Journal: Journal of Translational Medicine

Article Title: Lupus serum IgG induces microglia activation through Fc fragment dependent way and modulated by B-cell activating factor

doi: 10.1186/s12967-019-02175-0

Figure Lengend Snippet: Cytokine expression levels in the brains measured 48 h after injection of SLE-serum, healthy-serum or ACSF. IL-1β ( a ), TNF-α ( b ), IL-6 ( c ), IL-4 ( d ) and IL-10 ( e ) expression in brains measured 48 h under different conditions. Data are presented as mean ± SEM with n = 6 mice per group. **p < 0.01

Article Snippet: As shown in Fig. b, the SLE sera pre-treated withanti-IL-1 (#AF-201, R&D, Minnesota, USA), anti-IL-6or anti-TNF-α (#10395-R508, #10602-MM0N1, Sino Biological, Beijing, China) induced similar percentage of MHC-II expression in microglia as did non-pretreated SLE sera.

Techniques: Expressing, Injection

Journal: Cell reports

Article Title: Stromal-initiated changes in the bone promote metastatic niche development

doi: 10.1016/j.celrep.2015.12.016

Figure Lengend Snippet: A. Primary osteoblasts were obtained from the femurs of 6-week-old FASST mice. To activate the p27Kip1 transgene and induce senescence, FASST osteoblasts were treated with 10μM tamoxifen (4-OHT) for 3 days. SA-β-Gal staining was carried out on 4-OHT or vehicle (veh) treated osteoblasts on day 3. 4-OHT treatment resulted in a significant increase in enlarged, flattened cells that stained positive for SA-β-Gal (blue) compared to vehicle treated cells (lower left panel), scale bar is 200 μm (*p<0.05, two-tailed, Student t test. one of three representative experiments is shown). The induction of the senescence associated secretory phenotype (SASP) factor IL-6 was also quantitated by reverse transcriptase PCR (qRT-PCR) (lower right panel). Senescent osteoblasts expressed significantly more IL-6 mRNA compared to vehicle treated cells (SEM, *p<0.05, two-tailed, Student t test; one of three representative experiment is shown).

Article Snippet: For IL-6 neutralization in this assay, senescent or non-senescent osteoblasts were co-cultured with BMMs and 5pg/μl of IL-6 neutralization antibody (Cat#554400, BD Biosciences, San Jose, CA 95131) or 5pg/μl of IgG control (Cat#40041B, BioLegend, San Diego, CA 92121) was added to the medium.

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

Journal: Cell reports

Article Title: Stromal-initiated changes in the bone promote metastatic niche development

doi: 10.1016/j.celrep.2015.12.016

Figure Lengend Snippet: A. Quantification of osteoclasts following co-culture with non-senescent (NS) or senescent (SEN) osteoblasts in the presence of a control (IgG) or IL-6 neutralizing antibody (α-IL-6). Bone marrow derived macrophages were cultured with non-senescent or senescent osteoblasts for 4 days in the presence of 5 pg α-IL-6. The antibody was replaced every other day. TRAP staining was carried out after 4 days co-culture and osteoclast numbers were quantitated and are displayed as TRAP-positive osteoclasts per field of view (TRAP+OC/FOV) (SEM, *p<0.05, two-tailed, Student t test; one of two representative experiment is shown).

Article Snippet: For IL-6 neutralization in this assay, senescent or non-senescent osteoblasts were co-cultured with BMMs and 5pg/μl of IL-6 neutralization antibody (Cat#554400, BD Biosciences, San Jose, CA 95131) or 5pg/μl of IgG control (Cat#40041B, BioLegend, San Diego, CA 92121) was added to the medium.

Techniques: Co-Culture Assay, Derivative Assay, Cell Culture, Staining, Two Tailed Test

Journal: Oncogene

Article Title: CCL18 signaling from tumor-associated macrophages activates fibroblasts to adopt a chemoresistance-inducing phenotype

doi: 10.1038/s41388-022-02540-2

Figure Lengend Snippet: A Representative images of H&E staining and CD10/GPR77 immunofluorescent staining in serial sections of the pre-treatment breast cancer biopsies of chemosensitive ( n = 3) and chemoresistant ( n = 3) patients. B , C Western blotting ( B ) for α-SMA, FAP, CD10, and GPR77 and flow cytometric analysis ( C ) for CD10 and GPR77 in primary normal breast fibroblasts (NBFs) treated with pre-treatment tumor conditional medium (CM). D Cytokine arrays of pre-treatment tumor CM, squares indicate the cytokines with significant changes. E Signal intensity of indicated cytokines in the cytokine arrays and their relative fold change between the chemoresistant ( n = 3) and chemosensitive tumors ( n = 3) F Flow cytometric analysis for CD10 and GPR77 in NBFs treated with chemosensitive or chemoresistant tumor CM added without or with neutralizing antibodies against IL6(αIL6), IL8(αIL8) or CCL18(αCCL18). The patients with complete remission (CR) or partial remission (PR) were classified as chemosensitive, while those with stable disease (SD) or progressive disease (PD) were chemoresistant.

Article Snippet: Tumor CM was supplemented with CCL18 neutralizing antibody, IL6 neutralizing antibody (10 µg/mL, BD, #554543) or IL8 neutralizing antibody (10 µg/mL, BD, #554726) prior to the experiments.

Techniques: Staining, Western Blot

Journal: Oncogene

Article Title: CCL18 signaling from tumor-associated macrophages activates fibroblasts to adopt a chemoresistance-inducing phenotype

doi: 10.1038/s41388-022-02540-2

Figure Lengend Snippet: A The growth inhibition rate of docetaxel (up) and cisplatin (down) on MCF-7, BT474 or BT549 cultured alone (Ctrl) or co-cultured with NBFs underwent indicated treatment ( n = 3). B The growth inhibition rate of docetaxel on MCF-7 (up) and SK-BR3 (down) cells cultured alone (Ctrl) or co-cultured with NBFs underwent indicated treatment ( n = 3). C , D Representative western blotting ( C ) and quantification ( D ) for cleavage of caspase-3 and PARP in SK-BR3 cells cultured alone (Ctrl) or co-cultured with NBFs underwent indicated treatment and challenged with cisplatin ( n = 3). E , F The percentage of CD44 + CD24 - ( E ) and ALDH1 + ( F ) cells in MCF-7 cells cultured alone (UT) or co-cultured with NBFs underwent indicated treatment. G – I The apoptosis after cisplatin treatment ( G ), the ALDH1 + ( H ) and the CD44 + CD24 - ( I ) proportion of MCF-7 cells cultured alone or co-cultured with untreated or CCL18-treated NBFs transduced with GFP shRNA or IL-6 and IL-8 shRNA ( n = 3). Data expressed as mean ± SEM, *** P < 0.001 compared with NBFs co-cultured with untreated TAMs ( A ), untreated NBFs ( B , D – F ) or CCL18-treated NBFs without shRNA transduction ( G – I ) by two-tailed one-way ANOVA with Dunnett’s multiple comparison test.

Article Snippet: Tumor CM was supplemented with CCL18 neutralizing antibody, IL6 neutralizing antibody (10 µg/mL, BD, #554543) or IL8 neutralizing antibody (10 µg/mL, BD, #554726) prior to the experiments.

Techniques: Inhibition, Cell Culture, Western Blot, Transduction, shRNA, Two Tailed Test

Journal: Oncogene

Article Title: CCL18 signaling from tumor-associated macrophages activates fibroblasts to adopt a chemoresistance-inducing phenotype

doi: 10.1038/s41388-022-02540-2

Figure Lengend Snippet: A Representative western blotting for α-SMA, FAP, CD10 and GPR77 in untreated (UT) or CCL18-treated NBFs transduced with shRNA against GFP or PITPNM3 ( n = 3). B Representative images(up) and diameters(down) of collagen gel contraction assay for NBFs with indicated treatment ( n = 3). C Representative western blotting for total expression and phosphorylation of IKK, IκB and SMAD2/3 in NBFs with indicated treatment ( n = 3). D (left)Representative images of p65 immunofluorescent staining and (right) quantification of p65 nuclear translocation in NBFs with indicated treatment ( n = 3). Scale bars (white line), 50 μm; Scale bars (yellow line), 5 μm. E Localizations of p65 to the promoters of CD10 and GPR77 genes in indicated fibroblasts were analyzed by ChIP assay using anti-p65 Ab or control IgG. F , G QRT–PCR ( F ) and representative western blotting ( G ) for CD10 and GPR77 expression in untreated (UT) or CCL18-activated NBFs treated with DMSO, JSH-23 or BAY117082 ( n = 3). H , I ELISA for IL-8 ( H ) and IL-6 ( I ) levels in the conditioned medium of untreated, CCL18 or TGF-β treated NBFs in the presence of JSH-23, BAY117082 or transduced with GFP or p65 shRNAs ( n = 3). JSH-23, an NF-κB inhibitor. BAY117082, an IKK inhibitor. Data expressed as mean ± SEM, *** P < 0.001 compared with NBFs co-cultured with untreated CCL18-activated NBFs ( B , F , H , I ) or TGF-β treated NBFs ( E ) by two-tailed one-way ANOVA with Dunnett’s multiple comparison test.

Article Snippet: Tumor CM was supplemented with CCL18 neutralizing antibody, IL6 neutralizing antibody (10 µg/mL, BD, #554543) or IL8 neutralizing antibody (10 µg/mL, BD, #554726) prior to the experiments.

Techniques: Western Blot, Transduction, shRNA, Collagen Gel Contraction Assay, Expressing, Staining, Translocation Assay, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Cell Culture, Two Tailed Test

Journal: Oncogene

Article Title: CCL18 signaling from tumor-associated macrophages activates fibroblasts to adopt a chemoresistance-inducing phenotype

doi: 10.1038/s41388-022-02540-2

Figure Lengend Snippet: A MCF-7 cells were implanted alone or co-injected with NBFs into the mammary fat pads of NOD/SCID mice, and recombinant CCL18 was administrated twice a week and the tumor formation were monitored for up to three months. Xenograft formation rates were shown ( n = 12 per group). Green part represented the proportion of mice fail to develop tumors and red part represented the proportion of mice that developed tumors successfully. * P < 0.05, ** P < 0.01 by Fisher’s exact test. B Quantification for CD10 + GPR77 + fibroblasts(black) and ALDH1 + tumor cells(red) by immunofluorescent staining in the harvested tumors in A ( n = 5 per group). C Quantification for the density of IL6 + fibroblasts(black) and IL8 + fibroblasts(red) determined by immunofluorescent staining in the harvested tumors in A ( n = 5 per group). D , E MCF-7 cells were implanted alone or co-injected with NBFs and docetaxel was administrated one week after implantation concomitantly with or without recombinant CCL18. D Tumor growth was monitored for 7 weeks (mean ± SEM, n = 8 per group). E Representative images for TUNEL + cells in the xenografts related to D (mean ± SEM, n = 8 per group). Scale bars, 25 μm. F-H , MCF-7 cells were implanted without or with NBFs and TAMs into the mammary fat pads of NOD/SCID mice and neutralizing antibody against CCL18 was used to block CCL18 signaling. Docetaxel was administrated 1 week after implantation. F Tumor growth was monitored for 7 weeks (mean ± SEM, n = 8 per group). G Quantification ( n = 5 per group) and ( H ) representative immunofluorescent images for CD163 + CCL18 + macrophages, CD10 + GPR77 + fibroblasts and TUNEL + EPCAM + tumor cells in the harvested tumors in F . Scale bars in H 50 μm. Data expressed as Mean ± SEM, *** P < 0.001 compared with xenografts formed by untreated MCF-7( A , D , E ), xenografts formed by MCF-7 cells co-implanted with NBFs without CCL18 treatment ( B , C ) or xenografts formed by MCF-7 cells co-implanted with NBFs and TAMs without CCL18 neutralizing antibody treatment ( F , G ) by two-tailed one-way ANOVA with Dunnett’s multiple comparison test.

Article Snippet: Tumor CM was supplemented with CCL18 neutralizing antibody, IL6 neutralizing antibody (10 µg/mL, BD, #554543) or IL8 neutralizing antibody (10 µg/mL, BD, #554726) prior to the experiments.

Techniques: Injection, Recombinant, Staining, TUNEL Assay, Blocking Assay, Two Tailed Test