Journal: Journal of Neuroscience

Article Title: Notch Signaling Alters Sensory or Neuronal Cell Fate Specification of Inner Ear Stem Cells

doi: 10.1523/jneurosci.6366-10.2011

Figure Lengend Snippet: Figure 3. Inhibition of -secretase increased hair cell differentiation by upregulating Math1. a, Expression of Math1 in inner ear stem cells was increased after treatment with a -secretaseinhibitorandcouldbeblockedbysiRNAtoMath1butnotbyanontargetingsiRNA based on qRT-PCR. b, Differentiation of hair cells (myosin VIIa, green) induced by the -secretase inhibitor was unaffected by nontargeting siRNA but was inhibited by siRNA di- rectedatMath1.LabeledsiRNA(red,arrowheads)wasaddedtofollowtransfection.Blue,DAPI. Scalebar,15m.c,Thepercentageofhaircellsobtainedaftertreatmentwiththe-secretase inhibitorwassignificantlydecreasedbyMath1RNAi.Thepercentagewasreducedtothelevelof the untreated control but was not decreased by nontargeting RNAi. Error bars, SE; *p 0.05, **p 0.01.

Article Snippet: The same concentration of nontargeting RNAi (Dharmacon, ON-TARGETplus nontargeting siRNA, size-matched and selected for lack of an effect on gene expression) was used in parallel as a control, and gene silencing was assessed by quantitative (q) reverse transcriptase (RT)-PCR. qRT-PCR.

Techniques: Inhibition, Cell Differentiation, Expressing, Quantitative RT-PCR, Control

Journal: Respiratory Research

Article Title: Altered maturation and activation state of circulating monocytes is associated with their enhanced recruitment in pulmonary arterial hypertension

doi: 10.1186/s12931-025-03182-0

Figure Lengend Snippet: Decreased BMPR2 is linked to decreased CD14 expression. (A) RT-qPCR of BMPR2 gene expression in classical monocytes of IPAH patient. (B) RT-qPCR of CD14 gene expression in classical monocytes of IPAH patients. (C) RT-qPCR of CD14 gene expression in THP1 monocyte cell line, where BMPR2 was decreased via treatment with siRNA targeting BMPR2 (si BMPR2 ) vs. non-targeting siRNA (siControl). (D-G) RT-qPCR of STAT1 , IFN-α , IFN-β and IFN-γ gene expression in THP1 monocyte cell line, where BMPR2 was decreased via treatment with shRNA targeting BMPR2 (sh BMPR2 ) vs. non-targeting shRNA (shControl) (H) Schematic outlining the breeding strategy used to generate mice with tomato (Td) fluorescent monocyte-specific tag and a conditional, monocyte specific Bmpr2 deletion (MON- Bmpr2 −/− , Cx3cr1 Td Bmpr2 −/− ), and Control mice (Control; Cx3cr1Td ). Details are provided in the Methods. (I) RT-qPCR of Ly6c (mouse CD14 ) gene expression in circulating monocytes of the MON- Bmpr2 −/− mouse, compared to Control. n = 3 groups, each consisting of pooled blood from 3 mice. (J) LY6C protein in MON- Bmpr2 −/− bone marrow derived macrophages (mBMDM) assessed via western blot, with representative blot image. (K) Representative microscopic images of mBMDM stained with CD68 (green), DAPI (blue) and tamoxifen induced Td (red). MON- Bmpr2 −/− mBMDM were consistently smaller compared to Control. Scale bar = 50 μm, 20 μm (zoom). (L) Representative microscopy images with small pulmonary artery (white dashed outline) zoom insert, of lung sections of mice, labeled with DAPI (blue) and showing Td (red) labelled macrophages following hypoxia. Scale bar = 100 μm, 50 μm (zoom). With quantification of macrophages per field of view (FOV). Bars represent mean ± SEM. * p < 0.05, ** p < 0.01, by Mann-Whitney non-parametric t-test (A, B, J). For E, F, H, I, n = 3 biological replicates; + denotes the minimum achievable P -value for n = 3 was reached by the non-parametric t test

Article Snippet: THP1 cells at a density of 5 × 10 5 cells/mL were cultured overnight in a 6-well plate, followed by siRNA transfection with siRNA BMPR2 (SMARTpool ON-TARGETplus, Origene, Rockville, MD, Cat#: L-001230-00-005) or siRNA Control (ON-TARGETplus non-targeting Control siRNA, Origene, Cat#: D-001230-01) using the TransIT-TKO reagent (Mirus, Madison, WI Cat# MIR2150) according to the manufacturer’s recommendations.

Techniques: Expressing, Quantitative RT-PCR, Gene Expression, shRNA, Control, Derivative Assay, Western Blot, Staining, Microscopy, Labeling, MANN-WHITNEY

Journal: mBio

Article Title: Rotavirus NSP1 Requires Casein Kinase II-Mediated Phosphorylation for Hijacking of Cullin-RING Ligases

doi: 10.1128/mBio.01213-17

Figure Lengend Snippet: Effect of CKII inhibition on NSP1 phosphorylation. (A) HEK293T cells were cotransfected with a vector expressing HALO-tagged NSP1 (H-NSP1) and a 10 or 50 μM siRNA pool targeting the CKII RNA or representing a scrambled siRNA control pool. At 48 h p.t., cells were collected and protein samples were analyzed by immunoblot assay with antibodies specific for the HALO tag, p-NSP1, CKIIα, and PCNA. Band intensities from two independent experiments using 50 mM concentration of siRNA pools were determined with an Azure digital imager. (B) Levels of OSU NSP1 phosphorylation were calculated by dividing p-NSP1 intensity values by those for H-NSP1 and normalizing the results to 100% for the control siRNA sample. (C) CKII levels were calculated by dividing CKII intensity values by those for PCNA and normalizing the results to 100% for the control siRNA sample. (D) HT29 cells were infected with OSU at an MOI of 5 and treated with TBB or DMSO at 2 h p.i. At 4 and 9 h p.i., cells were collected and lysed, and proteins in the samples were resolved by electrophoresis and examined by immunoblot assay with antibodies recognizing OSU NSP1, p-NSP1, p-IκB, β-TrCP, or PCNA. (E) PK15 cells were infected with OSU at an MOI of 5 and mock treated or treated with TBB. Cells were harvest at 9 h p.i., and proteins in the samples were examined by immunoblot assay with antibodies to p-NSP1, rotavirus VP6, and PCNA.

Article Snippet: For RNA interference experiments, CKII-directed siRNA (CSNK2A1 siRNA, L-003475-00; Dharmacon SMARTpool ON-TARGETplus) or scrambled control siRNA (D-001810-10; ON-TARGETplus nontargeting pool) was added to siLentFect (1703360; BioRad) transfection mixtures to a final concentration of 10 or 50 nM, along with 1 μg of plasmid DNA, according to the manufacturer’s instructions.

Techniques: Inhibition, Phospho-proteomics, Plasmid Preparation, Expressing, Control, Western Blot, Concentration Assay, Infection, Electrophoresis