoptosis (SubG1/SubG1 control) in CASP3-infected cells growing under anoikis-inducing conditions ( lower panel). ( B ) RPE-1 cells were infected with either lentivirus expressing a shRNA to silence the expression of caspase 3 or a scramble shRNA (control). Experiments were carried out with RPE-1 infected cells previously selected in puromycin for 12 d. The diagram to the left shows CASP3 mRNA levels measured by real time PCR after puromycin selection. HPRT mRNA was used as the normalization control. On the right , levels of apoptotic cells in anoikis-inducing conditions are shown. Results are shown as the averages ± standard errors of the means from three independent experiments. The data were subjected to two-tailed Student's t -test. (**) P < 0.01, (***) P < 0.001. ( C ) Number of colonies after plating 2 × 10 4 cells from B per well in six-well plates with a bottom layer of 0.5% agar and a top layer of 0.35% agarose. Colonies were photographed and counted after 4 wk. Results are shown as the averages ± standard errors of the means from three independent experiments. The data were subjected to two-tailed Student's t -test. (**) P < 0.01.

Article Snippet: Immortalized human retinal pigment epithelial cell line hTERT RPE-1 (Clontech) was maintained in DMEM/F12 supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine, and antibiotics (50 units/mL penicillin, 50 μ/mL streptomycin).

Techniques: Infection, Expressing, Western Blot, Flow Cytometry, shRNA, Real-time Polymerase Chain Reaction, Selection, Two Tailed Test

Journal: RNA

Article Title: Novel small RNA expression libraries uncover hsa-miR-30b and hsa-miR-30c as important factors in anoikis resistance

doi: 10.1261/rna.039461.113

Figure Lengend Snippet: Hsa-miR-30a and -30d do not confer anoikis resistance in RPE-1 cells. ( A ) Western blot analysis of pro-CASP3 in RPE-1 cells infected with lentivirus expressing full-length miR-30a, miR-30d, or their corresponding empty vectors as control. Actin (ACTB) was used as the loading control. On the right , densitometric analysis of pro-CASP3 is shown. ACTB protein was used as a normalization control. ( B ) Flow cytometry analysis of the levels of apoptosis (SubG1/SubG1 control) in infected cells growing under anoikis-inducing conditions. ( C ) Endogenous levels of mature miR-30a/d ( left panel) and miR-30b/c ( right panel) expressed in RPE-1 and MDA-MB-231 (clone 4175) cells. miR-16 and miR-324 were used as normalization controls for miR-30b/c and miR-30a/d, respectively. Similar results were observed when human U6 snRNA primer and U6-snRNA ( RNU6B ) were used as controls. Results are shown as the averages ± standard errors of the means from three independent experiments. The data were subjected to two-tailed Student's t -test. (*) P < 0.05.

Article Snippet: Immortalized human retinal pigment epithelial cell line hTERT RPE-1 (Clontech) was maintained in DMEM/F12 supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine, and antibiotics (50 units/mL penicillin, 50 μ/mL streptomycin).

Techniques: Western Blot, Infection, Expressing, Flow Cytometry, Two Tailed Test

Journal: EMBO Molecular Medicine

Article Title: An essential role for decorin in bladder cancer invasiveness

doi: 10.1002/emmm.201302655

Figure Lengend Snippet: Decorin is necessary and sufficient for increased invasiveness of MB49-I cells

Article Snippet: Decorin overexpression in MB49 cells was achieved using the pCMV6-Entry plasmid containing cDNA of mouse Decorin (NM_007833) purchased from Origene (Rockville, MD, USA).

Techniques:

Journal: The Journal of Biological Chemistry

Article Title: Receptor for Activated C-Kinase (RACK1) Homolog Cpc2 Facilitates the General Amino Acid Control Response through Gcn2 Kinase in Fission Yeast *

doi: 10.1074/jbc.M112.445270

Figure Lengend Snippet: Cpc2, but not snoU24b RNA, is important for Gcn2 regulation. A, small nucleolar RNA U24b is encoded within the intron of cpc2 mRNA. Gray box, coding sequence; dashed line, intron; solid line, untranslated region. The position encoding Trp43 is indicated by an arrowhead. B, gene expression levels of cpc2, snoU24b, and cdk9+ (control) were analyzed by quantitative RT-PCR from wild-type (YT3033), cpc2Δ (YT2307), and cpc2_W43* (YT4299) cells. All data are normalized to the expression level of 18 S rRNA, and the average expression level of two independent experiments is shown as the relative fold to those in wild-type cells. C, 10-fold serial dilutions of wild-type (JK317), cpc2Δ (YT3540), and cpc2_W43* (YT4299) cells were spotted on EMM plates ± 10 mm 3AT. D, immunoblots of anti-FLAG-Gcn2 immunoprecipitates from Flag:gcn2 (YT3372) and Flag:gcn2 cpc2_W43* (YT4309) cells treated with 10 mm 3AT, probed with anti-phospho-Gcn2 and anti-FLAG (control) antibodies.

Article Snippet: To construct expression plasmids, cDNA of S. pombe cpc2 , S. cerevisiae asc1 , and human RACK1 ( GNB2L1 ) were cloned into the NdeI-SmaI site in the plasmid pREP2 ( 39 ). table ft1 table-wrap mode="anchored" t5 caption a7 Strain Genotype JK317 h − leu1-32 ura4-D18 YT2307 h − cpc2 :: ura4 + leu1-32 ura4-D18 YT2313 h − cpc2 : 2HA6His : ura4 + leu1-32 ura4-D18 YT2453 h − hri2 :: ura4 + leu1-32 ura4-D18 YT2459 h − hri2 :: ura4 + gcn2 :: Kan r leu1-32 ura4-D18 YT2465 h − rpS3 : Flag : ura4 + leu1-32 ura4-D18 YT2824 h − cpc2 :: ura4 + gcn2 :: Kan r leu1-32 ura4-D18 YT3033 h − leu1-32 YT3173 h − hri2 :: ura4 + cpc2 :: ura4 + leu1-32 ura4-D18 YT3360 h − gcn2 :: ura4 + leu1-32 ura4-D18 YT3372 h − 5Flag : gcn2 leu1-32 ura4-D18 YT3540 h − cpc2 :: Kan r leu1-32 ura4-D18 YT3542 h − cpc2 _ DE leu1-32 ura4-D18 YT3559 h − 5Flag : gcn2 cpc2 _ DE : ura4 + leu1-32 ura4-D18 YT3598 h − 5Flag : gcn2 cpc2 :: ura4 + leu1-32 ura4-D18 YT3648 h − 5Flag : gcn2 :: ura4 + leu1-32 ura4-D18 YT3656 h − 5Flag : gcn2 _ K585R leu1-32 ura4-D18 YT3657 h − 5Flag : gcn2 _ T818/823A leu1-32 ura4-D18 YT4251 h − atf1 :: ura4 + leu1-32 ura4-D18 YT4254 h − 5Flag : gcn2 atf1 :: ura4 + leu1-32 ura4-D18 YT4279 h + / h − 5Flag : gcn2 / 12myc : gcn2 ade6-M210 / ade6-M216 leu1-32 / leu1-32 ura4-D18 / ura4-D18 YT4280 h + / h − 5Flag : gcn2 / 12myc : gcn2 cpc2 :: ura4 + / cpc2 :: ura4 + ade6-M210 / ade6-M216 leu1-32 / leu1-32 ura4-D18 / ura4-D18 YT4281 h + / h − 12myc : gcn2 / gcn2 + ade6-M210 / ade6-M216 leu1-32 / leu1-32 ura4-D18 / ura4-D18 YT4299 h − cpc2 _ W43 * leu1-32 ura4-D18 YT4309 h − 5Flag : gcn2 cpc2 _ W43 * leu1-32 ura4-D18 YT4376 h + / h − 5Flag : gcn2 / gcn2 + ade6-M210 / ade6-M216 leu1-32 / leu1-32 ura4-D18 / ura4-D18 YT4386 h − Flag : cpc2 leu1-32 ura4-D18 YT4388 h − Flag : cpc2 _ W43 * leu1-32 ura4-D18 YT4389 h − cpc2 :: Kan r :2HA6His : ura4 + leu1-32 ura4-D18 YT4390 h − cpc2 _ W43 *: 2HA6His : ura4 + leu1-32 ura4-D18 Open in a separate window S. pombe strains used in this study RT-PCR Total RNA was prepared as described previously ( 40 ). cDNA was synthesized using an RNA PCR kit (Takara) with random 9-mer primers.

Techniques: Sequencing, Expressing, Quantitative RT-PCR, Western Blot