Journal: Nature

Article Title: STING inhibits the reactivation of dormant metastasis in lung adenocarcinoma

doi: 10.1038/s41586-023-05880-5

Figure Lengend Snippet: (a) Metastasis-free survival Kaplan-Meier plots of athymic mice intracardially inoculated with 1×105 H2030-BrM or H2087-LCC cells. One set of mice inoculated with H2087-LCC cells was treated with anti-asialo-GM1 antibody to deplete NK cells 30 days after cell inoculation. n=9 (H2030-BrM) or 10 (H2087-LCC) mice per group, log-rank test. (b) Flow cytometry analysis of Lineage−CD45+NKP46+ NK cells in blood from athymic mice that were treated with IgG control or anti-asialo-GM1 antibody for 4 days. Lineage− : TCRβ−CD3−CD19−B220−CD11c−Ly6G−F4/80−. n=3 mice per group. Mean ± s.e.m., two-sided unpaired t-test. (c) Petal charts of metastasis incidence in specific organs of 10 athymic mice intracardially inoculated with 1×105 H2087-LCC cells, treated with or without anti-asialo-GM1 antibody. Metastasis progression was monitored by BLI for 160 days after cell inoculation. (d) Petal charts of metastasis incidence in specific organs after intracardiac inoculation of 2×104 KPad1 cells into 8–9 B6-albino mice or NSG mice. Metastasis progression was monitored by BLI for 60 days (B6-albino mice) or 28 days (NSG mice) after cell inoculation. (e) Flow cytometry analysis of Lineage−CD45+NKP46+ NK cells, CD45+CD3+CD4+, and CD45+CD3+CD8+ T cells in blood from B6-albino mice that were treated for 3 days with IgG control, anti-NK1.1, anti-CD4, or anti-CD8 antibodies. n=3 mice per group. Mean ± s.e.m., two-sided unpaired t-test. (f) Metastasis-free survival Kaplan-Meier plots of NSG mice inoculated intracardially with KPad1 cells, or B6-albino mice inoculated intracardially with KPad1 cells or KP-482T1 cells, and treated with the indicated antibodies to deplete specific types of immune cells. 1×105 cells were inoculated per mouse. Antibody treatments started 12 days after inoculation. n=8 (KPad1/control, KPad1/αCD4), 7 (KPad1/αNK1.1, KPad1/αCD8, KP-482T1), or 9 (NSG) mice per group, log-rank test. *, p=0.03 (KPad1/αNK1.1 vs. KPad1/control), *, p=0.0146 (KPad1/αCD8 vs. KPad1/control), **, p=0.0094 (KPad1/αCD4 vs. KPad1/control), ***, p=0.0002 (KP-482T1 vs. KPad1/control), ****, p<0.0001 (KPad1/NSG vs. KPad1/control in B6-albino). (g) Gene set enrichment analysis (GSEA) showing pathways enriched in dormant H2087-LCC cells that were induced to proliferate in culture compared to cells from spontaneous metastatic outbreaks, analyzed from previous scRNA-seq data sets. (h-i) Metastasis-free survival of athymic mice (h) or B6-albino mice (i) intracardially inoculated with 2.5×105 H2087-LCC (h) or KPad1 cells (i), expressing a scrambled RNA control or various sgRNA pools. n=9 (Scrambled), 10 (NK ligands and MHC class I), 12 (STING pathway), 10 (LPS and RNA-sensing pathways), or 11 (Interferon response, Complement pathway) mice per group (h); n=7 (Scrambled, LPS and RNA-sensing pathways), 8 (Complement pathway), or 9 (other groups) mice per group (i). (j) BLI quantification of lung metastatic colonies after intravenous inoculation of 2.5×105 KPad1 cells expressing a scrambled control or different sgRNA pools in B6-albino mice. Tissues above the dotted line were harvested 57 days after cell inoculation, then subjected to sgRNA recovery and analysis. n=9 (Additional MHC class I) or 10 (other groups) mice per group. (k-l) Gene rank based on the average fold enrichment of sgRNAs in multi-organ metastases from H2087-LCC (k) or KPad1 (l) cells. (m) Gene rank based on average fold change of sgRNAs enriched in the lung colonies formed after tail-vein inoculation of 2.5×105 KPad1 cells into B6-albino mice. For k-m, genes with average log2FC >0 are plotted and genes targeted by the top 10 enriched sgRNAs are listed.

Article Snippet: Single-cell suspensions were then incubated with rat antibodies against mouse Ly6G, CD19, CD3ε, CD8α, CD4, and Ter-119 antibodies (Bio X Cell, clones 1A8, 1D3, 17A2, 2.43, GK1.5 and TER-119, respectively, 1:100), washed twice in PBS supplemented with 2% FBS, then incubated with BioMag goat anti-rat IgG beads (Qiagen, Cat# 310107, 5mg per spleen) to remove antibody-bound cells.

Techniques: In Vivo, CRISPR, Flow Cytometry, Control, Expressing

Journal: Nature

Article Title: STING inhibits the reactivation of dormant metastasis in lung adenocarcinoma

doi: 10.1038/s41586-023-05880-5

Figure Lengend Snippet: (a) Schematic of flow cytometry or immunofluorescence analysis of immune cells in bone metastasis. (b) Flow cytometry analysis and quantification of CD45+ leukocytes, CD45+CD19−TCRβ−XCR1+CD11c+ cDC1 cells, CD45+NK1.1+TCRβ−CD49b+NKp46+ NK cells, CD45+TCRβ+NK1.1−CD8+ T cells, and CD45+TCRβ+NK1.1−CD4+ T cells in KP-482T1 metastasis-bearing femurs after Dox-induced expression of STING for 2 days or 7 days. Cell numbers per gram of femur tissue were counted and normalized to control (-Dox). Metastasis-bearing femurs were collected 2 weeks after intracardiac inoculation of 2×104 KP-482T1 cells in B6-albino mice. n=7 mice per group. Mean ± s.e.m., two-sided unpaired t-test. p values comparing each Dox treatment group with control (-Dox). (c-f) Percentage of NK cells degranulating (CD107a+) (c) or producing IFNγ (d), and percentage of CD44+CD8+ T cells producing IFNγ (e) or TNF (f), isolated from KP-482T1 metastasis-bearing femurs after Dox-induced expression of STING for 2 days or 7 days, and then cultured ex vivo with phorbol 12-myristate 13-acetate (PMA) and ionomycin for 4h. n=5 (+Dox 7 days) or 6 (other groups) mice per group. Mean ± s.e.m. (g) Schematic of the NK cell-mediated killing assay, and the percentage of WT or STING KO H2087-LCC cells killed by incubation with naïve NK cells for 4h at the indicated effector:target ratios. n=3 per group. Mean ± s.e.m. (h) Schematic of the trans-well migration assay, and the number of NK cells migrated into cell culture media conditioned by WT or STING knockout H2087-LCC cells. n=3 per group. Mean ± s.e.m., two-sided unpaired t-test.

Article Snippet: Single-cell suspensions were then incubated with rat antibodies against mouse Ly6G, CD19, CD3ε, CD8α, CD4, and Ter-119 antibodies (Bio X Cell, clones 1A8, 1D3, 17A2, 2.43, GK1.5 and TER-119, respectively, 1:100), washed twice in PBS supplemented with 2% FBS, then incubated with BioMag goat anti-rat IgG beads (Qiagen, Cat# 310107, 5mg per spleen) to remove antibody-bound cells.

Techniques: Flow Cytometry, Immunofluorescence, Expressing, Control, Isolation, Cell Culture, Ex Vivo, Incubation, Migration, Knock-Out

Journal: Nature

Article Title: STING inhibits the reactivation of dormant metastasis in lung adenocarcinoma

doi: 10.1038/s41586-023-05880-5

Figure Lengend Snippet: (a) Rationale for experimental design. (b-c) qRT-PCR analysis of CXCL10 and CCL5 mRNA levels in WT or STING knockout KPad1 (b) or H2087-LCC (c) cells treated with 33 μM MSA-2 for 4h. Mean ± s.e.m., representative of 2 independent experiments. Each dot represents a technical replicate of the assay. (d-e) Treatment was performed as in (b, c) but with 10 μM (KPad1) or 50 μM (H2087-LCC) ADU-S100. Mean ± s.e.m., representative of 2 independent experiments. Each dot represents a technical replicate of the assay. (f-j) Metastasis-free survival plots of B6-albino mice intracardially inoculated with 2.5×105 KPad1 cells, treated with both MSA-2 and IgG control (f, n=10 mice) or individual antibodies to deplete NK cells (g, n=10 mice for vehicle or 8 mice for MSA-2), CD4+ T cells (h, n=10 mice), CD8+ T cells (i, n=10 mice), or a combination of these antibodies (j, n=10 mice for vehicle or 9 mice for MSA-2). Mice were administered antibodies (200 μg/mouse) once weekly for 3 weeks starting 6 days after cell inoculation, and vehicle or MSA-2 (50 mg/kg of body weight) once weekly for 2 weeks starting 9 days after cell inoculation. Log-rank test. (k) Metastasis-free survival plots of B6-albino mice intracardially inoculated with 2.5×105 KPad1 cells pre-treated with 33 μM MSA-2 for 24h before inoculation. n=10 mice per group. (l) Metastasis-free survival plots of C57BL/6J mice intravenously inoculated with 2.5×105 WT or Sting knockout KPad1 cells and treated with vehicle or ADU-S100 (1.25 mg/kg of body weight) once weekly by intratracheal delivery until the study endpoint. ADU-S100 treatment started 5 days after cell inoculation. n=15 mice (control) or 8 mice (ADU-S100). Log-rank test. (m) Quantification of WT or STING knockout H2087-LCC cell numbers in lungs from athymic mice treated with vehicle or ADU-S100 (6.25 mg/kg of body weight) once weekly for 4 weeks by intratracheal delivery. ADU-S100 treatment started 1 week after intravenous inoculation of 1×105 cells. Lungs were harvested 5 weeks after inoculation. n=5 mice per group. Mean ± s.e.m., two-sided unpaired t-test.

Article Snippet: Single-cell suspensions were then incubated with rat antibodies against mouse Ly6G, CD19, CD3ε, CD8α, CD4, and Ter-119 antibodies (Bio X Cell, clones 1A8, 1D3, 17A2, 2.43, GK1.5 and TER-119, respectively, 1:100), washed twice in PBS supplemented with 2% FBS, then incubated with BioMag goat anti-rat IgG beads (Qiagen, Cat# 310107, 5mg per spleen) to remove antibody-bound cells.

Techniques: Quantitative RT-PCR, Knock-Out, Control

Journal: Molecular and Cellular Biology

Article Title: Mutual Balance of Histone Deacetylases 1 and 2 and the Acetyl Reader ATAD2 Regulates the Level of Acetylation of Histone H4 on Nascent Chromatin of Human Cells

doi: 10.1128/mcb.00421-19

Figure Lengend Snippet: Figure 1. Deletion of HDAC1 is associated with a compensatory increase in the 914

Article Snippet: 425 426 Antibodies 427 Antibodies were as follows: mouse -HDAC1 Cat. No. 5356 (Cell Signaling); rabbit -428 HDAC1 Cat. No. NB100-56340SS (Novus Biologicals); mouse -HDAC2 Cat. No. 5113 (Cell 429 Signaling); rabbit -HDAC2 Cat. No. 57156 (Cell Signaling); -PCNA Cat. No. sc-56 (Santa 430 Cruz); -Nucleolin Cat. No. 396400 (Life Technologies); rabbit -ATAD2 Cat. No.61369 (Active 431 Motif); rabbit -ATAD2 Cat. No 23894-AP (Proteintech); -biotin (mouse) Cat. No. MB-9100 432 (Vector Laboratories); -biotin (rabbit) Cat. NoA150-109A (Bethyl); -H4K12ac Cat. No. 39165 433 on F ebruary 9, 2020 at V LU N D U LU B 2 http://m cb.asm .org/ D ow nloaded from (Active Motif); -H4K5ac Cat. No.39699 (Active Motif); -H3K9ac Cat. No. 9649 (Cell 434 Signaling); rabbit -BrdU Cat. No. GTX128091 (GeneTex); rat -BrdU/CldU Cat. No. NB500-435 169 (Novus Biologicals); mouse -BrdU/IdU Cat. No.347580 (BD Biosciences); mouse -436 RNA/DNA hybrid, clone S9.6 Cat. No. MABE1095 (Millipore-Sigma), rabbit -RNA/DNA hybrid, 437 clone S9.6 Cat. No. Ab01137-23.0 (Absolute Antibody); rabbit -RNAseH1 Cat. No. 15606-1-AP 438 (Proteintech), goat -V5-FITC Cat. No. A190-119F (Bethyl).

Techniques:

Journal: Molecular and Cellular Biology

Article Title: Mutual Balance of Histone Deacetylases 1 and 2 and the Acetyl Reader ATAD2 Regulates the Level of Acetylation of Histone H4 on Nascent Chromatin of Human Cells

doi: 10.1128/mcb.00421-19

Figure Lengend Snippet: Figure 5. Ectopic expression of HDAC2 reverses elevated HDAC1 and histone acetylation 1007

Article Snippet: 425 426 Antibodies 427 Antibodies were as follows: mouse -HDAC1 Cat. No. 5356 (Cell Signaling); rabbit -428 HDAC1 Cat. No. NB100-56340SS (Novus Biologicals); mouse -HDAC2 Cat. No. 5113 (Cell 429 Signaling); rabbit -HDAC2 Cat. No. 57156 (Cell Signaling); -PCNA Cat. No. sc-56 (Santa 430 Cruz); -Nucleolin Cat. No. 396400 (Life Technologies); rabbit -ATAD2 Cat. No.61369 (Active 431 Motif); rabbit -ATAD2 Cat. No 23894-AP (Proteintech); -biotin (mouse) Cat. No. MB-9100 432 (Vector Laboratories); -biotin (rabbit) Cat. NoA150-109A (Bethyl); -H4K12ac Cat. No. 39165 433 on F ebruary 9, 2020 at V LU N D U LU B 2 http://m cb.asm .org/ D ow nloaded from (Active Motif); -H4K5ac Cat. No.39699 (Active Motif); -H3K9ac Cat. No. 9649 (Cell 434 Signaling); rabbit -BrdU Cat. No. GTX128091 (GeneTex); rat -BrdU/CldU Cat. No. NB500-435 169 (Novus Biologicals); mouse -BrdU/IdU Cat. No.347580 (BD Biosciences); mouse -436 RNA/DNA hybrid, clone S9.6 Cat. No. MABE1095 (Millipore-Sigma), rabbit -RNA/DNA hybrid, 437 clone S9.6 Cat. No. Ab01137-23.0 (Absolute Antibody); rabbit -RNAseH1 Cat. No. 15606-1-AP 438 (Proteintech), goat -V5-FITC Cat. No. A190-119F (Bethyl).

Techniques: Expressing

Journal: Molecular and Cellular Biology

Article Title: Mutual Balance of Histone Deacetylases 1 and 2 and the Acetyl Reader ATAD2 Regulates the Level of Acetylation of Histone H4 on Nascent Chromatin of Human Cells

doi: 10.1128/mcb.00421-19

Figure Lengend Snippet: Figure 12. A working model of the relationship between HDAC1, HDAC2, ATAD2, 1147

Article Snippet: 425 426 Antibodies 427 Antibodies were as follows: mouse -HDAC1 Cat. No. 5356 (Cell Signaling); rabbit -428 HDAC1 Cat. No. NB100-56340SS (Novus Biologicals); mouse -HDAC2 Cat. No. 5113 (Cell 429 Signaling); rabbit -HDAC2 Cat. No. 57156 (Cell Signaling); -PCNA Cat. No. sc-56 (Santa 430 Cruz); -Nucleolin Cat. No. 396400 (Life Technologies); rabbit -ATAD2 Cat. No.61369 (Active 431 Motif); rabbit -ATAD2 Cat. No 23894-AP (Proteintech); -biotin (mouse) Cat. No. MB-9100 432 (Vector Laboratories); -biotin (rabbit) Cat. NoA150-109A (Bethyl); -H4K12ac Cat. No. 39165 433 on F ebruary 9, 2020 at V LU N D U LU B 2 http://m cb.asm .org/ D ow nloaded from (Active Motif); -H4K5ac Cat. No.39699 (Active Motif); -H3K9ac Cat. No. 9649 (Cell 434 Signaling); rabbit -BrdU Cat. No. GTX128091 (GeneTex); rat -BrdU/CldU Cat. No. NB500-435 169 (Novus Biologicals); mouse -BrdU/IdU Cat. No.347580 (BD Biosciences); mouse -436 RNA/DNA hybrid, clone S9.6 Cat. No. MABE1095 (Millipore-Sigma), rabbit -RNA/DNA hybrid, 437 clone S9.6 Cat. No. Ab01137-23.0 (Absolute Antibody); rabbit -RNAseH1 Cat. No. 15606-1-AP 438 (Proteintech), goat -V5-FITC Cat. No. A190-119F (Bethyl).

Techniques: