Journal: Breast Cancer Research : BCR

Article Title: STAT5 is activated in macrophages by breast cancer cell-derived factors and regulates macrophage function in the tumor microenvironment

doi: 10.1186/s13058-021-01481-0

Figure Lengend Snippet: Tumor cell-derived GM-CSF activates STAT5 in macrophages. A qRT-PCR analysis for GM-CSF in TNBC (Hs578T and MDA-MB-231), ER + (MCF7) and HER2 + (BT-474) human breast cancer cells relative to expression in MCF-10A cells. B ELISA analysis for GM-CSF in CM collected from MCF-10A, MDA-MB-231, Hs578T, MCF7, and BT-474 cells. C ELISA analysis for GM-CSF in CM collected from 4T1 cells and B/B-stimulated HC11, HC11/R1, and HC11/R1-LM cells relative to EtOH controls. D Immunoblot analysis for pSTAT5, TSTAT5, and β-tubulin in BMDMs treated with No CM, tumor CM (4T1 or HC11/R1 BB), or tumor CM incubated for 1 h at 37 °C with 2.5 µg/mL neutralizing GM-CSF antibody (⍺GM-CSF Ab)

Article Snippet: In experiments neutralizing GM-CSF, rat anti-mouse GM-CSF (BioTechne) was incubated with tumor cell CM at a concentration of 2.5 µg/mL for 1 h at 37 °C prior to treatment of BMDMs.

Techniques: Derivative Assay, Quantitative RT-PCR, Expressing, Enzyme-linked Immunosorbent Assay, Western Blot, Incubation

Journal: Breast Cancer Research : BCR

Article Title: STAT5 is activated in macrophages by breast cancer cell-derived factors and regulates macrophage function in the tumor microenvironment

doi: 10.1186/s13058-021-01481-0

Figure Lengend Snippet: STAT5 in macrophages differentially regulates expression of adaptive immunity-related and tumor-promoting genes. A Heatmap of differentially regulated genes comparing STAT5 fl/fl or STAT5 cKO macrophages treated with No CM, recombinant GM-CSF (rmGM-CSF), or 4T1 CM. B Gene ontology analysis showing pathways altered based on genes that are downregulated in STAT5 cKO macrophages stimulated with rmGM-CSF (left panel) and 4T1 CM (right panel). C , D Gene ontology analysis showing biological processes downregulated and upregulated in 4T1 CM-treated STAT5 cKO BMDMs, respectively. E Positively enriched (red) and negatively enriched (blue) GSEA pathways in 4T1 CM-treated STAT5 cKO BMDMs. F The changes in expression of Epithelial to Mesenchymal Transition (top) and Interferon Gamma Response (bottom) genes in 4T1 CM-treated STAT5 cKO BMDMs analyzed by GSEA

Article Snippet: In experiments neutralizing GM-CSF, rat anti-mouse GM-CSF (BioTechne) was incubated with tumor cell CM at a concentration of 2.5 µg/mL for 1 h at 37 °C prior to treatment of BMDMs.

Techniques: Expressing, Recombinant

Journal: Protein & Cell

Article Title: The chemical reprogramming of unipotent adult germ cells towards authentic pluripotency and de novo establishment of imprinting

doi: 10.1093/procel/pwac044

Figure Lengend Snippet: Single-cell RNA-sequencing roadmap of SSC reprogramming. (A) Schematic for the reprogramming of SSC. Days 16–20, Oct4 -EGFP positive (OG(+)) colonies appeared. SSCM, spermatogonial stem cell culture medium. (B) Bright field (top) and fluorescence images (bottom) of Oct4 -EGFP SSCs (days 0 and 10), Oct4 -EGFP positive (OG(+)) colonies in the early stage of reprogramming (day 19), and gPSC-1 (p1) in the late stage of reprogramming. Scale bar, 200 μm. (C) Uniform manifold approximation and projection (UMAP) plot showing the transcriptome data of reprogramming cells, gPSCs, and ESCs. Reprogramming cells are colored by the day of reprogramming. (D) UMAP plot showing the transcriptome data of reprogramming cells from day 0 to day 27, cells are colored by clusters identified from graphed-based unsupervised clustering. (E) UMAP plot showing the RNA velocity vector field of the early stage of SSC reprogramming. (F) PAGA analysis showing the trajectories of the early stage of SSC reprogramming, each circle represents an individual cluster, the size of circles indicates the number of cells in clusters. (G) Cell trajectory of the successful branch of SSC reprogramming, inferred by Moonocle2. Arrows indicate the developmental order of these cells. (H) Enriched GO terms and P values of differential expressed genes of RPG1/2/3/4 clusters. (I) Violin plots showing the expression level of KEGG pathway ‘Glycolysis/Gluconeogenesis’ (mmu00010) and representative glycolysis-related genes. Dunn’s non-parametric test was performed for pairwise multiple comparison of activity between adjacent clusters, P values were adjusted by Bonferroni method, **** P < 0.0001. (J) Extracellular lactic acid level plotted over time during SSC reprogramming. Unpaired two-tailed Student’s s -test, * P < 0.05. (K) Bar plot showing the number of OG(+) colonies on day 23 of SSC reprogramming treated with 2-DG at different concentration. Unpaired two-tailed Student’s t -test, * P < 0.05. (L) Violin plots showing the expression levels of DNA methylation related genes. (M) Schematic showing the stage division of gPSC-, CiPSC-, and iPSC-reprogramming. (N) Bubble plots showing GO terms and their representative genes upregulated in stage 1 (left) and stage 2 (right) of three reprogramming processes, corresponding to (M). GO terms and representative genes are indicated by colors. See also and .

Article Snippet: The primary antibodies used in this study were listed: Goat anti-GFRa1 (R&D, AF714), Mouse anti-ZBTB16 (Santa, sc-28319), Rabbit anti-DDX4 (Abcam, ab13840), Mouse anti-POU5F1 (OCT4) (Abcam, ab19857), Mouse anti-POU5F1 (OCT4) (Santa, sc-5279), Mouse anti-SSEA1(Sigma-Aldrich, MAB4301), Rabbit anti-NANOG (Abcam, ab70482), Rabbit anti-RHOX5 (Abcam, ab31922), Rabbit anti-PRDM14 (Cell Signaling Technology, 83527), Goat anti-OTX2 (R&D, AF1979), Rabbit anti-DPPA3 (Abcam, ab19878), Rabbit anti-L1TD1(Biorbyt, orb35537), Mouse anti-TFAP2C (Santa, sc-12762), Rabbit anti-DNMT3B (proteintech, 26971-1-AP), Rabbit anti-DNMT1 (abclonal, A16729), Mouse anti-UHRF1 (Santa, sc-373750), and Mouse anti-ACTIN (proteintech, CL594-66009).

Techniques: RNA Sequencing, Stem Cell Culture, Fluorescence, Plasmid Preparation, Expressing, Comparison, Activity Assay, Two Tailed Test, Concentration Assay, DNA Methylation Assay

Journal: Protein & Cell

Article Title: The chemical reprogramming of unipotent adult germ cells towards authentic pluripotency and de novo establishment of imprinting

doi: 10.1093/procel/pwac044

Figure Lengend Snippet: A robust SSC reprogramming system induced by small-molecule compounds. (A) Bar plots showing the number of OG(+) colonies on day 19 treated with individual chemical at 10 µmol/L or 50 μg/mL during SSC reprogramming. Unpaired two-tailed Student’s s -test was performed between DMSO and each chemical group, * P < 0.05. ns, not significant. (B) Bar plots showing the numbers of OG(+) colonies on day 19 treated with individual chemical at different concentration. Unpaired two-tailed Student’s t -test, * P < 0.05. (C) Schematic of SSC reprogramming with the original (top) and 5C (bottom) treatments. (D) AP staining (top) and fluorescence images (bottom) showing the reprogramming cells on day 19 in DMSO and 5C groups. Scale bar, 50 μm. AP staining, alkaline phosphatase staining. (E) Bar plot showing the number of OG(+) colonies at different time points in DMSO and 5C groups. (F) Line chart showing the extracellular lactic acid level plotted over time in DMSO and 5C groups. Unpaired two-tailed Student’s t -test, * P < 0.05, ** P < 0.01, *** P < 0.001, ns, not significant. (G) Bright field and fluorescence images (top) of gPSCs (p1) from DMSO and 5C groups, respectively. Bar plot (bottom) showing the numbers of gPSC colonies (p1) from these 2 groups. Unpaired two-tailed Student’s t -test; **** P < 0.0001. (H) Bar plot showing the numbers of OG(+) colonies on day 19 in the indicated groups, the original condition (control), DMSO, 5C and 5C withdrawing different chemicals. Unpaired two-tailed Student’s t -test, * P < 0.05, ** P < 0.01, **** P < 0.0001, ns, not significant. (I) Immunofluorescence of OCT4, NANOG and SSEA1 (red) in 5C-gPSC-1. Scale bar, 20 μm. (J) H&E staining of paraffin sections of teratoma derived from subcutaneous injection of 5C-gPSC-1 into nude mice. Scale bar, 100 μm. (K) Bright field and fluorescence images showing E12.5 embryo of KM mouse and chimaera embryo produced by the diploid blastocyst injection of CAG-EGFP 5C-gPSC-2, 2 out of 6 embryos were chimaeras after blastocyst injection. Scale bar, 2 mm. (L) Bright filed and fluorescence images of the E12.5 genital ridges derived from diploid blastocyst injection of 5C-gPSC-1, 7 out of 10 pairs of genital ridges were GFP positive after blastocyst injection. Scale bar, 500 μm. (M) Chimaeras produced from B6D2F1 (black coat color) 5C-gPSC-1. (N) Summary of chimeric assays of diploid blastocyst injection using 5C-gPSC-1.

Article Snippet: The primary antibodies used in this study were listed: Goat anti-GFRa1 (R&D, AF714), Mouse anti-ZBTB16 (Santa, sc-28319), Rabbit anti-DDX4 (Abcam, ab13840), Mouse anti-POU5F1 (OCT4) (Abcam, ab19857), Mouse anti-POU5F1 (OCT4) (Santa, sc-5279), Mouse anti-SSEA1(Sigma-Aldrich, MAB4301), Rabbit anti-NANOG (Abcam, ab70482), Rabbit anti-RHOX5 (Abcam, ab31922), Rabbit anti-PRDM14 (Cell Signaling Technology, 83527), Goat anti-OTX2 (R&D, AF1979), Rabbit anti-DPPA3 (Abcam, ab19878), Rabbit anti-L1TD1(Biorbyt, orb35537), Mouse anti-TFAP2C (Santa, sc-12762), Rabbit anti-DNMT3B (proteintech, 26971-1-AP), Rabbit anti-DNMT1 (abclonal, A16729), Mouse anti-UHRF1 (Santa, sc-373750), and Mouse anti-ACTIN (proteintech, CL594-66009).

Techniques: Two Tailed Test, Concentration Assay, Staining, Fluorescence, Control, Immunofluorescence, Derivative Assay, Injection, Produced

Journal: Protein & Cell

Article Title: The chemical reprogramming of unipotent adult germ cells towards authentic pluripotency and de novo establishment of imprinting

doi: 10.1093/procel/pwac044

Figure Lengend Snippet: 5C-gPSCs had true pluripotency identified by tetraploid complementation assay. (A) 5C-gPSC 4N-comp pups and their corresponding placentae. Note: * Pups survived beyond 4 days postnatal; # Pup died within 4 days postnatal. (B) Simple sequence length polymorphism (SSLP) analysis for lineage identification covers markers from different chromosomes, and the 4N-comp mice showed a polymorphic pattern similar with that from the F1-5 ESC, 5C-gPSC-S4, or gPSC-S2 cells originating from B6D2F1 mice, and different from the ICR, C57, or DBA mice. M denotes molecular mass marker. (C) Genotyping analysis of Oct4 -EGFP from F1-5 ESC, 5C-gPSC-S4, or gPSC-S2 4N-comp mice. (D) Bright field (left), fluorescence (middle) and merged (right) images of E3.5 Oct4 -EGFP blastocysts flushed from an ICR female mouse mated with an 5C-gPSC-S4 4N-comp mouse. Scale bar, 100 μm. (E) An 11-week-old 5C-gPSC-S4 4N-comp mouse with a uniformly black coat and its F 1 progeny from its mating to an ICR female mouse. (F) Bar plot showing the developmental rate of full-term 4N-comp pups derived from PSCs, numbers of animals obtained per total number of transferred embryos are shown. Groups are indicated by colors. See for details. (G) Dot plot showing the body weight of 4N-comp mice born by Caesarean section in this study on day 0 postnatal. Groups are indicated by colors. Unpaired two-tailed Student’s t -test, * P < 0.05, ns, not significant. (H) Survival curves of 4N-comp mice obtained in this study. n, total numbers of 4N-comp mice derived from each group. Groups are indicated by colors. (I) Growth curves of 4N-comp mice obtained in this study at the indicated weeks postnatal. Groups are indicated by colors. (J) Bar plot showing the DNA methylation level of the whole genome (left) and total ICRs (right) in ESCs (p10), 5C-gPSCs (p25), gPSCs (p25), respectively. Unpaired two-tailed Student’s t -test, P values were shown above the lines, ** P < 0.01, ns, not significant. (K) Heatmaps showing the mean methylation levels of the paternal ICRs in SSCs (p20), gPSCs (p25), 5C-gPSCs (p25), ESCs (p10). The color key from dark blue to red indicates low to high levels, respectively. (L) Line plot shows the mean methylation levels of Dlk1-Dio3 region in SSCs (p20), 5C-gPSCs (p25), ESCs (p10), gPSCs (p25), respectively. Grey shadows indicate gene regions, the red shadow indicates IG- Gtl2 ICR region. (M) Box plots show the mean DNA methylation levels across gene bodies (from transcription start site (TSS) to transcription end site (TES)) and the 50-bp flanking regions of each miRNA encoded locus in Dlk1-Dio3 region in ESCs, gPSCs and 5C-gPSCs. One dot represents an miRNA encoded locus, the same loci in distinct PSCs are connected by grey lines. Paired two-tailed Student’s t -test was performed between ESCs and other PSCs. (N) Heatmaps showing the mean methylation levels of the maternal ICRs in SSCs (p20), gPSCs (p25), 5C-gPSCs (p25), ESCs (p10). The color key from dark blue to red indicates low to high levels, respectively. (O) qPCR analysis of relative expression of Igf2r , Kcnq1ot1 , Peg3 , and Snrpn normalized to the geometric mean of Rps2 and Gapdh in ESCs, gPSCs, and 5C-gPSCs. Unpaired two-tailed Student’s t -test, ** P < 0.01, *** P < 0.001, ns, not significant. See also .

Article Snippet: The primary antibodies used in this study were listed: Goat anti-GFRa1 (R&D, AF714), Mouse anti-ZBTB16 (Santa, sc-28319), Rabbit anti-DDX4 (Abcam, ab13840), Mouse anti-POU5F1 (OCT4) (Abcam, ab19857), Mouse anti-POU5F1 (OCT4) (Santa, sc-5279), Mouse anti-SSEA1(Sigma-Aldrich, MAB4301), Rabbit anti-NANOG (Abcam, ab70482), Rabbit anti-RHOX5 (Abcam, ab31922), Rabbit anti-PRDM14 (Cell Signaling Technology, 83527), Goat anti-OTX2 (R&D, AF1979), Rabbit anti-DPPA3 (Abcam, ab19878), Rabbit anti-L1TD1(Biorbyt, orb35537), Mouse anti-TFAP2C (Santa, sc-12762), Rabbit anti-DNMT3B (proteintech, 26971-1-AP), Rabbit anti-DNMT1 (abclonal, A16729), Mouse anti-UHRF1 (Santa, sc-373750), and Mouse anti-ACTIN (proteintech, CL594-66009).

Techniques: Sequencing, Marker, Fluorescence, Derivative Assay, Two Tailed Test, DNA Methylation Assay, Methylation, Expressing

Journal: Protein & Cell

Article Title: The chemical reprogramming of unipotent adult germ cells towards authentic pluripotency and de novo establishment of imprinting

doi: 10.1093/procel/pwac044

Figure Lengend Snippet: A reversed developmental trajectory of germ cell in SSC reprogramming. (A) UMAP plot showing the integration of single-cell transcriptome data from successful branch of SSC reprogramming (DMSO- and 5C groups), mouse in vivo germ cells and gonadal somatic cells from E6.5 to PND5.5. Grey dots indicate in vivo germ cells or germline somatic cells, colored dots or triangles with black outline indicate reprogramming cells or ESCs. (B) Scmap projection of the successful branch of SSC reprogramming (left, 5C group; right, DMSO group) and mouse in vivo germ cells. The color key from bright yellow to dark blue indicates low to high ratio of projected cells. SPG, spermatogonia; in vivo GC dev., in vivo germ cell development. (C) Heatmap showing the expression of DEGs of in vivo germ cell development and their expression dynamics in SSC reprogramming, GO terms enriched in both processes were highlighted. (D) Violin plots showing the expression levels of canonical marker genes of in vivo germ cells in the successful branch of SSC reprogramming. (E) Immunofluorescence of Oct4 -EGFP (green) co-stained with RHOX5 (top, red) and L1TD1 (bottom, red), respectively, in domed colonies during SSC reprogramming. Scale bar, 50 μm. (F) Immunofluorescence of Oct4 -EGFP (green) co-stained with PRDM14 (pink) and TFAP2C (red) in domed colonies during SSC reprogramming. Scale bar, 50 μm. (G) Immunofluorescence of Oct4 -EGFP (green) co-stained with OTX2 (red) in domed colonies during SSC reprogramming. Scale bar, 50 μm. (H) Immunofluorescence of Oct4 -EGFP (green) co-stained with PRDM14 (pink) and OTX2 (red) in domed colonies at different time points during SSC reprogramming. Note, * The diameters of detected colonies. Scale bar, 50 μm. (I) Boxplot showing the number of PRDM14(+)OTX2(–), PRDM14(+)OTX2(+), PRDM14(–)OTX2(+) colonies (left) and their percentage (right) in OG(+) colonies at different time points during SSC reprogramming, corresponding to . See also .

Article Snippet: The primary antibodies used in this study were listed: Goat anti-GFRa1 (R&D, AF714), Mouse anti-ZBTB16 (Santa, sc-28319), Rabbit anti-DDX4 (Abcam, ab13840), Mouse anti-POU5F1 (OCT4) (Abcam, ab19857), Mouse anti-POU5F1 (OCT4) (Santa, sc-5279), Mouse anti-SSEA1(Sigma-Aldrich, MAB4301), Rabbit anti-NANOG (Abcam, ab70482), Rabbit anti-RHOX5 (Abcam, ab31922), Rabbit anti-PRDM14 (Cell Signaling Technology, 83527), Goat anti-OTX2 (R&D, AF1979), Rabbit anti-DPPA3 (Abcam, ab19878), Rabbit anti-L1TD1(Biorbyt, orb35537), Mouse anti-TFAP2C (Santa, sc-12762), Rabbit anti-DNMT3B (proteintech, 26971-1-AP), Rabbit anti-DNMT1 (abclonal, A16729), Mouse anti-UHRF1 (Santa, sc-373750), and Mouse anti-ACTIN (proteintech, CL594-66009).

Techniques: In Vivo, Expressing, Marker, Immunofluorescence, Staining

Journal: Protein & Cell

Article Title: The chemical reprogramming of unipotent adult germ cells towards authentic pluripotency and de novo establishment of imprinting

doi: 10.1093/procel/pwac044

Figure Lengend Snippet: Identification of key regulators in SSC reprogramming. (A) Volcano plots showing differentially expressed genes (DEGs, | log (fold change) | ≥ 1, adjusted P < 0.01) in RPG1/2/3/4. Red and blue dots indicate upregulated and downregulated genes, respectively. Transcription factors are highlighted in bold. (B) Heatmap showing the regulon activities from SCENIC analysis in the successful branch of SSC reprogramming and representative enriched GO terms of indicated regulons. The color key from blue to red indicates low to high regulon activity. (C) Heatmaps showing the expression pattern of Rhox5 related genes, target genes of Prdm14 and Otx2 , respectively. The color key from blue to red indicates low to high expression level. (D) Schematic of the gene editing by CRISPR-CasRx in SSC reprogramming. (E) Fluorescence images (left) and boxplot (right) showing the number of OG(+) colonies reprogrammed from empty vector (sgNC)–, vectors targeting Rhox5 (sg Rhox5 1# and sg Rhox5 2#)-transfected SSCs in 5C condition on day 19. Scale bar, 50 μm. Unpaired two-tailed Student’s t -test, ** P < 0.01, *** P < 0.001. NC, negative control. (F) (i) Immunofluorescence of Oct4 -EGFP (green) co-stained with PRDM14 (top, pink), TFAP2C (top, red), and OTX2 (bottom, red) in domed colonies reprogrammed from sgNC- and sg Rhox5 2#-transfected SSCs, respectively. Scale bar, 50 μm. (ii) Dot plots showing the intensity of PRDM14, TFAP2C, and OTX2 corresponding to (i), Unpaired two-tailed Student’s t -test, **** P < 0.0001. See also .

Article Snippet: The primary antibodies used in this study were listed: Goat anti-GFRa1 (R&D, AF714), Mouse anti-ZBTB16 (Santa, sc-28319), Rabbit anti-DDX4 (Abcam, ab13840), Mouse anti-POU5F1 (OCT4) (Abcam, ab19857), Mouse anti-POU5F1 (OCT4) (Santa, sc-5279), Mouse anti-SSEA1(Sigma-Aldrich, MAB4301), Rabbit anti-NANOG (Abcam, ab70482), Rabbit anti-RHOX5 (Abcam, ab31922), Rabbit anti-PRDM14 (Cell Signaling Technology, 83527), Goat anti-OTX2 (R&D, AF1979), Rabbit anti-DPPA3 (Abcam, ab19878), Rabbit anti-L1TD1(Biorbyt, orb35537), Mouse anti-TFAP2C (Santa, sc-12762), Rabbit anti-DNMT3B (proteintech, 26971-1-AP), Rabbit anti-DNMT1 (abclonal, A16729), Mouse anti-UHRF1 (Santa, sc-373750), and Mouse anti-ACTIN (proteintech, CL594-66009).

Techniques: Activity Assay, Expressing, CRISPR, Fluorescence, Plasmid Preparation, Transfection, Two Tailed Test, Negative Control, Immunofluorescence, Staining

Journal: Cell host & microbe

Article Title: Legionella-infected macrophages engage the alveolar epithelium to metabolically reprogram myeloid cells and promote antibacterial inflammation

doi: 10.1016/j.chom.2020.07.019

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: InVivoMAb Rat anti-Mouse GM-CSF (clone MP1-22E9) , Bio X Cell , Cat#BE0259; RRID: AB_2687738.

Techniques: Virus, Recombinant, Reverse Transcription, Membrane, Isolation, Enzyme-linked Immunosorbent Assay, Plex Assay, Flow Cytometry, Software

Journal: Cell reports

Article Title: Cystatin C Plays a Sex-Dependent Detrimental Role in Experimental Autoimmune Encephalomyelitis

doi: 10.1016/j.celrep.2020.108236

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Rat anti-mouse GM-CSF, BV421 (Clone MP1–22E9) , BD , Cat # 564747.

Techniques: Recombinant, Enzyme-linked Immunosorbent Assay, Knock-Out, Software

Journal: Breast Cancer Research : BCR

Article Title: STAT5 is activated in macrophages by breast cancer cell-derived factors and regulates macrophage function in the tumor microenvironment

doi: 10.1186/s13058-021-01481-0

Figure Lengend Snippet: Tumor cell-derived GM-CSF activates STAT5 in macrophages. A qRT-PCR analysis for GM-CSF in TNBC (Hs578T and MDA-MB-231), ER + (MCF7) and HER2 + (BT-474) human breast cancer cells relative to expression in MCF-10A cells. B ELISA analysis for GM-CSF in CM collected from MCF-10A, MDA-MB-231, Hs578T, MCF7, and BT-474 cells. C ELISA analysis for GM-CSF in CM collected from 4T1 cells and B/B-stimulated HC11, HC11/R1, and HC11/R1-LM cells relative to EtOH controls. D Immunoblot analysis for pSTAT5, TSTAT5, and β-tubulin in BMDMs treated with No CM, tumor CM (4T1 or HC11/R1 BB), or tumor CM incubated for 1 h at 37 °C with 2.5 µg/mL neutralizing GM-CSF antibody (⍺GM-CSF Ab)

Article Snippet: In experiments neutralizing GM-CSF, rat anti-mouse GM-CSF (BioTechne) was incubated with tumor cell CM at a concentration of 2.5 µg/mL for 1 h at 37 °C prior to treatment of BMDMs.

Techniques: Derivative Assay, Quantitative RT-PCR, Expressing, Enzyme-linked Immunosorbent Assay, Western Blot, Incubation

Journal: Breast Cancer Research : BCR

Article Title: STAT5 is activated in macrophages by breast cancer cell-derived factors and regulates macrophage function in the tumor microenvironment

doi: 10.1186/s13058-021-01481-0

Figure Lengend Snippet: STAT5 in macrophages differentially regulates expression of adaptive immunity-related and tumor-promoting genes. A Heatmap of differentially regulated genes comparing STAT5 fl/fl or STAT5 cKO macrophages treated with No CM, recombinant GM-CSF (rmGM-CSF), or 4T1 CM. B Gene ontology analysis showing pathways altered based on genes that are downregulated in STAT5 cKO macrophages stimulated with rmGM-CSF (left panel) and 4T1 CM (right panel). C , D Gene ontology analysis showing biological processes downregulated and upregulated in 4T1 CM-treated STAT5 cKO BMDMs, respectively. E Positively enriched (red) and negatively enriched (blue) GSEA pathways in 4T1 CM-treated STAT5 cKO BMDMs. F The changes in expression of Epithelial to Mesenchymal Transition (top) and Interferon Gamma Response (bottom) genes in 4T1 CM-treated STAT5 cKO BMDMs analyzed by GSEA

Article Snippet: In experiments neutralizing GM-CSF, rat anti-mouse GM-CSF (BioTechne) was incubated with tumor cell CM at a concentration of 2.5 µg/mL for 1 h at 37 °C prior to treatment of BMDMs.

Techniques: Expressing, Recombinant