Journal: Scientific Reports

Article Title: Plac1 Is a Key Regulator of the Inflammatory Response and Immune Tolerance In Mammary Tumorigenesis

doi: 10.1038/s41598-018-24022-w

Figure Lengend Snippet: Plac1 expression and lentivirus-mediated reduction of Plac1 in EO771 cells. ( a ) EO771 mouse mammary tumor cells expressed high levels of Plac1 in comparison to mouse placenta. ( b ) EO771 cells were transduced with lentiviruses expressing crambled RNA (Scr) or four Plac1 shRNAs designated sh81, sh187, sh300 and sh490; sh490 inhibited RNA expression >98%, and these cells were designated EO771/shPlac1. ( c ) EO771/Scr and EO771/shPlac1 cells were grown as monolayers, and the number of viable cells were quantified by sulforhodamine B staining. Shown is the mean ± S.D. of triplicate analysis of three samples. The growth of EO771/shPlac1 cells differed significantly ( P < 0.001) from EO771/Scr cells by the two-sided Student’s t test. ( d ) qRT-PCR analysis of immune cell-related gene expression downregulated in EO771/shPlac1 cells. Shown is the mean ± S.D. of triplicate analysis of three samples. Significant differences between EO771/Scr and EO771/shPlac1 cells were obtained for CD274 ( P < 0.01), Plac1 ( P < 0.01), Cxcl1 ( P < 0.001), Ccl5 ( P < 0.001) and Lif ( P < 0.001) using the two-tailed Student’s t test; values for Ccl7 were not significantly different ( P > 0.05). ( e ) Heatmap of gene expression as determined by Affymetrix microarray analysis of EO771/Scr (Ctl) and EO771/shPlac1 (sh) cells. Shown are immune cell-related transcripts (Table ) representing ≥3.0-fold change in expression.

Article Snippet: EO771 cells at an inoculum of 1 × 10 6 cells/0.1 ml were injected into the no. 4 mammary gland of C57BL/6 or SCID mice (Taconic), and tumor growth was monitored daily.

Techniques: Expressing, Comparison, Transduction, RNA Expression, Staining, Quantitative RT-PCR, Gene Expression, Two Tailed Test, Microarray

Journal: Scientific Reports

Article Title: Plac1 Is a Key Regulator of the Inflammatory Response and Immune Tolerance In Mammary Tumorigenesis

doi: 10.1038/s41598-018-24022-w

Figure Lengend Snippet: Growth of EO771/Scr and EO771/shPlac1 cells in syngeneic and SCID mice. ( a ) Syngeneic C57BL/6 mice or ( b ) SCID mice at five weeks of age, were inoculated in the mammary gland with 1 × 10 6 cells. Tumor size was measured by calipers in two dimensions. Tumor growth for EO771/Scr and EO771/shPlac1 cells in syngeneic mice differed significantly (P = 0.040) by the unpaired Student’s t test. There was no significant difference (P > 0.05) in tumor growth between the two cell lines in SCID mice. Shown is the mean ± SD, N = 5 per group. ( c ) H&E staining and Plac1 IHC in isografts of EO771/Scr and EO771/shPlac1 cells. Magnification 400X.

Article Snippet: EO771 cells at an inoculum of 1 × 10 6 cells/0.1 ml were injected into the no. 4 mammary gland of C57BL/6 or SCID mice (Taconic), and tumor growth was monitored daily.

Techniques: Staining

Journal: Scientific Reports

Article Title: Plac1 Is a Key Regulator of the Inflammatory Response and Immune Tolerance In Mammary Tumorigenesis

doi: 10.1038/s41598-018-24022-w

Figure Lengend Snippet: Growth of EO771 cells in syngeneic mice following treatment with a Cxcr 2 antagonist. ( a ) Syngeneic 57BL/6 mice were inoculated in the mammary gland with 1 × 10 6 at five weeks of age, and injected i.p. daily with vehicle (blue) or 2 mg/kg (red) or 20 mg/kg (green) SB225002 beginning 11 days after cell inoculation. SB225002 completely suppressed tumor growth after 14 days. Differences between vehicle- and 2 mg/kg SB225002-treated mice were not significantly different (P = 0.145); differences between vehicle- and 20 mg/kg SB225002-treated mice were significantly different (P = 0.005) by the unpaired two-tailed Student’s t test. Shown is the mean ± SD, N = 5 per group. ( b ) Immune gene expression in tumors 17 days after treatment with 20 mg/kg SB225002. Shown is the relative expression in control and SB225002-treated mice in comparison to their changes in EO771/shPlac1 cells (Table ). ( c ) FACS analysis of immune cell tumor infiltrates in isografts after treatment with vehicle or SB225002 as in ( b ). SB225002 treatment reduced the percentage of immune cell tumor infiltrates of CD11b + /Gr-1 + myeloid-derived suppressor cells ( MDSC ) and Foxp3 + /CD25 + T cells ( Treg ), and increased the percentages of CD8 + /CD4 + T cells ( T) , CD3 + /NK1.1 + NK cells ( NK ) and F4/80 + /CD80/86 + macrophages ( Mϕ ) and CD11c + /CD80/86 + dendritic cells ( DC ). Numbers in parentheses ( ) represent the percentages of each cell population. ( d ) Bar graph represents the mean±SD of the percent distribution of immune cell tumor infiltrates as in ( c ); P values were determined by the unpaired two-tailed Student’s t test, N = 4 per group. ( e ) CD8 + T cell infiltration determined by IHC in tumor isografts from vehicle-treated ( EO771/Ctl ) and SB225002-treated ( EO771/SB ) mice. Infiltration of CD8 + T cells increased after treatment with 20 mg/kg SB225002. Magnification 600X. ( f ) Macrophage ( F4/80 ) and Treg cell ( Foxp3 ) infiltration, Plac1 expression and apoptosis by cleaved caspase-3 expression ( Caspase ) in tumor isografts from vehicle-treated ( EO771/Ctl ) and SB225002-treated ( EO771/SB ) mice. Infiltration of macrophages and Treg cells were reduced and apoptosis was increased after treatment with 20 mg/kg SB225002. Magnification 400X

Article Snippet: EO771 cells at an inoculum of 1 × 10 6 cells/0.1 ml were injected into the no. 4 mammary gland of C57BL/6 or SCID mice (Taconic), and tumor growth was monitored daily.

Techniques: Injection, Two Tailed Test, Gene Expression, Expressing, Control, Comparison, Derivative Assay

Journal: Scientific Reports

Article Title: Plac1 Is a Key Regulator of the Inflammatory Response and Immune Tolerance In Mammary Tumorigenesis

doi: 10.1038/s41598-018-24022-w

Figure Lengend Snippet: Lentivirus-mediated reduction of Cxcl1 in EO771 cells. ( a ) EO771 cells were transduced with lentiviruses expressing scrambled RNA (Scr) or three Cxcl1 shRNAs designated sh118, sh174, sh218; sh174 inhibited RNA expression >99% (EO771/shCxcl1). ( b ) EO771/Scr and EO771/shCxcl1 cells were grown as monolayers, and the number of viable cells were determined by sulforhodamine B staining. Shown is the mean ± S.D. of triplicate analysis from three samples, which were significantly different ( P < 0.001) by the two-tailed Student’s t test. ( c ) Growth of EO771/Scr and EO771/shCxcl1 cells in syngeneic mice. Mice at five weeks of age were inoculated in the mammary gland with 1x10 6 cells, and tumor size was measured by calipers in two dimensions. Differences in tumor growth between EO771/Scr and EO771/shCxcl1 cells were significantly different (P = 0.006) by the unpaired two-tailed Student’s t test; N = 5. ( d ) qRT-PCR analysis of genes downregulated in EO771/shCxcl1 cells. Shown is the mean ± SD of triplicate analysis of 3 samples. Significant differences between EO771/Scr and EO771/shCxcl1 cells were obtained for Plau ( P < 0.02), C3 ( P < 0.01), Ly6a ( P < 0.01), Ccl7 ( P < 0.001) and Il23a ( P < 0.01) by the two-sided Student’s t test; differences for CD68 were not significantly different ( P > 0.05).

Article Snippet: EO771 cells at an inoculum of 1 × 10 6 cells/0.1 ml were injected into the no. 4 mammary gland of C57BL/6 or SCID mice (Taconic), and tumor growth was monitored daily.

Techniques: Transduction, Expressing, RNA Expression, Staining, Two Tailed Test, Quantitative RT-PCR

Journal: Scientific Reports

Article Title: Plac1 Is a Key Regulator of the Inflammatory Response and Immune Tolerance In Mammary Tumorigenesis

doi: 10.1038/s41598-018-24022-w

Figure Lengend Snippet: Expression of immune-related genes in E0771/shCxcl1 cells. Shown are ≥3-fold changes in gene expression with a raw score ≥300 in EO771/shCxcl1 or E0771/Scr cells.

Article Snippet: EO771 cells at an inoculum of 1 × 10 6 cells/0.1 ml were injected into the no. 4 mammary gland of C57BL/6 or SCID mice (Taconic), and tumor growth was monitored daily.

Techniques: Expressing, Gene Expression

Journal: Scientific Reports

Article Title: Plac1 Is a Key Regulator of the Inflammatory Response and Immune Tolerance In Mammary Tumorigenesis

doi: 10.1038/s41598-018-24022-w

Figure Lengend Snippet: Gene expression common to EO771/shPlac1 and EO771/shCxcl1 cells. Shown is the ratio between EO771/shPlac1 or EO771/shCxcl1 cells to EO771/Scr control cells for genes with ≥3.0-fold changes in expression and a raw score ≥300.

Article Snippet: EO771 cells at an inoculum of 1 × 10 6 cells/0.1 ml were injected into the no. 4 mammary gland of C57BL/6 or SCID mice (Taconic), and tumor growth was monitored daily.

Techniques: Gene Expression, Control, Expressing

Journal: Scientific Reports

Article Title: Plac1 Is a Key Regulator of the Inflammatory Response and Immune Tolerance In Mammary Tumorigenesis

doi: 10.1038/s41598-018-24022-w

Figure Lengend Snippet: Cxcl1 rescue of EO771/sh490 cells. ( a ) EO771/Scr and EO771/sh490 cells expressing eGFP were transduced with a lentivirus expressing Cxcl1 and mCherry, and selected for 35 days in 3.5 mg/ml G418. The merged photo shows cells co-expressing eGFP and mCherry (yellow). Magnification 200X. ( b ) qRT-PCR for Plac1 and Cxcl1 in EO771/Scr, EO771/sh490 and EO771/sh490/Cxcl1 cells. Shown is the mean ± S.D. of triplicate determinations.( c ) EO771/sh490/Cxcl1 cells were grown in 96-well plates at an initial density of 5,000 cells per well in media supplemented with 3.5 mg/ml G418. Cell density was determined by sulforhodamine B staining. Shown is the mean ± SD of triplicate determinations. ( d ) Syngeneic C57BL/6 mice were inoculated in the mammary gland with 1 × 10 6 at five weeks of age. There was a significant difference in the growth EO771/sh490 cells (P = 0.021) and EO771/sh490/Cxcl1 cells (P = 0.034) vs. EO771/Scr cells by the unpaired two-tailed Student’s t test. Shown is the mean ± SD, N=6 per group.

Article Snippet: EO771 cells at an inoculum of 1 × 10 6 cells/0.1 ml were injected into the no. 4 mammary gland of C57BL/6 or SCID mice (Taconic), and tumor growth was monitored daily.

Techniques: Expressing, Transduction, Quantitative RT-PCR, Staining, Two Tailed Test

Journal: Oncogenesis

Article Title: LIMK2 promotes the metastatic progression of triple-negative breast cancer by activating SRPK1

doi: 10.1038/s41389-020-00263-1

Figure Lengend Snippet: a Representative images for LIMK2 expression analysis in normal breast tissue or triple-negative breast cancer (TNBC) samples in a tissue microarray (TMA) from US Biomax at ×20 magnification. Scale bar, 50 μm. b (Left) Relative fraction of TNBC samples from US Biomax TMA scored by LIMK2 staining intensity: 0, no staining (not shown); +1, weak; +2, moderate; or +3, high. (Right) Percentage of LIMK2-positive cells in TNBC samples from US Biomax TMA: 0–25%, 25–50%, 51–75%, or 76–100%. Unpaired t -test with Welch’s correction was used to compare the LIMK2 expression between normal adjacent breast tissues and breast carcinoma. c Representative images for LIMK2 expression analysis in normal breast tissue or TNBC samples in three independent TNBC TMAs from Yale Tissue Microarray Facility (YTMA311, YTMA341, and YTMA347) at ×20 and ×40 magnification. Scale bar, 50 μm for ×20 and 25 μm for ×40. d (Left) Relative fraction of TNBC samples from three independent TNBC TMAs (YTMA311, YTMA341, and YTMA347) from Yale Tissue Microarray Facility scored by LIMK2 staining intensity: 0, no staining (not shown); +1, weak; +2, moderate; or +3, high. (Right) Percentage of LIMK2-positive cells in TNBC samples from three independent TNBC TMAs (YTMA311, YTMA341, and YTMA347) from Yale Tissue Microarray Facility: 0–25%, 25–50%, 51–75%, or 76–100%). ** P < 0.01.

Article Snippet: Scale bar, 50 μm for ×20 and 25 μm for ×40. b (Left) Relative fraction of TNBC samples from US Biomax TMA scored based on SRPK1 staining intensity: 0, no staining (not shown); +1, weak; +2, moderate; or +3, high. (Right) percentage of SRPK1-positive cells in TNBC samples from US Biomax TMA: 0–25%, 25–50%, 51–75%, or 76–100%.

Techniques: Expressing, Microarray, Staining

Journal: Oncogenesis

Article Title: LIMK2 promotes the metastatic progression of triple-negative breast cancer by activating SRPK1

doi: 10.1038/s41389-020-00263-1

Figure Lengend Snippet: a Representative images for SRPK1 expression analysis in normal breast tissue and triple-negative breast cancer (TNBC) samples on a tissue microarray (TMA) from US Biomax at 20× and 40× magnification. Scale bar, 50 μm for ×20 and 25 μm for ×40. b (Left) Relative fraction of TNBC samples from US Biomax TMA scored based on SRPK1 staining intensity: 0, no staining (not shown); +1, weak; +2, moderate; or +3, high. (Right) percentage of SRPK1-positive cells in TNBC samples from US Biomax TMA: 0–25%, 25–50%, 51–75%, or 76–100%. Unpaired t -test with Welch’s correction was used to compare the SRPK1 expression between normal adjacent breast tissues and breast carcinoma. c Representative images for SRPK1 expression analysis in normal breast tissue or TNBC samples from three independent TNBC TMAs from the Yale Tissue Microarray Facility (YTMA311, YTMA341, and YTMA347) at ×20 magnification. Scale bar, 50 μm. d (Left) Relative fraction of TNBC samples from three independent TNBC TMAs (YTMA311, YTMA341, and YTMA347) from the Yale Tissue Microarray Facility scored by SRPK1 staining intensity: 0, no staining (not shown); +1, weak; +2, moderate; or +3, high. (Right) percentage of SRPK1-positive cells in TNBC samples from three independent TNBC TMAs (YTMA311, YTMA341, and YTMA347) from the Yale Tissue Microarray Facility: 0–25%, 25–50%, 51–75%, or 76–100%. **** P < 0.0001.

Article Snippet: Scale bar, 50 μm for ×20 and 25 μm for ×40. b (Left) Relative fraction of TNBC samples from US Biomax TMA scored based on SRPK1 staining intensity: 0, no staining (not shown); +1, weak; +2, moderate; or +3, high. (Right) percentage of SRPK1-positive cells in TNBC samples from US Biomax TMA: 0–25%, 25–50%, 51–75%, or 76–100%.

Techniques: Expressing, Microarray, Staining

Journal: Frontiers in Pharmacology

Article Title: Gallic acid suppresses the progression of triple-negative breast cancer HCC1806 cells via modulating PI3K/AKT/EGFR and MAPK signaling pathways

doi: 10.3389/fphar.2022.1049117

Figure Lengend Snippet: GA inhibited HCC1806 cells viability and affected cells morphology, and affected HCC1806 cells proliferation and cycle distribution. (A) The MDA-MB-468 cells viability was detected by MTT assay. (B) The HCC1806 cell inhibitory rate was detected by MTT assay. (C) The morphology changes of HCC1806 cells treated with different concentrations of GA were observed under an inverted microscope. (D,E) Effect of GA on the colony formation rate of HCC1806 cells. (F,G) The cell cycle distribution of HCC1806 were assessed by flow cytometry. Date were expressed as means ± SD. Compared with the control group, ** p < 0.01.

Article Snippet: The human TNBC HCC1806 and MDA-MB-468 cells line were purchased form Jiangsu KeyGEN BioTECH Corp., Ltd. (Nanjing, China).

Techniques: MTT Assay, Inverted Microscopy, Flow Cytometry, Control

Journal: Frontiers in Pharmacology

Article Title: Gallic acid suppresses the progression of triple-negative breast cancer HCC1806 cells via modulating PI3K/AKT/EGFR and MAPK signaling pathways

doi: 10.3389/fphar.2022.1049117

Figure Lengend Snippet: GA induced HCC1806 cells apoptosis accompanied by ROS accumulation and MMP depolarization. (A–C) Hoechst 33258 staining and flow cytometry were performed to assess the effect of GA on HCC1806 cells apoptosis. (D,E) The depolarization ratio of HCC1806 cells were detected by flow cytometry. (F,G) The ROS level of HCC1806 cells were assessed by a fluorescence microscope. Date were expressed as means ± SD. Compared with the control group, ** p < 0.01.

Article Snippet: The human TNBC HCC1806 and MDA-MB-468 cells line were purchased form Jiangsu KeyGEN BioTECH Corp., Ltd. (Nanjing, China).

Techniques: Staining, Flow Cytometry, Fluorescence, Microscopy, Control

Journal: Frontiers in Pharmacology

Article Title: Gallic acid suppresses the progression of triple-negative breast cancer HCC1806 cells via modulating PI3K/AKT/EGFR and MAPK signaling pathways

doi: 10.3389/fphar.2022.1049117

Figure Lengend Snippet: GA induces HCC1806 cells apoptosis via the mitochondrial pathway. (A–D) Effect of GA on the expression of Bax, Bcl-2, cleaved-Caspase-3, cleaved-Caspase-9 and P53 proteins in HCC1806 cells. (E) Effect of GA on the expression of Bax, Bcl-2, Caspase-3, Caspase-9 and P53 mRNA in HCC1806 cells. Date were expressed as means ± SD. Compared with the control group, * p < 0.05, ** p < 0.01.

Article Snippet: The human TNBC HCC1806 and MDA-MB-468 cells line were purchased form Jiangsu KeyGEN BioTECH Corp., Ltd. (Nanjing, China).

Techniques: Expressing, Control

Journal: Frontiers in Pharmacology

Article Title: Gallic acid suppresses the progression of triple-negative breast cancer HCC1806 cells via modulating PI3K/AKT/EGFR and MAPK signaling pathways

doi: 10.3389/fphar.2022.1049117

Figure Lengend Snippet: GA induces HCC1806 cells apoptosis via the PI3K/Akt/EGFR and MAPK signaling pathways. (A–D) Effect of GA on the expression of P-PI3K, P-AKT, P-EGFR, P-ERK1/2, P-JNK, and P-P38 proteins in HCC1806 cells. (E,F) Effect of GA on the expression of PI3K, AKT, EGFR, ERK1/2, JNK and P38 mRNA in HCC1806 cells. Date were expressed as means ± SD. Compared with the control group, * p < 0.05, ** p < 0.01.

Article Snippet: The human TNBC HCC1806 and MDA-MB-468 cells line were purchased form Jiangsu KeyGEN BioTECH Corp., Ltd. (Nanjing, China).

Techniques: Protein-Protein interactions, Expressing, Control