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: Cell Reports

Article Title: Systematic analysis of SARS-CoV-2 infection of an ACE2-negative human airway cell

doi: 10.1016/j.celrep.2021.109364

Figure Lengend Snippet:

Article Snippet: RT-PCR was performed on 1μg of RNA using the iScript gDNA Clear cDNA Synthesis Kit (Bio-Rad) and analyzed by qPCR using PowerUp SYBR Green Master Mix (Applied Biosystems) on a QuanStudio 5 machine.

Techniques: Virus, Control, Recombinant, Transfection, Sequencing, Modification, Saline, RNAscope, Multiplex Assay, cDNA Synthesis, SYBR Green Assay, Infection, Software, Plasmid Preparation

Journal: eLife

Article Title: Retinoic acid signaling mediates peripheral cone photoreceptor survival in a mouse model of retina degeneration

doi: 10.7554/eLife.76389

Figure Lengend Snippet:

Article Snippet: Sequence-based reagent , Mm Nrl RNAscope Probe , Advanced Cell Diagnostics , 475,011 , .

Techniques: Recombinant, Expressing, Plasmid Preparation, Sequencing

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: eLife

Article Title: Deletion of KCNQ2/3 potassium channels from PV+ interneurons leads to homeostatic potentiation of excitatory transmission

doi: 10.7554/eLife.38617

Figure Lengend Snippet: ( a ) Top, representative voltage responses from a +150 pA current injection step (1 s) in PV- and SST-like interneurons in either the CA1 region of the hippocampus (P12–P17) or L2/3 of the somatosensory cortex (P8–P11). For L2/3 recordings, cells were also confirmed by immunoreactivity against SST antibodies. Bottom, summary graphs showing the effect of deleting KCNQ2 and KCNQ3 channels on action potential number from CA1 PV-like (control n = 8/6; IN:Kcnq2/3 null n = 8/5), SST-like (control n = 19/8; IN:Kcnq2/3 null n = 8/4), and L2/3 (PV-like: control n = 10/7; IN:Kcnq2/3 null n = 8/4; SST-like: control n = 10/6; IN:Kcnq2/3 null n = 5/4) interneurons (Vh=-75 to −77 mV). For CA1 PV-like cells (P16–P25), F (9,126) =2.849, p=0.0043; for L2/3 PV-like cells, F (9,144) =3.845, p=0.0002); for CA1 SST-like cells (P15–P19), F (9,225) =0.601, p=0.7955; and for L2/3 SST-like cells, F (9,117) =0.326, p=0.965. Significance was determined using a two-factor mixed ANOVA. See showing that indeed SST cells express KCNQ2 and KCNQ3 mRNA. ( b ) Top, representative voltage responses to a series of current injection steps (1 s) in PV + and SST + interneurons in the CA1 region of the hippocampus (Vh=-75 to −77 mV). Bottom left, summary graph showing the effect of deleting KCNQ2 and KCNQ3 channels on action potential number from CA1 PV + cells (control n = 15/8; PV:Kcnq2/3 null n = 14/7; F (9,243) =3.558 with p=0.0004). Middle left, summary graph showing that loss of KCNQ2/3 channels decreases PV + input resistance (control, n = 15/8; PV:Kcnq2/3 null, n = 14/7; df = 27 t=−2.54 p=0.017 unpaired Student’s t-test). See also regarding PV + Kcnq2/3 null neurons diversity of intrinsic properties. Middle right, summary graph showing the effect of deleting KCNQ2 and KCNQ3 channels on action potential number from CA1 SST + cells (control n = 6/2; SST:Kcnq2/3 null n = 8/4; F (9,108) =0.729 with p=0.6814). Bottom right, summary graph showing loss of KCNQ2/3 channels did not decrease SST + input resistance (control n = 6/2; SST:Kcnq2/3 null, n = 8/4; df = 12 t=−0.42 p=0.68 unpaired Student’s t-test). ‘n’ designates number of cells followed by number of animals. Each data point represents recording from one neuron. Data in summary graphs are represented as mean and s.e.m. 10.7554/eLife.38617.010 Figure 2—source data 1. Source data for .

Article Snippet: Sequence-based reagent , somatostatin mRNA probe (mouse); Mm-Sst-C1 , ACDBio , Cat#:404631 , (1:50).

Techniques: Injection, Control

Journal: eLife

Article Title: Deletion of KCNQ2/3 potassium channels from PV+ interneurons leads to homeostatic potentiation of excitatory transmission

doi: 10.7554/eLife.38617

Figure Lengend Snippet:

Article Snippet: Sequence-based reagent , somatostatin mRNA probe (mouse); Mm-Sst-C1 , ACDBio , Cat#:404631 , (1:50).

Techniques: Sequencing, RNAscope, Multiplex Assay, Software