Journal: The Journal of Cell Biology

Article Title: Bnip3 and AIF cooperate to induce apoptosis and cavitation during epithelial morphogenesis

doi: 10.1083/jcb.201111063

Figure Lengend Snippet: Bnip3 cooperates with AIF to induce apoptosis and cavitation. (A) 4-d AIF y/+ and AIF y/− EBs were analyzed by immunoblotting for AIF. Actin was used as a loading control. (B) EBs were cultured for 1–4 d and analyzed by immunoblotting for cleaved caspase-3 (cas-3) and actin. Ablation of AIF inhibited caspase-3 activation. (C) Live-phase micrographs show cavitation delay in AIF y/− EBs cultured for 4, 5, and 7 d. After 10 d, most of the AIF y/− EBs were cavitated similar to AIF y/+ EBs. Bars, 100 µm. (D) 4-d EBs were immunostained for cleaved caspase-3. F-actin was stained with rhodamine-phalloidin to show the apical actin belt. Ablation of AIF inhibited apoptosis of the core cells. 5-d EBs were immunostained for the apical marker MUPP1. Apical polarization of the AIF y/− epiblast was not affected despite delayed lumen clearance. (E) AIF y/− ES cells were stably transfected with Bnip3 shRNA (Bnip3 knockdown [KD]) or GFP. 5-d EBs were analyzed by immunoblotting for Bnip3 and cleaved caspase-3. Bnip3 silencing in AIF y/− EBs further inhibited caspase-3 activation. (F) AIF y/+ EBs expressing GFP and AIF y/− EBs stably transfected with Bnip3 shRNA or GFP were cultured for 4, 5, and 7 d. EB cavitation was quantitated by phase microscopy. EB cavitation was significantly delayed in the absence of AIF. Knockdown of Bnip3 in AIF y/− EBs nearly blocked cavitation. n = 6 independent experiments with a total of 529–808 EBs counted for each group. Error bars represent the mean ± SD. *, P < 0.01 versus AIF y/+ GFP; # , P < 0.01 versus AIF y/− GFP.

Article Snippet: Four pGFP-V-RS–based shRNA vectors targeting to mouse Bnip3 and four pRFP-V-RS–based vectors targeting to Bim together with scrambled controls were purchased from OriGene.

Techniques: Western Blot, Cell Culture, Activation Assay, Staining, Marker, Stable Transfection, Transfection, shRNA, Expressing, Microscopy

Journal: Cancer Research

Article Title: Chemotherapy Alters Monocyte Differentiation to Favor Generation of Cancer-Supporting M2 Macrophages in the Tumor Microenvironment

doi: 10.1158/0008-5472.can-12-3542

Figure Lengend Snippet: Figure 1. Survival of monocytes upon treatment with cisplatin and carboplatin. CxCa, cervical cancer cell line; OvCa, ovarian cancer cell line. A, example of survival of mo- DC, APC-TSN (TSN CASKI), and M2-TSN (TSN CAOV3) upon treatment with increasing doses of cisplatin. B, survival of different APC upon chemotherapy treatment. Tumor APC/M2-like macrophages reflect a mean of 3 experiments with 5 different APC-TSN and M2-TSN.

Article Snippet: The following factors were used to culture APC: 500 U/mL IL-4 (Invitrogen), 800 U/mL granulocyte macrophage colony-stimulating factor (GM-CSF; Immunotools), 25 ng/mL macrophage colony-stimulating factor (M-CSF; R&D), 1 to 10 ng/mL PGE2 (Sigma-Aldrich), and 1 to 50 ng/mL IL-6 (Immunotools).

Techniques:

Journal: Cancer Research

Article Title: Chemotherapy Alters Monocyte Differentiation to Favor Generation of Cancer-Supporting M2 Macrophages in the Tumor Microenvironment

doi: 10.1158/0008-5472.can-12-3542

Figure Lengend Snippet: Figure 2. Platinum-based chemotherapy treatment of cervical and ovarian cell lines alters APC differentiation. Mean (with SEM) of 3 experiments. Treated tumor APC/M2-like macrophages were compared with untreated tumor APC/M2-like macrophages with a paired t test; , P < 0.05; , P < 0.01; , P < 0.001. A, typical example of a flow cytometric analysis. Numbers in quadrants represent the percentage of cells within the live gate. Phenotype of mo-DC is not affected by chemotherapy following direct incubation with chemotherapy. B, bar plots showing the percentage of CD1aþ, CD1aCD14, and CD14þ cells induced by TSN of untreated and treated tumor cells. C, the production of IL-12 and IL-10 of the different types of APC depicted in B is shown. M2-like macrophages predominantly produce IL-10, whereas tumor APCs produce IL-12 and lower amounts of IL-10 compared with mo-DC. This effect is significantly enhanced upon chemotherapy treatment.

Article Snippet: The following factors were used to culture APC: 500 U/mL IL-4 (Invitrogen), 800 U/mL granulocyte macrophage colony-stimulating factor (GM-CSF; Immunotools), 25 ng/mL macrophage colony-stimulating factor (M-CSF; R&D), 1 to 10 ng/mL PGE2 (Sigma-Aldrich), and 1 to 50 ng/mL IL-6 (Immunotools).

Techniques: Incubation

Journal: Cancer Research

Article Title: Chemotherapy Alters Monocyte Differentiation to Favor Generation of Cancer-Supporting M2 Macrophages in the Tumor Microenvironment

doi: 10.1158/0008-5472.can-12-3542

Figure Lengend Snippet: Figure 4. Platinum-based chemotherapy treatment of cancer cell lines influences STAT phosphorylation in APC. A, examples of pSTAT1, pSTAT3, and pSTAT6 expression of monocytes cultured in the presence of (un)treated supernatant of cancer cells. B, overview of pSTAT levels in M2-like macrophages. Mean (with SEM) of 3 experiments carried out on 3 different donors. Treated tumor APC/M2-like macrophages were compared with untreated tumor APC/M2-like macrophages with a paired t test; , P < 0.05; , P < 0.01; , P < 0.001. C, pSTAT1 and 6 are PGE2 dependent, whereas pSTAT3 is IL-6 dependent. Dotted lines indicate pSTAT levels in mo-DC.

Article Snippet: The following factors were used to culture APC: 500 U/mL IL-4 (Invitrogen), 800 U/mL granulocyte macrophage colony-stimulating factor (GM-CSF; Immunotools), 25 ng/mL macrophage colony-stimulating factor (M-CSF; R&D), 1 to 10 ng/mL PGE2 (Sigma-Aldrich), and 1 to 50 ng/mL IL-6 (Immunotools).

Techniques: Phospho-proteomics, Expressing, Cell Culture