EMBO/SourceData huggingface dataset:
Figure 2(A) Bim−/− and Bim+/+ MEFs were cultured in 6-well dishes. Cells were treated with vehicle or Bafilomycin A1 (Baf) for 4 hr. Blots were probed with the indicated antibodies. LC3-II/tubulin in wild-type control MEFs is set as 1. The relative value of LC3-II/tubulin in Bim-knockout MEFs is shown (n = 5). Data are mean ± SD. ∗∗∗p < 0.001.(B) HeLa cells were transfected with control siRNA and Bim siRNA. After 48 hr, cells were treated with vehicle or Baf for 4 hr. Blots were probed with the indicated antibodies and analyzed as described in (A). ∗∗∗p < 0.001.(C) Bim−/− and Bim+/+ MEFs were transfected with GFP-LC3. The percentages of cells with GFP-LC3 vesicles were assessed. Data are shown as mean ± SD. ∗∗p < 0.01.(D) HeLa cells were transfected with control plasmid (pMKO)/GFP-LC3 or pMKO-BimshRNA/GFP-LC3 (to ensure that GFP-LC3-positive cells also contain BimshRNA or control plasmid, the ratio of pMKO or pMKO-BimshRNA/GFP-LC3 is 3:1). The percentages of cells with GFP-LC3 vesicles were assessed. Data are shown as mean ± SD. ∗∗∗p < 0.001.(E) HeLa cells were transfected with control plasmid (pMKO) or pMKO-BimshRNA. Blots were probed with the indicated antibodies. Data are shown as mean ± SD. ∗∗∗p < 0.001.(F) HeLa cells were transfected with control plasmid (pMKO)/GFP-PX or pMKO-BimshRNA/GFP-PX (3:1). The percentages of cells with GFP-vesicles were assessed. Data are shown as mean ± SD. ∗∗p < 0.01.(G) DFCP1-GFP cells were transfected with control siRNA or Bim siRNA. The numbers of GFP puncta were assessed. Data are shown as mean ± SD.∗p < 0.05.Images represent DFCP1-GFP vesicles in cells transfected with control siRNA and Bim siRNA. Arrows mark DFCP1-GFP puncta.(H) HeLa cells were treated with control siRNA or Bim siRNA. Blots were probed with the indicated antibodies. Data are shown as mean ± SD. ∗∗p < 0.01.(I) Bim(EL)/control siRNA, Bim(EL)/Bim siRNA, Bim(EL)EE/control siRNA, Bim(EL)EE/Bim siRNA, vector/control siRNA, and vector/Bim siRNA were transfected into HeLa cells. Blots were probed with the indicated antibodies. Data are shown as mean ± SD. ∗∗p < 0.01.(J) HeLa cells were transfected with control plasmid or Bim(EL)EE. Blots were probed with the indicated antibodies. Data are shown as mean ± SD. ∗∗p < 0.01.(K) Bax/Bak DKO MEFs were transfected with control plasmid /GFP-LC3, wild-type Bim(EL)/GFP-LC3 (3:1), or Bim (EL) EE/GFP-LC3. The percentages of cells with GFP-LC3 vesicles were assessed. Data are shown as mean ± SD. ∗∗p < 0.01.(L) Two independent control vector (pcDNA3) or Bim(EL)EE stably expressing HeLa cell clones were transfected with GFP-LC3 respectively. The percentages of cells with GFP-LC3 vesicles were assessed. Data are shown as mean ± SD. ∗∗∗p < 0.001.(M) HeLa cells were transfected with control plasmid/GFP-PX or Bim(EL)EE/GFP-PX (3:1). The percentages of cells with GFP-vesicles were assessed. Data are shown as mean ± SD. ∗∗p < 0.01. Fluorescent microscope images show GFP-PX vesicles.
Figure 3(A) Lysates from spleen-derived T cells of wild-type (Bim+/+) and Bim-knockout (Bim−/−) mice were immunoblotted and probed with indicated antibodies. Statistics were from three knockout and control mice. Data are shown as mean ± SD. ∗∗∗p < 0.001.(B) T cells from Bim+/+ and Bim−/− mice were cultured with/without Baf for 4 hr preceding harvest, immunoblotted, and probed with the indicated antibodies.(C) Spleen-derived T cells from Bim+/+ or Bim−/− mice were stained with rabbit anti-LC3 antibody. Representative confocal images and quantification from triplicates (n = 100-110) are shown. Data are shown as mean ± SD. ∗∗∗p < 0.0001.(D) Bim+/+ and Bim−/− mouse liver lysates were immunoblotted and probed with the indicated antibodies (n = 3). Data are shown as mean ± SD. ∗∗p < 0.01.(E) Bim+/+ and Bim−/− mouse liver lysates were immunoblotted and probed with the indicated antibodies (n = 3). Data are shown as mean ± SD. ∗∗∗p < 0.001.(F) RNA from Bim wild-type or Bim-knockout mouse livers was analyzed by qRT-PCR for p62/beta-actin mRNA. The mean ± SD from three mice per group is shown. NS, not significant.
Figure 4(A) HeLa cells were treated with control siRNA, Bim siRNA + control siRNA, control siRNA + Beclin 1 siRNA, or Bim siRNA + Beclin 1 siRNA. Blots were probed as indicated. Data are shown as mean ± SD. ∗∗p < 0.01.(B) HeLa cells were treated with control siRNA, Bim siRNA + control siRNA, control siRNA + Beclin 1 siRNA, or Bim siRNA + Beclin 1 siRNA. After 24 hr, cells were transfected with GFP-PX for 24 hr. GFP-PX vesicles were assessed with a confocal microscope. Data are shown as mean ± SD. ∗∗p < 0.01. Arrows label cells with increased number/size of GFP-PX vesicles.(C) GFP-mRFP-LC3 HeLa cells were treated with control, Bim siRNA, or Bcl-2 siRNA and then analyzed with Cellomics microscope. Data are shown as mean ± SD. ∗∗∗p < 0.001.(D) GFP-mRFP-LC3 stable HeLa cells were treated with control siRNA, Bim siRNA + control siRNA, control siRNA + Beclin 1 siRNA, or Bim siRNA + Beclin 1 siRNA and then analyzed with Cellomics microscope and matching confocal images are shown. Data are shown as mean ± SD. ∗∗∗p < 0.001.
Figure 5(A) Bim(EL)EE/empty vector (IP negative control) or Bim(EL)EE/Beclin 1-Flag (in duplicate) were transfected into HeLa cells. After 20 hr, one set of cells with Bim(EL)EE/Beclin 1-Flag were starved in HBSS for 2 hr. Anti-Flag antibody (M2) was used for immunoprecipitation, and blots were probed as indicated. Data are shown as mean ± SD. ∗∗∗p < 0.001.(B) Panel i: HeLa cells were treated with control siRNA or Bim siRNA. After 48 hr, one set of transfections were starved in HBSS for 2 hr. Blots were probed as indicated. LC3-II/tubulin ratio of siRNA-transfected cells is set as 1 (n = 3). Data are shown as mean ± SD. ∗∗∗p < 0.001; NS, not significant. Panel ii: HeLa cells were treated with control siRNA or Bim siRNA. After 48 hr, one set of transfections were treated with Baf for 2 hr; one set of transfections were starved and treated with Baf for 2 hr. Blots were probed as indicated and analyzed as in (Bi). ∗∗p < 0.01; NS, not significant.(C) HeLa cells were transfected with control plasmid /GFP-LC3 (3:1), or Bim(EL)EE/GFP-LC3 (3:1). After 20 hr, one set of transfected cells were starved in HBSS for 2 hr and then fixed. The percentages of cells with GFP-LC3 vesicles were assessed. Data are shown as mean ± SD. ∗∗p < 0.01. NS, not significant.(D) BimS-Beclin 1 binding is not sensitive to HBSS starvation. BimEL-EE (EL)/vector (IP negative control), BimEL-EE (EL)/Beclin 1-Flag (two replicates), BimL-EE (L)/Beclin 1-Flag (two replicates) or BimS-EE (S)/Beclin 1-Flag (two replicates) were transfected into HeLa cells. After 20 hr, one of EL-Beclin 1-Flag transfections, one of L-Beclin-Flag transfections and one of S-Beclin 1-Flag transfections were subjected to 2 hr HBSS starvation. Anti-Flag (M2) was used for immunoprecipitation.(E) HBSS starvation increases Bim T116 phosphorylation. Bim(EL)EE or BimEE(EL)-T116A were transfected into HeLa cells. After 20 hr, one set of Bim(EL)EE transfections and one of Bim(EL)EE-T116A were starved for 2 hr in HBSS. Blots were probed with the indicated antibodies.(F) Bim T116 phosphorylation disables Bim-Beclin 1 interaction. Bim(EL)EE /vector (IP negative control), Bim(EL)EE/Beclin 1-Flag (in duplicate), or Bim(EL)EE-T116E (phospho-mimic, designated as T116E here)/Beclin-Flag (in duplicate) were transfected into HeLa cells. After 20 hr, one set of Bim(EL)EE/Beclin 1-Flag transfections and one of Bim(EL)EE-T116E/Beclin 1-Flag transfections were starved for 2 hr in HBSS (Starv). Anti-Flag (M2) was used for immunoprecipitation.(G) HeLa cells were transfected with control plasmid /GFP-LC3, Bim(EL)-EE/GFP-LC3 (3:1), or Bim(EL)-EE T116E/GFP-LC3 (3:1). After 20 hr, one set of transfected cells were starved in HBSS for 2 hr and the cells were then fixed. The percentages of cells with GFP-LC3 vesicles were assessed. Data are shown as mean ± SD. ∗∗p < 0.01; NS, not significant.
Figure 6(A) Bim-147 does not bind to Beclin 1. Beclin 1-Flag/vector (IP negative control), Beclin 1-Flag/Myc-BimEL-1-147aa (Myc-147), or Beclin 1-Flag/Myc-BimEL-ΔBH3 (Myc-ΔBH3) were transfected into HeLa cells. Anti-Myc (Rabbit) was used for immunoprecipitation.(B) Bim-147 is competent for LC8 binding. LC8/vector (IP negative control), LC8/Myc-BimEL-1-147aa (Myc-147), or LC8/Myc-BimEL-ΔBH3 (Myc-ΔBH3) were transfected into HeLa cells. Anti-Myc (Rabbit) was used for immunoprecipitation.(C) Mutating LC8 binding consensus sites within Bim disrupts the Bim-LC8 interaction. Bim(EL)EE/vector (IP negative control), Bim(EL)EE/Myc-LC8, Bim(EL)EE-S109A, S113A, T114A (AAA)/ Myc-LC8, or Bim(EL)EE-T116E/ Myc-LC8 were transfected into HeLa cells. Anti-Myc (Rabbit) was used for immunoprecipitation. Data are shown as mean ± SD. ∗∗∗p < 0.001.(D) Mutating LC8 binding consensus sites within Bim disrupts the Bim-Beclin 1 interaction. Bim(EL)EE/vector (IP negative control), Bim(EL)EE/Beclin 1-Flag, Bim(EL)EE-S109A, S113A, T114A (AAA)/Beclin 1-Flag, or Bim(EL)EE-T116E/Beclin 1-Flag were transfected into HeLa cells. Anti-Flag (M2) was used for immunoprecipitation. Data are shown as mean ± SD. ∗∗∗p < 0.001.(E) Bim-S does not bind to LC8. BimEL-EE (EL)/vector (IP negative control), BimEL-EE (EL)/Myc-LC8, BimL-EE (L)/Myc-LC8, or BimS-EE (S)/Myc-LC8 were transfected into HeLa cells. Anti-Myc (Rabbit) was used for immunoprecipitation.(G) LC8 enhances Bim-Beclin 1 interaction in vitro. His-Bim, His-LC8 and GST-Beclin 1 were expressed in BL21(DE3) E. coli and purified. Two micrograms of His-Bim was combined with 2 μg GST without or with 1 μg His-LC8 (as controls); 2 μg His-Bim was combined with 2 μg GST-Beclin 1 without or with 1 μg His-LC8. The mixtures were incubated in buffer A for 3 hr. Glutathione beads were then used to pull down GST or GST-Beclin 1. The pulldown products were detected with anti-Bim and anti-Beclin 1.(H) LC8 siRNA knockdown reduces the Bim-Beclin 1 interaction. Control siRNA or LC8 siRNA were transfected into HeLa cells. After 48 hr, HA-Bim(EL)EE/vector (IP negative control) or HA-Bim(EL)EE/Beclin 1-Flag was transfected into control siRNA transfected HeLa cells; HA-Bim(EL)EE/Beclin 1-Flag was transfected into LC8 siRNA-transfected HeLa cells. Anti-Flag (M2) was used for immunoprecipitation. Note that in the presence of BimEE, endogenous LC8 was pulled down by Beclin 1 when cells were treated with control siRNA. Data are shown as mean ± SD. ∗∗∗p < 0.001.
Figure 7(A) HeLa cells were transfected with vector (vec) /GFP-LC3, BimEL-EE (EL)/GFP-LC3, BimL-EE (L)/GFP-LC3, BimS-EE (S)/GFP-LC3, or BimEL-EE-AAA (AAA)/GFP-LC3 (3:1). The percentages of cells with GFP-LC3 vesicles were assessed. Data are shown as mean ± SD. ∗∗∗p < 0.001; ∗∗p < 0.01; NS, not significant. Note that BimEL-EE-S109A, S113A, T114A is designated as BimEL-EE-AAA.(B) Bim bridges the Beclin 1-LC8 interaction. Dynein light chain1 (Myc-LC8)/empty vectors (IP negative control), Myc-LC8/Beclin 1-Flag/empty vector, Myc-LC8/Beclin 1-Flag/BimEL-EE (EL), Myc-LC8/Beclin 1-Flag/BimL-EE (L), Myc-LC8/Beclin 1-Flag/BimS-EE (S), and Myc-LC8/Beclin 1-Flag/BimEL-EE-S109A, S113A, T114A (AAA) were transfected into HeLa cells. Anti-Flag (M2) was used for immunoprecipitation.(C) Starvation reduces the ability of Bim to bridge the Beclin 1-LC8 interaction. Myc-LC8/empty vectors (IP negative control), Myc-LC8/Beclin 1-Myc/empty vector, or Myc-LC8/Beclin 1-Flag/HA-BimEL-EE (HA-Bim) (two replicates) were transfected into HeLa cells. After 20 hr, one set of cells with Myc-LC8/Beclin 1-Flag/HA-HA-Bim was starved in HBSS for 2 hr. Anti-Flag (M2) antibody was used for immunoprecipitation.(D) HeLa cells were cultured with DMEM with 10% serum (Control) or starved (Starve) in HBSS for 2 hr. The fixed cells were stained with Beclin 1 and tubulin antibodies and analyzed by confocal microscopy. White boxes show the areas where Beclin 1 is enriched. Yellow boxes show enlarged areas.(E) HeLa cells were treated with control siRNA or LC8 siRNA. After 24 hr, cells were split. Vector/GFP-LC3 or Bim(EL)EE/GFP-LC3 (3:1) were transfected into the control siRNA-transfected or LC8 siRNA-transfected cells. The percentages of cells with GFP-LC3 vesicles were assessed. Data are shown as mean ± SD. ∗∗p < 0.01; NS, not significant.(F) HeLa cells were treated with control siRNA, Bim siRNA, or LC8 siRNA for 48 hr. Cells were then fixed in 37°C, 4% PFA for 10 min. Cells were stained with Beclin 1 and tubulin antibodies and analyzed by confocal microscopy. White boxes/triangle show areas where Beclin 1 is enriched. Yellow boxes show enlarged areas. Colocalizations were quantified from images in 12-15 cells with Volocity program (Colocalization coefficient Mx). Data are shown as mean ± SD. ∗∗∗p < 0.001.
Figure 2 (C and D) Distant organ metastasis was examined by bioluminscent images of 4T1 tumor-bearing mice after tumor resection with (n=13 mice) or without (n=10 mice) tumor-draining lymph node dissection on week 5. The bioluminescent signal (pseudocolor) was recorded as photons per second per centimeter squared per steradian (p/s/cm2/sr) and the luminescent image was overlaid on the photographic image. For the additional details, see the methods section. (E and I) Summary table of distant organ metastasis derived from each surgery group. (E and I) Summary table of distant organ metastasis derived from each surgery group.(G and H) Distant organ metastasis was examined by bioluminscent images of inguinal lymph node pre-removed tumor-bearing mice (n=9 mice) or sham surgery tumor-bearing mice (n=5 mice) after tumor resection on week 5. The bioluminescent signal (pseudocolor) was recorded as photons per second per centimeter squared per steradian (p/s/cm2/sr) and the luminescent image was overlaid on the photographic image. For the additional details, see the methods section.
Figure 3 Figure 3. Up-regulation of Il-17rb contributes to the aggressive malignancy phenotypes of breast cancer cell derived from tumor-draining lymph node (A) Gene expression profiles were shown at 4T1PT to 4T1LN cells. Five genes encoding cell surface proteins were identified among up-regulated genes. (B) mRNA expression of each candidate gene in 4T1PT and 4T1LN cells was determined by RT-qPCR. Gapdh was used as an internal control.(C and D) Il-17rb, Gpr56, and Scara5 expression in 4T1PT and 4T1LN cells were examined by Western blotting analysis.(C and D) Il-17rb, Gpr56, and Scara5 expression in 4T1PT and 4T1LN cells were examined by Western blotting analysis.(E) Western blotting and RT-qPCR analysis of Il-17rb, Gpr56, and Scara5 expression in 4T1 cells transduced with Il-17rb, Gpr56, Scara5, or control LacZ shRNA lentivirus respectively.(F) Soft-agar colony forming activity was examined in lentivirus transduced shIl-17rb, shGpr56, shScara5, or shLacZ 4T1 cells (5x102 cells/well, n = 6 wells per group).(G) Western blotting analysis was used to detect ectopic expression of Il-17rb in 4T1 cells infected with either lentiviruses carrying Il-17rb cDNA or empty vector.(H) Soft-agar colony forming activity was examined using control or Il-17rb over-expressing 4T1 cells (5x102 cells/well, n = 6 wells per group).(I) Western blotting analysis was used to examine Il-17rb in a representative Il-17rbDel 4T1 clone derived from Il-17rb knocked-out 4T1 cells using a CRISPR/Cas9 system.(J and K) Tumorigenesis assays were determined in BALB/c mice (n = 3 mice per group) orthotopically injected with 5x102 4T1 cells from WT or Il-17rbDel clone. Tumor mass (J) and weight (K) were measured on day 28. Scale bar: 1 cm.(L and M) BALB/c mice (n = 3 mice per group) were injected with 5x105 4T1 cells from WT or Il-17rbDel clone via tail-vein. Lung colonization was examined by lung morphology (L) and the numbers of tumor nodule (M) on day 21. Scale bar: 1 cm.
Figure 4(B and C) Inguinal lymph node-injected BALB/c mice were sacrificed at the indicated week after initial injection. Total cells isolated from inguinal lymph node tissues were transwell co-cultured with 4T1 cells. Inguinal lymph node tissues came from un-injection BALB/c mice as control. After 5-day co-culture, 4T1 cells at lower well were examined in the RT-qPCR (B)analyses of Il-17rb expression. Gapdh was used as an internal control or as a loading control.(B and C) 4T1-injected BALB/c mice were sacrificed at the indicated week after initial injection. Total cells isolated from inguinal lymph node tissues were transwell co-cultured with 4T1 cells. Inguinal lymph node tissues came from un-injection BALB/c mice as control. After 5-day co-culture, 4T1 cells at lower well were examined in the Western blotting (C) analyses of Il-17rb expression. Gapdh was used as an internal control or as a loading control.(D) Soft-agar colony forming activity was examined using co-cultured 4T1 cells at lower well (5x102 cells/well, n = 4 wells per group).(E and F) 4T1-injected BALB/c mice were sacrificed at three weeks post initial injection. Each lymphocyte subsets isolated from inguinal lymph node tissues by FACS sorting were transwell co-cultured with 4T1 cells. After 5-day co-culture, 4T1 cells at lower well were used for the RT-qPCR (E)analyses of Il-17rb expression. Gapdh was used as internal control or as a loading control.(E and F) 4T1-injected BALB/c mice were sacrificed at three weeks post initial injection. Each lymphocyte subsets isolated from inguinal lymph node tissues by FACS sorting were transwell co-cultured with 4T1 cells. After 5-day co-culture, 4T1 cells at lower well were used for the Western blotting (F) analyses of Il-17rb expression. Gapdh was used as internal control or as a loading control.(G) Percentage of CD4+Foxp3+ Tregs of total CD4+ cells in inguinal LN, spleen and peripheral blood of BALB/c mice (n = 3 mice per group) injected with 4T1 (Blue) or γ-irradiated 4T1 (Red) cancer cells was analyzed by FACS.(H) 4T1 cells co-cultured with the indicated lymphocyte populations isolated from inguinal LN of 4T1 tumor-bearing mice by FACS sorting. After 5-day co-culture, 4T1 cells at lower well were analyzed for Il-17rb expression by RT-qPCR analysis. Gapdh was used as internal control.
Figure 5 Figure 5. Depletion of Tregs in the tumor-draining lymph node microenvironment abolishes Il-17rb induction and enhanced malignancy in breast cancer cells. (A) 4T1-injected BALB/c mice were treated with control IgG, anti-CD4 or anti-CD25 neutralizing antibody at the day 15 and day 18 after initial injection. Mice were sacrificed at three weeks after initial injection. Depletion of CD4+Foxp3+ Tregs in inguinal LNs was confirmed by FACS analysis. (B) Western blotting analysis of Il-17rb expression was performed in 4T1LN or 4T1PT cells collected from control IgG-, anti-CD4, or anti-CD25 antibody-treated 4T1 tumor-bearing mice.(C and D) BALB/c mice were orthotopically injected with 5x102 4T1LN or 4T1PT cells collected from control IgG-, anti-CD4, or anti-CD25 antibody-treated 4T1 tumor-bearing mice. (n = 3 mice per group). Tumor mass (D) and tumor weight (E) were measured on day 28. Scale bar: 1 cm.(C and D) BALB/c mice were orthotopically injected with 5x102 4T1LN or 4T1PT cells collected from control IgG-, anti-CD4, or anti-CD25 antibody-treated 4T1 tumor-bearing mice. (n = 3 mice per group). Tumor mass (D) and tumor weight (E) were measured on day 28. Scale bar: 1 cm.(E) NSG mice (n = 5 mice per group) were injected with 5x102 4T1LN or 4T1PT cells collected from control IgG-, anti-CD4, or anti-CD25 antibody-treated 4T1 tumor-bearing mice. Distant organ metastasis was examined by bioluminscent images on day 21. The bioluminescent signal (pseudocolor) was recorded as photons per second per centimeter squared per steradian (p/s/cm2/sr) and the luminescent image was overlaid on the photographic image.
Figure 6 Figure 6. TGF-β1 secreted from Tregs induces Il-17rb expression of breast cancer cells via Smad2/3/4 signaling. (A) CD4+CD25+ Tregs were isolated from inguinal LN of BALB/c mice injected with 4T1 cells for three weeks, and were transwell cocultured with 4T1 cells in the presence of control IgG or neutralizing antibodies against IL-10 or TGF-β1. After 5-day coculture, 4T1 cells at lower well were analyzed for Il-17rb expression by RT-qPCR analysis. Gapdh was used as internal control. (B and C) Western blotting analyzed Il-17rb in 4T1 cells treated with recombinant TGF-β1 (B)proteins for 5 days.(B and C) Western blotting analyzed Il-17rb in 4T1 cells treated with recombinant IL-10 (C) proteins for 5 days.(D) Western blotting analyzed Il-17rb in shLacZ or shTgfbr1 4T1 cells treated with recombinant TGF-β1 for 5 days.(E) 4T1-injected BALB/c mice were sacrificed at three weeks post initial injection. Total cells isolated from inguinal lymph node tissues were transwell co-cultured with 4T1 cells. After 5-day coculture, soft-agar colony forming activity was determined in co-cultured shLacZ or shTgfbr1 4T1 cells at lower well. (5x102 cells/well, n = 6 wells per group).(F-H) Western blotting analyzed Il-17rb in shLacZ, shSmad2 (F)4T1 cells treated with recombinant TGF-β1 for 5 days.(F-H) Western blotting analyzed Il-17rb in shLacZ,shSmad3 (G)4T1 cells treated with recombinant TGF-β1 for 5 days.(F-H) Western blotting analyzed Il-17rb in shLacZ, shSmad4 (H) 4T1 cells treated with recombinant TGF-β1 for 5 days. (I and J) WT 4T1 (I) or Il-17rbDel 4T1 (J) cells were treated with or without recombinant TGF-β1 for 5 days. After treatment, nuclear translocation of NF-κB p65 was assayed by Western blotting in cells treated with recombinant Il-17b for 2 hours. Nuclear Matrix Protein p84 was used as a loading control of nuclear extracts.f triplicate measurement. In (B, C, D, F, G, H, I, and J), the intensity of each band was quantified using the Image J software. Gapdh, α-tubulin, or p84 was used as a loading control. Relative expression (RE) of protein levels in each sample to control 4T1 cells or Il-17rbDel 4T1 clone is indicated.
Figure 7Figure 7. IL-17RB expression is correlated with the elevated prevalence of regulatory T cells in the tumor-draining lymph nodes of breast cancer patients. (A and C) Representative IHC images of IL-17RB (A) and Foxp3 (C) were taken in breast specimens from primary tumors and their paired LN metastasis specimens. Scale bar: 10 μmFigure 7. IL-17RB expression is correlated with the elevated prevalence of regulatory T cells in the tumor-draining lymph nodes of breast cancer patients. (A and C) Representative IHC images of IL-17RB (A) and Foxp3 (C) were taken in breast specimens from primary tumors and their paired LN metastasis specimens. Scale bar: 10 μm.
Figure 8 Figure 8. Elevated IL-17RB expression in cancer cells derived from tumor-draining lymph nodes in breast cancer patients correlates with aggressive growth nature. (A) Freshly collected humanbreast cancer (hBC) specimens (PT, primary tumors; LN, paired LN metastasis from the same breast cancer patient) were digested with collagenase-containing buffer overnight. Depletion of hematopoietic cells by CD45 Dyna beads (Invitrogen) enriched the EpCAM+ cells for following xenografts. Percentages of EpCAM+ cells in human primary breast cancer cells were confirmed by FACS analysis (Left panel: before enrichment; Right panel: after enrichment). All of the histograms were shown that they were EpCAM expressing (black line), compared to isotype control (gray filled). (B) Tumorigenesis assay were determined in NOD/SCID/IL2Rγnull mice orthotopically injected with hBCPT and hBCLN cells. Representative data on day 180 were shown.(D and E) H&E stainings and IHC stainings of pan-keratin were shown in the representative cases of fat-pad sections from (B). Scale bar: 1 mm (left panel), 100 μm (right panel).(D and E) H&E stainings and IHC stainings of pan-keratin were shown in the representative cases of fat-pad sections from (B). Scale bar: 1 mm (left panel), 100 μm (right panel).(F) IHC stainings of IL-17RB were shown in the representative cases of fat-pad sections from (B). Scale bar: 10 μm.(G) The correlation of tumor volumes and IL-17RB expression (composite IHC score) in hBCLN cells-injected NOD/SCID/IL2Rγnull mice.