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 4. NF-kB p65 Mediates linc-AAM Expression in Macrophages (A) Quantitative real-time PCR analysis of linc-AAM in RAW264.7 cells treated by AEPS for 1 h after pre-incubation of PDTC or Bay11-7082 (NF-kB inhibitor) for 1 h (n = 3). (B) Quantitative real-time PCR analysis of linc-AAM in RAW264.7 cells treated by AEPS for 1 h after pre-incubation of (STAT3 inhibitor) or fludarabine (STAT1 inhibitor) for 1 h (n = 3). (C) Quantitative real-time PCR analysis of linc-AAM in BMDCs treated by AEPS for 1 h after pre-incubation of PDTC for 1 h (n = 3). (D) Quantitative real-time PCR analysis of linc-AAM in RAW264.7 cells expressing shRNA targeting NF-kB p65 or STAT3, or control (Scramble) (n = 3). (E) Quantitative real-time PCR analysis of linc-AAM in RAW264.7 cells transfected with NF-kB p65 or STAT3 overexpressing vector or Ctrl (empty vector) (n = 3). (F) Luciferase (Luc) reporter assay: the linc-AAM promoter region (–3,893 to +995 nt relative to TSS, WT) or deletion variants were cloned upstream of firefly Luc coding region. Next, the Luc plasmid and indicated transcription factor (NF-kB p65 and STAT3) expression plasmid were co-transfected into HEK293T cells, and the Luc activity was determined. Data were normalized to Renilla Luc activity (n = 3). (G) Eleven regions (A–K) across linc-AAM gene locus were analyzed by multisite ChIP-qPCR for H3K4me3 histone modification and NF-kB p65 binding in RAW264.7 cells treated with AEPS for 1 h. Values were normalized to input samples and are presented as percentage of input chromatin (% input) (n = 3). (H) Luc reporter assay: linc-AAM promoter regions (–3,876 to –3,513 nt and –2,659 to –1,813 nt) were cloned upstream of Luc and co-transfected with the NF-kB p65 expression plasmids or empty plasmids (Ctrl) into HEK293T cells for detecting the Luc activity (n = 3). Data are presented as mean ± SEM. *p < 0.05, **p < 0.01, and ***p < 0.001.

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: Expressing, Real-time Polymerase Chain Reaction, Incubation, shRNA, Control, Transfection, Plasmid Preparation, Luciferase, Reporter Assay, Clone Assay, Activity Assay, ChIP-qPCR, Binding Assay

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 5. linc-AAM Interacts with hnRNPL to Regulate the Expression of IRGs (A) Coomassie blue staining of linc-AAM-associated proteins. RNA pull-down assay was carried out using desthiobiotinylated linc-AAM or its antisense. Eluted fractions were resolved by SDS-PAGE followed by Coomassie blue staining. Proteins highlighted by the red boxes in the linc-AAM lane were subjected to mass spectrometry. (B) Western blot analysis of proteins from linc-AAM and its antisense pull-down assays. (C) Western blotting of hnRNPL in samples pulled down by desthiobiotinylated full-length or truncated linc-AAM mutants (D1: 1–750; D2: 1–1,600). (D–F) Cross-linked RIP and native RIP analysis of linc-AAM. Total cell lysates of RAW264.7 cells were IPed with control IgG or anti-hnRNPL, anti-hnRNPK, anti- hnRNPU, anti-NF-kB p65, anti-STAT1, and anti-STAT3 antibody and then the complexes were analyzed for the presence of linc-AAM by PCR (D) and quantitative real-time PCR (E and F). Signals were normalized to 10% input samples and presented as percentage of input chromatin (% input) (n = 3). (G) Schematic of CACACA motifs in the region 50 750–1,599 nt of linc-AAM. The nucleotide mutations in CACACA tracts are highlighted. (H) linc-AAM binds hnRNPL through two CACACA motifs (826–831 and 1,472–1,477 nt) embedded in its 50 750–1,599 nt region. The RNA-protein binding assay was performed using desthiobiotinylated WT or CACACA tract mutants of the whole-length linc-AAM, captured using streptavidin beads, and subjected to western blot against hnRNPL. (I) Western blot analysis of hnRNPL in RAW264.7 cells transfected with Ctrl-siRNA (Scramble) or different siRNAs targeting hnRNPL for 24 h. (J) Quantitative real-time PCR analysis of hnRNPL in RAW264.7 cells transfected with Ctrl-siRNA (Scramble) or different siRNAs targeting hnRNPL for 24 h (n = 3). (K and L) Quantitative real-time PCR analysis of linc-AAM (K), and IL-6 (L) in linc-AAM KD or Ctrl RAW264.7 cells transfected with Ctrl-siRNA (Scramble) or different siRNA targeting hnRNPL for 24 h, followed by 4 h of AEPS treatment (n = 3). (M and N) Quantitative real-time PCR analysis of linc-AAM (M), IL-6, and COX-2 (N) in linc-AAM KD RAW264.7 cells expressing WT or the three point mutants of the full-length linc-AAM (two of them are defective in hnRNPL binding) were stimulated with AEPS for 1 h (M) or 4 h (N) (n = 3). Data are presented as mean ± SEM. *p < 0.05, **p < 0.01, and ***p < 0.001. See also Figure S5.

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: Expressing, Staining, Pull Down Assay, SDS Page, Mass Spectrometry, Western Blot, Control, Real-time Polymerase Chain Reaction, Protein Binding, Transfection, Binding Assay