Journal: Cell Biology and Toxicology

Article Title: SENP3 mediated DeSUMOylation of macrophage derived CCL17 accelerates atherosclerosis via regulation of Treg

doi: 10.1007/s10565-025-10099-3

Figure Lengend Snippet: Macrophages stimulated by oxLDL affect the chemotaxis of Treg through CCL17 and CCL22 competition. Macrophages were stimulated with oxLDL, and Treg cells were co-cultured with macrophage supernatant. A Immunofluorescence detection of co-localization of CCL22 and macrophages (CD68) in plaque tissue of AS mice. B The content of CCL22 in the supernatant of macrophages was detected by ELISA. C Transwell detected the effect of macrophages on the chemotaxis of Treg. ( D - E ) Transwell detected the migration of Treg cells. * p < 0.05, ** p < 0.01, *** p < 0.001. N = 3

Article Snippet: Prior to the transwell migration assay, the induced Tregs were treated with recombinant mouse CCL17 (MCE, HY-P71891A) or recombinant mouse CCL22 (MCE, HY-P7248) or anti-CCL17 (R&D, Catalog #: MAB529) and anti-CCL22 antibodies (R&D, Catalog #: MAB529).

Techniques: Chemotaxis Assay, Cell Culture, Immunofluorescence, Enzyme-linked Immunosorbent Assay, Migration

Journal: PLoS ONE

Article Title: LncRNA HOTAIR promotes proliferation, invasion and migration in NSCLC cells via the CCL22 signaling pathway

doi: 10.1371/journal.pone.0263997

Figure Lengend Snippet: A, B: the expression of HOTAIR and CCL22 mRNA were detected by RT-qPCR. C: CCL22 protein analyzed by ELISA assay. ****, P<0.001.

Article Snippet: The concentration of CCL22 in the supernatant was tested according to the instructions of ELISA kit operation (CUSABIO, CSB-E04660h).

Techniques: Expressing, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

Journal: PLoS ONE

Article Title: LncRNA HOTAIR promotes proliferation, invasion and migration in NSCLC cells via the CCL22 signaling pathway

doi: 10.1371/journal.pone.0263997

Figure Lengend Snippet: A: The HOTAIR and CCL22 mRNA expression examined by RT-qPCR B: Cell apoptosis detected by Flow cytometry. C: The cell proliferation tested by MTS assay. D: The CCL22 protein expression tested by ELISA. E: The cell migration and invasion detected by Transwell assay. **, P<0.05; ***, P<0.01; ****, P<0.001;ns, no significance.

Article Snippet: The concentration of CCL22 in the supernatant was tested according to the instructions of ELISA kit operation (CUSABIO, CSB-E04660h).

Techniques: Expressing, Quantitative RT-PCR, Flow Cytometry, MTS Assay, Enzyme-linked Immunosorbent Assay, Migration, Transwell Assay

Journal: Scientific Reports

Article Title: A transcription factor PU.1 is critical for Ccl22 gene expression in dendritic cells and macrophages

doi: 10.1038/s41598-018-37894-9

Figure Lengend Snippet: Analysis of cis -element in the Ccl22 promoter. JAWSII cells ( a ) and RAW264.7 cells ( b ) were transfected with reporter plasmids for renilla luciferase as an internal control and with reporter plasmids for firefly luciferase containing various lengths of the mouse Ccl22 promoter. ( c ) Sequences of the −27/−1 region of the mouse Ccl22 promoter. Two Ets motifs, designated Site1 and Site 2, are indicated in bold. ( d ) JAWSII cells were transfected with reporter plasmids of WT or mutant promoters lacking the Ets motif(s) at the indicated sites. All results are shown as means + S.D.s ( n = 3). Similar results were obtained in three independent experiments. * p < 0.05.

Article Snippet: The concentration of mouse CCL22 protein was determined using an ELISA kit (MCC220, R&D systems, Minneapolis, MN).

Techniques: Transfection, Luciferase, Control, Mutagenesis

Journal: Scientific Reports

Article Title: A transcription factor PU.1 is critical for Ccl22 gene expression in dendritic cells and macrophages

doi: 10.1038/s41598-018-37894-9

Figure Lengend Snippet: Effects of PU.1 knockdown on Ccl22 expression in mouse DCs and macrophages. JAWSII ( a ), RAW264.7 ( b ), BMDCs ( c , e ), and BMDMs ( d and f ) were transfected with either negative control siRNA (siNega) or Spi1 siRNA (siSpi1). At 48 h after transfection, relative mRNA levels were determined by quantitative RT-PCR after normalizing to mouse Gapdh mRNA levels. Data are expressed as the ratio of the expression level of the respective control siRNA-transfected cells. Western blotting analyses using anti-PU.1 Ab and anti-β-actin Ab were performed to evaluate the effect of Spi1 siRNA on PU.1 protein levels in BMDCs ( c right) and BMDMs ( d right). ( e , f ) The concentration of the CCL22 protein produced from either siNega or siSpi1 transfected BMDCs ( e ) or BMDMs ( f ) was determined as described in the Materials and Methods . Results are shown as means + S.D.s ( n = 3). Similar results were obtained in three independent experiments. * p < 0.05.

Article Snippet: The concentration of mouse CCL22 protein was determined using an ELISA kit (MCC220, R&D systems, Minneapolis, MN).

Techniques: Knockdown, Expressing, Transfection, Negative Control, Quantitative RT-PCR, Control, Western Blot, Concentration Assay, Produced

Journal: Scientific Reports

Article Title: A transcription factor PU.1 is critical for Ccl22 gene expression in dendritic cells and macrophages

doi: 10.1038/s41598-018-37894-9

Figure Lengend Snippet: Analysis of the PU.1 binding region in the mouse Ccl22 promoter. ChIP assay was performed using either goat IgG (gIgG) or anti-PU.1 Ab (PU.1) in BMDCs ( a ), BMDMs ( b ), splenic DCs ( c ), and peritoneal macrophages ( d ). The amounts of immunoprecipitated chromatin were determined by quantitative PCR amplifying the indicated region of the Ccl22 promoter. Data are expressed as the percentage of the input for each ChIP assay. Results are shown as means + S.D.s ( n > 3 ). Similar results were obtained in more than three independent experiments. * p < 0.05.

Article Snippet: The concentration of mouse CCL22 protein was determined using an ELISA kit (MCC220, R&D systems, Minneapolis, MN).

Techniques: Binding Assay, Immunoprecipitation, Real-time Polymerase Chain Reaction

Journal: Scientific Reports

Article Title: A transcription factor PU.1 is critical for Ccl22 gene expression in dendritic cells and macrophages

doi: 10.1038/s41598-018-37894-9

Figure Lengend Snippet: Involvement of IRFs in the expression of Ccl22 in BMDCs. ( a , b ) BMDCs were transfected with negative control siRNA (siNega), Irf4 siRNA (siIrf4) ( a ), or Irf8 siRNA (siIrf8) ( b ). At 48 h after transfection, relative mRNA levels were determined by quantitative RT-PCR after normalizing to mouse Gapdh mRNA levels. Data are expressed as the ratio of the expression level of the respective control siRNA-transfected cells. Western blotting analyses were performed using transfectants, which were harvested at 48 h after transfection (right in a , b ). ( c ) ChIP assay was performed by using either goat IgG (gIgG) or anti-IRF4 Ab (IRF4) in BMDCs. The amounts of immunoprecipitated chromatin were determined by quantitative PCR targeting the −17/+52 region of the Ccl22 promoter. Data are expressed as the percentage of the input for each ChIP assay. ( d ) mRNA level (left) and protein level (right) of PU.1 in control (siNega) or Irf4 knockdown cells (siIrf4). ( e ) PU.1 binding degree to −17/+52 in control (siNega) or Irf4 knockdown cells (siIrf4) determined by ChIP assay. Results are shown as means + S.D.s ( n = 3 ). Similar results were obtained in three independent experiments. * p < 0.05.

Article Snippet: The concentration of mouse CCL22 protein was determined using an ELISA kit (MCC220, R&D systems, Minneapolis, MN).

Techniques: Expressing, Transfection, Negative Control, Quantitative RT-PCR, Control, Western Blot, Immunoprecipitation, Real-time Polymerase Chain Reaction, Knockdown, Binding Assay

Journal: Scientific Reports

Article Title: A transcription factor PU.1 is critical for Ccl22 gene expression in dendritic cells and macrophages

doi: 10.1038/s41598-018-37894-9

Figure Lengend Snippet: Involvement of PU.1 in the expression of Ccl22 in mature BMDCs. ( a ) BMDCs were stimulated with 1 μg/ml LPS for the indicated period. Relative Ccl22 and Ccl17 mRNA levels were determined by quantitative RT-PCR after normalizing to mouse Gapdh mRNA levels. Data are expressed as the ratio of the expression level of the non-stimulated cells. ( b ) ChIP assay was performed using either goat IgG (gIgG) or anti-PU.1 Ab (α-PU.1) in BMDCs stimulated with 1 μg/ml LPS for 24 hours. The amounts of immunoprecipitated chromatin were determined by quantitative PCR targeting the −17/+52 region of the Ccl22 promoter. Data are expressed as the ratio of α-PU.1 to gIgG (α-PU.1/gIgG). ( c ) BMDCs were transfected with either negative control siRNA (siNega) or Spi1 siRNA (siSpi1). At 24 hours after transfection, cells were stimulated with 1 μg/ml LPS and further incubated for 24 hours. Relative mRNA levels were determined by quantitative RT-PCR after normalizing to mouse Gapdh mRNA levels. Data are expressed as the ratio of the expression level of the control siRNA-transfected immature BMDCs. Results are shown as means + S.D.s ( n > 3 ). Similar results were obtained in more than three independent experiments.

Article Snippet: The concentration of mouse CCL22 protein was determined using an ELISA kit (MCC220, R&D systems, Minneapolis, MN).

Techniques: Expressing, Quantitative RT-PCR, Immunoprecipitation, Real-time Polymerase Chain Reaction, Transfection, Negative Control, Incubation, Control

Journal: Scientific Reports

Article Title: A transcription factor PU.1 is critical for Ccl22 gene expression in dendritic cells and macrophages

doi: 10.1038/s41598-018-37894-9

Figure Lengend Snippet: Involvement of PU.1 in the expression of the human CCL22 gene. ( a ) mRNA levels of CCL22 and SPI1 in SPI1 siRNA (siSPI1) or control siRNA (siNega) introduced THP-1 cells. ( b ) Alignment of nucleotide sequences of human and mouse CCL22 genes. ( c ) ChIP assay was performed using either control IgG (gIgG) or anti-PU.1 Ab (α-PU.1) in THP-1 cells. The amounts of immunoprecipitated chromatin were determined by quantitative PCR targeting the −153/−76 (including cis -elements) or −2106/−2046 ( cis -control) of the CCL22 promoter. Results are shown as means + S.D.s of three independent experiments.

Article Snippet: The concentration of mouse CCL22 protein was determined using an ELISA kit (MCC220, R&D systems, Minneapolis, MN).

Techniques: Expressing, Control, Immunoprecipitation, Real-time Polymerase Chain Reaction

Journal: Scientific Reports

Article Title: MicroRNA-205-5p inhibits skin cancer cell proliferation and increase drug sensitivity by targeting TNFAIP8

doi: 10.1038/s41598-021-85097-6

Figure Lengend Snippet: TNFAIP8 expression is upregulated in BCC, SCC, and melanoma patient skin tissues. ( A ) Relative immunohistochemical TNFAIP8 expression in indicated skin cancer tissues is presented. ( B ) Immunohistochemical expression levels of TNFAIP8 protein from normal skin (n = 6), BCC (n = 6), H-SCC (n = 4), M-SCC (n = 4), L-SCC (n = 4), nevus (n = 6), and melanoma (n = 6) tissues were quantified using ImageJ software ( https://imagej.nih.gov/ij/ ) and plotted. The data represent mean ± SEM from 4 to 6 skin cancer tissues. * P < 0.05 relative to normal skin tissues. ( C ) Expression of TNFAIP8 transcripts between cutaneous melanoma (n = 45) and melanoma precursor (n = 18) samples were analyzed and presented from the Oncomine dataset ( https://www.oncomine.org/resource/login.html accessed October 17, 2020). *** P < 0.001 relatives to cutaneous melanoma tissues. ( D , E ) Expression of wild-type B-RAF, mutant B-RAF V600E , and TNFAIP8 in HaCaT, SK-MEL-2, A431, A375, and A2058 cells were analyzed by western blotting. Fifty micrograms of lysates from normal and skin cancer cells were immunoblotted with B-RAF, TNFAIP8, GAPDH, and β-actin antibodies. Immunoreactive bands were visualized using ECL chemiluminescence detection reagents after exposing the blots on X-ray films ( D ) or by scanning the blots with an Odyssey CLx Imager ( E ). The immunoblot scans were converted into grayscale and presented. ( F ) Expression of TNFAIP8 mRNA from indicated normal skin cells and skin cancer cells were analyzed by RT/qPCR as described in the “ ” section.

Article Snippet: Anti-β-actin antibody from Sigma (St. Louis, MO) and anti-TNFAIP8 antibody from Proteintech Group (Rosemont, IL) was also purchased.

Techniques: Expressing, Immunohistochemical staining, Software, Mutagenesis, Western Blot, Quantitative RT-PCR

Journal: Scientific Reports

Article Title: MicroRNA-205-5p inhibits skin cancer cell proliferation and increase drug sensitivity by targeting TNFAIP8

doi: 10.1038/s41598-021-85097-6

Figure Lengend Snippet: TNFα-induced TNFAIP8 expression in skin cancer cells ( A ) Schematic represents TNFAIP8 isoform-specific forward and reverse primer design. ( B ) The expression of different variants/isoforms of TNFAIP8 in normal HaCaT and skin cancer cells was analyzed by RT-PCR. NC–negative control (no cDNA). ( C ) HaCaT, A431, A375, and A2058 cells were treated with vehicle or TNFα (10–50 ng/ml) for 30 h, and cell lysates were immunoblotted with TNFAIP8 or β-actin antibodies. Immunoreactive bands were visualized using ECL chemiluminescence detection reagents and the blots were scanned using an Odyssey CLx imager. The immunoblot scans were converted into grayscale and presented. ( D ) Similarly, normal and skin cancer cells were treated with vehicle or TNFα (10-50 ng/ml) for 30 h, and TNFAIP8 mRNA expression was analyzed by RT/qPCR as described in the “ ” section. * P < 0.05, ** P < 0.01, *** P < 0.001 relative to control/vehicle-treated cells. ( E ) HaCaT, A431, A375 and, A2058 cells were treated with vehicle or TNFα (10–50 ng/ml) for 48 h and, relative cell survival was analyzed by MTT assay as described in the “ ” section. Results are representative of three independent experiments. * P < 0.05, *** P < 0.001 relative to control/vehicle-treated cells.

Article Snippet: Anti-β-actin antibody from Sigma (St. Louis, MO) and anti-TNFAIP8 antibody from Proteintech Group (Rosemont, IL) was also purchased.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Negative Control, Western Blot, Quantitative RT-PCR, Control, MTT Assay

Journal: Scientific Reports

Article Title: MicroRNA-205-5p inhibits skin cancer cell proliferation and increase drug sensitivity by targeting TNFAIP8

doi: 10.1038/s41598-021-85097-6

Figure Lengend Snippet: TNFAIP8 regulates cell proliferation and migration in skin cancer cells. ( A ) Western blotting analysis of TNFAIP8-Myc tagged protein expression in HaCaT and skin cancer cells. Immunoreactive bands were developed using ECL chemiluminescence detection reagents and the blots were scanned using an Odyssey CLx imager. The immunoblot scans were converted into grayscale and presented. (B) Effect of TNFAIP8-Myc protein overexpression on HaCaT, A431, A375 and, A2058 on cell survival was measured by MTT assay. Results are representative of three independent experiments. * P < 0.05 relative to EV transfected cells. ( C ) The effect of overexpression of TNFAIP8-Myc on cell colony formation was analyzed and plotted (upper and lower panels) as described in the “ ” section. * P < 0.05 relative to EV transfected cells. ( D ) HaCaT, A431, A375 and, A2058 cells were transfected with control siRNA or TNFAIP8 siRNA (100 nM) for 30 h, and cell lysates were western blotted with anti-TNFAIP8 and β-actin antibodies. Western blots were developed using ECL chemiluminescence detection reagents and the blots were scanned using an Odyssey CLx imager. The immunoblot scans were converted into grayscale and presented. TNFAIP8 and β-actin protein levels were quantified using ImageJ software ( https://imagej.nih.gov/ij/ ) and presented below. ( E ) The effect of TNFAIP8 knockdown by siRNA on cell survival was analyzed by MTT assay. The experiments were independently performed three times. * P < 0.05 relative to control siRNA transfected cells. ( F ) Control siRNA and TNFAIP8 siRNA-transfected HaCaT, A431, A375 and A2058 cells (3000 cells/well) were re-plated in 6-well plates in triplicate for 7–10 days and skin cancer cell colony formation was analyzed and plotted (upper and lower panels) as described in the “ ” section. * P < 0.05 relative to control siRNA transfected cells. ( G ) Wound healing assay: HaCaT, A431, A375 and A2058 cells were transfected with control siRNA or TNFAIP8 siRNA for 48 h. The effect of the TNFAIP8 knockdown on cell migration was analyzed using the wound healing assay. Representative images of the wound healing assay (top) and the calculated scratch area (bottom) are shown. * P < 0.05 relative to control siRNA transfected cells.

Article Snippet: Anti-β-actin antibody from Sigma (St. Louis, MO) and anti-TNFAIP8 antibody from Proteintech Group (Rosemont, IL) was also purchased.

Techniques: Migration, Western Blot, Expressing, Over Expression, MTT Assay, Transfection, Control, Software, Knockdown, Wound Healing Assay

Journal: Scientific Reports

Article Title: MicroRNA-205-5p inhibits skin cancer cell proliferation and increase drug sensitivity by targeting TNFAIP8

doi: 10.1038/s41598-021-85097-6

Figure Lengend Snippet: miR-205-5p targets the 3′UTR of TNFAIP8 and inhibits TNFAIP8 expression. ( A ) The binding site of miR-205-5p in the 3′UTR of TNFAIP8 was analyzed by TargetScan ( http://www.targetscan.org/vert_72/ ) and presented. We also generated a wild type and mutated binding nucleotides of miR-205-5p to the 3′UTR of TNFAIP8. Wild-type miR-205-5p and mutant miR-205-5p mimics are shown. ( B ) Relative expression of miR-205-5p in HaCaT, A431, A375, A2058, and SK-MEL-2 cell lines was analyzed by RT/qPCR as described in the “ ” section. ( C ) Luciferase reporter assay: A431 and A2058 cells were transfected with TNFAIP8-3′UTR-Luciferase reporter construct (0.5 µg) for 18 h and then transfected with NC mimic or wild-type miR-205-5p mimic or mutant miR-205-5p mimic for an additional 24 h. Transfected cells were lysed, and luciferase activity was measured. Results are representative of two independent experiments. ** P < 0.01, *** P < 0.001 compared with NC or mutant-type miR-205-5p mimic. ( D , E ) Normal or skin cancer cells were transfected with NC or miR-205-5p mimic for 48 h and the overexpression of miR-205-5p mimic and TNFAIP8 transcripts were analyzed by RT/qPCR. *** P < 0.001 compared with NC-transfected cells. * P < 0.05 compared with NC-transfected cells. ns-no significance. ( F ) HaCaT, A431 and A2058 cell lines were transfected with NC mimic or miR-205-5p mimic for 40 h and expression of endogenous TNFAIP8 protein were analyzed by western blotting. Western blots were developed using ECL chemiluminescence detection reagents, the blots were exposed to X-Ray films, scans were converted into grayscale and presented. ( G ) HaCaT, 431 and A2058 cell lines were grown on a coverslip and transfected with Cy3-labeled NC mimic or Cy3-labeled miR-205-5p mimic and the effect of Cy3-labeled miR-205-5p mimic on endogenous TNFAIP8 expression (green) was visualized by immunofluorescence as described in the “ ” section.

Article Snippet: Anti-β-actin antibody from Sigma (St. Louis, MO) and anti-TNFAIP8 antibody from Proteintech Group (Rosemont, IL) was also purchased.

Techniques: Expressing, Binding Assay, Generated, Mutagenesis, Quantitative RT-PCR, Luciferase, Reporter Assay, Transfection, Construct, Activity Assay, Over Expression, Western Blot, Labeling, Immunofluorescence

Journal: Scientific Reports

Article Title: MicroRNA-205-5p inhibits skin cancer cell proliferation and increase drug sensitivity by targeting TNFAIP8

doi: 10.1038/s41598-021-85097-6

Figure Lengend Snippet: miR-205-5p targets TNFAIP8 and reduces TNFAIP8-mediated cell proliferation and autophagy. ( A ) HaCaT, A431 and A2058 cells were transfected with NC mimic or miR-205-5p mimic for 48 h and relative cell survival was analyzed by MTT assay. The experiments were independently repeated three times. * P < 0.05 compared with NC mimic transfected cells. ( B ) NC mimic or miR-205-5p mimic transfected cells were re-plated in 6-well plates for 7–10 days and cell colony formation was analyzed and plotted (upper and lower panels) as described in the “ ” section. The experiment was repeated twice in triplicates. * P < 0.05 relative to NC mimic transfected cells. ( C ) HaCaT, A431 and A2058 cells were transfected EV or TNFAIP8-Myc plasmids for 30 h and lysates were western blotted with anti-Myc, anti-LC3β I/II, and anti-β-actin antibodies. Western blots were developed using ECL chemiluminescence detection reagents, the blots were exposed on X-ray films, converted into grayscale, and presented. ( D ) HaCaT, lanoma A431 and A2058 cells were transfected NC mimic or miR-205-5p mimic for 30 h and lysates were western blotted with anti-TNFAIP8, anti-LC3β I/II, anti-p62, and anti-β-actin antibodies. Western blots were developed using ECL chemiluminescence detection reagents, exposed on X-Ray films, converted into grayscale, and presented. ( E ) HaCaT, A431 and A2058 cells were grown on a coverslip transfected with Cy3-labeled NC mimic or Cy3-labeled miR-205-5p mimic and the effect of Cy3-labeled miR-205-5p mimic on endogenous LC3β I/II expression (green) and related puncta formation were visualized by immunofluorescence (upper panels) as described in the “ ” section (upper panel). LC3β I/II and related puncta were measured from individual cells (n = 10) and plotted (lower panels). ** P < 0.01, *** P < 0.001 relatives to Cy3 labeled-NC mimic transfected cells.

Article Snippet: Anti-β-actin antibody from Sigma (St. Louis, MO) and anti-TNFAIP8 antibody from Proteintech Group (Rosemont, IL) was also purchased.

Techniques: Transfection, MTT Assay, Western Blot, Labeling, Expressing, Immunofluorescence

Journal: Scientific Reports

Article Title: MicroRNA-205-5p inhibits skin cancer cell proliferation and increase drug sensitivity by targeting TNFAIP8

doi: 10.1038/s41598-021-85097-6

Figure Lengend Snippet: miR-205-5p targets TNFAIP8 in skin cancer cells and increase sensitivity to vemurafenib. ( A ) HaCaT cells, and skin cancer SK-MEL-2, A375 and A2058 cell lines were treated with increasing concentrations of vemurafenib for 48 h and relative cell survival/proliferation was measured by MTT assay. The experiments were independently repeated three times. ** P < 0.01, *** P < 0.001 compared to vehicle-treated cells. ( B ) HaCaT cells and skin cancer SK-MEL-2, A375 and A2058 cell lines were transfected with NC mimic or miR-205-5p mimic for 24 h and treated with vemurafenib or vehicle for an additional 48 h and relative cell survival were measured by MTT assay as described in the “ ” section. * P < 0.05, ** P < 0.01, *** P < 0.001 compared to NC mimic vehicle-treated cells or NC mimic and vemurafenib-treated cells. ns-no significance. ( C ) Melanoma A375 cells were transfected with NC mimic or miR-205-5p mimic for 18 h and then cells were treated with vemurafenib for an additional 24 h. Fifty micrograms of lysates were immunoblotted with anti-cleaved-PARP and anti-β-tubulin antibodies. Western blots were developed using ECL chemiluminescence detection reagents, the blots were scanned using Azure C-500 Biosystem, scans were converted into grayscale and presented. ( D ) Melanoma A375 cells were transfected with EV of TNFAIP8-Myc plasmid for 18 h first and then cells were treated with vemurafenib or paclitaxel for 24 h as indicated and fifty micrograms lysates were immunoblotted with anti-Myc, anti-cleaved-PARP, and anti-β-actin antibodies. Western blots were developed using ECL chemiluminescence detection reagents and the blots were scanned using an Odyssey CLx imager. The immunoblot scans were converted into grayscale and presented. ( E ) Fifty micrograms of lysates from A375 and A2058 cells or A375R and A2058R (vemurafenib resistant) cells were immunoblotted with anti-TNFAIP8, anti-LC3β I/II, and anti-β-actin antibodies. Western blots were developed using ECL chemiluminescence detection reagents, the blots were exposed on X-Ray films, scans were converted into grayscale and presented. ( F ) Relative cell proliferation/cell survival rate of A375 and A2058 cells, or A375R and A2058R (vemurafenib resistant) cells were measured by using WST-1 reagent (Cayman Chemical) (upper panel) or MTT assay reagent (lower panel) and plotted. ** P < 0.01, *** P < 0.001 compared to A375 and A2058 parent cells. Results are representative of three independent experiments. Vem. Vemurafenib.

Article Snippet: Anti-β-actin antibody from Sigma (St. Louis, MO) and anti-TNFAIP8 antibody from Proteintech Group (Rosemont, IL) was also purchased.

Techniques: MTT Assay, Transfection, Western Blot, Plasmid Preparation

Journal: Cell reports

Article Title: The IRAK1/IRF5 axis initiates IL-12 response by dendritic cells and control of Toxoplasma gondii infection

doi: 10.1016/j.celrep.2024.113795

Figure Lengend Snippet: (A) Volcano plot depicting T. gondii -infected versus uninfected CD11c + DCs (WT). The horizontal dashed line represents a —log false discovery rate of 2, and the vertical dashed lines represent a log 2 fold change of −1 and +1. (B) Heatmap showing the expression and hierarchical clustering of selected genes in CD11c + DCs from the indicated strains, uninfected or infected with T. gondii Me49 tachyzoites (MOI 3, 4 h). (C–F) Quantification of IL-12 p40 (C), CCL5 (D), TNF (E), and CCL22 (F) in culture supernatants of CD11c + DCs of the indicated strains infected with T. gondii Me49 (MOI 3) for 4 and 24 h. In (A) and (B), Data were pooled from four independent experiments. In (C)–(F), data were pooled from four independent experiments and are presented as mean ± SEM; each independent experiment consisted of one technical replicate. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 (ANOVA with Tukey’s multiple-comparisons test).

Article Snippet: Cytokine and chemokine quantification was performed from culture supernatants, peritoneal lavage or serum, according to the manufacturer instructions: IL-12 p40 Mouse uncoated ELISA kit (Invitrogen, 88–7120-88), Mouse CCL5/Rantes DuoSet ELISA (R&D Systems, DY478), Mouse TNF-alpha DuoSet ELISA (R&D Systems, DY410), Mouse Interferon-gamma DuoSet ELISA (R&D Systems, DY485), and Mouse CCL22 DuoSet ELISA (R&D Systems, DY439).

Techniques: Infection, Expressing

Journal: Cell reports

Article Title: The IRAK1/IRF5 axis initiates IL-12 response by dendritic cells and control of Toxoplasma gondii infection

doi: 10.1016/j.celrep.2024.113795

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Cytokine and chemokine quantification was performed from culture supernatants, peritoneal lavage or serum, according to the manufacturer instructions: IL-12 p40 Mouse uncoated ELISA kit (Invitrogen, 88–7120-88), Mouse CCL5/Rantes DuoSet ELISA (R&D Systems, DY478), Mouse TNF-alpha DuoSet ELISA (R&D Systems, DY410), Mouse Interferon-gamma DuoSet ELISA (R&D Systems, DY485), and Mouse CCL22 DuoSet ELISA (R&D Systems, DY439).

Techniques: Recombinant, Modification, Saline, Protease Inhibitor, Marker, Western Blot, Extraction, Enzyme-linked Immunosorbent Assay, Software

Journal: Science Advances

Article Title: Macrophage-derived chemokine CCL22 establishes local LN-mediated adaptive thermogenesis and energy expenditure

doi: 10.1126/sciadv.adn5229

Figure Lengend Snippet: ( A ) GO analysis of macrophage response to LNR. ( B ) Heatmaps of macrophage-secreted chemokines in the iWAT from mice receiving sham or LNR at 6°C for 7 days ( n = 4 per group). Red and blue represent the fold increase and decrease in a gene, respectively (see color scale). ( C ) mRNA expression of macrophage-secreted chemokines in iWAT from mice described in (B) ( n = 8 per group). ( D ) Ccl22 mRNA expression in iWAT from mice described in (B) at 23° or 6°C for 7 days ( n = 6 per group). ( E ) Ccl22 mRNA expression in iWAT from mice described in (B) exposed to 6°C for different hours ( n = 6). ( F ) Serum CCL22 levels from mice described in (B) exposed to 6°C for different days ( n = 6). ( G ) Serum CCL22 levels from mice described in (B) ( n = 6 per group). ( H ) Ccl22 mRNA expression in iWAT SVF cells or mature adipocytes (MAs). ( I ) Ccl22 mRNA expression in M0, M1, and M2 macrophages derived from 10-week-old C57BL/6 bone marrows ( n = 8 per group). ( J and K ) mRNA expression (J) and IF (K) of UCP1 in beige adipocytes ( n = 6 per group). Scale bars, 25 μm. ( L to N ) Immunofluorescence of UCP1 (L), mRNA expression of thermogenic genes (M), and immunoblots of UCP1 (N) in iWAT ( n = 3 to 6 per group). Scale bars, 50 μm. ( O to R ) Immunoblots and quantification of UCP1 [(O) and (P)], mRNA expression (Q) of thermogenic genes, and IF of UCP1 and H&E staining (R) in iWAT ( n = 3 to 6 per group). Scale bars, 50 μm. Data information: Results are presented as means ± SEM. [(C), (D), (G) to (J), (M), (P), and (Q)] * P ≤ 0.05, ** P < 0.01, and *** P < 0.001 by nonpaired Student’s t test. [(E) and (F)] * P ≤ 0.05 and ** P < 0.01 nonpaired Student’s t test compared with before cold stimulation.

Article Snippet: The plasma CCL22 levels were measured by a CCL22/MDC PicoKine ELISA kit (EK0447, Boster Bio).

Techniques: Expressing, Derivative Assay, Immunofluorescence, Western Blot, Staining

Journal: Science Advances

Article Title: Macrophage-derived chemokine CCL22 establishes local LN-mediated adaptive thermogenesis and energy expenditure

doi: 10.1126/sciadv.adn5229

Figure Lengend Snippet: ( A ) Pearson’s correlation coefficient analysis of serum CCL22 concentration and fat mass ( n = 28 per group). ( B to F ) Body weight (B), energy consumption rate [(C) and (D)], and RER [(E) and (F)] ( n = 8 per group). Ten-week-old C57BL/6 male mice received saline or rCCL22 (20 μg/kg per day) at 6°C for 14 days with HFD. ( G to I ) Immunofluorescence (G), immunoblots (H) of UCP1, and mRNA expression (I) of thermogenic genes in iWAT ( n = 3 to 6 per group). Scale bar, 50 μm. ( J to L ) Immunofluorescence (J), immunoblots, and quantification [(K) and (L)] of UCP1 in iWAT ( n = 3 per group). SVF cells were extracted from HFD-fed iWAT, cultured with vehicle or rCCL22 (10 ng/ml) for 4 days, and then induced to beige adipocytes for 5 days. Scale bars, 25 μm. ( M ) Schematic diagram: The 6-month trial of obese adults participated in ADF: a 3-month weight loss period followed by a 3-month weight maintenance period. ( N and O ) Pearson’s correlation coefficient analysis of human serum CCL22 levels, body weight (N), and fat mass (O) ( n = 40). ( P ) Human serum CCL22 levels ( n = 20). ( Q ) Schematic diagram: Human SVFs from iWAT were cultured at 31°C for 5 days, treated with vehicle or CCL22 (10 ng/ml) for 4 days, and then induced to beige adipocytes. ( R to T ) Immunoblots and quantification [(R) and (S)] of UCP1 and mRNA expression (T) of thermogenic genes in human beige adipocytes ( n = 3 to 6 per group). Data information: Results are presented as means ± SEM. [(A), (N), and (O)] Two-tailed Pearson’s correlation coefficient analysis. [(F), (I), (L), (S), and (T)] * P ≤ 0.05 and ** P < 0.01 by nonpaired Student’s t test. (P) ** P < 0.01 by nonpaired Student’s t test compared with month 0 body weight. (B) * P ≤ 0.05 by two-way analysis of variance (ANOVA) followed by post hoc Bonferroni tests.

Article Snippet: The plasma CCL22 levels were measured by a CCL22/MDC PicoKine ELISA kit (EK0447, Boster Bio).

Techniques: Concentration Assay, Saline, Immunofluorescence, Western Blot, Expressing, Cell Culture, Two Tailed Test

Journal: Science Advances

Article Title: Macrophage-derived chemokine CCL22 establishes local LN-mediated adaptive thermogenesis and energy expenditure

doi: 10.1126/sciadv.adn5229

Figure Lengend Snippet: Primer sequences for qPCR gene expression analysis.

Article Snippet: The plasma CCL22 levels were measured by a CCL22/MDC PicoKine ELISA kit (EK0447, Boster Bio).

Techniques: Gene Expression