heat inactivated fcs 20 ng ml 1 il  (Miltenyi Biotec)

Journal: bioRxiv

Article Title: Interferon-α subtype treatment induces the repression of SRSF1 in HIV-1 target cells and affects HIV-1 post integration steps

doi: 10.1101/2021.06.11.448031

Figure Lengend Snippet: a) - b) Differentiated THP-1 cells were treated with the indicated IFN subtype at a concentration of 10 ng/ml. 24 h post treatment, cells were harvested, RNA isolated and subjected to RT-qPCR for measurement of relative a) ISG15 and b) SRSF1 mRNA expression levels. ACTB was used as loading control. Unpaired t tests were calculated to determine whether the difference between the group of samples reached the level of statistical significance (* p<0.05, ** p<0.01 and *** p<0.001). c) Correlation between x-fold repression of SRSF1 mRNA levels and x-fold induction of ISG15 mRNA levels. IFNα subtypes 2, 4, 6 and 14 were excluded from correlation and are marked in red. Pearson correlation coefficient (r) and p-value (p) are indicated.

Article Snippet: Jurkat, CEM-SS, CEM-T4 and THP-1 cells were maintained in Roswell Park Memorial Institute (RPMI) 1640 medium (Gibco) supplemented with 10% (Jurkat, CEM-SS and CEM-T4) or 20% (THP-1) (v/v) heat-inactivated fetal calf serum (FCS) and 1% (v/v) Penicillin-Streptomycin (10.000 U/ml, Gibco).

Techniques: Concentration Assay, Isolation, Quantitative RT-PCR, Expressing

Journal: bioRxiv

Article Title: Interferon-α subtype treatment induces the repression of SRSF1 in HIV-1 target cells and affects HIV-1 post integration steps

doi: 10.1101/2021.06.11.448031

Figure Lengend Snippet: a) – h) SRSF1 levels in HIV-1 host cells are repressed upon IFN treatment. Differentiated THP-1 macrophages and Jurkat T-cells were treated with the indicated IFN subtype over a period of 48 h at a concentration of 10 ng/ml before cells were harvested and RNA was isolated. Relative mRNA expression levels of ISG15 and SRSF1 were measured via RT-qPCR. ISG15 mRNA levels were measured after 4 h for a) THP-1 cells and d) Jurkat cells. SRSF1 mRNA levels were measured at the indicated time points in THP-1 cells after treatment with b) IFNα2 and c) IFNα14 and in Jurkat cells after treatment with e) IFNα2 and f) IFNα14. ACTB was used as loading control. Unpaired t-tests were calculated to determine whether the difference between the group of samples reached the level of statistical significance (* p<0.05, ** p<0.01 and *** p<0.001). g) Differentiated THP-1 macrophages were treated with IFNα2 or IFNα14 for the indicated amount of time at a concentration of 10 ng/ml before cells were harvested. Proteins were separated by SDS-PAGE, blotted and analyzed with an antibody specific to SRSF1. GAPDH was used as loading control. h) Comparison of time-dependent SRSF1-repression after IFNα2-treatment on mRNA and protein level. i) – j) Repression of SRSF1 mRNA levels in primary human macrophages . Monocyte-derived macrophages (MDMs) were treated with IFNα14 over a period of 48 h at a concentration of 10 ng/ml before cells were harvested and RNA isolated. Relative mRNA expression levels of ISG15 and SRSF1 were measured via RT-qPCR. i) ISG15 mRNA levels were measured 4 h post treatment. j) SRSF1 mRNA levels were measured at the indicated time points. GAPDH was used as loading control. Unpaired t-tests were calculated to determine whether the difference between the group of samples reached the level of statistical significance (* p<0.05, ** p<0.01 and *** p<0.001). Time points 24 h and 48 h only include two biological replicates. k) – l) SRSF1 repression in THP-1 cells is type I IFN specific . Differentiated THP-1 cells were treated with IFNγ over a period of 48 h at a concentration of 10 ng/ml before cells were harvested and RNA was isolated. Relative mRNA expression levels of IRF1 and SRSF1 were measured via RT-qPCR. k) IRF1 mRNA levels were measured after 4 h. l) SRSF1 mRNA levels were measured at the indicated time points. GAPDH was used as loading control. Unpaired t-tests were calculated to determine whether the difference between the group of samples reached the level of statistical significance (* p<0.05, ** p<0.01 and *** p<0.001).

Article Snippet: Jurkat, CEM-SS, CEM-T4 and THP-1 cells were maintained in Roswell Park Memorial Institute (RPMI) 1640 medium (Gibco) supplemented with 10% (Jurkat, CEM-SS and CEM-T4) or 20% (THP-1) (v/v) heat-inactivated fetal calf serum (FCS) and 1% (v/v) Penicillin-Streptomycin (10.000 U/ml, Gibco).

Techniques: Concentration Assay, Isolation, Expressing, Quantitative RT-PCR, SDS Page, Derivative Assay

Journal: bioRxiv

Article Title: Interferon-α subtype treatment induces the repression of SRSF1 in HIV-1 target cells and affects HIV-1 post integration steps

doi: 10.1101/2021.06.11.448031

Figure Lengend Snippet: a) – b) Changes in newly transcribed mRNAs upon treatment with IFNα14. Differentiated THP-1 macrophages were treated with IFNα14 for 8 h or 24 h before labeling with 4sU for 30 min at a concentration of 500 µM. Cells were then harvested and total RNA isolated. Freshly transcribed RNA was labeled, purified and separated as described elsewhere . Relative mRNA expression levels of a) ISG15 and b) SRSF1 were measured via RT-qPCR. 2 biological replicates were pooled for qPCR analysis. GAPDH was used as loading control. c) – d) HIV-1 counteracts repression of SRSF1 upon IFN-treatment . Differentiated THP-1 macrophages were infected with the R5-tropic NL4-3 (AD8) at an MOI of 1. 16 h post infection, cells were treated with the indicated IFN subtype over a period of 48 h at a concentration of 10 ng/ml. Cells were then harvested, RNA isolated and subjected to RT-qPCR. Relative mRNA expression levels of SRSF1 in THP-1 cells after treatment with c) IFNα2 or d) IFN14. ACTB was used as loading control. Unpaired t-tests were calculated to determine whether the difference between the group of samples reached the level of statistical significance (* p<0.05, ** p<0.01 and *** p<0.001). e) – f) TLR7/8 agonist R848 induces repression of SRSF1 mRNA expression levels . Differentiated THP-1 macrophages were infected with the R5-tropic NL4-3 (AD8) at an MOI of 1 or mock infected. 16 h post infection, cells were treated with Resiquimod (R848) at a concentration of 30 µM for 8 h or 24 h respectively. Cells were then harvested, RNA isolated and subjected to RT-qPCR. e) Relative mRNA expression levels of SRSF1 after treatment with R848. f) Total viral mRNA levels were measured via RT-qPCR using a primer pair amplifying a sequence in Exon7. GAPDH was used as loading control. Unpaired t-tests were calculated to determine whether the difference between the group of samples reached the level of statistical significance (* p<0.05, ** p<0.01 and *** p<0.001).

Article Snippet: Jurkat, CEM-SS, CEM-T4 and THP-1 cells were maintained in Roswell Park Memorial Institute (RPMI) 1640 medium (Gibco) supplemented with 10% (Jurkat, CEM-SS and CEM-T4) or 20% (THP-1) (v/v) heat-inactivated fetal calf serum (FCS) and 1% (v/v) Penicillin-Streptomycin (10.000 U/ml, Gibco).

Techniques: Labeling, Concentration Assay, Isolation, Purification, Expressing, Quantitative RT-PCR, Infection, Sequencing

Journal: The Journal of Clinical Investigation

Article Title: A DOCK8-WIP-WASp complex links T cell receptors to the actin cytoskeleton

doi: 10.1172/JCI85774

Figure Lengend Snippet: (A and B) Representative images of splenic CD4+ T cells from WT, Dock8–/–, and Dock8pri/pri mice plated on anti-CD3– and ICAM-1–coated glass chambers (A) or a superantigen-treated endothelial cell monolayer (B) and stained for F-actin (phalloidin, pseudocolored green) and pCasL (pseudocolored red). Orange indicates merging. The extracellular striated pattern observed in B (phalloidin panel on the left) is due to the F-actin fluorescence contributed by the endothelial cell cytoskeleton. Original magnification, ×100. Scale bar: 5 μm. (C–G) Quantitative analysis of actin foci (C and E), pCasL intensity (D and F), and synaptic area (G) in T cells. Each data point represents a value obtained for a single cell. Similar results (A–G) were obtained in 2 independent experiments. Error bars in C–G represent the mean ± SEM. ***P < 0.001, **P < 0.01, and *P < 0.05, by Student’s t test.

Article Snippet: Briefly, in vitro polarized WT, Dock8 –/– , and Dock8 pri/pri Th1 cells (5 × 10 5 in 100 μl) were suspended in Dulbecco’s PBS containing 0.1% (w/v) BSA and 20 mM HEPES, pH 7.4, and were drawn across immobilized murine E-selectin-Fc (20 μg/ml), ICAM-1-Fc (20 μg/ml), and VCAM-1-Fc (5 μg/ml) chimeric proteins (R&D Systems) initially at a shear stress level of 1.0 dynes/cm 2 , and subsequently, the flow rate was reduced at 3 minutes to 0.8 dynes/cm 2 and finally to 0.5 dynes/cm 2 3 minutes later ( 59 ).

Techniques: Staining, Fluorescence

Journal: The Journal of Clinical Investigation

Article Title: A DOCK8-WIP-WASp complex links T cell receptors to the actin cytoskeleton

doi: 10.1172/JCI85774

Figure Lengend Snippet: (A) FACS analysis of CD44, PSGL-1, LFA-1, and VLA-4 surface expression by CD4+ Th1 cells from Dock8–/–, Dock8pri/pri, and WT control mice. Results are representative of 3 independent experiments using 2 mice each per strain. (B) Accumulation of CD4+ Th1 cells over E-selectin–, ICAM-1 plus 250 ng/ml SDF-1α–, and VCAM-1–coated surfaces under a range of shear stress conditions. (C) Representative photomicrographs and pooled results of spreading of CD4+ Th1 cells over ICAM-1. Arrows indicate fully spread cells scored with 2 points, and solid triangles indicate partially spread cells scored with 1 point. Round, unspread cells were scored with no points to calculate the mean spreading score of cells adherent to ICAM-1 in 5 fields of view at 0.5 dyn/cm2. Original magnification, ×20. Scale bar: 50 μm. Duplicate coverslips were assessed in 3 independent studies. (D) Th1 cell transmigration across a TNF-α–treated MHEC monolayer under 0.8 dyn/cm2 shear stress normalized to the percentage of WT Th1 cells. Pooled results in B–D represent 3 independent experiments each using CD4+ Th1 cells from 1 mouse per strain. ***P < 0.001, **P < 0.01, and *P <0.05, by Student’s t test.

Article Snippet: Briefly, in vitro polarized WT, Dock8 –/– , and Dock8 pri/pri Th1 cells (5 × 10 5 in 100 μl) were suspended in Dulbecco’s PBS containing 0.1% (w/v) BSA and 20 mM HEPES, pH 7.4, and were drawn across immobilized murine E-selectin-Fc (20 μg/ml), ICAM-1-Fc (20 μg/ml), and VCAM-1-Fc (5 μg/ml) chimeric proteins (R&D Systems) initially at a shear stress level of 1.0 dynes/cm 2 , and subsequently, the flow rate was reduced at 3 minutes to 0.8 dynes/cm 2 and finally to 0.5 dynes/cm 2 3 minutes later ( 59 ).

Techniques: Expressing, Shear, Transmigration Assay