Journal: Frontiers in Psychiatry

Article Title: Profiling prefrontal cortex protein expression in rats exhibiting an incubation of cocaine craving following short-access self-administration procedures

doi: 10.3389/fpsyt.2022.1031585

Figure Lengend Snippet: Changes in Akt1 and CaMKII expression and phosphorylation in the PL. While we did not detect any time-dependent or group differences in the PL expression of total Akt1 (A) , both 2-h and Mixed rats exhibited a time-dependent increase total p(Ser473)-Akt1 expression (B) and the 2-h rats exhibited a time-dependent increase in the relative expression of p(Ser473)-Akt1 (C) . (D) We detected no group or time-dependent changes in total CaMKII expression within the PL. However, the Mixed group demonstrated a significant time-dependent increase in both total (E) and relative (F) p(Thr286)-CaMKII expression. Data are normalized to the average protein densities of the Sham controls tested on WD3 and represented as means ± SEMs of the number of rats indicated. + p < 0.05, compared to WD3.

Article Snippet: The following mouse primary antibodies were also employed: Homer1b/c (1:1,000 dilution; Santa Cruz Biotechnology; sc-25271), mGlu1 (1:1,000 dilution; BD Biosciences; 610965), p(Ser473)-Akt1 (1:1,000 dilution; Cell Signaling Technology; 4051), GluN2b (1:500 dilution; Invitrogen; MA1-2014), GluA2 (1:500 dilution; Synaptic Systems; 182 111), and CaMKII (1:500 dilution; Millipore; 05-532).

Techniques: Expressing

Journal: Frontiers in Psychiatry

Article Title: Profiling prefrontal cortex protein expression in rats exhibiting an incubation of cocaine craving following short-access self-administration procedures

doi: 10.3389/fpsyt.2022.1031585

Figure Lengend Snippet: Summary of the negative results from the immunoblotting study of the IL from Sham, 2-h- and Mixed-Access rats.

Article Snippet: The following mouse primary antibodies were also employed: Homer1b/c (1:1,000 dilution; Santa Cruz Biotechnology; sc-25271), mGlu1 (1:1,000 dilution; BD Biosciences; 610965), p(Ser473)-Akt1 (1:1,000 dilution; Cell Signaling Technology; 4051), GluN2b (1:500 dilution; Invitrogen; MA1-2014), GluA2 (1:500 dilution; Synaptic Systems; 182 111), and CaMKII (1:500 dilution; Millipore; 05-532).

Techniques: Western Blot

Journal: Theranostics

Article Title: Cancer-associated fibroblasts facilitate premetastatic niche formation through lncRNA SNHG5-mediated angiogenesis and vascular permeability in breast cancer

doi: 10.7150/thno.74753

Figure Lengend Snippet: ZNF281 transcriptionally regulates CCL2 and CCL5 expression in CAFs. (A) Gene set enrichment analysis (GSEA) of lncSNHG5 in the KEGG pathway using TCGA data. (B) Venn diagram showing the cytokines as ZNF281 putative targets in CAFs. (C-D) qRT-PCR and western blotting were used to test the mRNA (C) and protein (D) expression levels of CCL2, CCL5 and VEGFA in CAFs/shNC, CAFs/shZNF281, CAFs/sh lncSNHG5 and CAFs/sh lncSNHG5/ZNF281. (E-G) ELISA was used to detect CCL2 (E), CCL5 (F) and VEGFA (G) protein levels in supernatant from the indicated engineered CAFs. (H-I) Schematic diagram depicting the predicted binding sites and sequences of ZNF281 in the CCL2 (H) and CCL5 (I) promoters. The relative luciferase activity was detected in CAFs transfected with wild-type (WT) or mutant luciferase reporter plasmids of CCL2 or CCL5. (J-K) ChIP-qPCR assay to determine ZNF281 binding to the CCL2 (J) and CCL5 (K) promoters using an anti-ZNF281 antibody. IgG was used as a negative control. Data represent the mean ± SD (ns: no significance, ** P < 0.01, *** P < 0.001).

Article Snippet: A total of 50-80 μg protein was separated using 8-12% SDS-PAGE gel and electrically transferred to 0.22 μM PVDF membrane (Bio-Rad, USA), which was blocked with 5% nonfat milk at room temperature for 3 h and then incubated at 4 °C overnight with the special primary antibodies: Occludin (Abcam, ab216327, 1:1000), ZO-1 (Cell Signaling, #8193, 1:1000), ZNF281 (SAB, #43566, 1:1000), IGF2BP2 (Proteintech, 11601-1-AP, 1:2000), VEGFA (Abcam, ab1316, 1:1000), CCL2 (SAB, #45065, 1:500), CCL5 (SAB, #32935, 1:500), p-AKT (CST, #12694, 1:1000), AKT (CST, #9272, 1:1000), p-STAT3 (CST, #9131, 1:500), STAT3 (CST, #4904, 1:500), p-P38 (CST, #4631, 1:500), P38 (CST, #9212, 1:1000), p-VEGFR2 (Sangon, D155165, 1:800), VEGFR2 (Sangon, D151118, 1:800), and β-Actin (Biosharp, 1:1000).

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Enzyme-linked Immunosorbent Assay, Binding Assay, Luciferase, Activity Assay, Transfection, Mutagenesis, Negative Control

Journal: Theranostics

Article Title: Cancer-associated fibroblasts facilitate premetastatic niche formation through lncRNA SNHG5-mediated angiogenesis and vascular permeability in breast cancer

doi: 10.7150/thno.74753

Figure Lengend Snippet: Breast CAF-derived CCL2 and CCL5 activate P38 MAPK signaling in endothelial cells. (A-B) Western blotting was used to analyze p-P38, P38, p-VEGFR2, VEGFR2, ZO-1, and Occludin levels in HUVECs incubated with CM from NF/Ctrl, CCL2, CCL5, CCL2/CCL5 or CAF/shNC, shCCL2, shCCL5, shCCL2/shCCL5. The pixel intensity of p-P38 was normalized to that of β-Actin for the quantification of p-P38 levels (B). (C-D) Western blotting was used to analyze the levels of p-P38, P38, p-VEGFR2, VEGFR2, ZO-1 and Occludin in HUVECs incubated with FBS-free medium with DMSO, rCCL2, rCCL5, rCCL2 and rCCL5 or rCCL2 and rCCL5 combined with axitinib, RS102895, maraviroc, or cenicriviroc. Levels of p-P38 (D) were quantified by normalizing its relative pixel intensity to β-Actin. (E-F) Effects of CM from NF/Ctrl, CCL2, CCL5, CCL2/CCL5 or CAF/shNC, shCCL2, shCCL5, shCCL2/shCCL5 on tube formation (E) and permeability (F) of HUVECs. (G-H) Effects of rCCL2, rCCL5, rCCL2 and rCCL5 or rCCL2 and rCCL5 combined with axitinib, RS102895, maraviroc, or cenicriviroc on tube formation (G) and permeability (H) of HUVECs. Data are presented as the mean ± SD (* P < 0.05, ** P < 0.01, *** P < 0.001).

Article Snippet: A total of 50-80 μg protein was separated using 8-12% SDS-PAGE gel and electrically transferred to 0.22 μM PVDF membrane (Bio-Rad, USA), which was blocked with 5% nonfat milk at room temperature for 3 h and then incubated at 4 °C overnight with the special primary antibodies: Occludin (Abcam, ab216327, 1:1000), ZO-1 (Cell Signaling, #8193, 1:1000), ZNF281 (SAB, #43566, 1:1000), IGF2BP2 (Proteintech, 11601-1-AP, 1:2000), VEGFA (Abcam, ab1316, 1:1000), CCL2 (SAB, #45065, 1:500), CCL5 (SAB, #32935, 1:500), p-AKT (CST, #12694, 1:1000), AKT (CST, #9272, 1:1000), p-STAT3 (CST, #9131, 1:500), STAT3 (CST, #4904, 1:500), p-P38 (CST, #4631, 1:500), P38 (CST, #9212, 1:1000), p-VEGFR2 (Sangon, D155165, 1:800), VEGFR2 (Sangon, D151118, 1:800), and β-Actin (Biosharp, 1:1000).

Techniques: Derivative Assay, Western Blot, Incubation, Permeability

Journal: Theranostics

Article Title: Cancer-associated fibroblasts facilitate premetastatic niche formation through lncRNA SNHG5-mediated angiogenesis and vascular permeability in breast cancer

doi: 10.7150/thno.74753

Figure Lengend Snippet: The lncSNHG5/ZNF281 signaling axis increases CCL2 and CCL5 secretion to promote angiogenesis and vascular permeability to induce premetastatic niche formation. (A) MDA-MB-231 cells were mixed with NFs/Ctrl, CAFs/shNC, CAFs/shlncSNHG5, CAFs/shZNF281, CAFs/shlncSNHG5/ZNF281, CAFs/RS102895, CAFs/Maraviroc or CAFs/Cenicriviroc and orthotopically inoculated into nude mice for 2 weeks. The mice were injected with rhodamine-dextran before lung metastasis, and the vascular permeability of mouse lungs in each group is shown in the upper panel (scale bar, 100 µm). Representative images of fluorescent staining of ZO-1 (green), Occludin (red) and CD31 (pink) in lung tissue are shown in the lower panel (scale bar, 100 µm). (B) The fluorescence levels of rhodamine-dextran in mouse lungs were calculated using ImageJ software and normalized to DAPI levels. (C) The microvessel density in mouse lungs was quantified by counting the microvessel numbers under the microscope. (D-E) The serum CCL2 and CCL5 levels in each group of mice were detected by ELISA at 2 weeks after injection. (F) The protein changes in ZO-1, Occludin and phosphorylated or total p38 in mouse lung tissues before metastasis were assessed by western blotting (left panel). Levels of p-P38 were quantified as pixel intensity compared with β-actin (right panel). Data are presented as the mean ± SD (** P < 0.01, *** P < 0.001. **** P < 0.0001).

Article Snippet: A total of 50-80 μg protein was separated using 8-12% SDS-PAGE gel and electrically transferred to 0.22 μM PVDF membrane (Bio-Rad, USA), which was blocked with 5% nonfat milk at room temperature for 3 h and then incubated at 4 °C overnight with the special primary antibodies: Occludin (Abcam, ab216327, 1:1000), ZO-1 (Cell Signaling, #8193, 1:1000), ZNF281 (SAB, #43566, 1:1000), IGF2BP2 (Proteintech, 11601-1-AP, 1:2000), VEGFA (Abcam, ab1316, 1:1000), CCL2 (SAB, #45065, 1:500), CCL5 (SAB, #32935, 1:500), p-AKT (CST, #12694, 1:1000), AKT (CST, #9272, 1:1000), p-STAT3 (CST, #9131, 1:500), STAT3 (CST, #4904, 1:500), p-P38 (CST, #4631, 1:500), P38 (CST, #9212, 1:1000), p-VEGFR2 (Sangon, D155165, 1:800), VEGFR2 (Sangon, D151118, 1:800), and β-Actin (Biosharp, 1:1000).

Techniques: Permeability, Injection, Staining, Fluorescence, Software, Microscopy, Enzyme-linked Immunosorbent Assay, Western Blot

Journal: Theranostics

Article Title: Cancer-associated fibroblasts facilitate premetastatic niche formation through lncRNA SNHG5-mediated angiogenesis and vascular permeability in breast cancer

doi: 10.7150/thno.74753

Figure Lengend Snippet: The lncSNHG5-ZNF281-CCL2/CCL5 signaling axis facilitates BC metastasis. (A) A mixture of MDA-MB-231 cells and NFs/Ctrl, CAFs/shNC, CAFs/shlncSNHG5, CAFs/shZNF281, CAFs/shlncSNHG5/ZNF281, CAFs/RS102895, CAFs/Maraviroc or CAFs/Cenicriviroc was orthotopically injected into nude mice for 6 weeks. Representative images of pulmonary metastases in the lung surface (upper panel) and lung section (scale bar, 200 µm) by H&E staining (lower panel) are shown. (B) The number of lung metastatic nodules in each group of mice was counted under a microscope. (C) The maximal area of lung metastasis in each group was quantified using SlideViewer software. (D-E) qRT-PCR was used to detect the mRNA expression of CCL2 and CCL5 in human breast tumors with (16 cases) or without (76 cases) metastasis and adjacent normal tissues (60 cases). (F-G) ELISA was used to determine CCL2 and CCL5 proteins in healthy donors (22 cases) and breast cancer patients with (14 cases) or without (30 cases) metastasis. (H) Kaplan-Meier survival analysis of relapse-free survival based on the combined low or high levels of CCL2 and CCL5 using the GSE25066 dataset. (I) The diagnostic value of CCL2 and CCL5 for poor BC prognosis was evaluated using ROC curves. Data are presented as the mean ± SD (* P < 0.05, ** P < 0.01, *** P < 0.001. **** P < 0.0001).

Article Snippet: A total of 50-80 μg protein was separated using 8-12% SDS-PAGE gel and electrically transferred to 0.22 μM PVDF membrane (Bio-Rad, USA), which was blocked with 5% nonfat milk at room temperature for 3 h and then incubated at 4 °C overnight with the special primary antibodies: Occludin (Abcam, ab216327, 1:1000), ZO-1 (Cell Signaling, #8193, 1:1000), ZNF281 (SAB, #43566, 1:1000), IGF2BP2 (Proteintech, 11601-1-AP, 1:2000), VEGFA (Abcam, ab1316, 1:1000), CCL2 (SAB, #45065, 1:500), CCL5 (SAB, #32935, 1:500), p-AKT (CST, #12694, 1:1000), AKT (CST, #9272, 1:1000), p-STAT3 (CST, #9131, 1:500), STAT3 (CST, #4904, 1:500), p-P38 (CST, #4631, 1:500), P38 (CST, #9212, 1:1000), p-VEGFR2 (Sangon, D155165, 1:800), VEGFR2 (Sangon, D151118, 1:800), and β-Actin (Biosharp, 1:1000).

Techniques: Injection, Staining, Microscopy, Software, Quantitative RT-PCR, Expressing, Enzyme-linked Immunosorbent Assay, Diagnostic Assay