Journal: Journal of Clinical Investigation

Article Title: NLRC4 suppresses melanoma tumor progression independently of inflammasome activation

doi: 10.1172/jci86953

Figure Lengend Snippet: Figure 1. NLRC4 protects against B16F10 tumor growth in vivo. (A–F) WT and Nlrp6–/– (A), Nlrp12–/– (B), Nlrp3–/– (C), and Nlrc4–/– (D–F) mice were injected s.c. with 1 × 105 B16F10 cells. (A–C and E) Tumor mass was determined at 16 to 20 days after inoculation. (D) WT and Nlrc4–/– tumor areas (length × width) were measured every 2 to 3 days. (F) Representative images of excised WT and Nlrc4–/– B16F10 tumors. (A–E) Data are representative of 3 independent experiments with n = 5 mice per group. (A–C and E) *P ≤ 0.05, unpaired 2-tailed Student’s t test. (D) Error bars represent SEM. **P ≤ 0.01, ****P ≤ 0.0001, 2-way ANOVA with Šidák’s multiple comparisons test.

Article Snippet: Tissues were stained with mouse anti-human CD163 (catalog MCA1853T, ABD Serotech) and rabbit anti-human IPAF (NLRC4) antibody (catalog 3107, ProSci).

Techniques: In Vivo, Injection

Journal: Journal of Clinical Investigation

Article Title: NLRC4 suppresses melanoma tumor progression independently of inflammasome activation

doi: 10.1172/jci86953

Figure Lengend Snippet: Figure 2. Human primary melanoma is enriched with NLRC4+ macrophages. (A) Deidentified skin biopsies of normal skin (n = 3 patients), benign mela- nocytic nevi (n = 3 patients), primary invasive melanomas (n = 3 patients), and metastatic melanomas (n = 3 patients) were stained for CD163 (brown) and NLRC4 (brown). Images were taken with a ×40 objective. (B) The densities of five 20× high-power fields (each 3.14 mm2) were determined per patient sample. **P ≤ 0.01; ****P ≤ 0.0001, Mann–Whitney U test.

Article Snippet: Tissues were stained with mouse anti-human CD163 (catalog MCA1853T, ABD Serotech) and rabbit anti-human IPAF (NLRC4) antibody (catalog 3107, ProSci).

Techniques: Staining, MANN-WHITNEY

Journal: Journal of Clinical Investigation

Article Title: NLRC4 suppresses melanoma tumor progression independently of inflammasome activation

doi: 10.1172/jci86953

Figure Lengend Snippet: Figure 3. NLRC4-mediated protection against tumor growth is independent of inflammasome activation. (A–D) WT, Asc–/– (A and B), and Casp1–/– (C and D) mice were injected s.c. with 1 × 105 B16F10 cells. (A and C) Tumor area was measured every 2 to 3 days. (B and D) Tumor mass was determined at 17 to 20 days after inoculation. (A–D) Data are representative of 3 experiments with n ≥ 5 mice per group. (A and C) Error bars represent SEM. (B and D) Unpaired 2-tailed Student’s t test. (E) B16F10 tumors from WT and Nlrc4–/– mice on day 14 after inoculation were homogenized and immunoblotted with antibodies against caspase-1 and GAPDH. Each lane represents a tumor from an individual mouse. Data are representative of 2 independent experiments.

Article Snippet: Tissues were stained with mouse anti-human CD163 (catalog MCA1853T, ABD Serotech) and rabbit anti-human IPAF (NLRC4) antibody (catalog 3107, ProSci).

Techniques: Activation Assay, Injection

Journal: Journal of Clinical Investigation

Article Title: NLRC4 suppresses melanoma tumor progression independently of inflammasome activation

doi: 10.1172/jci86953

Figure Lengend Snippet: Figure 4. Expression of Nlrc4 in hematopoietic cells is important for controlling tumor growth. (A) Expression of Nlrc4 in BMDMs, fibroblasts, CD4+ T cells, CD8+ T cells, B16F10, and LLC cells was determined by qPCR and represented as expression relative to Nlrc4 expression in BMDMs. Data are represen- tative of 2 independent experiments. (B and C) WT and Nlrc4–/– mice were lethally irradiated and reconstituted with either WT or Nlrc4–/– BM (donor BM → recipient mouse). Mice were injected s.c. with 1 × 105 B16F10 cells. (B) Tumor area was measured every 2 to 3 days. (C) Tumor mass was determined at day 19 after inoculation. (B and C) Data are representative of 2 experiments with n ≥ 9 mice per experiment. (B) Error bars represent SEM. ##P ≤ 0.01 (for Nlrc4–/–

Article Snippet: Tissues were stained with mouse anti-human CD163 (catalog MCA1853T, ABD Serotech) and rabbit anti-human IPAF (NLRC4) antibody (catalog 3107, ProSci).

Techniques: Expressing, Irradiation, Injection

Journal: Journal of Clinical Investigation

Article Title: NLRC4 suppresses melanoma tumor progression independently of inflammasome activation

doi: 10.1172/jci86953

Figure Lengend Snippet: Figure 5. Absence of NLRC4 in macrophages alters the tumor cytokine and chemokine milieu. (A–F) WT and Nlrc4–/– mice were injected s.c. with 1 × 105 B16F10 cells. On day 12 after inoculation, total RNA was isolated from homogenized tumors and used to determine cytokine and chemokine expression via quantitative qPCR utilizing a PCR array. Selected genes from the array are displayed; data are pooled from 3 separate experiments (n = 3 mice per group). (G and H) WT and Nlrc4–/– mice were injected s.c. with 1 × 105 B16F10 cells; 14 days after inoculation, tumors were harvested, pooled, and FACS sorted based on CD45.2 and F4/80 staining. RNA was isolated from CD45.2- and CD45.2+F4/80+ cells and used to determine Cxcl9, Cxcl10, Cxcl13, and Cxcl16 expression by qPCR; data are representative of 2 independent experiments with n ≥ 5 pooled tumors per group. (I) WT and Nlrc4–/– BMDMs were challenged for 9 hours with B16F10 whole tumor homogenate. Cxcl9, Cxcl10, and Cxcl13 expression was determined by qPCR. Data are pooled from 3 independent experi- ments, and fold change in gene expression is relative to unstimulated samples. (J and K) WT and Nlrc4–/– BMDMs were challenged with 50 ng/ml LPS, 50 μg/ml LTA, 100 ng/ml FSL-1, and 1 μg/ml Pam3CSK4. Twenty hours later, supernatants were collected and levels of IL-6 (J) and IL-12p40 (K) determined by ELISA; data are representative of 3 independent experiments. (A–F and I) Error bars represent SEM. (J and K) Error bars represent SD. (I–K) *P ≤ 0.05, **P ≤ 0.01, and ***P ≤ 0.001, unpaired 2-tailed Student’s t test.

Article Snippet: Tissues were stained with mouse anti-human CD163 (catalog MCA1853T, ABD Serotech) and rabbit anti-human IPAF (NLRC4) antibody (catalog 3107, ProSci).

Techniques: Injection, Isolation, Expressing, Staining, Gene Expression, Enzyme-linked Immunosorbent Assay

Journal: Journal of Clinical Investigation

Article Title: NLRC4 suppresses melanoma tumor progression independently of inflammasome activation

doi: 10.1172/jci86953

Figure Lengend Snippet: Figure 6. NLRC4 regulates STAT3 and p38 MAPK signaling in the tumor microenvironment. (A and B) B16F10 tumors from WT and Nlrc4–/– mice at day 14 after inoculation were homogenized and immunoblotted for phospho-STAT3 and STAT3 (A), phospho-p38 MAPK and p38 MAPK (B), and GAPDH (A and B). Each lane represents a tumor from an individual mouse. (A and B) Densitometry of the ratio of phosphorylated to total protein is shown. (C) WT and Nlrc4–/– BMDMs were challenged for 4, 5, 6, 7, and 8 hours with 50 ng/ml LPS. Cell lysates were immunoblotted with antibodies against phospho-STAT3, STAT3, and GAPDH. (D) WT and Nlrc4–/– BMDMs were challenged for 15, 30, 60, and 90 minutes with 50 ng/ml LPS. Cell lysates were immunoblotted with antibodies against phospho-p38 MAPK, p38 MAPK, and GAPDH. (E) WT and Nlrc4–/– BMDMs were challenged for 5, 10, 15, 30, and 60 minutes with 10 ng/ ml recombinant IL-6. Cell lysates were immunoblotted with antibodies against phospho-STAT3, STAT3, and GAPDH. (C–E) Data are representative of 3 independent experiments. (A and B) *P ≤ 0.05, unpaired 2-tailed Student’s t test.

Article Snippet: Tissues were stained with mouse anti-human CD163 (catalog MCA1853T, ABD Serotech) and rabbit anti-human IPAF (NLRC4) antibody (catalog 3107, ProSci).

Techniques: Recombinant

Journal: Journal of Clinical Investigation

Article Title: NLRC4 suppresses melanoma tumor progression independently of inflammasome activation

doi: 10.1172/jci86953

Figure Lengend Snippet: Figure 7. NLRC4 is required for IFN-γ+ tumor-infiltrating effector CD4+ and CD8+ T cells. (A–I) WT and Nlrc4–/– mice were injected s.c. with 1 × 105 B16F10 cells. (A–D and F–I) On day 14 after inoculation, tumors were made into single-cell suspensions for flow cytometry; the CD45.2+ population was gated on and the frequency of Thy1.2+CD4+ T cells (A), Thy1.2+CD8+ T cells (B), F4/80+ macrophages (F), CD11c+MHCII+ dendritic cells (G), CD11b+GR1+ monocytes (H), and Foxp3+CD4+ regulatory T cells (I) determined. (C and D) Single-cell suspensions of tumor cells were stimulated with PMA and ionomycin, followed by intracellular cytokine staining for IFN-γ. Frequency of CD4+IFN-γ+ T cells (C) and CD8+IFN-γ+ T cells (D) was determined by flow cytometry. Data are representative of at least 3 independent experiments with n ≥ 3 mice per experiment. (E) IFN-γ gene expression in WT and Nlrc4–/– tumors on day 12 after inoculation was determined by qPCR; data are pooled from 3 independent experiments with n = 11 WT and n = 12 Nlrc4–/– mice. Error bars represent SEM. (A–I) *P ≤ 0.05, ***P ≤ 0.001, unpaired 2-tailed Student’s t test.

Article Snippet: Tissues were stained with mouse anti-human CD163 (catalog MCA1853T, ABD Serotech) and rabbit anti-human IPAF (NLRC4) antibody (catalog 3107, ProSci).

Techniques: Injection, Flow Cytometry, Staining, Gene Expression

Journal: Journal of Clinical Investigation

Article Title: NLRC4 suppresses melanoma tumor progression independently of inflammasome activation

doi: 10.1172/jci86953

Figure Lengend Snippet: Figure 8. Expression of NLRC4, and not caspase-1, in macrophages regulates B16F10 tumor growth. (A and B) Nlrc4–/– mice were challenged s.c. with 1 × 105 B16F10 cells alone, a mixture of 1 × 105 B16F10 cells and 5 × 104 WT BMDMs, or 1 × 105 B16F10 cells and 5 × 104 Nlrc4–/– BMDMs. Tumor area was measured every 2 to 3 days (A). Tumor mass was determined at 15 days after inoculation (B). Data are representative of 2 experiments with n = 4 mice per group (A) or pooled from 3 independent experiments (n = 12–15 mice per group; B). (C and D) Nlrc4–/– mice were challenged s.c. with 1 × 105 B16F10 cells alone or a mixture of 1 × 105 B16F10 cells and 5 × 104 Casp1–/– BMDMs. Tumor area was measured every 2 to 3 days (C) and tumor mass was determined at 18 days after inoculation (D). Data are representative of 2 experiments each with n = 6 mice per group (C) or pooled from 2 independent experiments (n = 12–13 mice per group; D). (A) Error bars represent SEM. *P ≤ 0.05 and **P ≤ 0.01 (for B16F10 plus WT BMDMs compared with B16F10 plus Nlrc4–/– BMDMs); #P ≤ 0.05 (for B16F10 compared with B16F10 plus WT BMDMs), 2-way ANOVA with Tukey’s HSD post-test for multiple comparisons. (B and D) *P ≤ 0.05, unpaired 2-tailed Student’s t test. (C) Error bars represent SEM. *P ≤ 0.05, 2-way ANOVA with Sidak’s multiple comparisons test.

Article Snippet: Tissues were stained with mouse anti-human CD163 (catalog MCA1853T, ABD Serotech) and rabbit anti-human IPAF (NLRC4) antibody (catalog 3107, ProSci).

Techniques: Expressing

Journal: Cellular & Molecular Biology Letters

Article Title: LncRNA DNM3OS regulates GREM2 via miR-127-5p to suppress early chondrogenic differentiation of rat mesenchymal stem cells under hypoxic conditions

doi: 10.1186/s11658-021-00269-6

Figure Lengend Snippet: Primer sequences used for RT-qPCR

Article Snippet: After finishing blocking in 0.1% BSA, the rMSCs were incubated with the primary antibody against GREM2 overnight at 4 °C, followed by incubation with CY3-conjugated goat anti-rabbit IgG (1:100 dilution; Boster Biological Technology) for 1 h at room temperature.

Techniques:

Journal: Cellular & Molecular Biology Letters

Article Title: LncRNA DNM3OS regulates GREM2 via miR-127-5p to suppress early chondrogenic differentiation of rat mesenchymal stem cells under hypoxic conditions

doi: 10.1186/s11658-021-00269-6

Figure Lengend Snippet: GREM2 was a target of miR-127-5p. A Nucleotide sequences of the predicted target site for miR-127-5p in the GREM2 3ʹ-UTR. B Luciferase reporter assays were performed to identify direct interaction between miR-127-5p and the GREM2 3ʹ-UTR. Wild type and mutant miR-127-5p target binding sequences in the GREM2 3ʹ-UTR were cloned into a reporter luciferase vector and co-transfected with the synthetized miR-127-5p (or NC) into rMSCs. *** p < 0.001, compared with NC; C GREM2 protein expression was measured in rMSCs from the control, induction, hypoxia, and hypoxia/induction groups. D GREM2 protein expression was measured in rMSCs from the control, induction, and si-SHP/hypoxia groups. E GREM2 protein expression was measured in rMSCs from the control, induction, and miR-127-5p inhibitor/induction groups. F GREM2 protein expression in rMSCs was measured by immunofluorescence. * p < 0.05, *** p < 0.001, compared with control; # p < 0.05, ### p < 0.001, compared with hypoxia or induction group

Article Snippet: After finishing blocking in 0.1% BSA, the rMSCs were incubated with the primary antibody against GREM2 overnight at 4 °C, followed by incubation with CY3-conjugated goat anti-rabbit IgG (1:100 dilution; Boster Biological Technology) for 1 h at room temperature.

Techniques: Luciferase, Mutagenesis, Binding Assay, Clone Assay, Plasmid Preparation, Transfection, Expressing, Control, Immunofluorescence

Journal: Cellular & Molecular Biology Letters

Article Title: LncRNA DNM3OS regulates GREM2 via miR-127-5p to suppress early chondrogenic differentiation of rat mesenchymal stem cells under hypoxic conditions

doi: 10.1186/s11658-021-00269-6

Figure Lengend Snippet: Overexpression of miR-127-5p promoted chondrogenic differentiation of rMSCs by regulating GREM2-mediated SMAD-dependent BMP signaling. The rMSCs were transfected with miR-127-5p mimics, followed by hypoxia intervention (1% O 2 ) and induction of chondrogenic differentiation for 14 days. A MiR-127-5p and GREM2 expression levels were determined by quantitative real-time PCR analysis. Results are expressed as mean ± standard deviation of data obtained from three independent experiments. *** p < 0.001, compared with control; ### p < 0.001, compared with induction group; p < 0.001, compared with hypoxia/induction; B Images from Alcian blue staining assays of rMSCs. C Levels of COL1A1, COL2A1, SOX9, and ACAN protein expression were detected in rMSCs. D Levels of GREM2, BMP2, p-SMAD1, and p-SMAD2 protein expression in rMSCs were detected by western blotting. E GREM2 protein expression in rMSCs was detected by immunofluorescence

Article Snippet: After finishing blocking in 0.1% BSA, the rMSCs were incubated with the primary antibody against GREM2 overnight at 4 °C, followed by incubation with CY3-conjugated goat anti-rabbit IgG (1:100 dilution; Boster Biological Technology) for 1 h at room temperature.

Techniques: Over Expression, Transfection, Expressing, Real-time Polymerase Chain Reaction, Standard Deviation, Control, Staining, Western Blot, Immunofluorescence

Journal: Cellular & Molecular Biology Letters

Article Title: LncRNA DNM3OS regulates GREM2 via miR-127-5p to suppress early chondrogenic differentiation of rat mesenchymal stem cells under hypoxic conditions

doi: 10.1186/s11658-021-00269-6

Figure Lengend Snippet: DNM3OS inhibited chondrogenic differentiation of rMSCs via miR-127-5p regulation of GREM2. The rMSCs were transfected with a DNM3OS overexpression plasmid, followed by induction of chondrogenic differentiation for 14 days. Accordingly, the rMSCs were classified into control, induction, hypoxia, hypoxia/induction, and DNM3OS/induction groups. A The putative binding site for miR-127-5p in the DNM3OS 3′-UTR is shown. B DNM3OS expression was determined by quantitative real-time PCR analysis. C A luciferase reporter assay was performed to identify direct action between DNM3OS and the GREM2 3ʹ-UTR. D Levels of DNM3OS, miR-127-5p, and GREM2 expression were determined by quantitative real-time PCR analysis. Results are expressed as mean ± standard deviation of data obtained from three independent experiments. ** p < 0.01, *** p < 0.001, compared with control; # p < 0.05, ### p < 0.001, compared with induction group; E Images from Alcian blue staining assays of rMSCs. (F) Levels of COL1A1, COL2A1, SOX9, and ACAN protein expression were detected in rMSCs. G Levels of GREM2, BMP2, p-SMAD1, and p-SMAD2 in rMSCs were detected by western blotting. H GREM2 protein expression in rMSCs was measured by immunofluorescence

Article Snippet: After finishing blocking in 0.1% BSA, the rMSCs were incubated with the primary antibody against GREM2 overnight at 4 °C, followed by incubation with CY3-conjugated goat anti-rabbit IgG (1:100 dilution; Boster Biological Technology) for 1 h at room temperature.

Techniques: Transfection, Over Expression, Plasmid Preparation, Control, Binding Assay, Expressing, Real-time Polymerase Chain Reaction, Luciferase, Reporter Assay, Standard Deviation, Staining, Western Blot, Immunofluorescence

Journal: Cellular & Molecular Biology Letters

Article Title: LncRNA DNM3OS regulates GREM2 via miR-127-5p to suppress early chondrogenic differentiation of rat mesenchymal stem cells under hypoxic conditions

doi: 10.1186/s11658-021-00269-6

Figure Lengend Snippet: DNM3OS knockdown weakened the inhibitory effect of hypoxia on chondrogenic induction. The rMSCs were transfected with si-DNM3OS, followed by induction of chondrogenic differentiation under hypoxic conditions (1% O 2 ) for 14 days. A Levels of miR-127-5p, DNM3OS, and GREM2 expression were determined by quantitative real-time PCR analysis. *** p < 0.001, compared with si-NC + hypoxia/induction group. B Levels of GREM2, BMP2, p-SMAD1, and p-SMAD2 in rMSCs were detected by western blotting. C Images from Alcian blue staining assays of rMSCs. (D) Levels of COL1A1, COL2A1, SOX9, and ACAN protein expression were detected in rMSCs. si-NC: negative control

Article Snippet: After finishing blocking in 0.1% BSA, the rMSCs were incubated with the primary antibody against GREM2 overnight at 4 °C, followed by incubation with CY3-conjugated goat anti-rabbit IgG (1:100 dilution; Boster Biological Technology) for 1 h at room temperature.

Techniques: Knockdown, Transfection, Expressing, Real-time Polymerase Chain Reaction, Western Blot, Staining, Negative Control