Journal: PLoS ONE

Article Title: STAT6 knockdown using multiple siRNA sequences inhibits proliferation and induces apoptosis of human colorectal and breast cancer cell lines

doi: 10.1371/journal.pone.0207558

Figure Lengend Snippet: (A) Measure of STAT6 mRNA level in HT-29 cells 24 hours post-transfection. The graph represents the mean ± SEM of multiple independent experiments (n) obtained by real-time PCR. Results were analysed by ΔΔCt method for relative quantifications. The fold change is represented by the Y axis, and values are normalized to control cells. (B) STAT6 protein level analysis at 2, 5 and 7 days post-transfection in HT-29 cells. The graph represents the mean of the percentage of STAT6 positive cells ± SEM of multiple independent experiments (n) obtained by flow cytometry. (C) Measure of STAT6 mRNA level in ZR-75-1 cells 3 days post-transfection. The graph represents the mean ± SEM of multiple independent experiments (n) obtained by real-time PCR. Results were analysed by ΔΔCt method for relative quantifications. The fold change is represented by the Y axis, and values are normalized to control cells. (D) STAT6 protein level analysis after 4 and 7 days of transfection in ZR-75-1 cells. The graph represents the mean of the percentage of STAT6 positive cells ± SEM of multiple independent experiments (n) obtained by flow cytometry. E) Representative histograms (Control: back; NT: grey; STAT6.1: red; STAT6.4: blue) of STAT6 protein analysis at 7 days post-transfection by flow cytometry in HT-29 cells (top) and ZR-75-1 (bottom) cells. STAT6 siRNA sequences and non-targeting siRNA were used at 100 nM as the final concentration. Control cells were non-transfected cells and STAT6 siRNA sequences 1 and 4 and non-targeting siRNA are denoted as STAT6.1, STAT6.4 and NT, respectively. The number of independent experiments (n) is set out in the Fig.

Article Snippet: Cells were washed twice with PBS/0.5%BSA (Bovine Serum Albumin) and stained with anti-STAT6 APC conjugated antibody (Miltenyi Biotec, 130-104-030) (20 μl/10 6 cells) for 30 min in the dark at 4°C.

Techniques: Transfection, Real-time Polymerase Chain Reaction, Control, Flow Cytometry, Concentration Assay

Journal: PLoS ONE

Article Title: STAT6 knockdown using multiple siRNA sequences inhibits proliferation and induces apoptosis of human colorectal and breast cancer cell lines

doi: 10.1371/journal.pone.0207558

Figure Lengend Snippet: (A and B) Number of live HT-29 cells measured at 5 and 7 days post-transfection, respectively. The graphs represent the mean ± SEM of multiple independent experiments (n). (C) The graph illustrates how HT-29 cells grew over time and represents the mean ± SEM of the independent experiments (n) shown in A and B. (D and E) Number of live ZR-75-1 cells measured at 4 and 7 days post-transfection, respectively. The graphs represent the mean ± SEM of multiple independent experiments (n). (F) The graph illustrates how ZR-75-1 cells grew over time and represents the mean ± SEM of the multiple independent experiments (n) shown in D and E. The number of live cells was calculated as detailed in the material and methods using NucleoCounter NC-100. The percentage of reduction of the number of live cells was calculated by comparison between the mean of NT vs . the mean of the transfection with STAT6 siRNA sequences. STAT6 and non-targeting (NT) siRNA sequences were used at 100 nM as the final concentration. Non-transfected cells served as negative control and STAT6 siRNA sequences 1 and 4 and non-targeting siRNA are denoted as STAT6.1, STAT6.4 and NT, respectively. The number of independent experiments (n) is set out in the Fig.

Article Snippet: Cells were washed twice with PBS/0.5%BSA (Bovine Serum Albumin) and stained with anti-STAT6 APC conjugated antibody (Miltenyi Biotec, 130-104-030) (20 μl/10 6 cells) for 30 min in the dark at 4°C.

Techniques: Transfection, Comparison, Concentration Assay, Negative Control

Journal: PLoS ONE

Article Title: STAT6 knockdown using multiple siRNA sequences inhibits proliferation and induces apoptosis of human colorectal and breast cancer cell lines

doi: 10.1371/journal.pone.0207558

Figure Lengend Snippet: (A) Apoptosis analysis in HT-29 cells. (B) Apoptosis analysis in ZR-75-1 cells. In both cases, the graphs represent from left to right: early (Annexin V + /PI - ), late (Annexin V + /PI + ) and total (Annexin V + ) apoptosis. The graphs represent the mean ± SEM of multiple independent experiments (n) obtained by flow cytometry. (C) Representative flow cytometry plots in HT-29 cells. (D) Representative flow cytometry plots in ZR-75-1 cells. The X axes represent Annexin V and the Y axes represent PI fluorescence intensity. Quadrants were set according to cells independently stained with Annexin V or PI. Apoptosis was studied at 7 days post-transfection in both cell lines. Data were analysed with Flowjo Software. STAT6 siRNA sequences and a non-targeting siRNA sequence were used at 100 nM as the final concentration. Non-transfected cells served as control cells and STAT6 siRNA sequences 1 and 4 and non-targeting siRNA are denoted as STAT6.1, STAT6.4 and NT, respectively. The number of independent experiments (n) is set out in the Fig.

Article Snippet: Cells were washed twice with PBS/0.5%BSA (Bovine Serum Albumin) and stained with anti-STAT6 APC conjugated antibody (Miltenyi Biotec, 130-104-030) (20 μl/10 6 cells) for 30 min in the dark at 4°C.

Techniques: Flow Cytometry, Fluorescence, Staining, Transfection, Software, Sequencing, Concentration Assay, Control

Journal: PLoS ONE

Article Title: STAT6 knockdown using multiple siRNA sequences inhibits proliferation and induces apoptosis of human colorectal and breast cancer cell lines

doi: 10.1371/journal.pone.0207558

Figure Lengend Snippet: STAT6 siRNA transfection was carried out at day 1 of cell culture with (A) STAT6.1 and (B) STAT6.4 at 100 nM. A second transfection was carried out in both cases with STAT6.1 and STAT6.4 at the same concentration 7 days after the first transfection. The graphs represent the number of live cells over time measured at day 7 and 14 days post-first transfection counted using NucleoCounter NC-100 as detailed in the material and methods section. The mean ± SEM of 3 independent experiments is represented in each graph. Control cells were non-transfected cells and STAT6 siRNA sequences 1 and 4 and non-targeting siRNA are denoted as STAT6.1, STAT6.4 and NT, respectively. The percentage of reduction of the number of live cells was calculated by comparison between the mean of NT vs . the mean of the individual transfection with STAT6 siRNA sequences, and sequential transfection with NT (NT+NT) vs . sequential transfection with STAT6.1 and STAT6.4.

Article Snippet: Cells were washed twice with PBS/0.5%BSA (Bovine Serum Albumin) and stained with anti-STAT6 APC conjugated antibody (Miltenyi Biotec, 130-104-030) (20 μl/10 6 cells) for 30 min in the dark at 4°C.

Techniques: Transfection, Cell Culture, Concentration Assay, Control, Comparison

Journal: PLoS ONE

Article Title: STAT6 knockdown using multiple siRNA sequences inhibits proliferation and induces apoptosis of human colorectal and breast cancer cell lines

doi: 10.1371/journal.pone.0207558

Figure Lengend Snippet: (A) STAT6 expression at protein level in HT-29 cells at day 2 and 7 post-transfection measured by flow cytometry. The graph represents the mean ± SEM of multiple independent experiments (n). Data was analysed using Flowjo Software. The percentage of STAT6 positive cells is represented on the Y axis. (B) Representative histograms (Control: back; NT: grey; STAT6.1: red; STAT6.4: blue) of STAT6 protein analysis at 7 days post-transfection by flow cytometry in HT-29 cells. (C) Number of live HT-29 cells measured at day 7 post-transfection by NucleoCounter NC100. The graph represents the mean ± SEM of multiple independent experiments (n). (D) The graph illustrates how HT-29 cells grew over time and represents the mean ± SEM of the independent number of experiments shown in C. (E, F and G) The graphs show early (E) (Annexin V + /PI - ), late (F) (Annexin V + /PI + ) and total (G) (Annexin V + ) apoptosis, and represent the mean ± SEM of multiple independent experiments (n) obtained by flow cytometry. Apoptosis was studied at 7 days post-transfection. Data were analysed with Flowjo Software. STAT6 siRNA sequences and a non-targeting siRNA sequence were used at 100 nM as the final concentration. Non-transfected cells served as control cells and STAT6 siRNA sequences 1 and 4 and non-targeting siRNA are denoted as STAT6.1, STAT6.4 and NT, respectively. The number of independent experiments (n) is set out in the Fig.

Article Snippet: Cells were washed twice with PBS/0.5%BSA (Bovine Serum Albumin) and stained with anti-STAT6 APC conjugated antibody (Miltenyi Biotec, 130-104-030) (20 μl/10 6 cells) for 30 min in the dark at 4°C.

Techniques: Expressing, Transfection, Flow Cytometry, Software, Control, Sequencing, Concentration Assay

Journal: Molecules : A Journal of Synthetic Chemistry and Natural Product Chemistry

Article Title: The Protective Effects of Astaxanthin on the OVA-Induced Asthma Mice Model

doi: 10.3390/molecules22112019

Figure Lengend Snippet: Effects on total cells and Th2-mediated cytokines of AST in BALF. C57BL/6 mice were sensitized and challenged with OVA and AST. In these mice, BALF were collected 24 h after the last challenge. ( A ) The total BALF cells were counted by trypan blue staining; The levels of ( B ) IL-4; ( C ) IL-5; and ( D ) IFN-γ in BALF were measured by ELISA. The data represent four mice per group. ‘−’ indicates the mean of four mice. ## p < 0.01 and ### p < 0.001 control vs. OVA. * p < 0.05, ** p < 0.01, and *** p < 0.001 OVA vs. AST. Group numbers (1 = control, 2 = OVA, 3 = AST 5 mg/kg, 4 = AST 10 mg/kg, 5 = AST 50 mg/kg).

Article Snippet: Purified rat anti-mouse IL-4, purified rat anti-mouse IL-5, purified rat anti-mouse IFN-γ, biotin rat anti-mouse IL-4, biotin rat anti-mouse IL-5, biotin rat anti-mouse IFN-γ purified rat anti-mouse IgE (R35-72), purified rat anti-mouse IgG1 (A85-3), purified rat anti-mouse IgG2a (R11-89), biotin rat anti-mouse IgE (R35-118), biotin rat anti-mouse IgG1 (A85-1), and biotin rat anti-mouse IgG2a (19-5) were purchased from BD Biosciences (San Diego, CA, USA).

Techniques: Staining, Enzyme-linked Immunosorbent Assay

Journal: PLoS ONE

Article Title: ‘Multi-Epitope-Targeted’ Immune-Specific Therapy for a Multiple Sclerosis-Like Disease via Engineered Multi-Epitope Protein Is Superior to Peptides

doi: 10.1371/journal.pone.0027860

Figure Lengend Snippet: (C57Bl.FoxP3GFPxSJL/J)F1 mice were immunized s.c. with PLP139-151 (100 µg) in CFA. On day 3, 5, and 7 post immunization, mice (n = 3/group) were injected i.v. with YMSPc (75 µg/mouse), PLP139-151 (100 µg/mouse), huPEP mix (total 140 µg/mouse), Y-DMP (75 µg/mouse), or with 0.5 µl PBS. The huPEP mix contained 1∶1 ratios of phPLP139-151, phPLP175-194, phMOG34-56, phMBP89-104, phMOBP15-36, phOSP55-80 and phOSP186-205 (20 µg each peptide), representing the major encephalitogenic epitopes of five human myelin proteins. A control treatment with non-relevant recombinant protein (Y-DMP) was also included to exclude the possibility that residual bacterial contaminants contributed to the efficacy of Y-MSPc. (Y-DMP is a diabetes mellitus-related recombinant artificial protein encompassing selected multiple epitopes of several antigens related to diabetes; The Y-DMP was constructed, expressed and purified similar to Y-MSPc). Three days later (10 days after immunization), draining LN cells from each treatment group were pooled and analyzed for: ( A ), Ex vivo recall proliferative response to PLP139-151 . The LN cells from each treatment group were cultured for 72 h in microtiter wells in triplicates (0.5×10 6 /well) in the absence or presence of PLP139-151 (5 µg/ml ). [H 3 ]Thymidine was added for the last 18 h. ( B–G ), Secretion of pro-inflammatory (B–D) and anti-inflammatory (E–G) cytokines . LN cells from each treatment group were cultured (5×10 6 /ml ) in triplicate cultures in the absence or presence of PLP139-151 (5 µg/ml ) for 48 h, and the supernatants were collected for measuring the secreted INF-γ, IL-2, IL-17, TGF-ß,, IL-4 and IL-10 cytokines by ELISA. For each treatment group, the net values are presented (after subtracting the values obtained in control cultures without PLP peptide). ( H ), Ex-vivo recall proliferative response to PLP139-151 in the presence of exogenous IL-2 . LN cells from the different treatment groups were cultured in microtiter wells in triplicates without or with PLP139-151 (5 µg/ml) [as in (A)] and in the absence or the presence of exogenous rIL-2 (3.5 U/ml) for 72 h, with [H 3 ]Thymidine added for the last 18 h. Calculating % reactivation: For each treatment group, the S.I. calculated for the recall proliferative response in the presence of exogenous IL-2 was divided by the S.I. calculated in the absence of exogenous IL-2. ( I ), Flow cytometry of regulatory T-cells induced following systemic administration of YMSPc, PLP139-151, huPEP mix, Y-DMP, or PBS . Draining LN cells from different treatment groups were co-stained with anti-CD4-APC and anti-CTLA-4-PE. The percentage of the FoxP3 (a) or CTLA-4 (b) expressing cells on gated CD4+ cells is shown. The FACS histograms are from one representative experiment, and the panels at the right end of Ia and Ib are the mean values +/−SD from three independent experiments. ( J ), Ex-vivo recall proliferative response to PLP139-151 by primed LN cells in the presence of neutralizing anti- TGF-ß , or anti-CTLA-4 antibodies. Draining LN cells from the different treatment groups were cultured without or with PLP139-151 (5 µg/ml) [as in (A)] and without or with added neutralizing antibodies anti- TGF-ß, or anti-CTLA-4 (10 µg/ml) , or respective isotype control antibodies. Calculating % reactivation: The S.I. calculated for the recall proliferative response in the presence of neutralizing antibodies or the respective isotype control antibodies was divided by the S.I. of the recall response in the absence of neutralizing or isotype control antibodies, respectively. ( K ), Percent apoptotic cells in CD4+ T cell population of different treatment groups . Apoptosis was determined by FACS analysis of Annexin V/7-AAD staining on gated CD4+ cells. The data presented in A, H, I (the panels a and b at the right end), J, and K, are the mean values from three independent experiments. Data presented in B–G are the mean +/−SD of triplicate cultures of pooled LN cells from one experiment representative of three independent experiments showing a similar pattern. The significance of the effect of Y-MSPc treatment compared to PBS control treatment was p = 0.0003 in A; p = 0,001 in B: p = 0.0001 in C: p = 0.02 in D: p = 0.0002 in E: p<0.00001 in H; p = 0.03 in Ia: p = 0.04 in Ib; and p = 0.05 in K. The significance of the effect of Y-MSPc treatment compared to huPEP mix treatment: *, p <0.05; **, p <0.005; ***, p <0.0005 (two tailed Student's t-test).

Article Snippet: The biotinylated antibodies used were rat anti-mouse IL-4 (18042D), rat antimouse IL-2 (18172D), rat anti-mouse IL-10 (18152D) and rat anti-mouse IFN-γ (18112D; all from PharMingen).

Techniques: Injection, Recombinant, Construct, Purification, Ex Vivo, Cell Culture, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Staining, Expressing, Two Tailed Test

Journal: International Journal of Molecular Sciences

Article Title: Schwann Cell Transplantation Subdues the Pro-Inflammatory Innate Immune Cell Response after Spinal Cord Injury

doi: 10.3390/ijms19092550

Figure Lengend Snippet: Cytokine profile of CD11b + cells is unaltered by SC implants after SCI. Representative images of flow cytometry analysis and pie charts of CD11b population dynamics at 14 days post-injury (7 days post-transplantation) show that CD11b immune cells have unaltered expression of pro- (TNF-α, IL-1β) and anti-inflammatory (IL-4, IL-10) cytokines comparatively among SCI controls ( A – E ) and SC-transplanted groups ( F – J ). Results are expressed as mean ± SD. For panels ( A – D , F – I ) the blue dots represent the cell population that were CD11b − or did not express the selected cytokines, whereas the green dots represent the CD11b + population that expressed CD11b and the cytokine of interest (top right quadrant).

Article Snippet: Antibodies used were rabbit anti-Iba1 polyclonal antibody (catalog # 019-19741; FUJIFILM Wako Chemicals U.S.A Corporation, Richmond, VA, USA), chicken polyclonal to Iba1 (catalog # ab139590; Abcam, Cambridge, MA, USA), chicken anti-GFP antibody (catalog # AB16901; EMD Millipore-Sigma, St. Louis, MO, USA), mouse CD68/clone CD68 (catalog # MCA341R; BIO-RAD, Hercules, CA, USA), chicken anti-green fluorescent protein (catalog # AB16901; Millipore-Sigma), rabbit Arginase-1 (catalog # GTX109242; Gen eTex), rabbit anti-CD163 antibody (catalog # ab87099; Abcam), rabbit anti-MRC1 antibody (catalog # HPA045134; Millipore-Sigma), rabbit anti-COX2/Cyclooxygenase 2 antibody (catalog # ab15191; Abcam), rabbit polyclonal Neurofilament NF-M, (catalog # RPCA-NF-M; Encore Biotechnology, Gainesville, FL, USA), mouse anti-rat Cd11b:Pacific Blue (catalog # MCA275PB; BIO-RAD), human/mouse Arginase-1/ARG1 APC-conjugated (catalog# IC5868A; R&D systems, Minneapolis, MN, USA), NOS2 Alexa Fluoro 488 (catalog# 610331; BD Bioscience, San Jose, CA, USA), anti-rat CD68:RPE (catalog # MCA341PE; BIO-RAD), CD38 monoclonal antibody (14.27), eFluor 660, (catalog # 50-0380-80; ThermoFisher Scientific, Waltham, MA, USA), anti-rat CD163:FITC (catalog # MCA342F; BIO-RAD), PE anti-mouse/rat TNF alpha (catalog # 506104; Biolegend, San Diego, CA, USA), IL-1β antibody (E7-2-hILβ) PE (catalog # sc-32294 PE; Santa Cruz, Dallas, TX, USA), BD Pharmingen™ PE mouse anti-rat IL-4 (catalog # 555082; BD Bioscience), PE mouse anti-rat IL-10 Clone A5-4 (catalog # 555088; BD Bioscience), IL12, PE rat anti-mouse (catalog # 562038; BD Bioscience), PE anti-mouse CD206 (MMR), (catalog # 141705, Biolegend), and purified mouse anti-rat CD32 clone D34-485 (catalog # 55027; BD Pharmingen, San Jose, CA, USA).

Techniques: Flow Cytometry, Transplantation Assay, Expressing

Journal: bioRxiv

Article Title: Development of attenuated coxsackievirus B3 vectored intranasal pre-emptive pan-coronavirus vaccine

doi: 10.1101/2023.11.22.568225

Figure Lengend Snippet: (A) Genome Schematic diagram of attenuated rCVB3 delivery vector based intranasal pre-emptive pan-coronavirus vaccines construction. (B) Cartoon models of rCVB3-EPI and rCVB3-RBD-trimer. (C) Western Blot analysis of the intranasal pre-emptive pan-coronavirus vaccines. Collection and analysis the protein in serial passages of the recombinant viruses (1-6 passages) from Vero culture cells. (D) Plaque morphology of viruses on Vero cells. For each recombinant virus, we randomly measured 10 Plaque diameter for statistical analysis. (E) Experimental schema of vaccination and sampling timeline. Nasal washes and serum were collected on day 28, n=6 /group. (F) SARS-CoV-2 S1 specific serum IgG antibodies levels on day 28, group rCVB3-RBD-trimer and rCVB3-EPI SARS-CoV-2 S1 specific serum IgG antibodies levels on day 28 compare to control group. IgG levels measured by ELISA and presented as OD450 nm values, n=4 / group. (G) The neutralizing activity of serum samples isolated from vaccinated mice (n =6 per group) against SARS-CoV-2 (Wuhan-Hu-1) pseudovirus. The vertical axis represents the pseudovirus inhibition rate of each immune serum at a serum dilution of 1:45. (H) SARS-CoV-2 S1-specific secretory IgA (sIgA) response in nasal lavage fluid of immunized mice was assessed on day 14 post booster immunization. n=6/group. (L-J) Percentages of IFN-γ + CD3 + and CD4 + splenic T cells by FCM analysis on day 14 after the last immunization. The percentage of IFN-γ + CD4 + Th cells among from splenocytes was measured under non-stimulation (NON), PMA stimulation (PMA), and SARS-CoV-2 peptide pool (PP) stimulation, respectively, for both experimental group and vaccine group (n=3/ group). (K-L) The percentage of IL-4 + Th cells and IFN-γ + Th cells among CD4 + T lymphocytes was determined under SARS-CoV-2 PP stimulation, respectively. Significance analysis was performed using a two-tailed t-test for unpaired samples. One-way analysis of variance (ANOVA) was also employed for significance analysis, data are represented as mean ± standard error of the mean (SEM) *, P<0.05; **, P<0.01; ***, P<0.001.

Article Snippet: Next, cells were fixed and permeabilized using the Fixation/Permeabilization Kit (554714, BD Pharmingen, USA) to enable intracellular staining with PE Rat Anti-Mouse IFN-γ (554412, BD Pharmingen, USA) and PE-Cy™7 Rat Anti-Mouse IL-4 (560699, BD Pharmingen, USA).

Techniques: Plasmid Preparation, Vaccines, Western Blot, Recombinant, Virus, Sampling, Enzyme-linked Immunosorbent Assay, Activity Assay, Isolation, Inhibition, Two Tailed Test