Journal: bioRxiv

Article Title: Elimination of senescent cells with senolytic host-directed therapy reduces tuberculosis progression in mice

doi: 10.1101/2025.03.28.645957

Figure Lengend Snippet: (a) Schematics of the experimental strategy to measure senescence markers (p21, p16 and SA-βGal activity) and SASP cytokines levels in mice lungs at 10 and 20 days p.i. Day 1 CFU= 55. (b) %p21+p16+ and (c) %SA-βGal+ (CellEvent Senescence green+) cells out of all live lung (uninfected and Mtb- infected mice) and Spleen ( Mtb- infected mice) cells at 10 days p.i. as determined by multicolor flow cytometry. (d) %p21+p16+ and (e) %SA-βGal+ cells out of different lung-cell types at 10 days p.i., as determined by multicolor flow cytometry. Gating for p16⁺, p21⁺, and SA-βGal⁺ events for each cell type were established using reference cells from Mtb -infected WT B6 mice (set at 1 %). (AMs: Alveolar macrophages, IMs: Interstitial macrophages) (f) Normalized concentration of SASP-cytokines in lung homogenates at 20 dpi and 4 wpi as measured by a LEGENDplex™ Mouse Inflammation Panel (13-plex). The data are means ± SEM. Each data point represents a mouse (n=11-12). (two-way ANOVA with Tukey’s (b-e) or Dunnett’s (f) multiple comparisons test).

Article Snippet: The PVDF membranes were blocked using 5% Blotting-grade blocker (Bio-Rad; 1706404) in TBST buffer-Tris-buffered saline (Quality Biological; 351086101)+ 0.1% Tween 20 (Sigma; P2287) for 1-2 hour and incubated overnight at 4°C with the primary antibodies from Mouse Reactive Senescence Marker Antibody Sampler Kit (Cell Signaling Technology-78551)-Phospho-Histone H2A.X (Ser139) (20E3) Rabbit mAb (1: 1000 dilution), Lamin B1 (E6M5T) Rabbit mAb (1: 1000 dilution), HMGB1 (D3E5) Rabbit mAb (1: 1000 dilution), p16 INK4A (E5F3Y) Rabbit mAb (1: 1000 dilution) and β-Actin (D6A8) Rabbit mAb (Cell Signaling Technology; 8457) (1:5000 dilution).

Techniques: Activity Assay, Infection, Flow Cytometry, Concentration Assay

Journal: bioRxiv

Article Title: Elimination of senescent cells with senolytic host-directed therapy reduces tuberculosis progression in mice

doi: 10.1101/2025.03.28.645957

Figure Lengend Snippet: (a) Representative H&E-stained images of Mtb -infected mice lungs treated with Vehicle or drugs, and respective ImageJ-based quantification. Yellow arrow indicates necrotic granuloma in Vehicle treated B6.Sst1S mice at 5 wpi. The data are means ± SEM. Each data point represents a mouse. Statistical analysis between two groups was done by unpaired two-tailed Student’s t test. Square represents necrotic granuloma formation in the lung. (b) Representative γH2A.X-Immunohistochemistry images of mice after lungs vehicle/ drugs treatment, and respective Violin plots to show ImageJ quantification of γH2A.X-stained area. Each data point represents a mouse. Statistical analysis between two groups was done by unpaired two-tailed Student’s t test. (c) %p21+βGal+ and %p16+βGal+ subpopulation observed in all live lung cells of Mtb -infected mice (n= 5-8). The data are means ± SEM. Each data point represents a mouse. Statistical analysis was calculated by two-way ANOVA with Dunnett’s multiple comparisons test. Bubble plot to show %p21+βGal+ and %p16+βGal+ subpopulation in different lung cell types in Mtb -infected (d) B6.Sst1S mice (n= 8) and (e) WT B6 old mice (n= 5) at indicated time point and treatment groups. The size of the bubble indicates %subpopulation out of all of particular cell types and color is adjusted p values relative to Veh group. Statistics are calculated by one-way ANOVA with Tukey’s multiple comparisons test ( p > 0.15: ns).

Article Snippet: The PVDF membranes were blocked using 5% Blotting-grade blocker (Bio-Rad; 1706404) in TBST buffer-Tris-buffered saline (Quality Biological; 351086101)+ 0.1% Tween 20 (Sigma; P2287) for 1-2 hour and incubated overnight at 4°C with the primary antibodies from Mouse Reactive Senescence Marker Antibody Sampler Kit (Cell Signaling Technology-78551)-Phospho-Histone H2A.X (Ser139) (20E3) Rabbit mAb (1: 1000 dilution), Lamin B1 (E6M5T) Rabbit mAb (1: 1000 dilution), HMGB1 (D3E5) Rabbit mAb (1: 1000 dilution), p16 INK4A (E5F3Y) Rabbit mAb (1: 1000 dilution) and β-Actin (D6A8) Rabbit mAb (Cell Signaling Technology; 8457) (1:5000 dilution).

Techniques: Staining, Infection, Two Tailed Test, Immunohistochemistry

Journal: The Journal of biological chemistry

Article Title: Estrogen-induced activation of Cdk4 and Cdk2 during G1-S phase progression is accompanied by increased cyclin D1 expression and decreased cyclin-dependent kinase inhibitor association with cyclin E-Cdk2.

doi: 10.1074/jbc.272.16.10882

Figure Lengend Snippet: FIG. 4. Effects of estradiol on cyclin protein expression. The experimental design was described in Fig. 2. Representative Western blots are shown for G1 phase cyclins (D1, D3, E) (A) and S/G2M phase cyclins (A, B1) (B). Controls for cyclin E are as described in Fig. 2.

Article Snippet: Antibodies used were rabbit polyclonal antisera to human cyclin D1 (29), human cyclin E (C-19; Santa Cruz Biotechnology), and human Cdk2 (M2; Santa Cruz Biotechnology).

Techniques: Expressing, Western Blot

Journal: The Journal of biological chemistry

Article Title: Estrogen-induced activation of Cdk4 and Cdk2 during G1-S phase progression is accompanied by increased cyclin D1 expression and decreased cyclin-dependent kinase inhibitor association with cyclin E-Cdk2.

doi: 10.1074/jbc.272.16.10882

Figure Lengend Snippet: FIG. 5. Estrogen induction of cyclin D1 and cyclin D3 mRNA expression. MCF-7 cells were pretreated with 10 nM ICI 182780 for 48 h and then treated with 100 nM E2 or vehicle (control) at time 0. At intervals thereafter, total cellular RNA was harvested. A, representative North- ern blots are shown for cyclin D1 and D3 mRNA from estradiol-treated and control cells. Arrows indicate the 4.5- and 1.5-kb cyclin D1 transcripts. B, graphical pres- entation of temporal changes in mRNA (open symbols) and protein (solid symbols, mean of two experiments) for cyclin D1 (left) and cyclin D3 (right).

Article Snippet: Antibodies used were rabbit polyclonal antisera to human cyclin D1 (29), human cyclin E (C-19; Santa Cruz Biotechnology), and human Cdk2 (M2; Santa Cruz Biotechnology).

Techniques: Expressing, Control

Journal: The Journal of biological chemistry

Article Title: Estrogen-induced activation of Cdk4 and Cdk2 during G1-S phase progression is accompanied by increased cyclin D1 expression and decreased cyclin-dependent kinase inhibitor association with cyclin E-Cdk2.

doi: 10.1074/jbc.272.16.10882

Figure Lengend Snippet: FIG. 8. Activation of Cdk4 and Cdk2 following estrogen treatment. MCF-7 cells were pretreated with 10 nM ICI 182780 for 48 h and then treated with 100 nM E2 or vehicle (control) at time 0. At intervals thereafter, whole cell lysates were prepared and immunoprecipitated with antibodies to either Cdk4, cyclin E, or Cdk2, and then the kinase activity of the immunoprecipitates was determined by phosphorylation of GST-pRB773–923

Article Snippet: Antibodies used were rabbit polyclonal antisera to human cyclin D1 (29), human cyclin E (C-19; Santa Cruz Biotechnology), and human Cdk2 (M2; Santa Cruz Biotechnology).

Techniques: Activation Assay, Control, Immunoprecipitation, Activity Assay, Phospho-proteomics

Journal: The Journal of biological chemistry

Article Title: Estrogen-induced activation of Cdk4 and Cdk2 during G1-S phase progression is accompanied by increased cyclin D1 expression and decreased cyclin-dependent kinase inhibitor association with cyclin E-Cdk2.

doi: 10.1074/jbc.272.16.10882

Figure Lengend Snippet: FIG. 9. Composition of cyclin D1-as- sociated and cyclin E-associated complexes. MCF-7 cells were pretreated with 10 nM ICI 182780 for 48 h and then treated with 100 nM E2 or vehicle (control) at time 0. At intervals thereafter, whole cell lysates were prepared and immuno- precipitated with anti-cyclin D1 anti- serum or anti-cyclin E antibodies, and then these immunoprecipitates were sep- arated by SDS-PAGE and transferred to nitrocellulose membranes. A, cyclin D1 antiserum immunoprecipitates. The same filter was sequentially Western blotted for cyclin D1, Cdk4, p21, and p27. A rep- resentative blot is shown for each. B, rel- ative levels of cyclin D1 (E), Cdk4 (G), p21 (M), and p27 (f) were determined by den- sitometry and are expressed relative to the vehicle treated controls. Points repre- sent the mean of two separate experi- ments. C, cyclin E immunoprecipitates. The same filter was sequentially Western blotted for cyclin E, Cdk2, p21, and p27. A representative blot is shown for each. The asterisk marks the more mobile form of Cdk2 that is phosphorylated on Thr-160.

Article Snippet: Antibodies used were rabbit polyclonal antisera to human cyclin D1 (29), human cyclin E (C-19; Santa Cruz Biotechnology), and human Cdk2 (M2; Santa Cruz Biotechnology).

Techniques: Control, SDS Page, Western Blot

Journal: The Journal of biological chemistry

Article Title: Estrogen-induced activation of Cdk4 and Cdk2 during G1-S phase progression is accompanied by increased cyclin D1 expression and decreased cyclin-dependent kinase inhibitor association with cyclin E-Cdk2.

doi: 10.1074/jbc.272.16.10882

Figure Lengend Snippet: FIG. 10. Estrogen decreases inhibi- tory activity toward cyclin E-Cdk2. A, lysates were prepared from cells that were pretreated with antiestrogen and then treated with E2 (1) or vehicle (2). Active cyclin E-Cdk2 complexes that had been prepared from baculovirus-infected Sf9 cells were incubated with either lysis buffer only (labeled input) or with cell lysates. Lysates were also immunode- pleted with either anti-p27 antibodies, anti-p21 antibodies, or both and then in- cubated with recombinant cyclin E-Cdk2 complexes. Recombinant cyclin E-Cdk2 complexes were then recovered and as- sayed for histone (H1) kinase activity. The percentage of the input activity, de- fined as 100%, is also shown numerically below the autoradiograph. The same sam- ples were electrophoresed on a duplicate SDS-PAGE gel and then Western blotted for p21 and p27 to assess the binding of these proteins to recombinant cyclin E- Cdk2. B, the experiment described for panel A was repeated using either boiled lysis buffer (labeled input) or boiled ly- sates from cells treated with estradiol (1) or vehicle (2). C, MCF-7 cells were pre- treated with 10 nM ICI 182780 for 48 h and then treated with 100 nM estradiol or vehicle (control) at time 0. At intervals thereafter, whole cell lysates were pre- pared and assayed for cyclin E-Cdk2 in- hibitory activity as described for panel A.

Article Snippet: Antibodies used were rabbit polyclonal antisera to human cyclin D1 (29), human cyclin E (C-19; Santa Cruz Biotechnology), and human Cdk2 (M2; Santa Cruz Biotechnology).

Techniques: Activity Assay, Infection, Incubation, Lysis, Labeling, Recombinant, Autoradiography, SDS Page, Western Blot, Binding Assay, Control

Journal: The Journal of biological chemistry

Article Title: Estrogen-induced activation of Cdk4 and Cdk2 during G1-S phase progression is accompanied by increased cyclin D1 expression and decreased cyclin-dependent kinase inhibitor association with cyclin E-Cdk2.

doi: 10.1074/jbc.272.16.10882

Figure Lengend Snippet: FIG. 11. Cyclin E-Cdk2 activation is accompanied by loss of CDK inhibitor association and Cdk2 Thr-160 phosphorylation. MCF-7 cells were pretreated with 10 nM ICI 182780 for 48 h and then treated with 100 nM E2 or vehicle (control) at time 0. Whole cell lysates were prepared either 8 h after estrogen (8 h E2) or from control cells (ICI 182780). Lysates were then fractionated on a Superose 12 gel filtration column. A, fractions were precipitated with acetone and Western blot- ted for cyclin E (top panels) or assayed for cyclin E-Cdk2 histone (H1) kinase activity (bottom panel). The relative levels of cyclin E protein (E, G) and cyclin E-Cdk2 activity (M, f) in lysates from antiestrogen- pretreated cells and cells treated with estradiol for 8 h were determined by densitometry and are represented graphically. The elution of mark- ers of known molecular weight (ferritin, 440 kDa; catalase, 232 kDa; aldolase, 158 kDa) are indicated at the top of the graph. B, fractions 19 and 24 from the 8 h E2 lysate were immunoprecipitated with an anti- cyclin E antibody, and the immunoprecipitates were electrophoresed on a SDS-PAGE gel and transferred to a nitrocellulose filter. The same filter was then sequentially Western blotted for cyclin E, Cdk2, p21, and p27. A representative blot is shown for each. The asterisk marks the more mobile form of Cdk2 that is phosphorylated on Thr-160.

Article Snippet: Antibodies used were rabbit polyclonal antisera to human cyclin D1 (29), human cyclin E (C-19; Santa Cruz Biotechnology), and human Cdk2 (M2; Santa Cruz Biotechnology).

Techniques: Activation Assay, Phospho-proteomics, Control, Filtration, Western Blot, Activity Assay, Molecular Weight, Immunoprecipitation, SDS Page