mouse anti-hp1γ  (Merck KGaA)

Journal: Cellular & Molecular Biology Letters

Article Title: Senescence-associated reprogramming induced by interleukin-1 impairs response to EGFR neutralization

doi: 10.1186/s11658-022-00319-7

Figure Lengend Snippet: TRAP IL-1 decreases HP1y foci number in SW48, while increasing it in SW48 CXR. Sensitive and resistant SW48 cell lines (4 × 10 4 cells per well) were seeded on 13 mm coverslip. Twenty-four hours later, SW48 cells were treated for 4 days with control medium (10% FBS), IL-1 (α and β, 10 ng/ml each), and CTX (50 μg/ml) alone and in combination with TRAP IL-1 (20 μg/ml) ( a ), while SW48 CXR cells were treated with control medium (10% FBS + CTX 50 μg/ml), and TRAP IL-1 (20 μg/ml) ( b ). After fixation, permeabilization, and blocking with 2% BSA, cells were incubated with anti-HP1y antibody. The next day, cells were washed, incubated with secondary antibody FITC mouse, and stained with DAPI (nuclei). Finally, coverslips were mounted on slides and examined under a fluorescence microscope. Number of HP1y foci was determined and analyzed through ImageJ software from three different fields for each sample. Histograms shows the average of HP1y foci for each cell. Statistical analysis was carried out using two-way ANOVA with Tukey’s multiple comparisons test *** p < 0.001, **** p < 0.0001. Scale bar, 20 mm

Article Snippet: The following primary antibodies were used: anti-HP1y mouse monoclonal antibody (1:500 dilution; sc-398562, Santa Cruz Biotechnology), anti-p-yH2AX mouse monoclonal antibody (1:500; sc-517348, Santa Cruz Biotechnology), anti-SNAI1 mouse monoclonal antibody (1:800; sc-271977, Santa Cruz Biotechnology), anti-GAPDH (14C10) rabbit monoclonal antibody (1:1000, #2118 Cell Signaling Technology).

Techniques: Control, Blocking Assay, Incubation, Staining, Fluorescence, Microscopy, Software

Journal: Frontiers in Immunology

Article Title: Targeting Cbx3 /HP1γ Induces LEF-1 and IL-21R to Promote Tumor-Infiltrating CD8 T-Cell Persistence

doi: 10.3389/fimmu.2021.738958

Figure Lengend Snippet: Cbx3 /HP1γ deficiency modulates LEF-1 and IL-21R expression in CD8 + effector T cells. (A) Mouse Lef1 locus. -2 and 12: positions of first and last primer pairs, respectively. (B) Read-density tracks of Cbx3 /HP1γ peaks across Lef1 identified by ChIP-Seq using chromatin from wt day 5 activated/differentiated CD8 + T cells (pooled from 3 mice); y axis: number of reads per million mapped per 25-bp window; representative of 2 independent ChIP-Seq runs. (C) LEF-1 expression was evaluated by Western immunoblot using total protein lysates from activated/differentiated control and Cbx3 /HP1γ-deficient CD8 + T cells; 3/28+IL2: CD8 + T cells activated/differentiated for 5 days with plate-bound anti-CD3 and anti-CD28 plus IL-2; C: control (CD8α-Cre or wt); fl/+: Cbx3 fl/+ and fl/fl: Cbx3 fl/fl (CD8 + T-cell-restricted deletion of Cbx3/ HP1γ using the CD8α-Cre mouse); n = 3; representative of 3 experiments. (D) Mouse Il21r locus. -4 and 4: positions of first and last pair of primers, respectively. (E) Read-density tracks of Cbx3 /HP1γ peaks across Il21r were determined by ChIP-Seq as in (B) . (F) IL-21R expression levels on activated/differentiated CD8 + T cells by flow; numbers: percent cells; representative of 3 experiments; n = 3. (G) IL-21R mean fluorescence intensity (MFI) was evaluated from (F) . (H) Relative expression of Il21r in day 5 activated/differentiated CD8 + T cells was assessed by RT-qPCR and normalized to Gapdh ; Graphpad unpaired student t-test: **p ≤ 0.01; representative of 3 experiments. (I) Number of CD8 + IL-21R + T cells in cultures was calculated from (F) ; Graphpad unpaired student t-test: *p ≤ 0.05, **p ≤ 0.01.

Article Snippet: The Western blot and ChIP-tested mouse anti-HP1γ mAb (clone 42s2) was purchased from Millipore Sigma.

Techniques: Expressing, ChIP-sequencing, Western Blot, Fluorescence, Quantitative RT-PCR

Journal: Frontiers in Immunology

Article Title: Targeting Cbx3 /HP1γ Induces LEF-1 and IL-21R to Promote Tumor-Infiltrating CD8 T-Cell Persistence

doi: 10.3389/fimmu.2021.738958

Figure Lengend Snippet: Cbx3 /HP1γ regulates Lef1 and Il21r transcription initiation and chromatin remodeling. (A, B) Levels of Pol II S5 bound to Lef1 (A) and Il21r (B) were quantified by ChIP-qPCR using chromatin from day 5 activated/differentiated CD8 + T cells. TSS: transcription start site; numbers on X axis: positions of primers along the two loci; 150 bp products amplified by Lef1 or Il21r primers. (C, D) Levels of Pol II S2 bound to Lef1 (C) and Il21r (D) were quantified as in (A, B) . (E, F) H3K9me3 deposition on Lef1 (E) and Il21r (F) was quantified as in (A, B) . Graphpad unpaired student t-test: *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001; representative of 4 independent ChIPs using pooled chromatin from 3 mice.

Article Snippet: The Western blot and ChIP-tested mouse anti-HP1γ mAb (clone 42s2) was purchased from Millipore Sigma.

Techniques: Amplification

Journal: Frontiers in Immunology

Article Title: Targeting Cbx3 /HP1γ Induces LEF-1 and IL-21R to Promote Tumor-Infiltrating CD8 T-Cell Persistence

doi: 10.3389/fimmu.2021.738958

Figure Lengend Snippet: Cbx3 /HP1γ-deficient CD8 + effector T cells can persist and cause tumor rejection. (A) Ascites from control (CD8α-Cre or wt), Cbx3 Tg , Cbx3 fl/+ and Cbx3 fl/fl mice (X axis) were visualized on day 120 after IP injection of mouse ID8 ovarian tumor cells; Cbx3 Tg : Cbx3/ HP1γ T-cell-restricted expression driven by the human Cd2 promoter; Cbx3 fl/+ and Cbx3 fl/fl : CD8 + T-cell-restricted deletion of Cbx3/ HP1γ using the CD8α-Cre mouse. (B) Ovarian ascites of mice in (A) were measured; bars: group median; Graphpad unpaired student t-test: **p≤0.01, ***p≤0.001; each symbol = one mouse; n = 5-8; representative of 2 experiments. (C) Survival curves of ovarian tumor-bearing mice were determined; Graphpad log-rank (Mantel-Cox) test; n = 5-8. (D) B16 tumor cells were injected subcutaneously (sc) and growth was assessed starting on day 14 after tumor-cell injection then every 2 days through day 20; Graphpad two-way ANOVA: **p ≤ 0.01, ****p ≤ 0.0001; n = 5; representative of 3 experiments. (E) Survival curves of B16 melanoma tumor-bearing mice; Graphpad log-rank (Mantel-Cox) test; n = 5. (F) NB-9464 tumor cells were injected sc, NBL tumor volume was determined every 2 days starting on day 22 through day 28; Graphpad two-way ANOVA: ****p ≤ 0.0001; n = 5; representative of 2 experiments. (G) Survival curves of NBL tumor-bearing mice; Graphpad log-rank (Mantel-Cox) test, n = 5. (H, J, L) Frequencies of CD8 + NKG2D + T cells in ovarian ascites, B16 and NBL tumors from control (CD8α-Cre or wt), Cbx3 Tg , Cbx3 fl/+ and Cbx3 fl/fl mice; data were extracted from flow analysis ( Figure S2C, E, G ); bars: group median; Graphpad unpaired student t-test: **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001; each symbol = one mouse; n = 5-8; representative of 2-3 experiments. (I, K, M) Frequencies of CD4 + FOXP3 + Tregs in ovarian ascites, B16 and NBL tumors ( Figure S2D, F, H ); bars: group median; Graphpad unpaired student t-test: *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001; each symbol = one mouse; n = 5-8; representative of 2-3 experiments. (N, O) Apoptotic cells (brown) in B16 melanoma and NBL tumor sections were identified by TUNEL staining; representative of 4 tumors from each mouse strain.

Article Snippet: The Western blot and ChIP-tested mouse anti-HP1γ mAb (clone 42s2) was purchased from Millipore Sigma.

Techniques: Injection, Expressing, TUNEL Assay, Staining

Journal: Frontiers in Immunology

Article Title: Targeting Cbx3 /HP1γ Induces LEF-1 and IL-21R to Promote Tumor-Infiltrating CD8 T-Cell Persistence

doi: 10.3389/fimmu.2021.738958

Figure Lengend Snippet: Cbx3 /HP1γ-deficient CD8 + T cells remodel the tumor chemokine/receptor landscape. (A, B) Relative expression of chemokines and receptors mediating CD8 + T cells trafficking into B16 tumors was determined by RT-qPCR and normalized to Gapdh . (C, D) Relative expression of chemokines and receptors mediating CD8 + T cells trafficking into NBL tumors was evaluated by RT-qPCR and normalized to Gapdh . (E, F) Relative expression of chemokines and receptors inducing CD4 + Tregs homing into tumors was normalized to Gapdh . (G, H) Relative expression of chemokines and receptors inducing CD4 + Tregs homing into NBL tumors was assessed. X axis: mouse strains; Graphpad unpaired student t-test: *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001; ns, not significant; representative of 4 experiments; n = 4.

Article Snippet: The Western blot and ChIP-tested mouse anti-HP1γ mAb (clone 42s2) was purchased from Millipore Sigma.

Techniques: Expressing, Quantitative RT-PCR

Journal: Frontiers in Immunology

Article Title: Targeting Cbx3 /HP1γ Induces LEF-1 and IL-21R to Promote Tumor-Infiltrating CD8 T-Cell Persistence

doi: 10.3389/fimmu.2021.738958

Figure Lengend Snippet: LEF-1 and IL-21R are indispensable for halting tumor growth. (A) Western blot of LEF-1 expression was done using total protein lysates of day 5 activated/differentiated CD8 + T cells; C: Cbx3 /HP1γ-deficient; Lef1 fl/+ and Lef1 fl/fl : Cbx3 - Lef1 -deficient; representative of 4 experiments. (B) IL-21R expression was measured by flow; Cbx3 fl/fl : Cbx3 /HP1γ-deficient; Il21r +/- and Il21r -/- : Cbx3 - Il21r -deficient; representative of 4 experiments. (C) Ovarian ascites were measured on day 125 after tumor injection; bars: group median; Graphpad unpaired student t-test: ***p ≤ 0.001, ****p ≤ 0.0001; each symbol = one mouse; n = 3-5; Cbx3 fl/fl Lef1 fl/fl : Cbx3 - Lef1 -deficient; representative of 2 experiments. (D) B16 melanoma tumor burden was determined on day 18 after tumor injection; bars: group median; Graphpad unpaired student t-test: *p ≤ 0.05, ****p ≤ 0.0001; each symbol = mouse; for each genotype n = 4-7; Lef1 fl/fl : Lef1 ablation alone using CD8α-Cre mice; representative of 3 experiments. (E) On day 10 after injection of tumor cells, tumor bearing B6SJ/L mice (CD45.1 + ) were treated with in vitro -activated/differentiated CD8 + T cells (CD45.2 + ) from control, Cbx3 fl/fl , Cbx3 fl/fl , Il21r -/- ( Cbx3 - Il21r -deficient) or Il-21r -/- (germline deleted) mice; **p ≤ 0.01, ***p ≤ 0.001; n = 3-4 recipients; representative of 2 experiments. (F) NBL tumor burden was determined starting on day 25 after injection of tumor cells then every 2 days until day 33; Graphpad two-way ANOVA: ***p≤0.001; n = 2-5; representative of 2 experiments. (G) On day 14 after NBL injection, tumor bearing B6SJ/L mice were treated with in vitro -activated/differentiated CD8 + T cells from control, Cbx3 fl/fl or Cbx3 fl/fl , Il21r -/- ( Cbx3 - Il21r -deficient) mice; ****p ≤ 0.0001, n = 3-4 recipients; representative of 2 experiments.

Article Snippet: The Western blot and ChIP-tested mouse anti-HP1γ mAb (clone 42s2) was purchased from Millipore Sigma.

Techniques: Western Blot, Expressing, Injection, In Vitro

Journal: Frontiers in Immunology

Article Title: Targeting Cbx3 /HP1γ Induces LEF-1 and IL-21R to Promote Tumor-Infiltrating CD8 T-Cell Persistence

doi: 10.3389/fimmu.2021.738958

Figure Lengend Snippet: LEF-1 and IL-21R are required for CD8 + T cells to maintain their effector capacity. (A, B) Prf 1 and Gzmb relative expression in B16 melanoma tumors was determined by RT-qPCR; Graphpad unpaired student t-test: *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001; n = 3 tumors; representative of 3 experiments. (C–E) Prf1 , Gzmb and Ifng relative expression in NBL tumors was determined by RT-qPCR; Graphpad unpaired student t-test: *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001; n = 3 tumors; representative of 3 experiments. (F–H) Prf1 , Gzmb and Ifng relative expression in day 5 in vitro -activated/differentiated CD8 + T cells from Cbx3 /HP1γ-deficient and compound mutant mice was quantified by RT-qPCR; Graphpad unpaired student t-test: **p ≤ 0.01, ***p ≤ 0.001; representative of 3 experiments.

Article Snippet: The Western blot and ChIP-tested mouse anti-HP1γ mAb (clone 42s2) was purchased from Millipore Sigma.

Techniques: Expressing, Quantitative RT-PCR, In Vitro, Mutagenesis

Journal: The EMBO Journal

Article Title: DNA polymerase zeta contributes to heterochromatin replication to prevent genome instability

doi: 10.15252/embj.2020104543

Figure Lengend Snippet:

Article Snippet: Mouse monoclonal anti‐HP1γ , Millipore Sigma , MAB3450.

Techniques: Purification