mouse anti rac 1  (Cytoskeleton Inc)

Journal: Journal for immunotherapy of cancer

Article Title: RAC2 inhibition enhances tumor sensitivity to NK cell-mediated cytotoxicity.

doi: 10.1136/jitc-2024-010931

Figure Lengend Snippet: Figure 1 NK killing stress induces high expression of RAC2 in tumor cells. (A) Pathway enrichment analysis on RNA sequencing result from NK killing resistance genes. (B) A schematic diagram illustrating the flow cytometry sorting procedure employed for the identification of NK cell resistance genes within the Rho GTPase family. (C) A schematic diagram illustrating 16 genes from five subfamilies of the Rho GTPase family. (D–E) q-PCR was used to detect the expression levels of CDC42 (D) and RAC2 (E) in HeLa cells following co-culture with NK-92MI cells. The P-sup group refers to the supernatant from PBMCs that were stimulated with anti-human CD3/CD28 monoclonal antibodies for 2 days. HeLa cells cultured with this supernatant served as a positive control. The HeLa-ctrl group was used as a control to account for any stress induced by the flow cytometry sorting process; this group consisted of HeLa cells individually cultured under the same flow cytometry sorting conditions as the experimental group. (F–G) HeLa cells treated with RAC1, RAC2, RAC3, RHOG, and CDC42 gene-specific siRNAs were used to detect cell death rate by FACS following co-culture with NK-92MI cells at 6 hours. The results are representative of at least three independent experiments. Vertical bars indicate mean±SEM. Statistical significance between groups was assessed using the one-way analysis of variance with Bonferroni’s post-test, *p<0.05; **p<0.01; ***p<0.001. ctrl, control; E:T, effector-to-target; CFSE, Carboxyfluorescein succinimidyl ester; FACS, fluorescence-activated cell sorting; h, hours; NK, natural killer; PBMCs, peripheral blood mononuclear cells; q-PCR, quantitative PCR; Rho, Ras homology; RFU, relative fluorescence units; siRNA, small interfering RNA.

Article Snippet: The antibodies used for Western blot analysis included Anti- RAC2 (Santa Cruz Biotechnology, sc- 517424), AntiFlag- HRP (Sigma- Aldrich, sc- F1804), and anti-β-ACTIN (Santa Cruz Biotechnology, sc- 47778).

Techniques: Expressing, RNA Sequencing, Flow Cytometry, Co-Culture Assay, Bioprocessing, Cell Culture, Positive Control, Control, Fluorescence, FACS, Real-time Polymerase Chain Reaction, Small Interfering RNA

Journal: Journal for immunotherapy of cancer

Article Title: RAC2 inhibition enhances tumor sensitivity to NK cell-mediated cytotoxicity.

doi: 10.1136/jitc-2024-010931

Figure Lengend Snippet: Figure 2 RAC2 expression in tumor cells promotes tumor resistance to NK cytotoxicity. (A) Western blot and q-PCR were used to assess the efficiency of RAC2 knockout or knockdown in HeLa-sgRAC2 and HeLa-shRAC2 cell lines. (B) NK-92MI cells were co-cultured with Hela, Hela-sgRAC2, and Hela-shRAC2 cells, while the K562 co-culture group served as the positive control. The rate of tumor cell death following co-culture was assessed by FACS. (C) HeLa WT and HeLa-sgRAC2 cells, were co- cultured with primary human NK cells, and the rate of tumor cell death following co-culture was assessed by FACS. (D) Western blot and q-PCR were used to verify the knockout or knockdown efficiency of Rac2 in EL4-sgRac2 and EL4-shRac2 cell lines. (E) EL4, EL4-sgRac2, EL4-shRac2 cells were co-cultured with primary mouse NK cells, and the rate of tumor cell death after co-culture was assessed using FACS, with the EL4-shβ2m co-culture group serving as the positive control. (F) Western blot was used to verify the inhibition efficiency of RAC2 in EL4 cells following treatment with EHT-1864. Additionally, the cell death rate in tumor cells was evaluated using FACS after co-culture with primary mouse NK cells. The results are representative of at least three independent experiments. Vertical bars indicate mean±SEM. Statistical significance between groups was assessed using the one-way analysis of variance with Bonferroni’s post-test, *p<0.05; **p<0.01; ***p<0.001. CFSE, Carboxyfluorescein succinimidyl ester; E:T, effector-to-target; FACS, fluorescence-activated cell sorting; h, hours; NK, natural killer; q-PCR, quantitative PCR; WT, wild type.

Article Snippet: The antibodies used for Western blot analysis included Anti- RAC2 (Santa Cruz Biotechnology, sc- 517424), AntiFlag- HRP (Sigma- Aldrich, sc- F1804), and anti-β-ACTIN (Santa Cruz Biotechnology, sc- 47778).

Techniques: Expressing, Western Blot, Knock-Out, Knockdown, Cell Culture, Co-Culture Assay, Positive Control, Inhibition, Fluorescence, FACS, Real-time Polymerase Chain Reaction

Journal: Journal for immunotherapy of cancer

Article Title: RAC2 inhibition enhances tumor sensitivity to NK cell-mediated cytotoxicity.

doi: 10.1136/jitc-2024-010931

Figure Lengend Snippet: Figure 3 NK cells mediate the growth inhibition of RAC2-deficient tumors. (A–B) C57BL/6 (A) mice and BALB/c-nude (B) mice were injected subcutaneously with EL4 and EL4-sgRac2 cells, n=5 per group, and tumor growth of the mice was monitored every other day. (C) EL4, EL4-sgRac2 cells were subcutaneously inoculated on C57BL/6 mice with or without anti-NK1.1 mAb treatment (n=5 per group), and tumor growth of the mice was monitored every other day. Tumor growth curves were analyzed by two-way analysis of variance with Bonferroni’s post-test, ns, not significant; *p<0.05; **p<0.01; ***p<0.001. i.p., intraperitoneal injection; mAb, monoclonal antibody; ko, knockout; NK, natural killer; PBS, phosphate-buffered saline; wt, wild type.

Article Snippet: The antibodies used for Western blot analysis included Anti- RAC2 (Santa Cruz Biotechnology, sc- 517424), AntiFlag- HRP (Sigma- Aldrich, sc- F1804), and anti-β-ACTIN (Santa Cruz Biotechnology, sc- 47778).

Techniques: Inhibition, Injection, Knock-Out, Saline

Journal: Journal for immunotherapy of cancer

Article Title: RAC2 inhibition enhances tumor sensitivity to NK cell-mediated cytotoxicity.

doi: 10.1136/jitc-2024-010931

Figure Lengend Snippet: Figure 4 RAC2 knockout in human colon cancer cells enhanced the efficacy of adoptive NK therapy. (A–C) Western blot was used to verify the RAC2 knockout efficiency. While the rate of tumor cell death after co-culture with NK-92MI cells was determined by FACS in SW620 (A), HCT116 (B) and SW480 cells (C). (D) A schematic illustration depicting the in vivo xenograft mouse model of human HCT116 colon cancer cells used for the NK cell therapy with NK-92MI cells infusion. (E) HCT116- sgScr and sgRAC2 cells were s.c. inoculated into NOD/SCID mouse (n=12). When the tumor volume reached 100 mm3, six mice from each group received an adoptive transfer of NK-92MI cells or PBS via the tail vein. Tumor growth of the mice was monitored every other day. (F) Tumors were isolated, and tumor-infiltrating NK-92MI (human-CD45+ CD56+) cells were analyzed by flow cytometry (n=3). (G) Cell death within the tumor tissues from RAC2-WT/KO tumor-bearing mice was analyzed using the TUNEL assay following treatment with NK-92MI cells (n=3). Scale bars, 50 µm. The results are representative of at least three independent experiments. Vertical bars indicate mean±SEM. Tumor growth curves were analyzed by two-way analysis of variance with Bonferroni’s post-test. Statistical significance between groups was assessed using the one-way analysis of variance with Bonferroni’s post-test, or two-tailed unpaired t-test, ns, not significant; *p<0.05; **p<0.01; ***p<0.001. CFSE, carboxyfluorescein succinimidyl ester; DAPI, 4′,6-diamidino-2-phenylindole; FACS, fluorescence-activated cell sorting; i.v., intravenous; NK, natural killer; PBS, phosphate-buffered saline; s.c., subcutaneous; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling.

Article Snippet: The antibodies used for Western blot analysis included Anti- RAC2 (Santa Cruz Biotechnology, sc- 517424), AntiFlag- HRP (Sigma- Aldrich, sc- F1804), and anti-β-ACTIN (Santa Cruz Biotechnology, sc- 47778).

Techniques: Knock-Out, Western Blot, Co-Culture Assay, In Vivo, Adoptive Transfer Assay, Isolation, Flow Cytometry, TUNEL Assay, Two Tailed Test, Fluorescence, FACS, Saline

Journal: Journal for immunotherapy of cancer

Article Title: RAC2 inhibition enhances tumor sensitivity to NK cell-mediated cytotoxicity.

doi: 10.1136/jitc-2024-010931

Figure Lengend Snippet: Figure 5 RAC2 ablation increases tumor sensitivity to NK cytotoxicity depending on cell-cell contact. (A–B) EL4 and EL4- sgRac2 cells were co-cultured with primary mouse NK cells, HeLa and HeLa-sgRAC2 cells were co-cultured with NK-92MI cells, with (A) or without (B) transwell chamber method, and tumor cell death rate was determined by FACS. (C–D) Confocal microscopy was used to observe the cytoskeletal morphology and F-actin fluorescence intensity of EL4, EL4-sgRac2 and EL4-shRac2 cells (n=8) (C) and SW620-sgScr, SW620-sgRAC2-1 and SW620-sgRAC2-2 cells (n=10) (D). Scale bars, 5 µm. (E–F) Confocal microscopy was used to observe the Hela and Hela-sgRAC2 cells (spindle cells) co-cultured with NK-92MI cells (round cells), representative immunofluorescent images (E) and quantitative analysis (F) for F-actin fluorescence intensity in HeLa, HeLa-sgRAC2 cells (n=11). Scale bars, 5 µm. (G) The ratio of the F-actin fluorescence intensity at the effector–target conjugate interface to the total fluorescence intensity of the unconjugated membranes. The results are representative of at least three independent experiments. Vertical bars indicate mean±SEM. Statistical significance between groups was assessed using the two-tailed t-test, or one-way analysis of variance with Bonferroni’s post-test, ns, not significant; *p<0.05; **p<0.01; ***p<0.001. DAPI, 4′,6-diamidino-2-phenylindole; FACS, fluorescence-activated cell sorting; MFI, mean fluorescence intensity; NK, natural killer; WT, wild type.

Article Snippet: The antibodies used for Western blot analysis included Anti- RAC2 (Santa Cruz Biotechnology, sc- 517424), AntiFlag- HRP (Sigma- Aldrich, sc- F1804), and anti-β-ACTIN (Santa Cruz Biotechnology, sc- 47778).

Techniques: Cell Culture, Confocal Microscopy, Fluorescence, Two Tailed Test, FACS

Journal: Journal for immunotherapy of cancer

Article Title: RAC2 inhibition enhances tumor sensitivity to NK cell-mediated cytotoxicity.

doi: 10.1136/jitc-2024-010931

Figure Lengend Snippet: Figure 6 Aspartic acid 148 residue of RAC2 is critical for IS formation and NK killing sensitivity of tumor cells. (A) Schematic re-presentation of the Rac2 truncation and mutation construction, with the truncated or mutated sites in red. (B–D) Western blot showed the construct expression in EL4 cells, EL4-sgRac2 and EL4-sgRac2+RAC2 WT/CAAX-/D148E/D150G cells (B), and tumor cells were co-cultured with primary mouse NK cells, and the tumor cell death rate was determined by FACS (C). Confocal microscopy was used to observe the cytoskeletal morphology and F-actin fluorescence intensity in EL4, EL4- sgRac2, EL4-sgRac2+RAC2 WT and EL4-sgRac2+RAC2-D148G (n=5) (D). Scale bars, 10 µm. (E–F) Kaplan-Meier analysis of RAC2 expression in relation to clinical prognosis in patients with solid tumors, specifically COAD (E), and hematological malignancies, such as AML (F), as derived from TCGA dataset. The p value was calculated using the log-rank test. The results are representative of at least three independent experiments. Vertical bars indicate mean±SEM. Statistical significance between groups was assessed using the one-way analysis of variance with Bonferroni’s post-test, ns, not significant; *p<0.05; **p<0.01; ***p<0.001. AML, acute myeloid leukemia; COAD, colon adenocarcinoma; CFSE, carboxyfluorescein succinimidyl ester; DAPI, 4′,6-diamidino-2-phenylindole; FACS, fluorescence-activated cell sorting; IS, immunological synapse; MFI, mean fluorescence intensity; NK, natural killer; TCGA, The Cancer Genome Atlas; WT, wild type.

Article Snippet: The antibodies used for Western blot analysis included Anti- RAC2 (Santa Cruz Biotechnology, sc- 517424), AntiFlag- HRP (Sigma- Aldrich, sc- F1804), and anti-β-ACTIN (Santa Cruz Biotechnology, sc- 47778).

Techniques: Residue, Mutagenesis, Western Blot, Construct, Expressing, Cell Culture, Confocal Microscopy, Fluorescence, Derivative Assay, FACS