reference strains plasmids bc a1 002 gfp assay vector addgene pet 28b  (Addgene inc)

Journal: Theranostics

Article Title: Myeloid cell-specific β2-adrenergic receptor deletion improves early cardiac injury resolution via de-repression of Anxa1

doi: 10.7150/thno.123651

Figure Lengend Snippet: Annexin A1 expression is increased in β2AR-deficient myeloid cells post-MI. (A) RT-qPCR screening of various efferocytosis pathway genes was performed on total RNA isolated from whole bone marrow (BM) cells from FL/FL and LB2 mice, with the heatmap representing the differential expression pattern of Anxa1, Mertk, Axl, Stab1 and Stab2 genes between FL/FL and LB2 BM cells. RT-qPCR-detected Anxa1 expression in Veh or ISO-treated (10 µM for 16h) FL/FL versus LB2 BMDM (B, n = 3 each), prepared as described in Figure A. (C) Schematic of magnetic microbead column-based cardiac CD11b+ cell enrichment at 1 Day post-MI, after which gene expression of β2AR and Anxa1 were assessed via RT-qPCR (D), n = 3/group. Flow cytometry-based histogram analysis showing comparative AnxA1 protein expression via mean fluorescence intensity (MFI) among various phenotypically distinct myeloid cell types: general CD11b+ myeloid cells (E), Nu (F), Ly6C hi Mo (G), Mac (H) and Ly6C lo Mo (I) at 4 days post-MI FL/FL and LB2 hearts, n = 5/group. Data are Mean ± SEM of independent experiments. ns, non-significant, * p < 0.05; ** p < 0.01, **** p < 0.0001, Unpaired student's t-test.

Article Snippet: Bone marrow cells from the femurs of LB2 mice were isolated and transduced with either scrambled or Annexin A1 (Anxa1) shRNA using TR30030 pRFP-C-shLenti vector (Origene Technologies Inc. MD, USA) at an MOI of 150 ( ).

Techniques: Expressing, Quantitative RT-PCR, Isolation, Quantitative Proteomics, Gene Expression, Flow Cytometry, Fluorescence

Journal: Theranostics

Article Title: Myeloid cell-specific β2-adrenergic receptor deletion improves early cardiac injury resolution via de-repression of Anxa1

doi: 10.7150/thno.123651

Figure Lengend Snippet: β2AR signaling induces miR-374b-50-mediated repression of Anxa1. RT-qPCR screening of miR-196b (A, n = 4 each), miR-374b-5p (B, n = 3 each), miR-128a (C, n = 3 each) and miR-26a (D, n = 3 each) in BMDM treated with vehicle (0.002% L-ascorbic acid) or ISO (10 μM, 16h). RT-qPCR screening of miR-196b (E), miR-374b-5p (F), miR-128a (G) and miR-26a (H) in 4 day post-MI cardiac CD11b + cells, n = 5 FL/FL and n = 4 LB2. For each replicate, a minimum of 5 heart cells were combined. (I) Table showing sequence % identity between mouse (prefixed; mmu-) and human (prefixed; hsa-) miR-374b-5p. (J) Predicated alignment of miR-374b-5p with 3'-UTR of Anxa1. (K) TaqMan TM based RT-qPCR analysis of the effect of miR-374b-5p mimic on miR-374b-5p expression in HEK293T cells. (L) RT-qPCR analysis of the effect of miR-374b-5p mimic on ANXA1 gene expression in HEK293T cells. Data are Mean ± SEM of independent experiments. ns, non-significant, * p < 0.05, ** p < 0.01, *** p < 0.001, One way ANOVA with Tukey's post-hoc test (A-D, K) or Unpaired student's t-test (E-H).

Article Snippet: Bone marrow cells from the femurs of LB2 mice were isolated and transduced with either scrambled or Annexin A1 (Anxa1) shRNA using TR30030 pRFP-C-shLenti vector (Origene Technologies Inc. MD, USA) at an MOI of 150 ( ).

Techniques: Quantitative RT-PCR, Sequencing, Expressing, Gene Expression

Journal: Theranostics

Article Title: Myeloid cell-specific β2-adrenergic receptor deletion improves early cardiac injury resolution via de-repression of Anxa1

doi: 10.7150/thno.123651

Figure Lengend Snippet: Anxa1 downregulation reduces efferocytosis and ameliorative cardiac remodeling effects in myeloid cell-specific β2AR knockout mice. (A) BMDM underwent lentivirus transduction of various clones (A-D) of AnxA1 shRNA versus control (CTL) shRNA for 48 h, after which RT-qPCR was used to assess gene silencing efficiency, n = 3/group. (B) Schematic depicting the experimental design to test the impact of AnxA1 knockdown in LB2 bone marrow-derived Nu. Briefly, LB2 BM cells were transduced with lentivirus containing either CTL shRNA or AnxA1 shRNA (1:1 mixture of clones B and D), after which they were retro-orbitally injected into lethally irradiated LB2 mice to generate bone marrow transplant (BMT) mice featuring LB2 bone marrow with or without AnxA1 knockdown, as confirmed via RT-qPCR analysis after terminal experiments (C), n = 4 (CTL shRNA), n = 4 (AnxA1 shRNA). 4 weeks after BM reconstitution, CTL shRNA and AnaA1 shRNA Nu were enriched from the respective BMT mice using magnetic columns, after which they underwent efferocytosis assays with LB2 BMDM (as described in Figure ) and flow cytometry analysis, as shown via contour plots (D) of LB2 BMDM incubated without Nu (left) or with CMFDA + Nu from either CTL shRNA (middle) or Anxa1 shRNA (right) LB2 BMT mice. (E) Quantification of CTL shRNA versus Anxa1 shRNA LB2 Nu uptake efficiency into LB2 BMDM in terms of % efferocytosis, n = 4/each group. CTL shRNA and AnxA1 shRNA-transduced LB2 BMT mice were generated as described above and 4 weeks after BM reconstitution underwent MI surgery. Histograms depicting %EF (F) and %FS (G) at baseline, 3 and 14 days post-MI in CTL and AnxA1 shRNA LB2 BMT mice, n = 4 for CTL, n = 6 for AnxA1 shRNA. (H) MT staining of whole heart (top row) displaying infarct size of CTL and AnxA1 shRNA LB2 BMT mice at 14 days post-MI with quantification shown in (I), n = 4 for CTL and n = 5 for AnxA1 shRNA group, scale bar = 1000 µm, and 20X images (bottom row) displaying interstitial fibrosis in the border zone of the post-MI hearts with quantification as % area fibrosis (J), n =4 for CTL and n = 5 for AnxA1 shRNA group, scale bar = 100 µm. Data are Mean ± SEM of independent experiments. ns, non-significant, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, One way ANOVA with Tukey's post-hoc test (A), Unpaired student's t-test (C, E, I, J) or Two-way repeated measures ANOVA with Tukey's post-hoc test (F, G).

Article Snippet: Bone marrow cells from the femurs of LB2 mice were isolated and transduced with either scrambled or Annexin A1 (Anxa1) shRNA using TR30030 pRFP-C-shLenti vector (Origene Technologies Inc. MD, USA) at an MOI of 150 ( ).

Techniques: Knock-Out, Transduction, Clone Assay, shRNA, Control, Quantitative RT-PCR, Knockdown, Derivative Assay, Injection, Irradiation, Flow Cytometry, Incubation, Generated, Staining

Journal: Theranostics

Article Title: Myeloid cell-specific β2-adrenergic receptor deletion improves early cardiac injury resolution via de-repression of Anxa1

doi: 10.7150/thno.123651

Figure Lengend Snippet: Mechanism of myeloid cell-specific β2AR deletion-mediated post-MI injury resolution. Myocardial infarction triggers local ischemic damage and elevated catecholamines (epinephrine/norepinephrine; NE/EP). Stimulation of myeloid cell β2AR increases the abundance of miR-347b-5p leading to degradation of Anxa1 mRNA, resulting in decreased Anxa1 protein expression (A). In the absence of β2AR, miR-347b-5p cannot be induced, leading to de-repression of Anxa1 expression, and ultimately increased Nu efferocytosis (B). This enhanced Nu clearance during the early post-MI period leads to better injury resolution with less fibrosis and infarct expansion and better contractile function.

Article Snippet: Bone marrow cells from the femurs of LB2 mice were isolated and transduced with either scrambled or Annexin A1 (Anxa1) shRNA using TR30030 pRFP-C-shLenti vector (Origene Technologies Inc. MD, USA) at an MOI of 150 ( ).

Techniques: Expressing

Journal: PLOS Biology

Article Title: TDP-43 and other hnRNPs regulate cryptic exon inclusion of a key ALS/FTD risk gene, UNC13A

doi: 10.1371/journal.pbio.3002028

Figure Lengend Snippet: ( A ) Schematic representation of RNA pull-down system to identify proteins that bind UNC13A RNA. First, UNC13A RNA is in vitro transcribed from UNC13A minigene construct. Second, the RNA is labeled with a T4 RNA ligase, and the labeled RNA is then captured with streptavidin magnetic beads. The UNC13A RNA-streptavidin beads complex is mixed with either WT or TARDBP KO HeLa cell nuclei extract to elute the UNC13A RBPs, which are then assessed by western blot. ( B, C ) In vitro-transcribed RNA from WT UNC13A minigene (containing reference haplotype in UNC13A ) showed binding to TDP-43, hnRNP L, hnRNP A1, and hnRNP A2B1 by western blot ( B ) and mass spectrometry ( C ). Blots in B provided in Supporting information . Data used to generate the volcano plot in C can be found in . ( D ) In vitro-transcribed RNA from WT UNC13A minigene demonstrate binding of UNC13A cryptic exon to hnRNP L, hnRNP A1, and hnRNP A2B1 even in the absence of TDP-43 ( TARDBP KO HeLa cells), as shown in western blot. Blots in D provided in Supporting information . TARDBP KO HeLa cells treated with siRNAs against HNRNPL , HNRPNA1 , and HNRNPA2B1 were used as an additional negative control in the assay. Representative images of at least 2 independent experiments are shown. RBP, RNA-binding protein; siRNA, small interfering RNA; TDP-43, TAR DNA-binding protein-43.

Article Snippet: To generate Flag-tagged hnRNP A1 and hnRNP A2B1 overexpression constructs, an hnRNP A2B1 protein vector (pPM-N-D-C-HA) (Applied Biological Materials, Accession Number BC000506) and an hnRNP A1 protein vector (pPM-N-D-C-HA) (Applied Biological Materials, Accession Number BC002355) were used.

Techniques: In Vitro, Construct, Labeling, Magnetic Beads, Western Blot, Binding Assay, Mass Spectrometry, Negative Control, RNA Binding Assay, Small Interfering RNA

Journal: PLOS Biology

Article Title: TDP-43 and other hnRNPs regulate cryptic exon inclusion of a key ALS/FTD risk gene, UNC13A

doi: 10.1371/journal.pbio.3002028

Figure Lengend Snippet: In vitro-transcribed RNA from WT and CE SNP UNC13A minigenes were incubated with nuclear extracts from WT HeLa cells to assess their ability to bind the following proteins by western blot analyses after pull-down: TDP-43 ( A ), hnRNP L ( B ), hnRNP A1 ( C ), and hnRNP A2B1 ( D ). The graphs show reduced binding to CE SNP minigene by TDP-43 and other hnRNPs, as quantified by the signal intensity of the western blots using Image J. Graphs represent mean ± SEM of 6 independent assays. Statistical differences were assessed by Student’s t test, ** P < 0.005, **** P < 0.0001. Blots provided in Supporting information . Data used to generate graphs can be found in . CE, cryptic exon; SEM, standard error of mean; SNP, single-nucleotide polymorphism; TDP-43, TAR DNA-binding protein-43.

Article Snippet: To generate Flag-tagged hnRNP A1 and hnRNP A2B1 overexpression constructs, an hnRNP A2B1 protein vector (pPM-N-D-C-HA) (Applied Biological Materials, Accession Number BC000506) and an hnRNP A1 protein vector (pPM-N-D-C-HA) (Applied Biological Materials, Accession Number BC002355) were used.

Techniques: In Vitro, Incubation, Western Blot, Binding Assay

Journal: PLOS Biology

Article Title: TDP-43 and other hnRNPs regulate cryptic exon inclusion of a key ALS/FTD risk gene, UNC13A

doi: 10.1371/journal.pbio.3002028

Figure Lengend Snippet: ( A ) WT UNC13A minigene was expressed in WT HeLa cells treated with either control (siControl) or siRNAs against TARDBP (siTARDBP), HNRNPL (siHNRPL), HNRNPA1 (siHNRNPA1), or HNRNPA2B1 (siHNRNPA2B1). RNA was extracted, and RT-qPCR was performed to assess the expression levels of UNC13A cryptic (A), TARDBP , HNRNPL , HNRNPA1 , or HNRNPA2B1 RNA. (B, C) Flag-tagged TDP-43, hnRNP L, hnRNP A1, and hnRNP A2B1 were expressed in TARDBP KO HeLa cells transfected with UNC13A WT or CE SNP minigenes to evaluate the ability of other hnRNPs to repress UNC13A cryptic exon inclusion by RT-qPCR. A representative immunoblot confirming the expression of each Flag-tagged plasmid using a Flag antibody is shown in B. Blot provided in Supporting information . All graphs represent mean ± SEM of UNC13A cryptic RNA levels of 3 independent experiments. Statistical differences were assessed by one-way followed by Tukey’s multiple comparisons test (A) or two-way (C) ANOVA (ns: not significant, * P < 0.05, ** P < 0.005, **** P < 0.0001). Data used to generate graphs can be found in . CE, cryptic exon; hnRNP, heterogeneous nuclear ribonucleoprotein; SEM, standard error of mean; siRNA, small interfering RNA; SNP, single-nucleotide polymorphism;TDP-43, TAR DNA-binding protein-43.

Article Snippet: To generate Flag-tagged hnRNP A1 and hnRNP A2B1 overexpression constructs, an hnRNP A2B1 protein vector (pPM-N-D-C-HA) (Applied Biological Materials, Accession Number BC000506) and an hnRNP A1 protein vector (pPM-N-D-C-HA) (Applied Biological Materials, Accession Number BC002355) were used.

Techniques: Quantitative RT-PCR, Expressing, Transfection, Western Blot, Plasmid Preparation, Small Interfering RNA, Binding Assay