Journal: The Journal of Clinical Investigation

Article Title: Activating transcription factor 4 regulates osteoclast differentiation in mice

doi: 10.1172/JCI42106

Figure Lengend Snippet: (A) Schematic representation of a transgene construct. An 1,846-bp fragment of the mouse Trap promoter was used to drive expression of full-length mouse ATF4 cDNA. Atf4 transgene expression in RANKL-differentiated and undifferentiated BMMs, BMSCs, or calvarial OBLs was measured by quantitative real-time RT-PCR using transgene-specific primers as described in Methods. (B–D) In vitro OCL differentiation. BMMs from 4-week-old WT and Trap-Atf4-tg mice (founder no. 2360) were differentiated into OCLs for 5 days followed by (B) Western blot analysis of ATF4, NFATc1, PU.1, CSFR1, and β-actin for loading; (C) real-time RT-PCR analysis for Trap, Cat K, Mmp9, Rank, Spi1, and Csfr1 mRNAs; and (D) TRAP staining of the BMM cultures. (E) TRAP staining. Tibial sections from 4-week-old WT and Trap-Atf4-tg mice were stained for TRAP activity. (F) TRAP+ OCLs (arrows) on trabecular surfaces of WT and Trap-Atf4-tg tibiae. Oc.S/BS and Oc.Nb/BPm values for primary and secondary spongiosa are shown in Table ​Table2.2. (G) μCT analysis. Fixed nondemineralized femurs from 3-month-old male WT and Trap-Atf4-tg mice were used for μCT analysis as previously described (41). BV/TV, Tb.N, and Tb.Sp values are shown in Table ​Table3.3. n = 3–7. *P < 0.01 versus WT. Original magnification, ×100 (D and E); ×200 (F).

Article Snippet: Antibodies used were as follows: antibodies against NFATc1, c-Fos, TRAF6, RANK, and CSFR1 and anti-rabbit or anti-mouse antibodies conjugated with horseradish peroxidase from Santa Cruz Biotechnology Inc.; antibodies recognizing phosphorylated and total ERK1/2, p38 MAPK, JNK, AKT, Src, IκBα, and PU.1 from Cell Signaling Technology Inc.; and mouse monoclonal antibody against β-actin from Sigma-Aldrich.

Techniques: Construct, Expressing, Quantitative RT-PCR, In Vitro, Western Blot, Staining, Activity Assay

Journal: bioRxiv

Article Title: Persistent fibrosis and decreased cardiac function following cardiac injury in the Ctenopharyngodon idella (grass carp)

doi: 10.1101/627752

Figure Lengend Snippet: (A) Representative images of csfr1+ macrophages are shown in both the injured regions and regions of the heart far from the injured zone over time. (B) Quantification of csfr1+ cell density reveals a peak in macrophage density at 14 DPI, although there is no difference in density between injured and remote regions of the heart at any timepoint. (scale bar = 100 μm; *p < 0.05, **p < 0.01, ***p < 0.001 relative to uninjured fish).

Article Snippet: Therefore, csfr1+ (GTX128677, Genetex, 1:200 dilution) macrophage density was assessed by immunofluorescent staining of both injured and remote regions of the grass carp ventricle following injury to detect both the location and time-dependence of macrophage infiltration into the damaged tissue.

Techniques:

Journal: bioRxiv

Article Title: Hyperglycemia enhances cancer immune evasion by inducing alternative macrophage polarization through increased O-GlcNAcylation

doi: 10.1101/831610

Figure Lengend Snippet: Hyperglycemia (HyG) was induced in vivo in C57BL/6 mice with an i.p. injection of 150 mg/kg STZ. Euglyemic control mice (EuG) received vehicle in a single i.p. injection. Seven days after the STZ injection, (A) MC38 cancer cells were implanted subcutaneously in NOD SCID mice and tumor area was measured. (B) The macrophage reduction was carried out by injection of anti-CSFR1 antibody in HyG (n=6–10) and EuG (n=7–12) C57BL/6 mice. In total 7 injections, 1 mg before MC38 injection and 6 more injections (400 g) each 3 or 4 days. #. p values were calculated using Anova one-way with Tukey post-test. * P < 0.05, ** P < 0.01, *** P < 0.001, where * HyG vs. EuG and # HyG vs. HyG + Anti-CSFR1. Representative dot plots of macrophage reduction comparing (C) non-treated mice and (D) CSFR1 treated. (E) The frequency of CD11b + F4/80 + inside the CD45 + population (n=13–18). Results are expressed as mean ± S.D. p values were calculated using the Students’ t-test ****P<0.0001.

Article Snippet: Depletion of intra-tumoral macrophages was achieved by i.p. anti-CSFR1 antibody treatment (CD115, clone AFS98, BioXCell) with a total of 7 injections, 1 mg before tumor inoculation, followed by 6 injections of 400 µg each every 3 to 4 days over 3 weeks.

Techniques: In Vivo, Injection