Journal: Cell reports

Article Title: Yap/Taz inhibit goblet cell fate to maintain lung epithelial homeostasis

doi: 10.1016/j.celrep.2021.109347

Figure Lengend Snippet: (A) Representative YAP/TAZ and MUC5AC immunostaining of human airway sections (performed using an antibody recognizing YAP and TAZ). DAPI-stained nuclei are in blue. Nuclei within MUC5AC + cells are highlighted with a yellow dotted line and with a blue dotted line in MUC5AC − cells. A white dotted line marks the basal surface of the epithelium (scale bars, 10 μm). (B) Quantification of nuclear YAP/TAZ intensity in airway epithelial cells across multiple sections from two patient donors. Cells were scored as either MUC5AC-positive or -negative and the intensity of YAP/TAZ staining was measured within the nuclear area outlined by DAPI staining (minimum of n = 14; unpaired t test, ****p < 0.0001). (C) YAP/MUC5AC immunostaining of human ALI cultures imaged by confocal microscopy. A z stack view is shown in the top panels (scale bars,10 μm). (D) HBECs were transfected with control siRNA (siCTL) or siRNA targeting YAP/TAZ (siY/T). MUC5AC, SCGB1A1, YAP , and WWTR1/TAZ qPCR analysis of lysates collected 72 h after knockdown (n = 6; unpaired t test, **p = 0.001, ****p < 0.0001). (E) Heatmap of gene expression changes resulting from YAP/TAZ knockdown in HBECs analyzed by RNA sequencing (RNA-seq). 2 distinct patient isolates were treated with three independent siCTLs or siRNA targeting YAP/TAZ (siY/T), and global gene expression changes were examined by RNA-seq after 48 h of culture (n = 3 per condition, 2-fold change cutoff, FDR = 0.05). (F) Pathway enrichment of significantly upregulated and downregulated genes following YAP/TAZ depletion in human airway epithelial cells identified by GSEA. Both the −log 10 p value and the percentage representation within each gene set are displayed. In all bar plots data are represented as mean ± SEM. See also and and .

Article Snippet: Mouse Wwtr1 Taqman probe Mm01289583_m1 , ThermoFisher , 4331182.

Techniques: Immunostaining, Staining, Confocal Microscopy, Transfection, Control, Knockdown, Gene Expression, RNA Sequencing

Journal: Cell reports

Article Title: Yap/Taz inhibit goblet cell fate to maintain lung epithelial homeostasis

doi: 10.1016/j.celrep.2021.109347

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Mouse Wwtr1 Taqman probe Mm01289583_m1 , ThermoFisher , 4331182.

Techniques: Recombinant, Lysis, TUNEL Assay, RNAscope, Positive Control, Negative Control, Staining, Transfection, Purification, cDNA Synthesis, SYBR Green Assay, Knock-Out, Microarray, Knockdown, Control, Software

Journal: Molecular Therapy. Nucleic Acids

Article Title: Efficacy and safety of a SOD1-targeting artificial miRNA delivered by AAV9 in mice are impacted by miRNA scaffold selection

doi: 10.1016/j.omtn.2023.102057

Figure Lengend Snippet: AAV9-miR155-SOD1 shows greater artificial miRNA expression than AAV9-miR30a-SOD1 in vitro (A–E) NGN2 neurons were transduced with an artificial miRNA targeting SOD1 that was inserted into either a miR155 or miR30a scaffold, and a non-targeting (NT) control into the same scaffolds. Samples were analyzed 14 days post-transduction. (A) Illustration of artificial miRNA vectors and the complementary guide strand sequence targeting SOD1 (exon 5) and PCR amplicon targeting SOD1 (exon 4). (B) Reads per million (RPM) of artificial miRNA guide and passenger strands when expressed in either a miR155 scaffold or a miR30a scaffold as calculated by small RNA-seq. Data presented as mean ± SD (n = 4). (C) Real-time PCR shows percent SOD1 expression compared with NT control after artificial miRNA treatment. Expression levels were normalized to GAPDH prior to comparison. Data presented as mean ± SD (n = 4). Welch’s one-way ANOVA followed by Holm-Sidak’s multiple comparison post hoc (∗p < 0.05). (D) Heatmap of the top 30 expressing endogenous miRNAs in NGN2 neurons. Values in the heatmap represent log2 (RPM) (n = 4). (E) Differential mRNA expression in NGN2 neurons after artificial miRNA treatment based on RNA-seq analysis (n = 4). Two-tailed Wald test and p values were adjusted for multiple comparisons by implementing Benjamini-Hochberg false discovery rate. Blue dots indicate significant changes and gray dots highlight non-significant changes in transcript expression compared with control.

Article Snippet: For iPSC-NGN2 cells, human GAPDH (Hs99999905_m1) and custom probes targeting human SOD1 exon 4 (forward 5′-TGGTGTGGCCGATGTGTCTA-3′, reverse 5′-ATGATGCAATGGTCTCCTGAGA-3′, probe 5′-6FAM-TGAAGATTCTGTGATCTCA-MGB/NFQ-3′) were used in C. For wild-type spinal cord and cortex, mouse GAPDH (Mm99999915_g1) and mouse SOD1 (Mm01344233_g1) probes were used in B.

Techniques: Expressing, In Vitro, Transduction, Control, Sequencing, Amplification, RNA Sequencing, Real-time Polymerase Chain Reaction, Comparison, Two Tailed Test

Journal: Molecular Therapy. Nucleic Acids

Article Title: Efficacy and safety of a SOD1-targeting artificial miRNA delivered by AAV9 in mice are impacted by miRNA scaffold selection

doi: 10.1016/j.omtn.2023.102057

Figure Lengend Snippet: AAV9-miR155-SOD1 shows greater artificial miRNA expression than AAV9-miR30a-SOD1 in vivo (A and B) Mice were given a P0 ICV injection of either AAV9-miR155-SOD1, AAV9-miR30a-SOD1, or a vehicle control. Each amiR was given at two different dose levels, 8E+10GC or 16E+10GC. Expression levels of the artificial miRNA were measured 10 weeks post-injection. (A) RPM of artificial miRNA guide and passenger strands when expressed in either the miR155 or miR30a scaffold in the spinal cord and cortex using small RNA-seq. (B) qPCR shows percent SOD1 expression compared with control in the spinal cord or cortex after artificial miRNA treatment. SOD1 levels were normalized to GAPDH levels prior to comparison. Data presented as mean ± SD (n = 3–4). Generalized least squares test followed by a Tukey’s post hoc to adjust for multiple comparisons ( NS p > 0.05, ∗p < 0.05, ∗∗∗p < 0.001).

Article Snippet: For iPSC-NGN2 cells, human GAPDH (Hs99999905_m1) and custom probes targeting human SOD1 exon 4 (forward 5′-TGGTGTGGCCGATGTGTCTA-3′, reverse 5′-ATGATGCAATGGTCTCCTGAGA-3′, probe 5′-6FAM-TGAAGATTCTGTGATCTCA-MGB/NFQ-3′) were used in C. For wild-type spinal cord and cortex, mouse GAPDH (Mm99999915_g1) and mouse SOD1 (Mm01344233_g1) probes were used in B.

Techniques: Expressing, In Vivo, Injection, Control, RNA Sequencing, Comparison

Journal: Molecular Therapy. Nucleic Acids

Article Title: Efficacy and safety of a SOD1-targeting artificial miRNA delivered by AAV9 in mice are impacted by miRNA scaffold selection

doi: 10.1016/j.omtn.2023.102057

Figure Lengend Snippet: Suppression of SOD1 in spinal motor neurons after AAV9-amiR-SOD1 treatment (A and B) Mice were given a P0 ICV injection of either AAV9-miR155-SOD1, AAV9-miR30a-SOD1, or vehicle control. Each amiR was given at two different dose levels, 8E+10GC or 16E+10GC. Expression of SOD1 was measured in motor neurons 10 weeks post-injection. (A) RNA scope showing ChAT+ (red) and SOD1+ (brown) cells. Black boxes indicate regions shown at 40x optical magnification illustrating SOD1+ (brown) chromogen. (B) Percent SOD1 in ChAT+ cells after treatment. Percentage SOD1 in ChAT+ cells was normalized to the vehicle prior to comparison. Data presented as mean ± SD (n = 3–4). Generalized least squares test followed by a Tukey’s post hoc test to adjust for multiple comparisons ( NS p > 0.05, ∗p < 0.05).

Article Snippet: For iPSC-NGN2 cells, human GAPDH (Hs99999905_m1) and custom probes targeting human SOD1 exon 4 (forward 5′-TGGTGTGGCCGATGTGTCTA-3′, reverse 5′-ATGATGCAATGGTCTCCTGAGA-3′, probe 5′-6FAM-TGAAGATTCTGTGATCTCA-MGB/NFQ-3′) were used in C. For wild-type spinal cord and cortex, mouse GAPDH (Mm99999915_g1) and mouse SOD1 (Mm01344233_g1) probes were used in B.

Techniques: Injection, Control, Expressing, RNAscope, Comparison

Journal: Molecular Therapy. Nucleic Acids

Article Title: Efficacy and safety of a SOD1-targeting artificial miRNA delivered by AAV9 in mice are impacted by miRNA scaffold selection

doi: 10.1016/j.omtn.2023.102057

Figure Lengend Snippet: A greater number of adverse events are associated with the miR155 scaffold than with the miR30a scaffold (A–D) Mice were given a P0 ICV injection of either AAV9-miR155-SOD1, AAV9-miR30a-SOD1, or vehicle control. Each amiR was given at two different dose levels, 8E+10GC or 16E+10GC. (A) H&E staining highlighting various pathologies associated with either AAV9-miR30a-SOD1 or AAV9-miR155-SOD1 treatment within the sciatic nerve, spinal cord, or cerebellum. Scale bars represent 50 µm. (B) Scatterplot representing pathological severity related to AAV9-amiR-SOD1 treatment. (C) A longitudinal measure of serum pNF-H levels in mice. Data presented as mean ± SD (n = 4–8). Significance versus vehicle was determined using a generalized estimating equation (∗∗∗p < 0.001). (D) Survival of mice after AAV-amiR treatment (n = 4–10). Log rank test was used to determine if there was evidence in favor of a different survival between groups (p = 0.03).

Article Snippet: For iPSC-NGN2 cells, human GAPDH (Hs99999905_m1) and custom probes targeting human SOD1 exon 4 (forward 5′-TGGTGTGGCCGATGTGTCTA-3′, reverse 5′-ATGATGCAATGGTCTCCTGAGA-3′, probe 5′-6FAM-TGAAGATTCTGTGATCTCA-MGB/NFQ-3′) were used in C. For wild-type spinal cord and cortex, mouse GAPDH (Mm99999915_g1) and mouse SOD1 (Mm01344233_g1) probes were used in B.

Techniques: Injection, Control, Staining

Journal: Molecular Therapy. Nucleic Acids

Article Title: Efficacy and safety of a SOD1-targeting artificial miRNA delivered by AAV9 in mice are impacted by miRNA scaffold selection

doi: 10.1016/j.omtn.2023.102057

Figure Lengend Snippet: AAV9-miR30a-SOD1 can increase survival and reverse ALS-like phenotypes in SOD1-G93A mice (A–C) P0 SOD1-G93A mice were given a one-time ICV injection of AAV9-miR30a-SOD1 at one of five dose levels (0.5E+10GC, 1.5E+10GC, 4.0E+10GC, 8.0E+10GC, or 16.0E+10GC), or an NT-miR30a control. A longitudinal measure of (A) serum pNF-H and (B) CMAP signaling. Data expressed as mean ± SD (n = 6–13). Generalized estimating equation was used to determine significance as compared with the NT-control ( NS p > 0.05, ∗∗∗p < 0.001). (C) Survival of SOD1-G93A mice after amiR treatment (n = 6–13). Log rank test was used to determine significance as compared with the NT-control (∗∗∗p < 0.001).

Article Snippet: For iPSC-NGN2 cells, human GAPDH (Hs99999905_m1) and custom probes targeting human SOD1 exon 4 (forward 5′-TGGTGTGGCCGATGTGTCTA-3′, reverse 5′-ATGATGCAATGGTCTCCTGAGA-3′, probe 5′-6FAM-TGAAGATTCTGTGATCTCA-MGB/NFQ-3′) were used in C. For wild-type spinal cord and cortex, mouse GAPDH (Mm99999915_g1) and mouse SOD1 (Mm01344233_g1) probes were used in B.

Techniques: Injection, Control

Journal: Cell

Article Title: Tissue Repair Signals and In Vitro Culture: Inflammatory Cytokine TNFα Promotes the Expansion of Primary Hepatocytes in 3D Culture

doi: 10.1016/j.cell.2018.11.012

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Experimental Models: Cell Lines HEK293T ATCC CRL-1573 GFP expressing human umbilical vein endothelial cells(GFP-HUVECs) Angioproteomie cAP-0001GFP Experimental Models: Organisms/Strains C57BL/6J Jackson lab 000664 Axin2-CreERT2 Jackson lab 018867 Axin2-rtTA Jackson lab 016997 Rosa26-mTmG Jackson lab 007676 TetO-H2B:GFP Jackson lab 005104 Oligonucleotides RNAscope Probe: Mm-Fah-C2 (target region: 490-1364) Advanced Cell Diagnostics Cat#503431-C2 RNAscope Probe: Mm-Wnt9b (target region: 706-1637) Advanced Cell Diagnostics Cat#405091 RNAscope Probe: Mm-Wnt2 (target region: 857-2086) Advanced Cell Diagnostics Cat#313601 RNAscope Probe: Mm-Rspo3 (target region: 731-2164) Advanced Cell Diagnostics Cat#402011 RNAscope Probe: Mm-Rspo 1 (target region: 550-1679) Advanced Cell Diagnostics Cat#401991 RNAscope Probe: Mm-Rspo2 (target region: 537-1452) Advanced Cell Diagnostics Cat# 402001 RNAscope Probe: Mm-Rspo4 (target region: 1180-2202) Advanced Cell Diagnostics Cat#402021 RNAscope Probe: Mm-CD31/Pecam1-C2 (target region: 915-1827) Advanced Cell Diagnostics Cat#316721-C2 RNAscope Probe: Mm-CD45/Ptprc-C2 (target region: 2922-3866) Advanced Cell Diagnostics Cat# 318651-C2 RNAscope Probe: Mm-TNFa (target region: 41-1587) Advanced Cell Diagnostics Cat# 311081 Taqman Probe Alb Applied Biosystem Cat# 4331182 Mm00802090_m1 Taqman Probe Apoe Applied Biosystem Cat# 4331182 Mm01307193_g1 Taqman Probe: Cps1 Applied Biosystem Cat# 4331182 Mm01256489_m1 Taqman Probe: Cyp1a2 Applied Biosystem Cat# 4331182 Mm00487224_m1 Taqman Probe: Cyp2f2 Applied Biosystem Cat# 4331182 Mm00484087_m1 Taqman Probe: Cyp3a11 Applied Biosystem Cat# 4331182 Mm00731567_m1 Taqman Probe: Fah Applied Biosystem Cat# 4331182 Mm00487336_m1 Taqman Probe: Gck Applied Biosystem Cat# 4331182 Mm00439129_m1 Taqman Probe: Tat Applied Biosystem Cat# 4331182 Mm01244282_m1 Taqman Probe: Ttr Applied Biosystem Cat# 4331182 Mm00443267_m1 Recombinant DNA Plasmid :1-puro-CMV-TurboGFP Sigma Aldrich SHC003 Plasmid: psPAX2 Addgene 12260 Plasmid: pMD.2g Addgene 12259 Software and Algorithms Prism Graphpad 7.0 Graphpad software http://www.graphpad.com Adobe Photoshop CC 2018 Adobe http://www.adobe.com/product/photoshop.html Adobe Illustrator CC 2018 Adobe http://www.adobe.com/product/illustrator.html Image J NIH http://imagej.nih.gov/ij/ Cell Ranger version 2.1.1 10x Genomics https://support.10xgenomics.com/single-cell-gene-expression/software/downloads/latest Seurat R Package Butler et al., 2018 https://satijalab.org/seurat HCS Studio Cell Analysis Software Thermo Fisher https://thermofisher.com Other Open in a separate window KEY RESOURCES TABLE TNFα is a pro-inflammatory signal critical for fetal liver growth and liver regeneration after injury ( Beg et al., 1995 ; Kirillova et al., 1999 ; Yamada et al., 1997 ).

Techniques: Virus, Recombinant, RNAscope, Reverse Transcription, Enzyme-linked Immunosorbent Assay, RNA Sequencing, Expressing, Plasmid Preparation, Software, Cell Analysis

Journal: Cell

Article Title: Tissue Repair Signals and In Vitro Culture: Inflammatory Cytokine TNFα Promotes the Expansion of Primary Hepatocytes in 3D Culture

doi: 10.1016/j.cell.2018.11.012

Figure Lengend Snippet: Taqman™ q-PCR probes.

Article Snippet: Experimental Models: Cell Lines HEK293T ATCC CRL-1573 GFP expressing human umbilical vein endothelial cells(GFP-HUVECs) Angioproteomie cAP-0001GFP Experimental Models: Organisms/Strains C57BL/6J Jackson lab 000664 Axin2-CreERT2 Jackson lab 018867 Axin2-rtTA Jackson lab 016997 Rosa26-mTmG Jackson lab 007676 TetO-H2B:GFP Jackson lab 005104 Oligonucleotides RNAscope Probe: Mm-Fah-C2 (target region: 490-1364) Advanced Cell Diagnostics Cat#503431-C2 RNAscope Probe: Mm-Wnt9b (target region: 706-1637) Advanced Cell Diagnostics Cat#405091 RNAscope Probe: Mm-Wnt2 (target region: 857-2086) Advanced Cell Diagnostics Cat#313601 RNAscope Probe: Mm-Rspo3 (target region: 731-2164) Advanced Cell Diagnostics Cat#402011 RNAscope Probe: Mm-Rspo 1 (target region: 550-1679) Advanced Cell Diagnostics Cat#401991 RNAscope Probe: Mm-Rspo2 (target region: 537-1452) Advanced Cell Diagnostics Cat# 402001 RNAscope Probe: Mm-Rspo4 (target region: 1180-2202) Advanced Cell Diagnostics Cat#402021 RNAscope Probe: Mm-CD31/Pecam1-C2 (target region: 915-1827) Advanced Cell Diagnostics Cat#316721-C2 RNAscope Probe: Mm-CD45/Ptprc-C2 (target region: 2922-3866) Advanced Cell Diagnostics Cat# 318651-C2 RNAscope Probe: Mm-TNFa (target region: 41-1587) Advanced Cell Diagnostics Cat# 311081 Taqman Probe Alb Applied Biosystem Cat# 4331182 Mm00802090_m1 Taqman Probe Apoe Applied Biosystem Cat# 4331182 Mm01307193_g1 Taqman Probe: Cps1 Applied Biosystem Cat# 4331182 Mm01256489_m1 Taqman Probe: Cyp1a2 Applied Biosystem Cat# 4331182 Mm00487224_m1 Taqman Probe: Cyp2f2 Applied Biosystem Cat# 4331182 Mm00484087_m1 Taqman Probe: Cyp3a11 Applied Biosystem Cat# 4331182 Mm00731567_m1 Taqman Probe: Fah Applied Biosystem Cat# 4331182 Mm00487336_m1 Taqman Probe: Gck Applied Biosystem Cat# 4331182 Mm00439129_m1 Taqman Probe: Tat Applied Biosystem Cat# 4331182 Mm01244282_m1 Taqman Probe: Ttr Applied Biosystem Cat# 4331182 Mm00443267_m1 Recombinant DNA Plasmid :1-puro-CMV-TurboGFP Sigma Aldrich SHC003 Plasmid: psPAX2 Addgene 12260 Plasmid: pMD.2g Addgene 12259 Software and Algorithms Prism Graphpad 7.0 Graphpad software http://www.graphpad.com Adobe Photoshop CC 2018 Adobe http://www.adobe.com/product/photoshop.html Adobe Illustrator CC 2018 Adobe http://www.adobe.com/product/illustrator.html Image J NIH http://imagej.nih.gov/ij/ Cell Ranger version 2.1.1 10x Genomics https://support.10xgenomics.com/single-cell-gene-expression/software/downloads/latest Seurat R Package Butler et al., 2018 https://satijalab.org/seurat HCS Studio Cell Analysis Software Thermo Fisher https://thermofisher.com Other Open in a separate window KEY RESOURCES TABLE TNFα is a pro-inflammatory signal critical for fetal liver growth and liver regeneration after injury ( Beg et al., 1995 ; Kirillova et al., 1999 ; Yamada et al., 1997 ).

Techniques: Amplification