Journal: Scientific reports

Article Title: Altered functional properties of the codling moth Orco mutagenized in the intracellular loop-3.

doi: 10.1038/s41598-021-83024-3

Figure Lengend Snippet: Figure 3. Comparison of HEK cell responses expressing wild-type and mutagenized form of Orco. (a)— Universal Orco agonist, VUAA1, elicits dose-dependent Ca++ i increase in HEK293A cells expressing either CpomOrco (blue) or CpomOrcoQ417H (red). (b)—VUAA1 concentration dependencies. Data points represent the mean response amplitudes (± SE). Data were fit to a Hill equation for CpomOrco (blue smooth line) or CpomOrcoQ417H (red smooth line) respectively, providing maximum responses of ~ 6.3 ± 0.1 and ~ 3.9 ± 1.3 ∆F, EC50s of ~ 157.1 ± 3.58 and ~ 261.8 ± 165.6 µM and Hill coefficients of ~ 2.4 ± 0.1 and ~ 2.1 ± 1.9; total number of cells analysed: N = 123 and 94. (c)—Effects of Pear ester on the activity of CpomOrco+OR3 (blue) and CpomOrcoQ417H+OR3 (red) heteromers. (d)—Pear ester concentration dependencies. Data points represent the mean response amplitudes (± SE) of cells from at least three experiments. Data were fit to a Hill equation providing the following parameters: maximum responses of ~ 6.6 ± 0.3 and 3.7 ± 0.1 ∆F; EC50s of ~ 210 ± 14.2 and ~ 733.5 ± 26.9 µM; Hill coefficients of ~ 4.6 ± 2.4 and ~ 4.6 ± 0.4, for CpomOR3/CpomOrco (blue smooth line) or CpomOR3/CpomOrcoQ417H (red smooth line) respectively. Total number of cells analysed: N = 160 and 262. Traces in A and C represent the mean responses of cells from one experiment. Data in B and D were not normalized. Scales in B and D are different.

Article Snippet: HEK293 cells lines (HEK293A/HEK293T) were grown in HEK cell media [Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum (MP Biomedicals, Solon, OH, USA), 2.0 mM L-glutamine, and 100 μg/mL penicillin/streptomycin (Invitrogen)] at 37 °C and 5% CO2.

Techniques: Comparison, Expressing, Concentration Assay, Activity Assay

Journal: Scientific reports

Article Title: Altered functional properties of the codling moth Orco mutagenized in the intracellular loop-3.

doi: 10.1038/s41598-021-83024-3

Figure Lengend Snippet: Figure 4. Testing pH sensitivity of HEK cell expressing wild-type and mutagenized form of Orco. (a)— Decrease in fluorescence intensity of the pH sensitive probe, BCECF, possibly reflects acidification of cytoplasm in response to low pH extracellular conditions. (b)—Comparison of VUAA1 concentration dependencies obtained after 30 min incubation at low pHe for CpomOrco or CpomOrcoQ417H. Left panels—VUAA1 activated calcium responses. Right panels—VUAA1 concentration dependencies. Data points represent the mean response amplitudes (± SE). Data were fit to a Hill equation with the following parameters: maximum responses of ~ 1.3 ± 0.7 and ~ 1.8 ± 0.2 ∆F; EC50s of ~ 260 ± 187 and ~ 263.3 ± 45.3 µM; Hill coefficients of ~ 2.5 ± 4.3 and ~ 2.2 ± 0.5, for CpomOrco (blue smooth line, n = 84) or CpomOrcoQ417H (red smooth line, n = 63) respectively. Constraints were applied to fit greatly scattered data in B. Traces in B (left panels) represent the mean responses of cells from one experiment. Data in B (right panels) were not normalized. Concentration dependencies obtained in physiologically relevant control conditions were taken from Fig. 3.

Article Snippet: HEK293 cells lines (HEK293A/HEK293T) were grown in HEK cell media [Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum (MP Biomedicals, Solon, OH, USA), 2.0 mM L-glutamine, and 100 μg/mL penicillin/streptomycin (Invitrogen)] at 37 °C and 5% CO2.

Techniques: Expressing, Fluorescence, Comparison, Concentration Assay, Incubation, Control

Journal: Journal of translational medicine

Article Title: Unraveling resistance mechanisms in anti-CD19 chimeric antigen receptor-T therapy for B-ALL: a novel in vitro model and insights into target antigen dynamics.

doi: 10.1186/s12967-024-05254-z

Figure Lengend Snippet: Fig. 1 Proliferation and specific cytotoxic effects of CART-19 cells. A The design of the CAR-T cell construction experiments. B Morphological images of activated T cells clustered after 24 h and 72 h of incubation with TransAct CD3/28 beads. C Flow cytometric analysis of CAR expression on the surface of mock T, and CART-19 cells with biotin-conjugated anti-Fab antibody followed by PE-conjugated streptavidin. Gating was based on the same cells stained with isotype-matched antibody. The median fluorescence intensity (MFI) was calculated for CAR-T population in the PE fluorescence channel (right column). This result is the representative of three separate experiments using cells from healthy volunteer donors. D The phenotypic characterization of CART-19 cells by flow cytometry. The ratio of CD4+ / CD8+ T cells (left) and the proportion of TN/CM (right) are shown. E Growth curves of CAR-T cells. Data represent the mean ± s.d. of three separate experiments. F Cytolytic activities of CART-19 cells in cell assays. Nalm-6 cells were labeled with CFSE labeling reagent (Sigma-Aldrich, USA) and co-cultured with CART-19 cells at the E: T ratio of 1:1 for 30 h. The presence of CFSE-labeled cells was observed by mi croscopy. Bar, 100 μm. G Cytotoxic activity of mock NT and CART cells against Nalm-6 cells. The effector cells were co-cultured with target cells at E: T ratios of 1:5, 1:2, 1:1 and 5:1 with a total cell number of 1 × 106. H Dynamic changes of cytokine secretion profile of CART-19 cells during 24 h after co-culture with Nalm-6 cells at E: T ratios of 1:5 to 5:1. Data were visualized by heatmap. Concentrations (pg/ml) of cytokines and chemokines in the supernatant were detected by multiplex immunoassay and the values were log2 transformed

Article Snippet: To evaluate CAR expression after 7–10 days of culture, CART-19 cells were washed once and incubated with goat anti-human biotin conjugated anti-Fab antibody (Jackson ImmunoResearch, USA) for 30 min at room temperature.

Techniques: Incubation, Expressing, Staining, Fluorescence, Flow Cytometry, Labeling, Cell Culture, Activity Assay, Co-Culture Assay, Multiplex Assay, Transformation Assay

Journal: Journal of translational medicine

Article Title: Unraveling resistance mechanisms in anti-CD19 chimeric antigen receptor-T therapy for B-ALL: a novel in vitro model and insights into target antigen dynamics.

doi: 10.1186/s12967-024-05254-z

Figure Lengend Snippet: Fig. 5 Observation of CD19-BBζ-CAR expression in relapsed Nalm-6 cells and salvage treatment. A Detection of FMC63 and CD247 transcripts and 4-1BB gene of CAR in CD19+ Nalm-6 (red) and relapsed CD19− Nalm-6 cells (blue) by qRT-PCR. Data of left bar graph represent the relative quantification using ACTB as the internal reference. Error bars represent s.d. The data are the representative of three independent experiments. B Expression of CD19 and CAR on CD19+ Nalm-6 cells and relapsed CD19− Nalm-6 cells analyzed by flow cytometry (representative of 3 experiments). Merge Graphs, the blue dots represent CD19− Nalm-6 cells and the red dots represent Nalm-6 cells. C Confocal imaging of Nalm-6 cells and relapsed CD19− Nalm-6 cells using Alexa Flour 488-conjugated anti-CD19 antibody (green), Alexa Flour 647-conjugated anti-CAR19 antibody (red), and DAPI (blue). D Lentiviral integration sites of CAR transduced Nalm-6 cells were analyzed by linear-amplification mediated PCR (LAM-PCR) and visualized with Circos plots. The integration sites across the genome and genomic features were shown from outer to inner circle: (1) cytogenetic bands; (2) genes that harbor these integration sites along with a bar chart showing the reads of integration sites; (3) the distribution of integration sites, with colored circles representing different gene functional regions of the host sequence: purple for promoter region, green for intron region, and red for distal intergenic region. E Phenotype changes of Nalm-6 cells transduced with small amount of CD19 CAR lentiviruses detected by flow cytometry over time. Gating was based on the same cells stained with isotype-matched antibody. F Dynamics of CD19− B phenotype in relapsed cells after co-culture with different ratios (5×, 20×) of Nalm-6 cells. Gating was based on the same cells stained with isotype-matched antibody. G Relapsed CD19− Nalm-6 cells were tested by qPCR specific for VSV-G sequence. H Comparison of in vitro efficacy of CD19-, CD22-, CD19/CD22- and CD22×CD19- CAR T cells. Cocultures with the relapsed cells were performed at 1:5, 1:1, and 5:1 E: T ratios, and lysis efficacies were detected by the LDH release assay Declarations

Article Snippet: To evaluate CAR expression after 7–10 days of culture, CART-19 cells were washed once and incubated with goat anti-human biotin conjugated anti-Fab antibody (Jackson ImmunoResearch, USA) for 30 min at room temperature.

Techniques: Expressing, Quantitative RT-PCR, Quantitative Proteomics, Flow Cytometry, Imaging, Amplification, Functional Assay, Sequencing, Transduction, Staining, Co-Culture Assay, Comparison, In Vitro, Lysis, Lactate Dehydrogenase Assay

Journal: Frontiers in Pharmacology

Article Title: Oleoylethanolamide as a New Therapeutic Strategy to Alleviate Doxorubicin-Induced Cardiotoxicity

doi: 10.3389/fphar.2022.863322

Figure Lengend Snippet: OEA inhibited DOX-induced oxidative stress and apoptosis in cardiomyocytes through TRPV1 pathway. HL-1 cells were treated with 0.1% DMSO, OEA (30 μM), GW6471 (5 μM), capsazepine (10 μM), nonivamide (10 μM), fenofibrate (100 μM) for 30 min. Cells were then treated with DOX (2 μM) for 24 h. Levels of (A) GST (B) TBARS and (C) cell viability was assessed in HL-1 cardiomyocytes. (D) TUNEL positive cells shown in (E) were counted. (E) TUNEL staining of HL-1 cardiomyocytes. Data are expressed as mean ± SEM, n = 4. ***, p < 0.001 vs. control. ##, p < 0.01; ###, p < 0.001 vs. DOX + vehicle. $$$, p < 0.001 vs. DOX + OEA.

Article Snippet: Adult derived primary human cardiomyocytes (Celprogen, Cat # 36044-15) were cultured in complete growth media (Celprogen, Cat #M36044-15S) in an incubator at 37°C with 5% CO 2 atmosphere.

Techniques: TUNEL Assay, Staining

Journal: Frontiers in Pharmacology

Article Title: Oleoylethanolamide as a New Therapeutic Strategy to Alleviate Doxorubicin-Induced Cardiotoxicity

doi: 10.3389/fphar.2022.863322

Figure Lengend Snippet: OEA promotes TRPV1-mediated PI3K/ Akt signaling in cardiomyocytes. HL-1 cells were treated with 0.1% DMSO, OEA (30 μM), GW6471 (5 μM), capsazepine (10 μM) and nonivamide (10 μM) for 30 min. Cells were then treated with DOX (2 μM) for 24 h. PI3K, Akt, p-PI3K and p-Akt was determined using western blot analysis.

Article Snippet: Adult derived primary human cardiomyocytes (Celprogen, Cat # 36044-15) were cultured in complete growth media (Celprogen, Cat #M36044-15S) in an incubator at 37°C with 5% CO 2 atmosphere.

Techniques: Western Blot

Journal: Frontiers in Pharmacology

Article Title: Oleoylethanolamide as a New Therapeutic Strategy to Alleviate Doxorubicin-Induced Cardiotoxicity

doi: 10.3389/fphar.2022.863322

Figure Lengend Snippet: Inhibition of the PI3K-Akt signaling blocks the protective effects of OEA in cardiomyocytes. HL-1 cells were treated with 0.1% DMSO, OEA (30 μM) and LY294002 (10 μM) for 30 min. Cells were then treated with DOX (2 μM) for 24 h. Levels of (A) GST (B) TBARS and (C) cell viability was assessed in HL-1 cardiomyocytes. (D) TUNEL positive cells shown in (F) were counted. (E) Fluorescence quantification for caspase three in (G) were calculated. (F) TUNEL staining of HL-1 cardiomyocytes. (G) Immunofluorescence staining of caspase three in HL-1 cardiomyocytes. Data are expressed as mean ± SEM, n = 4. ***, p < 0.001 vs. control. #, p < 0.05; ##, p < 0.01; ###, p < 0.001 vs. DOX + vehicle. $, p < 0.05; $$, p < 0.01; $$$, p < 0.001 vs. DOX + OEA.

Article Snippet: Adult derived primary human cardiomyocytes (Celprogen, Cat # 36044-15) were cultured in complete growth media (Celprogen, Cat #M36044-15S) in an incubator at 37°C with 5% CO 2 atmosphere.

Techniques: Inhibition, TUNEL Assay, Fluorescence, Staining, Immunofluorescence

Journal: The Journal of Biological Chemistry

Article Title: DGCR8-dependent efficient pri-miRNA processing of human pri-miR-9-2

doi: 10.1016/j.jbc.2021.100409

Figure Lengend Snippet: Live-cell pri-miRNA processing reporter assay. A , schematic of fluorescent reporter vector construction. Venus and tdTomato mRNAs were transcribed under the Tet-On-responsive bidirectional promoter (P Tight-BI ). The 300-nt cDNA coding human pri-miR-9-1 was subcloned into a multicloning site (MCS) in the 3′-UTR of tdTomato mRNA. A microprocessor complex including Drosha and DGCR8 cleaves pri-miRNA to produce pre-miRNA from the 3′-UTR of tdTomato mRNA, which is destabilized because the poly(A) sequence is removed from the 3′-UTR. pA, poly(A) signal sequence. B , After transfecting the fluorescent reporter vector into HeLa Tet-On 3G cells, nuclear expression of Venus and tdTomato was observed by Opera Phenix, a high content cell imaging analyzer. The scale bar represents 200 μm. C , After transfecting the fluorescent reporter control, the pri-miRNA or pri-miR-9-1 reporter vector was transfected with pcDNA3.1 or the FLAG-DGCR8 expression vector into HeLa Tet-On 3G cells. The sums of the Venus fluorescent signal intensity and tdTomato fluorescent signal intensity in selected nuclei were shown in the graph. The relative sum of the Venus signal intensity to the sum of the tdTomato signal intensity was calculated for each well and relative values are shown. Error bars show the standard deviation (n = 3). D , fluorescence pri-miRNA processing reporter assay. Control, pri-miR-9-1, and pri-miR-9-1M were transfected with pcDNA3.1 or FLAG-DGCR8 expression vectors into HeLa Tet-On 3G and fluorescent signals were monitored from each cell. The relative sum of the Venus signal intensity to the sum of the tdTomato signal intensity was calculated in each well and shown. The error bar shows the standard deviation (n = 3). E , has-miR-9-5p was quantified by qRT-PCR with total RNA purified from HeLa Tet-On 3G cells transiently transfected with control, pri-miR-9-1, pri-miR-9-1M reporter, and FLAG-DGCR8 expression vectors. The error bar shows the standard deviation (n = 3). The asterisk indicates significant change ( t -test p < 0.001); NLS, nuclear localization signal; PEST, a peptide sequence that acts as a signal peptide for protein degradation.

Article Snippet: To quantify human DGCR8 mRNA, human Drosha mRNA, and human β-actin mRNA expression levels, real-time PCR was performed using the TaqMan Gene Expression assay (Life Technologies, DGCR8 ; Hs00256062_m1 and Hs00987085_m1, Drosha ; Hs00203008_m1, β-actin; Hs01060665_g1, pri-let-7b; Hs03302548-pri, pri-miR-100; Hs03302731-pri, pri-miR-15a; Hs03302582-pri, pri-miR-9-1; Hs03303201_pri, pri-miR-9-2; Hs03303202_pri, pri-miR-9-3; Hs03293595_pri, pri-miR-99a; Hs03302729-pri), TaqMan Gene Expression Master Mix (Life Technologies), and ViiA7 Real Time PCR or StepOnePlus system (Life Technologies) following the manufacturer’s instructions.

Techniques: Reporter Assay, Plasmid Preparation, Sequencing, Expressing, Imaging, Control, Transfection, Standard Deviation, Fluorescence, Quantitative RT-PCR, Purification

Journal: The Journal of Biological Chemistry

Article Title: DGCR8-dependent efficient pri-miRNA processing of human pri-miR-9-2

doi: 10.1016/j.jbc.2021.100409

Figure Lengend Snippet: Higher DGCR8 sensitivity of pri-miR-9-2 processing. A , fluorescent pri-miRNA processing reporter assay. Control, pri-miR-9-1, pri-miR-9-2, and pri-miR-9-3 were transfected with pcDNA3.1 or FLAG-DGCR8 expression vectors into HeLa Tet-On 3G cells, and fluorescent signals were monitored. The relative sum of the Venus signal intensity to the sum of the tdTomato signal intensity was calculated and shown in the graph. The error bar shows the standard deviation (n = 3). B , has-miR-9-5p was quantified by qRT-PCR with total RNA purified from HeLa Tet-On 3G cells transiently transfected with control, pri-miR-9-1, pri-miR-9-2, pri-miR-9-3 reporter, and FLAG-DGCR8 expression vectors. The error bar shows the standard deviation (n = 3). C , control, pri-miR-9-1, and pri-miR-9-1x2 (containing twice tandem repeat of pri-miR-9-1) reporter vectors were transfected with pcDNA3.1 control or DGCR8 expression vectors into HeLa Tet-On 3G cells and fluorescent signals monitored. The relative sum of the Venus signal intensity to the sum of the tdTomato signal intensity was calculated and is shown in the graph. The error bar shows the standard deviation (n = 3). D , control, pri-miR-9-2, and pri-miR-9-2x2 (containing twice tandem repeat of pri-miR-9-2) reporter vectors were transfected with pcDNA3.1 control or DGCR8 expression vectors into HeLa Tet-On 3G cells and fluorescent signals were monitored. The relative sum of the Venus signal intensity to the sum of the tdTomato signal intensity was calculated and is shown in the graph. The error bar shows the standard deviation (n = 3). E and F , the sum of the Venus fluorescent signal intensity in selected nuclei was calculated and is shown in the graph. The error bar shows the standard deviation (n = 3). The asterisk indicates significant change ( t -test ∗ p < 0.001).

Article Snippet: To quantify human DGCR8 mRNA, human Drosha mRNA, and human β-actin mRNA expression levels, real-time PCR was performed using the TaqMan Gene Expression assay (Life Technologies, DGCR8 ; Hs00256062_m1 and Hs00987085_m1, Drosha ; Hs00203008_m1, β-actin; Hs01060665_g1, pri-let-7b; Hs03302548-pri, pri-miR-100; Hs03302731-pri, pri-miR-15a; Hs03302582-pri, pri-miR-9-1; Hs03303201_pri, pri-miR-9-2; Hs03303202_pri, pri-miR-9-3; Hs03293595_pri, pri-miR-99a; Hs03302729-pri), TaqMan Gene Expression Master Mix (Life Technologies), and ViiA7 Real Time PCR or StepOnePlus system (Life Technologies) following the manufacturer’s instructions.

Techniques: Reporter Assay, Control, Transfection, Expressing, Standard Deviation, Quantitative RT-PCR, Purification

Journal: The Journal of Biological Chemistry

Article Title: DGCR8-dependent efficient pri-miRNA processing of human pri-miR-9-2

doi: 10.1016/j.jbc.2021.100409

Figure Lengend Snippet: pri-miR-9-2 has a DGCR8-responsive element in the 3’ wing region of pri-miR-9-2. A , schematic of deletion mutant reporters used in this study. The wing region around the stem–loop structures coding pre-miRNA was removed in the deletion mutant reporters. B , fluorescent pri-miRNA processing reporter assay. Control, pri-miR-9-2, pri-miR-9-2-200, pri-miR-9-2-100, pri-miR-9-2-200-1, and pri-miR-9-2-200-2 reporter vectors were transfected with pcDNA3.1 or FLAG-DGCR8 expression vectors into HeLa Tet-On 3G cells and fluorescent signals were monitored. The relative sum of the Venus signal intensity to the sum of the tdTomato signal intensity was calculated and is shown in the graph. The error bar shows the standard deviation (n = 3). C , schematic of deletion mutant reporters used in this study. The wing region around the stem–loop structures coding pre-miRNA was removed in the deletion mutant reporters. D , fluorescent pri-miRNA processing reporter assay. Control, pri-miR-9-1, pri-miR-9-1-200, and pri-miR-9-1-100 reporter vectors were transfected with pcDNA3.1 or FLAG-DGCR8 expression vectors into HeLa Tet-On 3G cells and fluorescent signals were monitored. The relative sum of the Venus signal intensity to the sum of the tdTomato signal intensity was calculated and is shown in the graph. The error bar shows the standard deviation (n = 3). E , alignment of human pri-miR-9-1 (300 nt) and pri-miR-9-2 (300 nt) to 35 other mammalian species by Genomic Evolutionary Rate Profiling on the UCSC Human Genome Browser. The red asterisk indicates the highly conserved region in the 3’-end wing of pri-miR-9-2. The asterisk indicates significant change (∗ p < 0.001 ∗∗ p < 0.005).

Article Snippet: To quantify human DGCR8 mRNA, human Drosha mRNA, and human β-actin mRNA expression levels, real-time PCR was performed using the TaqMan Gene Expression assay (Life Technologies, DGCR8 ; Hs00256062_m1 and Hs00987085_m1, Drosha ; Hs00203008_m1, β-actin; Hs01060665_g1, pri-let-7b; Hs03302548-pri, pri-miR-100; Hs03302731-pri, pri-miR-15a; Hs03302582-pri, pri-miR-9-1; Hs03303201_pri, pri-miR-9-2; Hs03303202_pri, pri-miR-9-3; Hs03293595_pri, pri-miR-99a; Hs03302729-pri), TaqMan Gene Expression Master Mix (Life Technologies), and ViiA7 Real Time PCR or StepOnePlus system (Life Technologies) following the manufacturer’s instructions.

Techniques: Mutagenesis, Reporter Assay, Control, Transfection, Expressing, Standard Deviation

Journal: The Journal of Biological Chemistry

Article Title: DGCR8-dependent efficient pri-miRNA processing of human pri-miR-9-2

doi: 10.1016/j.jbc.2021.100409

Figure Lengend Snippet: pri-miR-9-1 also has a DGCR8-responsive element in the region near the pre-miR-9-1. A , alignment analysis of human pri-miR-9-1-100 and human pri-miR-9-2-100 by the Clustal W method. The 5′-end ( black dashed box ), 3′-end ( blue dashed box ), and loop between miR-9-5p and miR-9-3p ( red dashed box ) are unique. The box residues match the consensus/majority exactly. B , schematic of chimera reporters used in this study. Unique sequences of pri-miR-9-1-100 and pri-miR-9-2-100 are shown in gray and red , respectively. pri-miR-9-1/2-102 (including pre-miR-9-1, and 5′- and 3′-ends of pri-miR-9-2-100) and pri-miR-9-1/2-98 (including pre-miR-9-2, and 5′- and 3′-ends of pri-miR-9-1-100) were constructed. C , fluorescence pri-miRNA processing reporter assay. Control, pri-miR-9-1, pri-miR-9-2, pri-miR-9-1-100, pri-miR-9-2-100, pri-miR-9-1/2-102, and pri-miR-9-1/2-98 reporter vectors were transfected with pcDNA3.1 or FLAG-DGCR8 expression vectors and fluorescent signals were monitored. The relative sum of the Venus signal intensity to the sum of the tdTomato signal intensity was calculated and is shown in the graph. The error bar shows the standard deviation (n = 4). The asterisk indicates significant change ( t -test p < 0.001).

Article Snippet: To quantify human DGCR8 mRNA, human Drosha mRNA, and human β-actin mRNA expression levels, real-time PCR was performed using the TaqMan Gene Expression assay (Life Technologies, DGCR8 ; Hs00256062_m1 and Hs00987085_m1, Drosha ; Hs00203008_m1, β-actin; Hs01060665_g1, pri-let-7b; Hs03302548-pri, pri-miR-100; Hs03302731-pri, pri-miR-15a; Hs03302582-pri, pri-miR-9-1; Hs03303201_pri, pri-miR-9-2; Hs03303202_pri, pri-miR-9-3; Hs03293595_pri, pri-miR-99a; Hs03302729-pri), TaqMan Gene Expression Master Mix (Life Technologies), and ViiA7 Real Time PCR or StepOnePlus system (Life Technologies) following the manufacturer’s instructions.

Techniques: Construct, Fluorescence, Reporter Assay, Control, Transfection, Expressing, Standard Deviation

Journal: The Journal of Biological Chemistry

Article Title: DGCR8-dependent efficient pri-miRNA processing of human pri-miR-9-2

doi: 10.1016/j.jbc.2021.100409

Figure Lengend Snippet: Exploration of pri-miRNA candidates possessing DRE. A , miRNA profiling of U251 MG (KO) cells treated with siNC#1, siNC#2 siDGCR8#1, and siDGCR8#2 was performed with the nCounter miRNA analysis system. Expression levels of top20 ranked miRNAs are shown in the graph. The bar graph indicates the average in each two technical replicates (n = 1). B , pri-miRNA expression levels of hsa-let-7b-5p, hsa-miR-99a-5p, hsa-miR-15a-5p, and hsa-miR-100-5p in U251 MG (KO) cells treated with siNC#1, siNC#2 siDGCR8#1, and siDGCR8#2 were quantified by qRT-PCR. The error bar represents SD using four biological replicates. C , pri-miRNA expression levels of hsa-let-7b-5p, hsa-miR-99a-5p, hsa-miR-15a-5p, and hsa-miR-100-5p in HeLa Tet-On 3G cells treated with siNC#1, siNC#2 siDGCR8#1, and siDGCR8#2 were quantified by qRT-PCR. The error bar represents SD using four biological replicates. D , pri-miRNA processing reporters containing pri-miR-9-2 (300 nt), pri-let-7b (300 nt), pri-miR-99a (300 nt), pri-miR-15a-16-1 (400 nt), and pri-miR-100 (300 nt) were constructed and the processing assay was performed with HeLa Tet-On 3G cells. The error bar represents SD using three biological replicates. E , pri-miRNA processing reporters containing pri-miR-9-2 (300 nt), pri-miR-9-2M (300 nt), pri-miR-17/92 (887 nt), pri-miR-409-412-369-410 (800 nt), and pri-miR-137 (500 nt) were constructed, and the processing assay was performed with HeLa Tet-On 3G cells. The error bar represents SD using three biological replicates. Asterisk s indicate significant change (∗ p < 0.01).

Article Snippet: To quantify human DGCR8 mRNA, human Drosha mRNA, and human β-actin mRNA expression levels, real-time PCR was performed using the TaqMan Gene Expression assay (Life Technologies, DGCR8 ; Hs00256062_m1 and Hs00987085_m1, Drosha ; Hs00203008_m1, β-actin; Hs01060665_g1, pri-let-7b; Hs03302548-pri, pri-miR-100; Hs03302731-pri, pri-miR-15a; Hs03302582-pri, pri-miR-9-1; Hs03303201_pri, pri-miR-9-2; Hs03303202_pri, pri-miR-9-3; Hs03293595_pri, pri-miR-99a; Hs03302729-pri), TaqMan Gene Expression Master Mix (Life Technologies), and ViiA7 Real Time PCR or StepOnePlus system (Life Technologies) following the manufacturer’s instructions.

Techniques: Expressing, Quantitative RT-PCR, Construct

Journal: The Journal of Biological Chemistry

Article Title: DGCR8-dependent efficient pri-miRNA processing of human pri-miR-9-2

doi: 10.1016/j.jbc.2021.100409

Figure Lengend Snippet: Role of DRE in DGCR8-dependent microprocessor activity. A , pri-miRNA processing reporters containing pri-miR-99a (300 nt) and pri-miR-99a+DRE (300 nt) were constructed, and the processing assay was performed with HeLa Tet-On 3G cells. The graph indicates plots for each cell obtained from Venus and tdTomato fluorescence signals. Slopes from linear regression are shown in each graph. The bar graph indicates slopes with or without DGCR8. The asterisk indicates significant change ( t -test p = 0.026). B , RNA expression levels of METTL3 and unprocessed pri-miR-9-2 in U251 MG (KO) cells treated with siNC#1, siNC#2 siMETTL3#1, and siMETTL3#2 were quantified by qRT-PCR. The asterisk indicates significant change ( t -test p < 0.01) C , Quantification of DGCR8-bound pri-miRNA was analyzed by CLIP-qRT-PCR assay. The asterisk indicates significant change ( t -test p < 0.05). D , Model of DRE in DGCR8-dependent microprocessor activity. DRE, DGCR8-responsive RNA element; CLIP, UV cross-linking and immunoprecipitatio.

Article Snippet: To quantify human DGCR8 mRNA, human Drosha mRNA, and human β-actin mRNA expression levels, real-time PCR was performed using the TaqMan Gene Expression assay (Life Technologies, DGCR8 ; Hs00256062_m1 and Hs00987085_m1, Drosha ; Hs00203008_m1, β-actin; Hs01060665_g1, pri-let-7b; Hs03302548-pri, pri-miR-100; Hs03302731-pri, pri-miR-15a; Hs03302582-pri, pri-miR-9-1; Hs03303201_pri, pri-miR-9-2; Hs03303202_pri, pri-miR-9-3; Hs03293595_pri, pri-miR-99a; Hs03302729-pri), TaqMan Gene Expression Master Mix (Life Technologies), and ViiA7 Real Time PCR or StepOnePlus system (Life Technologies) following the manufacturer’s instructions.

Techniques: Activity Assay, Construct, Fluorescence, RNA Expression, Quantitative RT-PCR

Journal: mBio

Article Title: Regulation of the Sae Two-Component System by Branched-Chain Fatty Acids in Staphylococcus aureus

doi: 10.1128/mbio.01472-22

Figure Lengend Snippet: Branched-chain fatty acids are required for Sae activation. (A to D) Strains carrying sae P1 -gfp reporter fusions were grown under three fluidizing conditions: 0.5 mM sBCFA supplementation (sBCFA), 0.1% benzyl alcohol (BnOH), or growth at 41°C (HT). After 5 h, optical density (OD) (A) and relative fluorescence units (RFUs) (B to D) were measured. (B) Fluorescence in sBCFA-supplemented medium, (C) Fluorescence in BnOH-supplemented medium, (D) Fluorescence at a higher temperature. <, fluorescence was undetectable compared to blank PBS. (E and F) Strains were grown in TSB with or without 0.5 mM sBCFAs, 10 mg mL −1 BSA, and 0.5 mM 15:0 anteiso-fatty acid (FA), before optical density (E) and relative fluorescence units (RFUs) (F) were measured. For all panels, bars represent mean from three trials ± standard deviation. ns, not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001, ANOVA with Tukey post hoc test. In panel A, significance is relative to the indicated strain in TSB.

Article Snippet: Cells carrying the indicated reporter fusions were grown from an OD 600 of 0.05 to specified times after their second dilution in TSB with or without supplements: 0.5 mM BCFAs, 0.1% benzyl alcohol (BnOH), 5 μg mL −1 human neutrophil peptide 1 (HNP-1), 10 mg mL −1 fatty-acid-free bovine serum albumin (BSA), or 0.5 mM 15:0 anteiso-fatty acid (Avanti).

Techniques: Activation Assay, Fluorescence, Standard Deviation

Journal: Neuro-Oncology

Article Title: Dissecting inherent intratumor heterogeneity in patient-derived glioblastoma culture models

doi: 10.1093/neuonc/now253

Figure Lengend Snippet: Floating cells display enhanced mesenchymal properties. (A) MGG6 NS and FC mRNA were analyzed by qRT-PCR for different mesenchymal markers including CD44, vimentin, YKL-40, N-cad, and MMP2. (B) Cell lysates from different cultures were analyzed by western blotting for pSTAT3, STAT3, CD44, C/EBPβ, and β-actin. (C) FACS analysis showing proportion of CD44 positive cells in FC and their corresponding NS from different GSC cultures. (D) MGG29 NS were fractionated into CD44high and CD44low subpopulation by FACS and predominant cells were established and labeled with GFP and mCherry, respectively. Left: fluorescence analysis of a mixture of these 2 subpopulations cultured in serum; middle: FC collected and analyzed for both GFP and mCherry; right: flow cytometry of CD44high cells in both GFP and mCherry populations. (E) NS and FC were differentiated using StemPro Chondrogenesis Differentiation Kit and stained with fast green and safranin O (positive, #; negative, +). (F) NS and FC were cultured as a monolayer and their ability to invade/migrate was evaluated using scratch healing assay. Bright field micrographs are showing for different groups. Scale bar, 50 μm.

Article Snippet: CD44 expression was measured in 500000 cells dissociated with 2 mM EDTA and incubated in 80 μL PBS/0.1% bovine serum albumin at 4°C for 30 min in the dark with mouse monoclonal anti-human CD44 antibody conjugated to fluorescein isothiocyanate (FITC, 1:10; Miltenyi Biotec) or respective mouse immunoglobulin G1 control in the presence of FcR blocking reagent (1:5).

Techniques: Quantitative RT-PCR, Western Blot, Labeling, Fluorescence, Cell Culture, Flow Cytometry, Staining

Journal: Clinical and Experimental Immunology

Article Title: MOSPD2 is a therapeutic target for the treatment of CNS inflammation

doi: 10.1111/cei.13448

Figure Lengend Snippet: Selection of lead anti‐motile sperm domain‐containing protein 2 (MOSPD2) monoclonal antibodies (mAbs). (a) Migration of primary CD14 + human monocytes towards stromal derived factor‐1 alpha + monocyte chemotactic factor 1 (SDF‐1+MCP‐1) and CD3 T cells towards SDF‐1 + regulated upon activation, normal T cell expressed and secreted (RANTES) in the presence of 10 μg/ml anti‐MOSPD2 mAb Clone‐s1 or ‐3 compared with matched isotype controls. One of three experiments is shown. P ‐values were calculated by Student’s t ‐test. Data are of triplicates and shown as mean ± standard error (s.e.). * P < 0·005; ** P < 0·001. (b) Linear epitope mapping of clones‐1 and ‐3 against the extracellular region of human MOSPD2. (c) Alignment of human and mouse MOSPD2. Differences are indicated by vertical lines. (d) Western blots of human embryonic kidney 293 (HEK) 293 cells transfected with human and mouse MOSPD2 and detected with anti‐human MOSPD2 mAbs clones‐1 or ‐3. One of two experiments is shown. (e) Fluorescence activated cell sorter (FACS) analysis for the recognition of anti‐human MOSPD2 mAbs clones‐1 and ‐3 of surface‐expressed mouse MOSPD2. One of two experiments is shown. (f) Migration of mouse splenic CD11b cells towards SDF‐1 + MCP‐1 in the presence of 10 μg/ml anti‐MOSPD2 mAb clone‐1 or clone‐3 compared with matched isotype controls. One of three experiments is shown. P ‐values were calculated by Student’s t ‐test. Data are of triplicates and shown as mean ± s.e. * P < 0·005; ** P < 0·001.

Article Snippet: Cells were washed in phosphate‐buffered saline (PBS) (Biological Industries, Beit HaEmek, Israel), and incubated at 4°C for 15 min in buffer containing PBS and 0·5% bovine serum albumin (BSA) with human CD14 microbeads (cat. no. 130‐050‐201; Miltenyi Biotec, Bergisch Gladbach, Germany).

Techniques: Selection, Bioprocessing, Migration, Derivative Assay, Activation Assay, Clone Assay, Western Blot, Transfection, Fluorescence