Journal: Frontiers in endocrinology

Article Title: Transcriptional Analysis of Sepsis-Induced Activation and Damage of the Adrenal Endothelial Microvascular Cells.

doi: 10.3389/fendo.2019.00944

Figure Lengend Snippet: FIGURE 3 | mRNA and protein verification of RNA sequencing analysis. Real-time PCR (qPCR) verification of expression of endothelial-specific genes (A) and expression of some of the top 100 most altered genes (B) with specific primers. Relative gene expression was determined by the 11Ct method with normalization of 18S ribosomal RNA. Saline-treated samples were depicted with open bars, and LPS-treated samples were depicted with black filled bars. N = 5 and statistics were calculated with the two-tailed non-parametric Mann-Whitney test. Values are mean with SEM. *p < 0.05, **p < 0.01. Images of fluorescent staining of thrombomodulin (Thmd) and Vascular adhesion molecule 1 (VCAM-1) (C) in adrenal tissue sections of mouse adrenal gland isolated from either Saline or LPS treated mice. Thmd and VCAM-1 were visualized using secondary antibodies coupled to Cy3 dye (red color). Western blot evaluation of VCAM-1 expression in adrenal glands isolated from mice treated either with Saline or LPS (N = 4) (D). Graphic representation of the VCAM-1 and GAPDH protein expression quantified in ImageJ Software based on the densitometric evaluation of protein bands (E). Results and evaluation of dot-blot based protein array (F) of different inflammatory-related molecules (G), cytokines (H) and chemokines (I). Saline-treated samples were depicted with open bars, and LPS-treated samples were depicted with gray filled bars. For each experimental group, two adrenals from three different animals (N = 6) were pooled and results are presented as double replicate.

Article Snippet: For the VCAM-1 and GAPDH detection following antibodies were used rabbit monoclonal anti-mouse VCAM-1 antibody (1:1000, clone EPR5047, ab134047, Abcam), a monoclonal rabbit anti-mouse GAPDH (1:1000; clone 14C10, #2118, Cell Signaling Technology), and goat anti-rabbit HRP conjugated secondary antibodies (1:6000, CST).

Techniques: RNA Sequencing, Real-time Polymerase Chain Reaction, Expressing, Gene Expression, Saline, Two Tailed Test, MANN-WHITNEY, Staining, Isolation, Western Blot, Software, Dot Blot, Protein Array

Journal: Cell reports

Article Title: Gα13 loss in Kras/Tp53 mouse model of pancreatic tumorigenesis promotes tumors susceptible to rapamycin.

doi: 10.1016/j.celrep.2022.110441

Figure Lengend Snippet: Figure 2. Human PDAC tumors with reduced Ga13 expression and tumors developing in the KPCGfl/fland KPCGfl/+ mice demonstrate increased mTOR signaling (A) Analysis of samples in cBioPortal with low and high expression of GNA13 for E-cadherin (CDH1), catenin-b1 (CTNNB1), claudin-7 (CLDN7), p-PDK1 (p-S241), mTOR, RPS6, and EEF2 at the protein level using the reverse-phase protein array (RPPA) data (n = 43 and 43, respectively). t test, mean ± SEM,*p % 0.05, **p % 0.01.

Article Snippet: Primary antibodies used include Ga13 (Santa Cruz #sc-293424, 1:1,000), E-cadherin (Cell Signaling #3195, RRID:AB_2291471, 1: 5,000), PDK1 (Cell Signaling #5662, AB_10839264, 1:1,000), p-PDK1 (Cell Signaling #3438, RRID:AB_2161134 1:1,000) mTOR (Cell Signaling #2983, RRID: AB_2105622, 1:1,000), p-mTOR(Abcam ab109268, RRID: AB_10888105, 1:1,000), RPS6(Cell Signaling #2217, RRID:AB_331355, 1:10,000), p-RPS6 (Cell Signaling #4858, RRID: AB_916156, 1:10,000), Gapdh (Millipore Sigma #MAB374, RRID: AB_2107445 1:5,000), and Hsp90 (Santa Cruz Biotechnology sc-7947, RRID: AB_2121235 1:5,000).

Techniques: Expressing, Protein Array

Journal: Cell reports

Article Title: Gα13 loss in Kras/Tp53 mouse model of pancreatic tumorigenesis promotes tumors susceptible to rapamycin.

doi: 10.1016/j.celrep.2022.110441

Figure Lengend Snippet: Figure 4. Ga13 loss sensitizes KPC tumors to the mTOR inhibitor rapamycin and reduces tumor growth in vivo (A) Syngeneic KPCG+/+ and KPCGfl/fltumors were analyzed for p-Pdk1, Pdk1, p-mTOR, mTOR, p-Rps6, and Rps6 by western blotting using Hsp90 as a loading control. (B and C) The KPCG+/+ and KPCGfl/flcells were implanted subcutaneously in the flank of B6 mice (6–8 weeks old) and treated with vehicle control or rapamycin (60 mg/kg daily; arrow indicates the start of treatment), once the tumors reached 100 mm3. The tumor sizes were measured using a caliper, harvested day 27,

Article Snippet: Primary antibodies used include Ga13 (Santa Cruz #sc-293424, 1:1,000), E-cadherin (Cell Signaling #3195, RRID:AB_2291471, 1: 5,000), PDK1 (Cell Signaling #5662, AB_10839264, 1:1,000), p-PDK1 (Cell Signaling #3438, RRID:AB_2161134 1:1,000) mTOR (Cell Signaling #2983, RRID: AB_2105622, 1:1,000), p-mTOR(Abcam ab109268, RRID: AB_10888105, 1:1,000), RPS6(Cell Signaling #2217, RRID:AB_331355, 1:10,000), p-RPS6 (Cell Signaling #4858, RRID: AB_916156, 1:10,000), Gapdh (Millipore Sigma #MAB374, RRID: AB_2107445 1:5,000), and Hsp90 (Santa Cruz Biotechnology sc-7947, RRID: AB_2121235 1:5,000).

Techniques: In Vivo, Western Blot, Control

Journal: British Journal of Cancer

Article Title: Antibody targeting of CD24 efficiently retards growth and influences cytokine milieu in experimental carcinomas

doi: 10.1038/bjc.2013.102

Figure Lengend Snippet: Effects of SWA11 mAb treatment on intratumoural cytokine milieu in A549 tumours. ( A ) The levels of 40 different cytokines were determined in tumour lysates using a cytokine protein arrays. The level of cytokines was quantified using ImageJ software. Representative arrays from two independent SWA11 mAb treatment experiments are shown. ( B ) Protein levels of CCL5/RANTES, CXCL9/MIG and CCL2/MCP-1 in tumour lysates ( n =5 per group) were determined by ELISA. Data are normalised to the total protein concentration in tumour lysates. * P <0.05.

Article Snippet: Relative levels of 40 different cytokines and chemokines in xenograft tumour lysates were evaluated using a mouse cytokine antibody array kit (Proteome Profiler, R&D Systems) according to the manufacturer's recommendations.

Techniques: Software, Enzyme-linked Immunosorbent Assay, Protein Concentration

Journal: British Journal of Cancer

Article Title: Antibody targeting of CD24 efficiently retards growth and influences cytokine milieu in experimental carcinomas

doi: 10.1038/bjc.2013.102

Figure Lengend Snippet: Effects of SWA11 mAb treatment on intratumoural cytokine milieu in SKOV3ip tumours. ( A ) The levels of 40 different cytokines were determined in tumour lysates using cytokine protein arrays. The level of cytokines was quantified using ImageJ software. Representative arrays from two independent SWA11 mAb treatment experiments are shown. ( B ) Protein levels of CCL5/RANTES, CXCL9/MIG and CCL2/MCP-1 in tumour lysates ( n =5–6 per group) were determined by ELISA. Data are normalised to the total protein concentration in tumour lysates.

Article Snippet: Relative levels of 40 different cytokines and chemokines in xenograft tumour lysates were evaluated using a mouse cytokine antibody array kit (Proteome Profiler, R&D Systems) according to the manufacturer's recommendations.

Techniques: Software, Enzyme-linked Immunosorbent Assay, Protein Concentration

Journal: British Journal of Cancer

Article Title: Antibody targeting of CD24 efficiently retards growth and influences cytokine milieu in experimental carcinomas

doi: 10.1038/bjc.2013.102

Figure Lengend Snippet: Effects of combined SWA11 mAb and gemcitabine treatment on A549 tumour growth and intratumoural cytokine milieu. ( A ) SCID beige mice with established xenograft A549 lung carcinomas received treatment with SWA11 mAb (10 mg kg −1 ) or IgG 2A (10 mg kg −1 ) followed by gemcitabine at a dose of 12 mg kg −1 (10% of maximal tolerated dose) 1 day later ( n =5 per group). Control animals received SWA11 mAb (10 mg kg −1 ), IgG 2A (10 mg kg −1 ) or gemcitabine (12 mg kg −1 ) alone ( n =5 per group). Treatment was repeated three times with a 5-day interval. External size of A549 tumours was measured with a caliper. * P <0.05. Note that part of the results from this experiment are also shown in for illustration. ( B ) Effects of SWA11 mAb and gemcitabine treatment on the levels of 40 different cytokines were determined in A549 tumour lysates using a cytokine protein arrays. The level of cytokines was quantified using ImageJ software.

Article Snippet: Relative levels of 40 different cytokines and chemokines in xenograft tumour lysates were evaluated using a mouse cytokine antibody array kit (Proteome Profiler, R&D Systems) according to the manufacturer's recommendations.

Techniques: Software

Journal: PLoS ONE

Article Title: A Jak1/2 inhibitor, baricitinib, inhibits osteoclastogenesis by suppressing RANKL expression in osteoblasts in vitro

doi: 10.1371/journal.pone.0181126

Figure Lengend Snippet: (A) Immunoblot images of adenovirus-mediated knockdown of murine Jak1 or Jak2. (B, C) Effects of shJak1_#1, #2, shJak2_#1 or #2 on osteoclast formation in co-cultures of osteoblasts and bone marrow cells treated with 1,25D 3 and PGE 2 (B) or in bone marrow macrophage cultures treated with RANKL and M-CSF (C) . (D, E) Effects of shJak1_#1, #2, shJak2_#1 or #2 on expression of RANKL mRNAs (D) and proteins (E) in osteoblasts. Osteoblasts were transfected with shCtrl (lane 1, lane 2), shJak1_#1 (lane 3), shJak1_#2 (lane 4), shJak2_#1 (lane 5) or shJak2_#2 (lane 6) and treated with (lane 2–6) or without (lane 1) 1,25D 3 and PGE 2 for 24 h. In B–D : error bars, s.e. (n = 3–4). ** P < 0.01, Student's t test. † † P < 0.01, Dunnett’s multiple comparisons test (vs shCtrl with 1,25D 3 and PGE 2 ). Original immunoblot images are shown in .

Article Snippet: Immunoblotting was performed using the following antibodies; anti-phospho Jak1 (Tyr1022/1023) rabbit IgG (3331; Cell Signaling Technology, Beverly, MA, 1:1000), anti-Jak1 rabbit IgG (3332; Cell Signaling Technology, 1:1000), anti-phospho Jak2 (Tyr1007/1008) rabbit IgG (3776; Cell Signaling Technology, 1:1000), anti-Jak2 rabbit IgG (3230; Cell Signaling Technology, 1:1000), anti-RANKL goat IgG (sc-7628; Santa Cruz Biotechnology, Santa Cruz, CA, 1:1000), anti-phospho Stat3 (Tyr705) rabbit IgG (9145; Cell Signaling Technology, 1:2000), anti-Stat3α rabbit IgG (8768; Cell Signaling Technology, 1:1000), anti-α tubulin mouse IgG (CP06; Calbiochem, San Diego, CA, 1:1000), donkey anti-rabbit IgG-HRP (NA934V; GE Healthcare, Little Chalfont, UK, 1:5000), goat anti-mouse IgG-HRP (170–6516; Bio-Rad Laboratories, Hercules, CA, 1:2000), and donkey anti-goat IgG-HRP (sc-2056; Santa Cruz Biotechnology, 1:5000).

Techniques: Western Blot, Knockdown, Expressing, Transfection

Journal: PLoS ONE

Article Title: A Jak1/2 inhibitor, baricitinib, inhibits osteoclastogenesis by suppressing RANKL expression in osteoblasts in vitro

doi: 10.1371/journal.pone.0181126

Figure Lengend Snippet: (A) Densitometric data of cytokine protein array in co-cultured medium of osteoblasts and bone marrow cells in the presence or absence of 1,25D 3 and PGE 2 . Two independent experiments were performed, and a representative result is shown. (B, C) Osteoblasts were stimulated by 1,25D 3 and PGE 2 for 0.5 (B) or 1 h (B, C) , and phosphorylation of Jak1, Jak2, and Stat3 were determined by immunoblotting. (D) Effects of 2.5 μM baricitinib on expression of Socs3 mRNA in osteoblasts in the presence of 1,25D 3 and PGE 2 . (E) An activator of Stat3, colivelin (Santa Cruz Biotechnology; 0.1 or 1 µM), rescued baricitinib-induced RANKL down-regulation in osteoblast cultures. error bars, s.e. (n = 3). ** P < 0.01, Student’s t test. Original immunoblot images are shown in .

Article Snippet: Immunoblotting was performed using the following antibodies; anti-phospho Jak1 (Tyr1022/1023) rabbit IgG (3331; Cell Signaling Technology, Beverly, MA, 1:1000), anti-Jak1 rabbit IgG (3332; Cell Signaling Technology, 1:1000), anti-phospho Jak2 (Tyr1007/1008) rabbit IgG (3776; Cell Signaling Technology, 1:1000), anti-Jak2 rabbit IgG (3230; Cell Signaling Technology, 1:1000), anti-RANKL goat IgG (sc-7628; Santa Cruz Biotechnology, Santa Cruz, CA, 1:1000), anti-phospho Stat3 (Tyr705) rabbit IgG (9145; Cell Signaling Technology, 1:2000), anti-Stat3α rabbit IgG (8768; Cell Signaling Technology, 1:1000), anti-α tubulin mouse IgG (CP06; Calbiochem, San Diego, CA, 1:1000), donkey anti-rabbit IgG-HRP (NA934V; GE Healthcare, Little Chalfont, UK, 1:5000), goat anti-mouse IgG-HRP (170–6516; Bio-Rad Laboratories, Hercules, CA, 1:2000), and donkey anti-goat IgG-HRP (sc-2056; Santa Cruz Biotechnology, 1:5000).

Techniques: Protein Array, Cell Culture, Phospho-proteomics, Western Blot, Expressing

Journal: Communications Biology

Article Title: Macrophage migration inhibitory factor is overproduced through EGR1 in TET2 low resting monocytes

doi: 10.1038/s42003-022-03057-w

Figure Lengend Snippet: a , b Cytokine profile arrays of supernatant collected from cord blood CD34 + cells infected with SCR - and TET2- shRNA-GFP lentiviruses, sorted on GFP expression, and induced to differentiate with stem cell factor (SCF), interleukin-3 (IL-3), Fms-related tyrosine kinase 3 ligand (FLT3L) and granulocyte-colony stimulating factor (G-CSF). a Representative cytokine array with supernatants collected at day 10 of differentiation. The rectangle points to MIF detection. b Quantification of MIF signals, normalized to positive controls. Data are mean +/− SEM of three independent experiments. Paired t test: * P < 0.05. c MIF concentrations determined by ELISA in the supernatant of cells induced to differentiate for indicated time. Data are mean +/− SEM of indicated independent experiments (day 5: n = 6; day 7: n = 5; day 8: n = 3; day 10: n = 7). Paired t test: * P < 0.05; *** P < 0.001. d RT-qPCR analysis of MIF mRNA expression in four TET2 -depleted ( TET2 shRNA, gray bars) and control ( SCR shRNA, black bars) human leukemic cell lines. Data are mean +/− SEM of three biological replicates. Unpaired t test: * P < 0.05; *** P < 0.001; **** P < 0.0001. e Immunoblot of SCR or TET2 shRNA infected leukemic cell lines sorted on GFP expression. Lower panels, quantification after actin normalization using Image J software. f MIF concentrations determined by ELISA in the supernatants of kasumi-1 ( n = 5), M07e ( n = 5), UT-7 ( n = 4) and TF-1 ( n = 5) cells transduced 24 h before with SCR (black squares) or TET2 (gray squares) shRNA. Data are mean +/− SEM of indicated biological replicates. Unpaired-t test: * P < 0.05; ** P < 0.01. g MIF concentrations determined by ELISA in the plasma of two Tet2 -deficient models (1–3 months): K427 knock-out model (wt/wt or Tet2 +/+ n = 4, wt/exc or Tet2 +/− n = 5 and exc/exc or Tet2 −/− n = 5); ANO knock-down model (wt/wt or Tet2 + /+ n = 5, wt/LacZ or Tet2 +/− n = 5 and LacZ/LacZ or Tet2 −/− n = 6). Data are mean +/− SEM of indicated biological replicates. Dunnett’s multiple comparison tests using wt/wt as control: * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: CD34 + collected supernatants were analyzed using human cytokine antibody array (panel A; R&D Systems).

Techniques: Infection, shRNA, Expressing, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Western Blot, Software, Knock-Out, Comparison