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 following Western blot and ChIP-tested antibodies were purchased from Cell Signaling: tri-methyl-histone H3 (Lys9) (D4W1U) rabbit mAb #13969, phospho-Rpb1 CTD (Ser2) (E1Z3G) rabbit mAb #13499, phospho-Rpb1 CTD (Ser5) (D9N5I) rabbit mAb #13523, LEF-1 (C12A5) rabbit mAb #2230, cleaved caspase-3 (Asp175) antibody #9661 and phospho-HP1γ (Ser83) Ab #2600.

Techniques: Expressing, ChIP-sequencing, Western Blot, Control, 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 following Western blot and ChIP-tested antibodies were purchased from Cell Signaling: tri-methyl-histone H3 (Lys9) (D4W1U) rabbit mAb #13969, phospho-Rpb1 CTD (Ser2) (E1Z3G) rabbit mAb #13499, phospho-Rpb1 CTD (Ser5) (D9N5I) rabbit mAb #13523, LEF-1 (C12A5) rabbit mAb #2230, cleaved caspase-3 (Asp175) antibody #9661 and phospho-HP1γ (Ser83) Ab #2600.

Techniques: ChIP-qPCR, 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 following Western blot and ChIP-tested antibodies were purchased from Cell Signaling: tri-methyl-histone H3 (Lys9) (D4W1U) rabbit mAb #13969, phospho-Rpb1 CTD (Ser2) (E1Z3G) rabbit mAb #13499, phospho-Rpb1 CTD (Ser5) (D9N5I) rabbit mAb #13523, LEF-1 (C12A5) rabbit mAb #2230, cleaved caspase-3 (Asp175) antibody #9661 and phospho-HP1γ (Ser83) Ab #2600.

Techniques: Control, 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 following Western blot and ChIP-tested antibodies were purchased from Cell Signaling: tri-methyl-histone H3 (Lys9) (D4W1U) rabbit mAb #13969, phospho-Rpb1 CTD (Ser2) (E1Z3G) rabbit mAb #13499, phospho-Rpb1 CTD (Ser5) (D9N5I) rabbit mAb #13523, LEF-1 (C12A5) rabbit mAb #2230, cleaved caspase-3 (Asp175) antibody #9661 and phospho-HP1γ (Ser83) Ab #2600.

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 following Western blot and ChIP-tested antibodies were purchased from Cell Signaling: tri-methyl-histone H3 (Lys9) (D4W1U) rabbit mAb #13969, phospho-Rpb1 CTD (Ser2) (E1Z3G) rabbit mAb #13499, phospho-Rpb1 CTD (Ser5) (D9N5I) rabbit mAb #13523, LEF-1 (C12A5) rabbit mAb #2230, cleaved caspase-3 (Asp175) antibody #9661 and phospho-HP1γ (Ser83) Ab #2600.

Techniques: Western Blot, Expressing, Injection, In Vitro, Control

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 following Western blot and ChIP-tested antibodies were purchased from Cell Signaling: tri-methyl-histone H3 (Lys9) (D4W1U) rabbit mAb #13969, phospho-Rpb1 CTD (Ser2) (E1Z3G) rabbit mAb #13499, phospho-Rpb1 CTD (Ser5) (D9N5I) rabbit mAb #13523, LEF-1 (C12A5) rabbit mAb #2230, cleaved caspase-3 (Asp175) antibody #9661 and phospho-HP1γ (Ser83) Ab #2600.

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

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: BMC Cell Biology

Article Title: The histone methyltransferase SUV420H2 and Heterochromatin Proteins HP1 interact but show different dynamic behaviours

doi: 10.1186/1471-2121-10-41

Figure Lengend Snippet: Identification of SUV420H2 and HP1 interacting domains by in vitro GST-pull down . ( A ) The heterochromatic targeting module of SUV420H2 interacts with HP1 proteins. GST-tagged truncation SUV420H2 [347–435], as well as control GST-GFP fusion were bound to glutathione-Sepharose and incubated with nuclear extracts from HEK293 cells expressing TAP-tagged HP1α, HP1β, or HP1γ as indicated. After extensive washings, proteins were separated on SDS-PAGE and Western blot probed to reveal TAP-tagged proteins. ( B ) The chromoshadow domain of HP1γ is required for its interaction with SUV420H2. GST-SUV420H2 [347–435], as well as control GST-HP1γ fusions were bound to glutathione-Sepharose and incubated with nuclear extracts from HEK293 cells expressing TAP-HP1γ truncations or TAP-GFP control. HP1γ [ΔKKK] harbours a deletion from lysine 105 to lysine 107 within the hinge region, whereas HP1γ [ΔCSD] contains the 114 N-terminus amino-acids of HP1γ but not the chromoshadow domain. Loss of the chromoshadow domain abolishes the interaction with the heterochromatic targeting module of SUV420H2 and HP1γ oligomerization. ( C ) The chromoshadow domain (CSD) of HP1γ interacts with the SUV420H2 [347–435] region. GST-HP1γ [CSD] consisting of the HP1γ chromoshadow domain (AA 115–183), as well as control GST-HP1γ and GST-GFP fusions were bound to glutathione-Sepharose and incubated with in vitro translated TAP-SUV420H2 [347–435] fusion protein or TAP-GFP control. Bound TAP-tagged proteins are revealed by Western blotting using the peroxidase-anti-peroxidase (PAP) antibody (Sigma) which recognizes the protein A moiety of the TAP tag.

Article Snippet: The following primary antibodies were used for Western blotting: mouse anti-HP1α antibody (2HP-2G9, Euromedex; used at a dilution of 1:2 000), mouse anti-HP1β antibody (1MOD-1A9, Euromedex; used at a dilution of 1:2 000), mouse anti-HP1γ antibody (2MOD-1G6, Euromedex; used at a dilution of 1:2 000), goat anti-CBP antibody (sc-9456, Santa Cruz Biotechnology; used at a dilution of 1:300).

Techniques: In Vitro, Control, Incubation, Expressing, SDS Page, Western Blot

Journal: BMC Cell Biology

Article Title: The histone methyltransferase SUV420H2 and Heterochromatin Proteins HP1 interact but show different dynamic behaviours

doi: 10.1186/1471-2121-10-41

Figure Lengend Snippet: FRAP analysis of GFP-HP1 shows that HP1 proteins are dynamic components of heterochromatin . ( A ) Nuclear localization of GFP-tagged proteins. GFP-HP1 expressing L929 cells were fixed with paraformaldehyde and localization of fusion protein was visualized with fluorescence microscopy. Dense foci seen by Hoechst staining represent pericentric heterochromatin regions. ( B ) Detection of GFP-HP1 and endogenous HP1 isoforms in stable cell lines and in L929 cells by Western blotting using specific antibodies for each isoform (α-HP1β, α-HP1γ and α-HP1α). ( C ) Time-lapse series of confocal images of living cells expressing GFP-HP1β, GFP-HP1γ or GFP-HP1α, as indicated. Heterochromatic foci were selected and photobleached. Images were recorded before (Prebleach) and at different time intervals after the bleach. Arrows indicate the photobleached areas. ( B ) Parameters of HP1 protein dynamics from FRAP curves. The half-time of fluorescence recovery (t1/2), the percentage of fluorescence recovery after 30 s, and the number of FRAP curves used are indicated. ( E ) Representation of the number of cells characterized by a given t1/2 value. A single area was photobleached per cell.

Article Snippet: The following primary antibodies were used for Western blotting: mouse anti-HP1α antibody (2HP-2G9, Euromedex; used at a dilution of 1:2 000), mouse anti-HP1β antibody (1MOD-1A9, Euromedex; used at a dilution of 1:2 000), mouse anti-HP1γ antibody (2MOD-1G6, Euromedex; used at a dilution of 1:2 000), goat anti-CBP antibody (sc-9456, Santa Cruz Biotechnology; used at a dilution of 1:300).

Techniques: Expressing, Fluorescence, Microscopy, Staining, Stable Transfection, Western Blot

Journal: BMC Cell Biology

Article Title: The histone methyltransferase SUV420H2 and Heterochromatin Proteins HP1 interact but show different dynamic behaviours

doi: 10.1186/1471-2121-10-41

Figure Lengend Snippet: Dynamics of HP1 varies during cell cycle . Distribution of t1/2 values deduced from FRAP experiments for GFP-HP1β (upper panels), GFP-HP1γ (middle panels), and GFP-HP1α (lower panels) after serum starvation ( A ) or nocodazole treatment ( B ). The bars indicate the number of cells presenting a given t1/2 value. The effect of the treatments on cell synchronization is measured by FACS analysis (a-f). Nuclear localization of GFP-tagged HP1 proteins (G) and heterochromatin foci revealed by Hoechst staining (H) are shown. ( C ) Detection of GFP-HP1 and endogenous HP1 isoforms by Western blotting using specific antibodies for each isoform (α-HP1β, α-HP1γ and α-HP1α) in absence of treatment (Control), in presence of Nocodazole (50 ng/ml) 24 hours or after 72 hours serum starvation.

Article Snippet: The following primary antibodies were used for Western blotting: mouse anti-HP1α antibody (2HP-2G9, Euromedex; used at a dilution of 1:2 000), mouse anti-HP1β antibody (1MOD-1A9, Euromedex; used at a dilution of 1:2 000), mouse anti-HP1γ antibody (2MOD-1G6, Euromedex; used at a dilution of 1:2 000), goat anti-CBP antibody (sc-9456, Santa Cruz Biotechnology; used at a dilution of 1:300).

Techniques: Staining, Western Blot, Control

Journal: Radiation Oncology (London, England)

Article Title: Increased SHP-1 expression results in radioresistance, inhibition of cellular senescence, and cell cycle redistribution in nasopharyngeal carcinoma cells

doi: 10.1186/s13014-015-0445-1

Figure Lengend Snippet: Effects of SHP-1 knockdown in CNE-1 cells and overexpression in CNE-2 cells on cell senescence. a Cell senescence was determined by β-galactosidase staining (magnification: ×400). Arrows: senescent cells. Heterochromatin markers H3K9Me3 and HP1γ location and protein expression were determined by immunofluorescence staining ( b ) (magnification: ×400; red: Alexa Fluor® 568) and western blot ( c ), respectively. β-actin was used as an inner control. Data are shown as mean ± SD. ** P < 0.01, *** P < 0.001 vs. CNE-1 or CNE-2; ## P < 0.01, ### P < 0.001 vs. CNE-1- scramble shRNA or CNE-2-empty vector

Article Snippet: The following primary antibodies were used: rabbit anti-human polyclonal antibody against histone H3 trimethylated at lysine 9 (H3K9Me3, #07-442, 1:1,000, Millipore corp., Billerica, MA, USA) and mouse anti-human polyclonal antibody against heterochromatin protein-1γ (HP1γ, #MAB3450, 1:4,000, Millipore corp., Billerica, MA, USA).

Techniques: Knockdown, Over Expression, Staining, Expressing, Immunofluorescence, Western Blot, Control, shRNA, Plasmid Preparation