Journal: International Journal of Oncology

Article Title: Sulforaphane and TRAIL induce a synergistic elimination of advanced prostate cancer stem-like cells

doi: 10.3892/ijo.2014.2335

Figure Lengend Snippet: Sulforaphane, but not TRAIL, strongly inhibits stem cell-associated signaling and differentiation and their combination enhances these effects. (A) PC3 cells were treated as described in . Twenty-four hours later, the proteins were isolated and a human pluripotent stem cell array was performed. The binding of proteins to antibodies spotted in duplicate on the membranes was detected using biotinylated secondary antibodies, streptavidin-HRP and chemiluminescence. After normalization to reference spots (positive control A1, A4 and F1; PBS-negative control E4), the pixel density was quantified using ImageJ software. (B) Proteins were also harvested, and a western blot analysis was performed to detect the expression of CXCR4, Jagged1, Notch 1, SOX2 and Nanog. The detection of β-actin served as a control for equal conditions. (C) For ALDH1 evaluation, PC3 cells were treated as described in ; the activity of ALDH1 was determined by a substrate assay and the turnover was analyzed by flow cytometry. The data are presented as the percentage of ALDH1-positive cells ± SD ( * p<0.05, ** p<0.01). (D) DU145 and PC3 cells were treated as described in . Twenty-four hours later, the medium was exchanged for NH AdipoDiff medium to induce adipocyte differentiation. After 14 days, the cells were stained with Oil Red O to detect the fat droplets in adipocytes. Representative images are shown, and the arrows indicate the red fat droplets. The scale bar indicates 10 μ m.

Article Snippet: Biotinylated goat anti-rabbit or anti-mouse IgG (Vector) served as the secondary Abs.

Techniques: Isolation, Binding Assay, Positive Control, Negative Control, Software, Western Blot, Expressing, Activity Assay, Flow Cytometry, Staining

Journal: bioRxiv

Article Title: Interferon-armed RBD dimer enhances the immunogenicity of RBD for sterilizing immunity against SARS-CoV-2

doi: 10.1101/2021.05.12.443228

Figure Lengend Snippet: (a) Schematic illustration of the I-R-F fusion protein. The structural elements contain a mouse IFNα4a (IFNα), receptor-binding domain (RBD), immunoglobulin Fc fragment (Fc). (b) Size exclusion chromatography of I-R-F was performed on a Superdex200 Increase Column. The ultraviolet absorption at 280mm is shown. The insert photograph presents the SDS-PAGE of the eluted protein samples. (c) The real-time binding kinetics between I-R-F and hACE2 was determined by the BIAcore T100 system. (d) Evaluation of the binding ability of monoclonal antibodies to I-R-F by ELISA. To determine the known RBD epitopes in I-F-R, ELISA Plate was coated with I-R-F. Then, 2-fold serially diluted monoclonal antibodies were detected. The anti-Pres1 XY007 monoclonal antibody was given as the control. The absorbance was read at 450-630. (e)The bioactivity of IFNα contained in I-R-F was measured by an anti-viral infection biological assay. (f) Binding of mouse IFNα-RBD-Fc to FcγR on RAW264.7 cells. Cells were incubated with serial dilutions of WT IgG fusion protein, mutant IgG fusion protein, or WT IgG fusion protein with anti-FcγR, followed by a fluorophore-conjugated anti-human IgG secondary antibody. Flow cytometry measured MFI (n=3). (g) BALB/c mice (n=8/group) were immunized intramuscularly with 10μg of I-R-F or equimolar RBD protein, mixed with alum adjuvant, respectively. Mice were re-immunized with the same dose of vaccine on day 14 post the first shot. PBS containing alum adjuvant was chosen as a negative control. Sera were collected on days 7, 14, 21, 28, 35, and 42 after initial immunization, and the IgG levels were measured by ELISA. (h) Groups of BALB/c mice (n=7/groups) were intramuscularly immunized twice on day 0 and day 14 with 10μg of alum-adjuvanted I-R-F, or equimolar RBD or alum alone as a control. Serum was collected at indicated time points, and the kinetics of the RBD-specific IgG antibody titers were determined by ELISA. The dashed line indicates the limit of detection. The data shown are presented as mean ± SEM. P-values were determined by one-way ANOVA with multiple comparison tests. ns (not significant), *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

Article Snippet: To detect the Ig subclasses, goat anti-mouse IgG1 (1:5000, Proteintech), goat anti-mouse IgG2a (1:5000, Proteintech) were added.

Techniques: Binding Assay, Size-exclusion Chromatography, SDS Page, Bioprocessing, Enzyme-linked Immunosorbent Assay, Control, Infection, Incubation, Mutagenesis, Flow Cytometry, Adjuvant, Negative Control, Comparison

Journal: bioRxiv

Article Title: Interferon-armed RBD dimer enhances the immunogenicity of RBD for sterilizing immunity against SARS-CoV-2

doi: 10.1101/2021.05.12.443228

Figure Lengend Snippet: (a). BALB/c mice (n=6/group) were i.m vaccinated with 10μg of I-P-F or equimolar of RBD, RBD-dimer, and R-F protein or PBS control with a signal vaccination regimen. The levels of RBD-specific IgG in serum on day 28 after immunization were determined by ELISA (b) BALB/c Mice (n=6/group) were intramuscularly vaccinated with 1μg of I-R-F or equimolar R-F plus IFNα protein without adjuvant and boosted with the same dose at a 14-day interval. Serum samples were collected on day 14 after the second immunization to evaluate the levels of RBD-specific IgG. (c) BALB/C mice (n=8/group) were immunized intramuscularly or subcutaneously with alum-adjuvanted 10μg of I-R-F and boosted on day 14 after initial immunization with equivalent dose. Serum samples were collected every week to determine the SARS-CoV-2-specific IgG antibody titers by ELISA. (d) BALB/C mice (n=8/group) were immunized intramuscularly with alum-adjuvanted 10μg of I-R-F by using a single dose (day0), two-dose (day0/14), and three-dose (day0/14/28) immunization procedures, respectively. Sera were collected on days 7, 14, 21, 28, 35, and 42 after the initial immunization and analyzed by ELISA to determine the IgG titer. (e) The neutralization antibody titers in serum described in on day 28 were determined by SARS-CoV-2 pseudovirus neutralization assay. The dashed line indicates the limit of detection. Data are shown as mean ± SEM. P-values were calculated by one-way ANOVA with multiple comparisons tests. ns (not significant), *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

Article Snippet: To detect the Ig subclasses, goat anti-mouse IgG1 (1:5000, Proteintech), goat anti-mouse IgG2a (1:5000, Proteintech) were added.

Techniques: Control, Enzyme-linked Immunosorbent Assay, Adjuvant, Neutralization

Journal: bioRxiv

Article Title: Interferon-armed RBD dimer enhances the immunogenicity of RBD for sterilizing immunity against SARS-CoV-2

doi: 10.1101/2021.05.12.443228

Figure Lengend Snippet: (a) The kinetics of the RBD-specific IgG antibody response. BALB/c mice (n=7/group) were immunized with a 1:10 series dilution of the vaccine, containing 10μg, 1μg, 0.1μg, 0.01μg, 0.001μg of I-R-F, respectively. Sera were collected to assess the levels of RBD-specific IgG. (b) The serum described in Fig 1e on day 28 was used to determine the neutralization activity with live SARS-CoV-2 by FRNT. The sera from different groups were serially diluted and mixed with 600 FFU of SARS-CoV-2, and the mixtures were then transferred to Vero E6 cells. The number of SARS-CoV-2 foci was counted in the next day. The FRNT 50 was defined as the reciprocal of serum dilution, which inhibits 50% of viral infection. (c) Comparison of the neutralizing antibody titers in sera between the I-R-F vaccinated mice (n=7) and the convalescent COVID-19 patients with different severity (n=6-13/group). The sera from 3 groups of COVID-19 convalescent patients and the I-P-F immunized mice as mentioned above in were serially diluted and mixed with live SARS-CoV-2. The mixtures were added to Vero E6 cells. The neutralizing titers were presented by FRNT 50 , which was the reciprocal of serum dilution neutralizing 50% of viral infection. (d) Mouse RBD-specific IgG induced by single immunization. Mice (n=8/group) were i.m. immunized once with alum-adjuvanted 10ug I-R-F (n=8), equimolar RBD protein, or alum alone, respectively. The levels of RBD-specific IgG in sera on days 7, 14, 21, 28, 35, and 42 after the first immunization were determined by ELISA. (e) Mouse RBD-specific IgG induced by vaccines without adjuvant. Mice (n=7 or 8) were vaccinated with no adjuvant 10μg I-R-F, equimolar RBD protein, or PBS control and boosted with the same dose 14-day after the initial immunization. Sera were collected every week after immunization and used to determine the IgG titers. (f) BALB/C mice (n=8/group) were immunized i.m. with alum-adjuvanted 0.1μg I-R-F, equimolar RBD protein, or alum alone, respectively, and boosted with the same dose at a 14-day interval. Sera were collected on days 7, 14, 21, 28, 35, and 42 after the initial immunization. RBD-specific IgG levels were analyzed by ELISA. The dashed line indicates the limit of detection. Data are shown as mean ± SEM. P-values were calculated by one-way ANOVA with multiple comparisons tests. ns (not significant), *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

Article Snippet: To detect the Ig subclasses, goat anti-mouse IgG1 (1:5000, Proteintech), goat anti-mouse IgG2a (1:5000, Proteintech) were added.

Techniques: Neutralization, Activity Assay, Infection, Comparison, Enzyme-linked Immunosorbent Assay, Vaccines, Adjuvant, Control

Journal: bioRxiv

Article Title: Interferon-armed RBD dimer enhances the immunogenicity of RBD for sterilizing immunity against SARS-CoV-2

doi: 10.1101/2021.05.12.443228

Figure Lengend Snippet: (a and b) C57BL/6 mice were immunized with 10 μg I-R-F, equimolar RBD, or PBS with a prime-boost vaccination regimen in a 14-day interval. Mice were sacrificed six months post the first vaccination, and splenocytes were collected. (a) ELISPOT assay was performed to determine RBD-specific B cells in the spleen. (b) The percentage of memory B cells in the spleen was analyzed. (c) BALB/c mice (n=8/group) were immunized with 10 μg of I-R-F, equimolar RBD, or PBS twice in a 14-day interval. PBS was performed as a negative control. Sera were collected on day 28 after the initial immunization and used to determine the IgG subclasses. (d-g) C57BL/6 mice were immunized with 10 μg I-R-F, equimolar RBD, or PBS with a prime-boost vaccination regimen in a 14-day interval. Mice were sacrificed, and splenocytes were collected 28 days post the first vaccination. ELISPOT assay was performed for IFN-γ (d) and IL-4 (e) secretion from mice splenocytes stimulated with RBD peptide pool. Splenocytes were incubated with an RBD peptide pool. (f) The percentages of IFN-γ + , IL-4 + , and TNF-α + CD4 + T cells were determined by ICCS. (g) The percentages of IFN-γ + , IL-2 + , and TNF-α + CD8 + T cells were determined by ICCS. The dashed line indicates the limit of detection. Data are presented as mean ± SEM. P-Values in (a-e) were calculated by one-way ANOVA with multiple comparisons tests. P-values in (f) and (g) were calculated by two-way ANOVA with multiple comparisons tests. ns (not significant), *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

Article Snippet: To detect the Ig subclasses, goat anti-mouse IgG1 (1:5000, Proteintech), goat anti-mouse IgG2a (1:5000, Proteintech) were added.

Techniques: Enzyme-linked Immunospot, Negative Control, Incubation

Journal: bioRxiv

Article Title: Interferon-armed RBD dimer enhances the immunogenicity of RBD for sterilizing immunity against SARS-CoV-2

doi: 10.1101/2021.05.12.443228

Figure Lengend Snippet: C57BL/6 mice (n=6/group) were vaccinated intramuscularly with 0.1μg of I-R-F or I-P-R-F using a two-dose immunization procedure. Sera were collected on days 7, 14, 21, 28, 35, 42 after the initial immunization. The RBD-specific IgG antibody titer was determined by ELISA. (b) The neutralization activity of vaccinated sera collected on day 28, as shown in (a), was evaluated using a pseudovirus neutralization assay. (c) BALB/C mice (n=8/group) were immunized i.m. with 1 μg of human IFNα-RBD-Fc (human I-R-F), human IFNα-Pan-RBD-Fc (human I-P-R-F), or equimolar RBD respectively, and boosted with the same dose at a 14-day interval. Sera were collected on days 0, 14, 21, 28, 35, and 42 after initial immunization and analyzed by ELISA. The dashed line indicates the limit of detection. Data are shown as mean ± SEM. P-values were calculated by one-way ANOVA with multiple comparisons tests in (a) and (c). P-values in (b) were analyzed with an unpaired t-test. P-values in (d-e) were determined using a paired t-test. ns (not significant), *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

Article Snippet: To detect the Ig subclasses, goat anti-mouse IgG1 (1:5000, Proteintech), goat anti-mouse IgG2a (1:5000, Proteintech) were added.

Techniques: Enzyme-linked Immunosorbent Assay, Neutralization, Activity Assay

Journal: bioRxiv

Article Title: Interferon-armed RBD dimer enhances the immunogenicity of RBD for sterilizing immunity against SARS-CoV-2

doi: 10.1101/2021.05.12.443228

Figure Lengend Snippet: Male and female rhesus macaques (n=8) were equally divided into four groups with a sex ratio of 1:1 and were immunized i.m. with a high dose (50ug) or a low dose (10ug) of I-P-R-F with or without alum as an adjuvant and boost with the same dose at a 14-day interval. The sera were collected every week are analyses for I-P-R-F immunogenicity and vaccine-induced neutralizing antibodies. (a) RBD-specific IgG titers in the high-dose group were determined by ELISA at indicated time points. (b) The dynamic IgG titers in sera from the low-dose vaccinated group were determined. (c-d) The kinetics of neutralization antibody titers in serum from I-P-R-F immunized animals were determined by SARS-CoV-2 pseudovirus and authentic virus neutralization assays. (e) Neutralization of SARA-CoV-2 pseudovirus by the anti-sera from I-P-R-F immunized rhesus macaques before the virus challenge. (f) Viral load in anal swabs of rhesus macaques challenged with live SARS-CoV-2. (g) Viral loads in the lungs. For each group of vaccinated macaques, 84 specimens from fourteen lung lobes were collected on day 7 post infection and subjected to determine the viral loads in the lungs. High dose/A: immunization with high does with adjuvant, High dose/A-: immunization with high does without adjuvant, Low dose/A: immunization with low does with adjuvant, Low dose/A-: immunization with low does without adjuvant. The dashed line indicates the limit of detection. Data are shown as mean ± SEM. P-values in (d) were analyzed with the unpaired t-test. P-values were calculated by one-way ANOVA with multiple comparisons tests in (e). ns (not significant), *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

Article Snippet: To detect the Ig subclasses, goat anti-mouse IgG1 (1:5000, Proteintech), goat anti-mouse IgG2a (1:5000, Proteintech) were added.

Techniques: Adjuvant, Immunopeptidomics, Enzyme-linked Immunosorbent Assay, Neutralization, Virus, Infection

Journal: bioRxiv

Article Title: Interferon-armed RBD dimer enhances the immunogenicity of RBD for sterilizing immunity against SARS-CoV-2

doi: 10.1101/2021.05.12.443228

Figure Lengend Snippet: Rhesus macaques (n=6/group) were administered twice with 10μg or 50μg of alum-adjuvanted I-P-R-F or alum alone as control via the intramuscular injection and challenged with SARS-CoV-2 intranasally on day 21 after the initial immunization. Sera were collected on days 0, 14, 21 (before virus infection), and day 28 (after infection) and subjected to antibody assays. (a) The SARS-CoV-2 specific IgG was determined by ELISA. (b) Neutralization antibody titers were analyzed. Body temperature (c) and body weight (d) were measured after the virus challenge. Viral loads in nose swabs (e) and tracheal brushes (f) following the virus challenge were determined by qPCR. (g) Viral load in various lung lobes of macaques challenged with SARS-CoV-2 on day 7 post infection. Dashed line in(a-b, e-g)indicates the limit of detection. Data are shown as mean ± SEM. P-values were calculated by one-way ANOVA with multiple comparisons tests. ns (not significant), *P<0.05, **P<0.01, ***P<0.001, ****P<0.000

Article Snippet: To detect the Ig subclasses, goat anti-mouse IgG1 (1:5000, Proteintech), goat anti-mouse IgG2a (1:5000, Proteintech) were added.

Techniques: Control, Injection, Virus, Infection, Enzyme-linked Immunosorbent Assay, Neutralization

Journal: Immunology and cell biology

Article Title: Mice lacking Axl and Mer tyrosine kinase receptors are susceptible to experimental autoimmune orchitis induction

doi: 10.1038/icb.2014.97

Figure Lengend Snippet: Lymphocyte infiltration. (a) Flow cytometry analysis. Testicular interstitial cells were isolated from control and immunized Axl−/−Mer−/− mice and gated for lymphocyte analyses. SSC, side scattering; FSC, forward scattering. (b) CD4+ cells. Testicular interstitial cells were labeled with fluorescein isothiocyanate (FITC)-conjugated anti-CD4 antibody (anti-CD4-FITC) and analyzed by flow cytometry. (c) CD8+ cells. Testicular interstitial cells were labeled with PE-Cy5-conjugated anti-CD8 antibody (anti-CD8-PE-Cy5) and analyzed using flow cytometry. (d) B cells. Testicular interstitial cells were labeled with PE-Cy5-conjugated anti-B220 antibody (anti-B220-PE-Cy5) and analyzed using flow cytometry. Absolute numbers of lymphocytes per testis were determined based on flow cytometry data (right panels). Flow cytometry density plots represent five mice. Data are mean values ±s.e.m. of five mice. **P<0.01. A full color version of this figure is available at the Immunology and Cell Biology journal online.

Article Snippet: The paraffin sections were stained with fluorescein isothiocyanate-conjugated streptavidin (Vector Laboratories, Burlingame, CA, USA) at room temperature for 30 min. After washing with PBS twice, the sections were counterstained with 4′, 6-diamidino-2-phenylindol for 10 min.

Techniques: Flow Cytometry, Isolation, Labeling

Journal: Immunology and cell biology

Article Title: Mice lacking Axl and Mer tyrosine kinase receptors are susceptible to experimental autoimmune orchitis induction

doi: 10.1038/icb.2014.97

Figure Lengend Snippet: Lymphocytes in renal lymph nodes (RLNs). (a) RLN size. WT and Axl−/−Mer−/− (AM−/−) mice were immunized. After 50 days, RLNs were collected for analysis. Images represent RLNs from five mice. Scale bar =1 mm. (b) Lymphocytes in RLNs of AM−/− mice. RLN cells were isolated from the control and immunized AM−/− mice. Cells were labeled with anti-CD4-FITC, anti-CD8-PE-Cy5 and anti-B220-PE-Cy5. Lymphocyte populations were gated (upper left panel). Ratios of CD4+ cells (upper right panel) and CD8+ and B cells (lower panels) were determined using flow cytometry. (c) Lymphocytes in RLNs of WT mice. RLN cells of control and immunized WT mice were analyzed as in b. Data are mean values ±s.e.m. of five mice. *P<0.05. FITC, fluorescein isothiocyanate; FSC, forward scattering; SSC, side scattering. A full color version of this figure is available at the Immunology and Cell Biology journal online.

Article Snippet: The paraffin sections were stained with fluorescein isothiocyanate-conjugated streptavidin (Vector Laboratories, Burlingame, CA, USA) at room temperature for 30 min. After washing with PBS twice, the sections were counterstained with 4′, 6-diamidino-2-phenylindol for 10 min.

Techniques: Isolation, Labeling, Flow Cytometry

Journal: Immunology and cell biology

Article Title: Mice lacking Axl and Mer tyrosine kinase receptors are susceptible to experimental autoimmune orchitis induction

doi: 10.1038/icb.2014.97

Figure Lengend Snippet: Autoantibody production and BTB permeability. (a) Autoantibodies in sera. Sera of control and immunized Axl−/−Mer−/− (AM−/−) mice were collected by tail bleeding. Male germ cells were isolated from 10-week-old WT mice. Western blots on germ cell lysates were performed using sera (1:500 dilution) of AM−/− mice as primary antibodies. (b) Distribution of autoantibodies. Testicular cryosections of 10-week-old WT mice were immunostained with sera (1:100 dilution) of control (left panel) and immunized (right panel) AM−/− mice. Insets in the upper right corners are negative controls. Negative controls were stained using sera of WT mice (without immunization) as primary antibodies. (c) Autoantibody deposition. Testicular sections of control (left panel) and immunized (middle panel) AM−/− mice were directly stained with anti-mouse IgG antibodies. Signals in germ cells (arrow) of immunized AM−/− mice indicate deposition of autoantibodies. Arrows indicate elongating spermatids. Testis of WT mice without immunization served as the negative control (upper right corners). Heavy (H) and light (L) chains of IgG in testes of immunized AM−/− mice were confirmed by Western blot using anti-mouse IgG antibodies (right panel). β-Actin was used as the loading control for western blot. (d) BTB permeability. Biotin was injected underneath the testicular capsules of control (lower panels) and immunized (upper panels) AM−/− mice. After 30 min, paraffin sections were stained with fluorescein isothiocyanate-conjugated streptavidin (left panels), after which the sections were counterstained with 4′, 6-diamidino-2-phenylindol (DAPI) (middle panels). Right panels are merged biotin and DAPI images. Arrowheads, arrows and asterisks indicate interstitial spaces, basal and adluminal compartments of the seminiferous tubules, respectively. Images represent at least three experiments of three mice. Scale bar =20 μm. A full color version of this figure is available at the Immunology and Cell Biology journal online.

Article Snippet: The paraffin sections were stained with fluorescein isothiocyanate-conjugated streptavidin (Vector Laboratories, Burlingame, CA, USA) at room temperature for 30 min. After washing with PBS twice, the sections were counterstained with 4′, 6-diamidino-2-phenylindol for 10 min.

Techniques: Permeability, Isolation, Western Blot, Staining, Negative Control, Injection

Journal: Journal of Cancer

Article Title: Reduced FANCE Confers Genomic Instability and Malignant Behavior by Regulating Cell Cycle Progression in Endometrial Cancer

doi: 10.7150/jca.86348

Figure Lengend Snippet: FANCE suppressed cell cycle progression in EC cells (A) The protein lysates of HEC-1-A OE-FANCE, HEC-1-B OE-FANCE, and corresponding NC cells were collected at the indicated time points after double-thymidine block and CDK4, CDK6, cyclinA2, cyclin B1, cyclin D1 and cyclin E expression were detected by WB. β-tubulin was used as the housekeeping protein. FANCE induced G2/M arrest and delayed entry to mitosis. (B) Analysis of EC cell cycle distribution with flow cytometry. (C) The proportion of cells in G1 phase was decreased and S and G2 phase was increased in HEC-1-A OE-FANCE and HEC-1-B OE-FANCE cells. ** p < 0.01, *** p < 0.001.

Article Snippet: Proteins were electrophoresed on Sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE, CWBIO), then transferred to polyvinylidene fluoride (PVDF) membranes, blocked, and incubated with primary anti-FANCE (Biobryt, 1:10000), anti-FANCD2 (1: 5000; Novus Biologicals), anti-GAPDH (1:10000, bioworld), anti-β-tubulin (1:10000, proteintech), anti-RAD51(1:5000, Abcam), anti-γH2AX (1:10000, Abcam), anti-cyclin dependent kinase 4 (CKD4) (1:5000, proteintech), anti-cyclin dependent kinase 6 (CDK6) (1:5000, proteintech), anti-cyclin D1 (1:5000, proteintech), anti-cyclin B1(1:5000, proteintech), anti-cyclin E(1:5000, proteintech) and anti-cyclin A2(1:5000, proteintech) for overnight at 4°C.

Techniques: Blocking Assay, Expressing, Flow Cytometry

Journal: Frontiers in Pharmacology

Article Title: Quercetin inhibits the cAMP/PKA/CREB/glycolysis axis to exert anti-acute lymphoblastic leukemia effects

doi: 10.3389/fphar.2025.1614973

Figure Lengend Snippet: The effect of Que on the growth of CEM and MOLT-4 cells. Cells were treated with Que at specified concentrations. (A,B) Transmission electron microscopy was used to observe the submicroscopic cellular structures (×30000 magnification; scale bar = 2 μm). (C,D) Western blotting was performed to evaluate the expression of apoptosis-related proteins, with β-actin and GAPDH serving as loading controls. (E,F) Cell cycle distribution was analyzed using a NovoCyte flow cytometer and NovoExpress software. (G,H) Western blotting was further employed to assess the expression of cell cycle-related proteins with β-actin as the loading control. Data are presented as the mean ± SD of three independent experiments. * p < 0.05,** p < 0.01,** p < 0.001,indicate statistical significance and highly significant differences, respectively.

Article Snippet: Antibodies against β-actin (66009-1-Ig, 1:20000), and GAPDH (10494-1-AP, 1:5000) were purchased from Proteintech (Wuhan, China).

Techniques: Transmission Assay, Electron Microscopy, Western Blot, Expressing, Flow Cytometry, Software, Control

Journal: Frontiers in Pharmacology

Article Title: Quercetin inhibits the cAMP/PKA/CREB/glycolysis axis to exert anti-acute lymphoblastic leukemia effects

doi: 10.3389/fphar.2025.1614973

Figure Lengend Snippet: Que exerts its anti-ALL effects through the cAMP/PKA/CREB signaling pathway. Cells were treated with Que at specified concentrations. (A–E) Transcriptomic sequencing analysis was performed. (A) Volcano plot: Differentially expressed genes were plotted as a volcano plot, with each point representing a gene. (B) Heatmap: Differentially expressed genes were visualized using a heatmap. (C) KEGG enrichment analysis: The top ten enriched pathways are shown. (D) Heatmap: Differentially expressed genes enriched in the glycolysis pathway were plotted as a heatmap. (E) GSEA analysis revealed enrichment in the glycolysis pathway. (F,G) Western blotting was used to analyze the protein levels of components in the cAMP/PKA/CREB signaling pathway, with GAPDH serving as a loading control. Data are presented as the mean ± SD of three independent experiments. * p < 0.05,** p < 0.01 indicate statistical significance and highly significant differences, respectively.

Article Snippet: Antibodies against β-actin (66009-1-Ig, 1:20000), and GAPDH (10494-1-AP, 1:5000) were purchased from Proteintech (Wuhan, China).

Techniques: Sequencing, Western Blot, Control

Journal: Frontiers in Pharmacology

Article Title: Quercetin inhibits the cAMP/PKA/CREB/glycolysis axis to exert anti-acute lymphoblastic leukemia effects

doi: 10.3389/fphar.2025.1614973

Figure Lengend Snippet: Sp-cAMP attenuates the glycolysis-inhibitory effects of Que in CEM and MOLT-4 cells. After treating CEM and MOLT-4 cells with specified concentrations of Que and Sp-cAMP, (A) the impact on cell proliferation was determined using the CCK-8 assay. (B–F) The effects on intracellular ROS levels, mitochondrial membrane potential, ATP content, glucose uptake, and lactate production were evaluated. (G-I) Western blot analysis was performed to examine the expression of apoptosis-related proteins, glycolysis-related proteins, and proteins in the cAMP/PKA/CREB pathway, with β-actin and GAPDH as loading controls. Data are presented as the mean ± SD of three independent experiments. * p < 0.05,** p < 0.01 indicate statistical significance and highly significant differences, respectively.

Article Snippet: Antibodies against β-actin (66009-1-Ig, 1:20000), and GAPDH (10494-1-AP, 1:5000) were purchased from Proteintech (Wuhan, China).

Techniques: CCK-8 Assay, Membrane, Western Blot, Expressing