Journal: Cell

Article Title: Mobilization-based chemotherapy-free engraftment of gene-edited human hematopoietic stem cells

doi: 10.1016/j.cell.2022.04.039

Figure Lengend Snippet:

Article Snippet: Anti-human CD184 , Miltenyi Biotec , Cat# 130-117-519; RRID:AB_2734059.

Techniques: Blocking Assay, Plasmid Preparation, Recombinant, Electroporation, CRISPR, Adjuvant, Enzyme-linked Immunosorbent Assay, Clinical Proteomics, Cell Culture, Gene Expression

Journal: Advanced Science

Article Title: Isoquercitrin Alleviates Diabetic Nephropathy by Inhibiting STAT3 Phosphorylation and Dimerization

doi: 10.1002/advs.202414587

Figure Lengend Snippet: Isoquercitrin inhibits STAT3 to improve endothelial and renal tubular epithelial cell injury. A–E) RT‐qPCR detection of mRNA expression levels of interleukin (IL)‐6, IL‐1β, monocyte chemoattractant protein‐1 (MCP‐1), intercellular adhesion molecule‐1 (ICAM‐1), and tumor growth factor beta (TGF‐β) in kidneys ( n = 4). F) Immunofluorescence detection of endothelial cell injury marker ET‐1 in kidneys ( n = 6). G) WB detection of Kim‐1 expression, a marker of renal tubular epithelial cell injury, in mouse kidneys ( n = 4). RT‐qPCR detection of lipocalin‐2 (Lcn‐2) (H) and tissue inhibitor of metalloproteinase 1 (TIMP‐1) (I) mRNA expression levels in kidneys ( n = 4). J) Immunohistochemistry and immunofluorescence detection of TNF‐α, p‐STAT3, α‐SMA, E‐cadherin, vimentin, and aquaporin 2 (AQP2) expression in kidney tubules ( n = 6). Electron microscopy observation of the morphology of mitochondria in renal tubular epithelial cells of mice (J) ( n = 3). Data are presented as the mean ± SEM. ** p < 0.01, *** p < 0.001, **** p < 0.0001. One‐way ANOVA followed by the Dunnett's post hoc test.

Article Snippet: Antibodies specific for syndecan‐1 (10593‐1‐AP; 1:1000 for WB), glypican‐1 (16700‐1‐AP; 1:1000 WB), BCL2 (68103‐1‐Ig; 1:5000 for WB), BAX (50599‐2‐lg; 1:2000 for WB), TGF‐β (21898‐1‐AP; 1:1000 for WB), collagen‐I (14695‐1‐AP; 1:1000 for WB), β‐tubulin (66240‐1‐Ig; 1:20 000 for WB), β‐actin (66009‐1‐Ig; 1:20 000 WB, 1:500 for immunofluorescence) were purchased from Proteintech (Wuhan, Hubei, China).

Techniques: Quantitative RT-PCR, Expressing, Immunofluorescence, Marker, Immunohistochemistry, Electron Microscopy

Journal: Advanced Science

Article Title: Isoquercitrin Alleviates Diabetic Nephropathy by Inhibiting STAT3 Phosphorylation and Dimerization

doi: 10.1002/advs.202414587

Figure Lengend Snippet: Isoquercitrin alleviates high glucose or IL‐6‐induced endothelial cell injury by inhibiting STAT3 activity. RPMI Medium 1640 containing 45 mM glucose was used as a high glucose (HG) culture medium to induce human umbilical vein endothelial cells (HUVECs). RPMI Medium 1640 with a glucose concentration of 11 mM was used to culture HUVECs in the normal glucose (NG) control group. Subsequently, isoquercitrin (ISO) at concentrations of 5, 10, and 20 µM was added to the culture medium for intervention. A) WB detection of the p‐STAT3, STAT3, and TGF‐β expression levels in HG‐stimulated HUVECs ( n = 4). B) The mRNA expression changes of cytokines IL‐6, TNF‐α, ICAM‐1, and MCP‐1 in HUVECs after HG stimulation and ISO intervention ( n = 3). C) WB detection of the expression of endothelial dysfunction marker inducible nitric oxide synthase (iNOS) and endothelial glycocalyx components syndecan‐1 and glypican‐1 in HUVECs ( n = 4). D) WB detection of endothelial cell marker VE‐cadherin and mesenchymal cell markers vimentin and α‐SMA in HUVECs ( n = 4). HUVECs were stimulated with 25 ng mL −1 IL‐6, whereas HUVECs without IL‐6 stimulation were the normal control (NC) group. E) WB detection of p‐STAT3, STAT3, IL‐6, and IL‐1β expression in HUVECs stimulated with IL‐6 ( n = 3). F) The mRNA expression changes of cytokines IL‐1β, TNF‐α, and MCP‐1 in HUVECs after IL‐6 stimulation and ISO intervention ( n = 6). STAT3 overexpression in HUVECs via plasmid transfection, with WB detection of transfection efficiency (G) ( n = 3). H) WB detection of the effects of IL‐6 and ISO on p‐STAT3 and STAT3 expressions in HUVEC cells under transfected or nontransfected conditions ( n = 6). I–K) RT‐qPCR detection of the effects of IL‐6 and ISO on the transcription levels of cytokines IL‐6, IL‐1β, and TNF‐α in HUVEC cells with or without transfection ( n = 4). Data are presented as the mean ± SEM. * /# p < 0.05, ** /## p < 0.01, *** /### p < 0.001, **** /#### p < 0.0001. In (C), (D), and (F), * indicates comparison with db/m , and # indicates comparison with db/db . One‐way ANOVA followed by the Dunnett's post hoc test.

Article Snippet: Antibodies specific for syndecan‐1 (10593‐1‐AP; 1:1000 for WB), glypican‐1 (16700‐1‐AP; 1:1000 WB), BCL2 (68103‐1‐Ig; 1:5000 for WB), BAX (50599‐2‐lg; 1:2000 for WB), TGF‐β (21898‐1‐AP; 1:1000 for WB), collagen‐I (14695‐1‐AP; 1:1000 for WB), β‐tubulin (66240‐1‐Ig; 1:20 000 for WB), β‐actin (66009‐1‐Ig; 1:20 000 WB, 1:500 for immunofluorescence) were purchased from Proteintech (Wuhan, Hubei, China).

Techniques: Activity Assay, Concentration Assay, Control, Expressing, Marker, Over Expression, Plasmid Preparation, Transfection, Quantitative RT-PCR, Comparison

Journal: Advanced Science

Article Title: Isoquercitrin Alleviates Diabetic Nephropathy by Inhibiting STAT3 Phosphorylation and Dimerization

doi: 10.1002/advs.202414587

Figure Lengend Snippet: Isoquercitrin inhibits STAT3 in renal tubular epithelial cells, reducing IL‐6‐induced pro‐inflammatory and profibrotic cytokines. Human kidney 2 cells (HK2) cells were stimulated with 25 ng mL −1 IL‐6, whereas HK2 cells without IL‐6 stimulation were the normal control (NC) group. Subsequently, interventions were conducted with 5, 10, and 20 µM of isoquercitrin. A) WB detection of p‐STAT3 and STAT3 expression in HK2 cells ( n = 4). RT‐qPCR detection of the mRNA expression of pro‐inflammatory cytokines IL‐6 (B), IL‐1β (C), and TNF‐α (D), macrophage chemoattractant protein MCP‐1 (E), and profibrotic cytokine TGF‐β (F) ( n = 4). G–J) Isoquercitrin inhibits EMT in renal tubular epithelial cells. Isoquercitrin downregulates the abnormally high expression of mesenchymal markers vimentin and α‐SMA, while restoring the expression of the epithelial marker E‐cadherin ( n = 6). Data are presented as the mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. One‐way ANOVA followed by the Dunnett's post hoc test.

Article Snippet: Antibodies specific for syndecan‐1 (10593‐1‐AP; 1:1000 for WB), glypican‐1 (16700‐1‐AP; 1:1000 WB), BCL2 (68103‐1‐Ig; 1:5000 for WB), BAX (50599‐2‐lg; 1:2000 for WB), TGF‐β (21898‐1‐AP; 1:1000 for WB), collagen‐I (14695‐1‐AP; 1:1000 for WB), β‐tubulin (66240‐1‐Ig; 1:20 000 for WB), β‐actin (66009‐1‐Ig; 1:20 000 WB, 1:500 for immunofluorescence) were purchased from Proteintech (Wuhan, Hubei, China).

Techniques: Control, Expressing, Quantitative RT-PCR, Marker

Journal: Advanced Science

Article Title: Isoquercitrin Alleviates Diabetic Nephropathy by Inhibiting STAT3 Phosphorylation and Dimerization

doi: 10.1002/advs.202414587

Figure Lengend Snippet: Schematic diagram showing the mechanism of isoquercitrin inhibiting STAT3 to ameliorate diabetic nephropathy. Isoquercitrin alleviates the injury of endothelial and renal tubular epithelial cells in diabetic nephropathy by inhibiting STAT3 phosphorylation and dimerization. Based on these actions, isoquercitrin reduces the expression of pro‐inflammatory and profibrotic cytokines such as IL‐1β, IL‐6, TNF‐α, ICAM‐1, MCP‐1, and TGF‐β, thereby alleviating renal inflammation and extracellular matrix accumulation.

Article Snippet: Antibodies specific for syndecan‐1 (10593‐1‐AP; 1:1000 for WB), glypican‐1 (16700‐1‐AP; 1:1000 WB), BCL2 (68103‐1‐Ig; 1:5000 for WB), BAX (50599‐2‐lg; 1:2000 for WB), TGF‐β (21898‐1‐AP; 1:1000 for WB), collagen‐I (14695‐1‐AP; 1:1000 for WB), β‐tubulin (66240‐1‐Ig; 1:20 000 for WB), β‐actin (66009‐1‐Ig; 1:20 000 WB, 1:500 for immunofluorescence) were purchased from Proteintech (Wuhan, Hubei, China).

Techniques: Phospho-proteomics, Expressing

Journal: Endocrinology

Article Title: Histone Deacetylase 6-Controlled Hsp90 Acetylation Significantly Alters Mineralocorticoid Receptor Subcellular Dynamics But Not its Transcriptional Activity.

doi: 10.1210/en.2015-2055

Figure Lengend Snippet: Figure 1. Hsp90 acetylation shifts naïve MR subcellular localization towards the nucleus in COS-7 cells. A, Representative images of COS-7 cells transfected with MR-147-YFP showing each category used for scoring the subcellular localization of the receptor in the absence of ligand. At the time of transfection cells were switched to culture medium supplemented with charcoal-stripped FCS. Cells were fixed 48h after transfection, mounted and images were taken in a confocal microscope. DAPI was included in the mounting medium to counterstain cell nuclei. N, exclusive nuclear localization; N C, predominant nuclear localization; N C, even distribution throughout cytosol and nucleus; N C, predominant cytosolic localization; C, exclusive cytosolic localization. B, Representative images of MR subcellular distribution in COS-7 cells cotransfected with Hsp90-WT-CFP or Hsp90–K295Q-CFP. C, Western blot analysis of transfected MR expression in COS-7 cells cotransfected with Hsp90-WT or K295Q. N.T., nontransfected cells. -Actin was used as a loading control. D, Quantitative analysis of MR-147-YFP subcellular localization in COS-7 cells cotransfected with an empty plasmid, Hsp90-WT or mutants K295Q or K295R. Values represent the average percentage of cells in each category from the total amount of cells scored in three independent experiments. *, P .05, one-way ANOVA.

Article Snippet: Immunohistochemical analysis of MR expression and Hsp90 acetylation Endogenous levels of acetylated Hsp90 were specifically detected using a polyclonal anti Hsp90- acetyl K294 antibody (Rockland Immunochemicals, Limerick, PA) on Sprague-Dawley male rats kidney and heart sections (43).

Techniques: Transfection, Microscopy, Western Blot, Expressing, Control, Plasmid Preparation

Journal: Endocrinology

Article Title: Histone Deacetylase 6-Controlled Hsp90 Acetylation Significantly Alters Mineralocorticoid Receptor Subcellular Dynamics But Not its Transcriptional Activity.

doi: 10.1210/en.2015-2055

Figure Lengend Snippet: Figure 3. HDAC6-controlled Hsp90 acetylation alters MR subcellular localization. A, Western blot analysis of HDAC6-WT and HDAC6-DN expression after transfection in COS-7 cells; lower panel, Hsp90 expression under the same conditions. B, Western blot analysis of transfected MR expression in COS-7 cells cotransfected with HDAC6-WT or HDAC6-DN. N.T., nontransfected cells. -Actin was used as a loading control. C, Representative confocal images of COS-7 cells cotransfected with MR-147-GFP and HDAC6-WT or HDAC6-DN. MR was detected by GFP emission; HDAC6 was detected by immunocytochemistry using a secondary antibody conjugated to Alexa-fluor 594. Nuclei were counterstained with DAPI. D, subcellular localization of MR-147-GFP in COS-7 cells cotransfected with HDAC6-WT, HDAC6-DN, or with HDAC6- DN Hsp90–K295R. Bars represent the average SE of MR subcellular localization from two independent experiments.*, P .05. Subcellular distribution categories were defined as in Figure 1A.

Article Snippet: Immunohistochemical analysis of MR expression and Hsp90 acetylation Endogenous levels of acetylated Hsp90 were specifically detected using a polyclonal anti Hsp90- acetyl K294 antibody (Rockland Immunochemicals, Limerick, PA) on Sprague-Dawley male rats kidney and heart sections (43).

Techniques: Western Blot, Expressing, Transfection, Control, Immunocytochemistry

Journal: Endocrinology

Article Title: Histone Deacetylase 6-Controlled Hsp90 Acetylation Significantly Alters Mineralocorticoid Receptor Subcellular Dynamics But Not its Transcriptional Activity.

doi: 10.1210/en.2015-2055

Figure Lengend Snippet: Figure 4. siRNA-mediated HDAC6 knockdown or pharmacological inhibition of type I/type II HDACs shift naïve MR towards the cell nucleus A, Western blot analysis of siRNA-mediated HDAC6 knockdown, showing endogenous HDAC6 expression in COS-7 cells transfected with control (scrambled) siRNA or anti-HDAC6 specific siRNA. Hsp90 expression in the same cells is also shown. Anti--actin was used as loading control. Bars represent average band intensity ( SE, n 3) corrected by -actin and normalized to control; *, P .01, Student´s t test. B, Immunofluorescence localization of HDAC6 in COS-7 cells cotransfected with scrambled siRNA or anti-HDAC6 siRNA. C, Subcellular localization of MR-147-GFP in COS-7 cells cotransfected with control (scrambled) siRNA or anti-HDAC6 specific siRNA, anti-HDAC6 siRNA and Hsp90–K295R, or in cells pretreated for 4h with 1 M with TSA. Bars represent the average SE of MR subcellular localization from two independent experiments. *, P .05. Subcellular distribution categories were defined as in Figure 1A.

Article Snippet: Immunohistochemical analysis of MR expression and Hsp90 acetylation Endogenous levels of acetylated Hsp90 were specifically detected using a polyclonal anti Hsp90- acetyl K294 antibody (Rockland Immunochemicals, Limerick, PA) on Sprague-Dawley male rats kidney and heart sections (43).

Techniques: Knockdown, Inhibition, Western Blot, Expressing, Transfection, Control, Immunofluorescence

Journal: Endocrinology

Article Title: Histone Deacetylase 6-Controlled Hsp90 Acetylation Significantly Alters Mineralocorticoid Receptor Subcellular Dynamics But Not its Transcriptional Activity.

doi: 10.1210/en.2015-2055

Figure Lengend Snippet: Figure 5. Mutation mimicking Hsp90 acetylation (Hsp90–K295Q) partially impairs MR-Hsp90 interaction. A, In situ proximity ligation assay (PLA) detecting MR-Hsp90 interactions. Cells were fixed and after the PLA reaction was finished coverslips were mounted in the presence of DAPI to counterstain nuclei. Left column shows representative images of the PLA reaction obtained in nontransfected COS-7 cells, cells cotransfected with MR-147-HA and wild type or K295Q Hsp90, or cells cotransfected with MR-147-HA and wild type Hsp90 and then treated with 10 nM aldosterone overnight. Middle column shows merged images with PLA signals and DAPI-stained nuclei. Right column shows PLA signal detection by analysis software. B, Quantitative analysis of PLA signals. Bars represent the number of discrete dots normalized by cell area SE (n 19–22). *, P .01, one-way ANOVA followed by Dunn´s multiple comparison test.

Article Snippet: Immunohistochemical analysis of MR expression and Hsp90 acetylation Endogenous levels of acetylated Hsp90 were specifically detected using a polyclonal anti Hsp90- acetyl K294 antibody (Rockland Immunochemicals, Limerick, PA) on Sprague-Dawley male rats kidney and heart sections (43).

Techniques: Mutagenesis, In Situ, Proximity Ligation Assay, Staining, Software, Comparison

Journal: Endocrinology

Article Title: Histone Deacetylase 6-Controlled Hsp90 Acetylation Significantly Alters Mineralocorticoid Receptor Subcellular Dynamics But Not its Transcriptional Activity.

doi: 10.1210/en.2015-2055

Figure Lengend Snippet: Figure 9. Immunohistochemical analysis of Hsp90 acetylation in rat kidney and heart shows predominant labeling in vascular smooth muscle cells. A, Representative micrograph of a paraffin-embedded section of rat kidney cortex stained with anti-Hsp90- acetyl-K295 antibody. Staining was restricted to renal arteries and arterioles (arrowheads). Renal tubules were negative for anti-Hsp90-acetyl-K295 staining (arrow). B, Rat kidney cortex stained with 12A7 anti-MR antibody. Staining is prominent in distal nephron (arrowheads) but negative in proximal tubules (arrow). C, Negative control of rat kidney cortex obtained omitting the primary anti-Hsp90-acetyl-K295 antibody. D, Representative micrograph of rat heart stained with anti-Hsp90-acetyl-K295 antibody. Staining was restricted to coronary arteries and arterioles (arrowheads). E, Rat heart stained with anti-MR antibody. Cardiomyocyte nuclei are clearly stained (arrowheads). F, Negative control of rat heart obtained omitting the primary anti-Hsp90-acetyl-K295 antibody. Bars in panels A and D, 50 m.

Article Snippet: Immunohistochemical analysis of MR expression and Hsp90 acetylation Endogenous levels of acetylated Hsp90 were specifically detected using a polyclonal anti Hsp90- acetyl K294 antibody (Rockland Immunochemicals, Limerick, PA) on Sprague-Dawley male rats kidney and heart sections (43).

Techniques: Immunohistochemical staining, Labeling, Staining, Negative Control

Journal: Cancers

Article Title: Sustaining the Activation of EGFR Signal by Inflammatory Cytokine IL17A Prompts Cell Proliferation and EGFR-TKI Resistance in Lung Cancer

doi: 10.3390/cancers15133288

Figure Lengend Snippet: Interleukin (IL)-17A induces proliferation of human non-small-cell lung cancer (NSCLC) cells harboring wild-type (WT) or mutant epidermal growth factor receptor (EGFR). ( A ) Western blot analysis of IL-17A expressions in PC9, HCC827, and A549 cells after transfecting a control vector (Neo) or IL-17A-expressing vector (IL-17A-His). ( B ) Dot blot analysis of secreted IL-17A using conditioned media of IL-17A-His-transfected PC9, HCC 827, and A549 cells. The uncropped blots are shown in the . ( C ) Proliferation rates of IL-17A-overexpressing PC9, HCC827, and A549 cells were measured by performing a CCK8 assay. Proliferation rates had significantly increased in IL-17A-His-transfected NSCLC cells after 24 (left) and 48 h (right) compared to control cells. ( D ) PC9, HCC827, and A549 cells were treated with 0, 1, 10, or 50 ng/mL recombinant human (rh)IL-17A for 24 and 48 h. The effect of rhIL-17A on proliferation of these NSCLC cells was evaluated by a CCK-8 assay. * p < 0.05, ** p < 0.01, *** p < 0.001 compared to the control group.

Article Snippet: Santa Cruz Biotechnology (Santa Cruz, CA, USA) provided antibodies for IL-17A (sc-374218), EGFR (sc-101), lysosomal-associated membrane protein 1 (LAMP1; sc-20011), and GAPDH (sc-32233), while Proteintech Group (Chicago, IL, USA) supplied the antibody for His-tag (66005-1-ig).

Techniques: Mutagenesis, Western Blot, Control, Plasmid Preparation, Expressing, Dot Blot, Transfection, CCK-8 Assay, Recombinant

Journal: Cancers

Article Title: Sustaining the Activation of EGFR Signal by Inflammatory Cytokine IL17A Prompts Cell Proliferation and EGFR-TKI Resistance in Lung Cancer

doi: 10.3390/cancers15133288

Figure Lengend Snippet: Interleukin (IL)-17A facilitates phosphorylation of epidermal growth factor receptor (EGFR) and Met and subsequently induces EGFR-tyrosine kinase inhibitor (TKI) resistance in human non-small-cell lung cancer (NSCLC) cells harboring mutant-EGFR. ( A , B ) Detection of phosphorylated (p)-EGFR and its downstream signaling by Western blotting after overexpression of IL-17A ( A ) and treatment with rhIL-17A at different concentrations and time points ( B ) in EGFR-mutant PC9 cells. ( C ) Western blot analysis of p-EGFR in PC9 cells after transducing shIL-17RC or control shRNA (shLuc) and treatment with rhIL-17A or the vehicle. Quantitative results of p-EGFR proteins were adjusted to GAPDH protein levels. ( D ) A CCK8 assay was performed to evaluate the proliferation inhibitory effect of afatinib treatment for 72 h in PC9 or HCC827 cells with or without IL-17A overexpression *** p < 0.001, ## p < 0.01, ### p < 0.001. ( E ) Proliferation rates of PC9 cells that received different concentrations of afatinib combined with or without rhIL-17A for 24 h. *** p < 0.001 compared to the control group. # p < 0.05, ## p < 0.01, ### p < 0.001 compared to the afatinib-treated only group. ( F ) Differential expression levels of phosphorylated receptor tyrosine kinases (RTKs) in PC9 cell lysates following 24 h treatment with rhIL-17A. An antibody array (R&D Systems) was used to detect 49 different phosphorylated human RTKs. The left panel shows representative array blots. The right panel shows a quantitative analysis of phosphorylated RTKs using a densitometer. Values are presented as the mean ± SD. n = 2. ( G , H ) Detection of phosphorylated (p)-Met by Western blotting after overexpression of IL-17A ( G ) and treatment with rhIL-17A at different concentrations and time points ( H ) in PC9 cells. ( I ) Detection of p-Met by Western blotting in PC9/IL-17A and PC9/Neo cells receiving different concentrations of SU11274. The uncropped blots are shown in the . ( J ) Proliferation rates of rhIL-17A-treated or vehicle-treated PC9 cells after receiving afatinib or afatinib+SU11274 for 48 h. * p < 0.05, ** p < 0.01, *** p < 0.001 compared to the control group. ### p < 0.001 compared to the afatinib-treated only group.

Article Snippet: Santa Cruz Biotechnology (Santa Cruz, CA, USA) provided antibodies for IL-17A (sc-374218), EGFR (sc-101), lysosomal-associated membrane protein 1 (LAMP1; sc-20011), and GAPDH (sc-32233), while Proteintech Group (Chicago, IL, USA) supplied the antibody for His-tag (66005-1-ig).

Techniques: Phospho-proteomics, Mutagenesis, Western Blot, Over Expression, Control, shRNA, CCK-8 Assay, Quantitative Proteomics, Ab Array

Journal: Cancers

Article Title: Sustaining the Activation of EGFR Signal by Inflammatory Cytokine IL17A Prompts Cell Proliferation and EGFR-TKI Resistance in Lung Cancer

doi: 10.3390/cancers15133288

Figure Lengend Snippet: Interleukin (IL)-17A synergizes with epidermal growth factor (EGF) to trigger EGF receptor (EGFR) activation via preventing EGF-mediated EGFR degradation in human non-small-cell lung cancer (NSCLC) cells harboring wild-type (WT)-EGFR. ( A , B ) Differential expression levels of phosphorylated kinases in A549 cell lysates following 10 min treatment with EGF or EGF + rhIL-17A. An antibody array (R&D Systems) was used to detect 37 different phosphorylated kinases and two related total proteins. Representative array blots are shown in ( A ). Quantitative analysis of phosphorylated kinases or total proteins using a densitometer are shown in ( B ). ( C ) Detection of phosphorylated (p)-EGFR and total EGFR by Western blotting after treatment of A549 cells with EGF or EGF+rhIL-17A for the indicated time points. ( D ) Western blot analysis of p-EGFR and total EGFR in A549 cells after transducing shIL-17RC or control shRNA (shLuc) and treatment with EGF or EGF+rhIL-17A for 10 min. ( E ) IL-17A-His-transfected A549 or control cells were treated with EGF (100 ng/mL) for the indicated time points, and expression levels of p-EGFR and total EGFR were further evaluated by a Western blot analysis. ( F ) A549/shLuc and A549/shIL-17RC cells were transfected with IL-17A-His or a control vector and treated with EGF for 10 min, and total EGFR was further detected. Quantitative results of p-EGFR and total EGFR proteins from ( C – F ) were, respectively, adjusted to total EGFR and β-actin or GAPDH protein levels. The uncropped blots are shown in the . ( G ) A CCK8 assay was performed to evaluate proliferation rates of A549 cells which were treated with the vehicle, EGF (100 ng/mL), rhIL-17A (10 ng/mL), or EGF+rhIL-17A for 24 h. * p < 0.05, compared to the EGF- or rhIL-17A-treated only group.

Article Snippet: Santa Cruz Biotechnology (Santa Cruz, CA, USA) provided antibodies for IL-17A (sc-374218), EGFR (sc-101), lysosomal-associated membrane protein 1 (LAMP1; sc-20011), and GAPDH (sc-32233), while Proteintech Group (Chicago, IL, USA) supplied the antibody for His-tag (66005-1-ig).

Techniques: Activation Assay, Quantitative Proteomics, Ab Array, Western Blot, Control, shRNA, Transfection, Expressing, Plasmid Preparation, CCK-8 Assay

Journal: Cancers

Article Title: Sustaining the Activation of EGFR Signal by Inflammatory Cytokine IL17A Prompts Cell Proliferation and EGFR-TKI Resistance in Lung Cancer

doi: 10.3390/cancers15133288

Figure Lengend Snippet: Interleukin (IL)-17A prevents epidermal growth factor (EGF)-mediated EGF receptor (EGFR) degradation via impairing the transport of EGFR to lysosomes in human non-small-cell lung cancer (NSCLC) cells harboring wild-type (WT)-EGFR. ( A , B ) Serum-starved A549 cells were pretreated with MG132 (20 µM) ( A ) or bafilomycin A1 (20 nM) ( B ) for 1 h, and then 100 ng/mL EGF was added for the indicated time points. Cell lysates from ( A , B ) were subjected to a Western blot analysis to detect phosphorylated (p)-EGFR and total EGFR expressions. The uncropped blots are shown in the . ( C ) Serum-starved A549 cells were treated with EGF (100 ng/mL) alone or EGF combined with rhIL-17A (10 ng/mL) for the indicated time points (10 and 30 min). Membrane EGFR levels were detected by a FASC analysis after staining with a mouse mAb to EGFR or mouse isotype control antibodies. The mean fluorescence intensity was a measure of the level of EGFR expression. *** p < 0.001 compared to the control group. ( D ) Different colocalization of lysosomal-associated membrane protein 1 (LAMP1) and endocytosed EGFR after 30 min of EGF or EGF+rhIL-17A treatment. Left panel: Cells were fixed, permeabilized, and stained with anti-EGFR (green), anti-LAMP1 (red), and DAPI (blue) for nuclear staining, and further examined by confocal microscopy. White arrows point to colocalization of EGFR and lysosomes. Orange arrows point to the nuclear translocation of EGFR. Right panel: Colocalization rate of EGFR and lysosome or nucleus was analyzed by MetaMorph software (version 7.8.4.0). * p < 0.05, ** p < 0.01 compared to the vehicle control group. # p < 0.05 compared to the EGF-treated alone group. ns: No Significance.

Article Snippet: Santa Cruz Biotechnology (Santa Cruz, CA, USA) provided antibodies for IL-17A (sc-374218), EGFR (sc-101), lysosomal-associated membrane protein 1 (LAMP1; sc-20011), and GAPDH (sc-32233), while Proteintech Group (Chicago, IL, USA) supplied the antibody for His-tag (66005-1-ig).

Techniques: Western Blot, Membrane, Staining, Control, Fluorescence, Expressing, Confocal Microscopy, Translocation Assay, Software

Journal: Cancer Communications

Article Title: CEBPB Expression in Tumor Cells Drives Immune Evasion in Colorectal Cancer via CTLA4 Up-regulation in T Cells

doi: 10.34133/cancomm.0013

Figure Lengend Snippet: WT p53 suppresses mRNA and protein levels of C/EBPβ in CRC cell lines. (A) Protein and mRNA expression of C/EBPβ in CT26 cells stably expressing shCtrl or sh Trp53 were analyzed by Western blotting and real-time PCR, respectively. (B) C/EBPβ protein expression was determined in CRC cell lines harboring TP53 WT, nonsense mutation, and missense mutation treated with nutlin-3a (10 μmol/l) for WT p53 induction or vehicle for 24 h (left), as well as CEBPB mRNA levels in TP53 WT cell lines (right). (C) C/EBPβ protein expression was compared in TP53 WT HCT116 and SNU1544 cells after transfection with siCtrl or si TP53 , followed by nutlin-3a (10 μmol/l) or vehicle treatment for 24 h. (D) C/EBPβ protein expression was determined in TP53 nonsense SNUC1 and SNU1411 cells after transient transfection with EV or TP53 WT, followed by 24 h of nutlin-3a (10 μmol/l) treatment. (E) C/EBPβ protein levels were determined in SNU1544 cells pretreated with nutlin-3a (10 μmol/l) for 16 h and stimulated with 8-Br-cAMP (100 μmol/l), an inducer of CEBPB transcription, for 8 h. (F) C/EBPβ protein levels were determined in SNU1411 cells transiently transfected with EV, TP53 WT, or TP53 R175H for 24 h. (G) C/EBPβ protein (left) and mRNA (right) levels were assessed in p53-null Caco-2 cells stably transduced with doxycycline-inducible TP53 WT or TP53 R175H following treatment with 1 μg/ml doxycycline for 48 h. (H) C/EBPβ protein stability was compared in shCtrl and sh Trp53 CT26 cells after CHX treatment (5 μg/ml) for the indicated times. (I) SNU1544 were treated with nutlin-3a (10 μmol/l) for 21 h, followed by MG132 (2 μmol/l) for 3 h to block proteasomal degradation. Protein expression was analyzed by Western blotting, representative of 3 independent experiments, and the values presented underneath or in the graph (H) were generated by densitometric analyses using ImageJ. mRNA levels were determined using real-time PCR. The bar graphs represent the mean ± SD; each dot represents an independent biological replicate. P values were calculated using 2-tailed Student’s t tests. Abbreviations: 8-Br-cAMP, 8-bromoadenosine 3′,5′-cyclic monophosphate; CEBPB , CCAAT enhancer binding protein beta; CHX, cycloheximide; CRC, colorectal cancer; doxy, doxycycline; EV, empty vector; SD, standard deviation; shCtrl, control short hairpin RNA; sh Trp53 , short hairpin RNA for Trp53 ; si TP53 , small interfering RNA for TP53 ; WT, wild type.

Article Snippet: For overexpression of genes, cDNA of human TP53 and LCN2 from HEK293T and mouse Lcn2 from CT26 were generated by PCR amplification, cDNA was obtained from human CEBPB (SC319561, OriGene) and mouse Cebpb (MR227563, OriGene), open reading frame clones were cloned into entry vector pDONR221 (12536017, Thermo Fisher Scientific), and entry vector with non-transcribed stuffer sequence (C125-E01, a gift from Dominic Esposito, Frederick National Laboratory for Cancer Research) was used for empty vector (EV) controls.

Techniques: Expressing, Stable Transfection, Western Blot, Real-time Polymerase Chain Reaction, Mutagenesis, Transfection, Transduction, Blocking Assay, Generated, Binding Assay, Plasmid Preparation, Standard Deviation, Control, shRNA, Small Interfering RNA

Journal: Cancer Communications

Article Title: CEBPB Expression in Tumor Cells Drives Immune Evasion in Colorectal Cancer via CTLA4 Up-regulation in T Cells

doi: 10.34133/cancomm.0013

Figure Lengend Snippet: Tumor cell C/EBPβ increases CTLA-4 expression in CD4 + T cells. (A) CT26 cells expressing Cebpb -oe and EV control were injected subcutaneously into immunocompetent BALB/c mice ( n = 4; representative of 3 independent experiments, top) and immunodeficient BALB/c nude mice ( n = 5; single experiment, bottom). (B) Subcutaneous tumors from EV and Cebpb -oe CT26 cells injected into BALB/c mice were collected approximately 21 days post-inoculation. The percentages of CD4 + and CD8 + cells among CD45 + cells, CTLA-4 + cells among CD4 + and CD8 + cells, Foxp3 + CTLA-4 + and Foxp3 − CTLA-4 + cells among CD4 + cells, and GzmB + and IFN-γ + cells among CD8 + cells were compared by flow cytometry. (C) Mouse splenic T cells isolated from BALB/c mice were activated with anti-CD3ε and anti-CD28, then co-cultured with EV or Cebpb -oe CT26 cells for 72 h. The culture ratio of T cells to CT26 cells is indicated. Flow cytometry was used to analyze CTLA-4 + and Foxp3 + levels among CD4 + cells, as well as CTLA-4 + , PD-1 + , GzmB + , and IFN-γ levels among CD8 + cells. Representative histograms of CTLA-4 + expression in CD4 + T are presented. (D) mRNA expression of immune checkpoint genes in Jurkat (left) and EL4 cells (right) after co-culture with indicated cells, measured by real-time PCR. (E) Proliferation analysis of CFSE-labeled, activated mouse splenic T cells co-cultured with indicated cells for 72 h. CFSE dilution in CD4 + T and CD8 + T cells were assessed by flow cytometry and presented as histograms (representative of 2 independent experiments). (F) Transwell migration assay of mouse splenic T cells (left) and EL4 (right) cultured for 24 h in CM from Cebpb -oe or EV CT26 cells or in fresh media, in the presence of CXCL12 (200 ng/ml). (G) Tumor growth curves of BALB/c mice bearing Cebpb -oe CT26 tumors treated with isotype control IgG, anti-PD-1, anti-CTLA-4, or the combination ( n = 5 per group). Tumor volumes are shown as mean ± SD; statistical comparisons were made using 2-tailed Student’s t tests based on the final tumor volume. Bar graphs represent the mean ± SD; each dot represents an independent biological replicate. P values were calculated using 2-tailed Student’s t tests. Abbreviations: CEBPB , CCAAT enhancer binding protein beta; Cebpb -oe, Cebpb -overexpression; CFSE, carboxyfluorescein succinimidyl ester; CM, conditioned media; CTLA-4, cytotoxic T-lymphocyte associated protein 4; CXCL12, C-X-C motif chemokine ligand 12; EV, empty vector; Foxp3, forkhead box protein P3; LAG3 , lymphocyte activating 3; PD-1, programmed cell death protein 1; PDCD1 , programmed cell death 1; SD, standard deviation.

Article Snippet: For overexpression of genes, cDNA of human TP53 and LCN2 from HEK293T and mouse Lcn2 from CT26 were generated by PCR amplification, cDNA was obtained from human CEBPB (SC319561, OriGene) and mouse Cebpb (MR227563, OriGene), open reading frame clones were cloned into entry vector pDONR221 (12536017, Thermo Fisher Scientific), and entry vector with non-transcribed stuffer sequence (C125-E01, a gift from Dominic Esposito, Frederick National Laboratory for Cancer Research) was used for empty vector (EV) controls.

Techniques: Expressing, Control, Injection, Flow Cytometry, Isolation, Cell Culture, Co-Culture Assay, Real-time Polymerase Chain Reaction, Labeling, Transwell Migration Assay, Binding Assay, Over Expression, Plasmid Preparation, Standard Deviation

Journal: Cancer Communications

Article Title: CEBPB Expression in Tumor Cells Drives Immune Evasion in Colorectal Cancer via CTLA4 Up-regulation in T Cells

doi: 10.34133/cancomm.0013

Figure Lengend Snippet: Lipocalin-2 mediates C/EBPβ-driven CTLA-4 up-regulation in T cells. (A) Venn diagram of genes up-regulated in CEBPB -oe compared to EV cells in CT26, SNU1544, and SNU81, analyzed by RNA sequencing, identifying 13 genes that were concurrently up-regulated. (B) CM from EV or Cebpb -oe CT26 cells were subjected to mouse cytokine array. Each cytokine is represented by duplicate spots, and those showing altered expression are indicated (left). The graph shows signal intensity relative to control spots, analyzed by densitometric quantification (right). (C) Protein levels of C/EBPβ and lipocalin-2 in SNU1544 and SW837 cells by Western blotting . (D) mRNA expression of CTLA4 in Jurkat cells after 24 h co-culture with indicated cells, measured by real-time PCR. (E) mRNA expression of immune checkpoint genes in Jurkat cells after co-culture with LCN2 -oe or EV SNU1544 cells. (F) mRNA expression of CTLA4 in Jurkat cells co-cultured with SW837 cells at a 1:1 ratio in the presence of lipocalin-2 neutralizing antibody (1 μg/ml) or isotype IgG control for 24 h. (G) Protein levels of CEBP/β and lipocalin-2 in CEBPB -oe and EV SNU1544 cells (left). mRNA levels of CTLA4 in Jurkat cells co-cultured with CEBPB -oe or EV SNU1544 cells in the presence of lipocalin-2 neutralizing antibody (1 μg/ml) or isotype IgG control for 24 h (right). (H) Protein levels of C/EBPβ and lipocalin-2 in EV, Cebpb -oe, and Lcn2 -oe CT26 cells (left). Flow cytometry analysis of CTLA-4 + in CD4 + and CD8 + cells from activated mouse splenic T cells co-cultured with EV or Lcn 2-oe CT26 cells for 72 h at the indicated culture ratio of T cells to CT26 cells (right). (I) Flow cytometry analysis of activated mouse splenic T cells co-cultured with Cebpb -oe CT26 at a 5:1 ratio for 48 h in the presence of lipocalin-2 neutralizing antibody (2 μg/ml) or isotype IgG control. The bar graphs represent the mean ± SD; each dot indicates a biological replicate. P values were calculated using 2-tailed Student’s t tests. Abbreviations: AKR1C1 , aldo-keto reductase family 1 member C1; C3 , complement C3; CDA , cytidine deaminase; CD14 , cluster of differentiation 14; CEBPB , CCAAT enhancer binding protein beta; CEBPB -oe, CEBPB overexpression; CM, conditioned media; CTLA4 , cytotoxic T-lymphocyte associated protein 4; CXCL1, C-X-C motif chemokine ligand 1; EV, empty vector; IL1RL1 , interleukin 1 receptor like 1; IRF7 , interferon regulatory factor 7; LCN2 , lipocalin-2; Lcn2 -oe, Lcn2 -overexpression; S100A8 , S100 calcium binding protein A8; S100A9 , S100 calcium binding protein A9; SBSN , suprabasin; SD, standard deviation; SERPINB2 , serpin family B member 2; SLPI , secretory leukocyte peptidase inhibitor.

Article Snippet: For overexpression of genes, cDNA of human TP53 and LCN2 from HEK293T and mouse Lcn2 from CT26 were generated by PCR amplification, cDNA was obtained from human CEBPB (SC319561, OriGene) and mouse Cebpb (MR227563, OriGene), open reading frame clones were cloned into entry vector pDONR221 (12536017, Thermo Fisher Scientific), and entry vector with non-transcribed stuffer sequence (C125-E01, a gift from Dominic Esposito, Frederick National Laboratory for Cancer Research) was used for empty vector (EV) controls.

Techniques: RNA Sequencing, Expressing, Control, Western Blot, Co-Culture Assay, Real-time Polymerase Chain Reaction, Cell Culture, Flow Cytometry, Binding Assay, Over Expression, Plasmid Preparation, Standard Deviation

Journal: Oncotarget

Article Title: Mesenchymal stem cells enhance tumorigenic properties of human glioblastoma through independent cell-cell communication mechanisms

doi: 10.18632/oncotarget.25346

Figure Lengend Snippet: ( A ) Basal TGFB1 protein levels secreted in conditioned medium (CM) by MSC derived from bone marrow (BMMSC1); umbilical cord (UCMSC3, UCMSC4 and UCMSC5) and adipose tissue (ATMSC1, ATMSC2 and ATMSC3). TGFB1 protein levels for U87MG and fibroblasts are shown for comparison. ( B ) Normalized TGFB1 expression in MSC from umbilical cord (UCMSC4). (C) TGFB1 knockdown significantly decreased TGFB1 protein levels in CM, and in exosomes of MSC. Total amount ( D ) and proliferation index ( E ) of viable U87MG cells cultured in the presence or absent of CM from transduced MSC. MSC Ctr. (MSC transduced with non-specific control plasmid); MSC shTGFB1 (MSC transduced with TGFB1 shRNA plasmid). Significance: * p ≤ 0.05, ** p ≤ 0.01, **** p ≤ 0.0001.

Article Snippet: TGFB1 protein levels in cell culture supernatants, as well as in cell extracts and exosomes, were quantified using a Quantikine human TGFB1 immunoassay (SB100B - R&D Systems, Minneapolis, MN, USA).

Techniques: Derivative Assay, Comparison, Expressing, Knockdown, Cell Culture, Transduction, Control, Plasmid Preparation, shRNA

Journal: Oncotarget

Article Title: Mesenchymal stem cells enhance tumorigenic properties of human glioblastoma through independent cell-cell communication mechanisms

doi: 10.18632/oncotarget.25346

Figure Lengend Snippet: ( A ) Total amount of viable U87MG cells in single cultures or co-cultures with MSC allowing direct cell-cell contact. ( B ) TGFB1 protein levels in CM from U87MG and MSC single cultures, and in CM from U87MG–MSC co-cultures systems. ( C ) Kaplan–Meier plots of tumor growth after subcutaneous injection of MSC, U87MG cells, or U87MG cells in combination with MSC, in nude mice. Representative tumor images are shown. MSC injection did not generate tumors. ( D ) Kaplan–Meier plots of tumor growth after subcutaneous injection of U87MG cells with transduced MSC in nude mice. Representative tumor images are shown. MSC Ctr. (MSC transduced with non-specific control plasmid); MSC shTGFB1 (MSC transduced with TGFB1 shRNA plasmid). Significance: * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001.

Article Snippet: TGFB1 protein levels in cell culture supernatants, as well as in cell extracts and exosomes, were quantified using a Quantikine human TGFB1 immunoassay (SB100B - R&D Systems, Minneapolis, MN, USA).

Techniques: Injection, Transduction, Control, Plasmid Preparation, shRNA

Journal: Oncotarget

Article Title: Mesenchymal stem cells enhance tumorigenic properties of human glioblastoma through independent cell-cell communication mechanisms

doi: 10.18632/oncotarget.25346

Figure Lengend Snippet: Tumor cells displayed significant chemoattraction to MSC, regardless the MSC-derived TGFB1 levels, showing increased migration ( A ) and invasion ( B ) compared with controls. ( C ) Presence of MSC enhanced U87MG 3-D cell invasion. GBM spheroids displayed significantly more protrusions in hydrogel matrix when co-cultured with MSC, regardless the MSC-derived TGFB1 levels. MSC did not show invasive properties in this assay. Photomicrographs at day 1, 4 and 7 for all groups. Scale bar: 400 µm. ( D ) Kinetics of 3-D cell invasion of U87MG, transduced MSC, and U87MG co-cultured with transduced MSC cells. MSC Ctr. (MSC transduced with non-specific control plasmid); MSC shTGFB1 (MSC transduced with TGFB1 shRNA plasmid). Significance: * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001.

Article Snippet: TGFB1 protein levels in cell culture supernatants, as well as in cell extracts and exosomes, were quantified using a Quantikine human TGFB1 immunoassay (SB100B - R&D Systems, Minneapolis, MN, USA).

Techniques: Derivative Assay, Migration, Cell Culture, Transduction, Control, Plasmid Preparation, shRNA