Journal: Scientific Reports

Article Title: Cube natural sea salt ameliorates obesity in high fat diet-induced obese mice and 3T3-L1 adipocytes

doi: 10.1038/s41598-020-60462-z

Figure Lengend Snippet: CNS regulates obesity related genes in A/D + HFD induced mice. (A) Adipo/lipogenesis related genes of mRNA expression levels of SREBP-1, PPARγ, FAS, and DGAT2 in liver tissue. (B) protein expression levels of the lipogenesis genes LPL and DGAT2 in the liver and (C) epididymal fat. Fold ratio: Gene expression/(GAPDH or Actin) × HFD numerical value (HFD fold ratio = 1). a–d Means with different letters are significantly different ( P < 0.05) by Duncan’s multiple range test. The grouping of gels/blots cropped from different gels/blots.

Article Snippet: Membranes were then captured using the primary antibodies SREBP-1 (sc-365513, Lot# D1513), C/EBPα (sc-365318, Lot# H0613), FAS (sc-1024, Lot# G0312), GPAT2 (sc-168448, Lot# A1811), DGAT1 (sc-26173, Lot# C2315), DGAT2 (sc-66859, Lot# A1215), CPT-1 (sc-139482, Lot# H2213), Actin (sc-8432, Lot# C3017) (Santa Cruz Biotechnology Inc., Dallas, TX, USA), and the respective secondary antibody (Santa Cruz Biotechnology Inc.).

Techniques: Expressing, Gene Expression

Journal: Scientific Reports

Article Title: Cube natural sea salt ameliorates obesity in high fat diet-induced obese mice and 3T3-L1 adipocytes

doi: 10.1038/s41598-020-60462-z

Figure Lengend Snippet: CNS treatment reduces lipid droplet and obesity related genes in differentiated 3T3-L1 adipocytes. (A) Oil red O staining, (B) 490 nm absorbance value, (C) mRNA expression levels of the adipo/lipogenesis-related genes PPARγ, SREBP-1, C/EBPα, LXRα, LPL, FAS, and DGAT2, and (D) protein expression levels of SREBP-1 in differentiated 3T3-L1 adipocytes. GS: Generally manufactured sea salt (mixture of concentrated old and new seawater) (1%), FS: Filtering processed sea salt (mixture of concentrated old and new seawater filtered through a charcoal and magnetic filter) (1%), CNS: Cube natural sea salt (sea salt made from only concentrated new seawater) (1%). Fold ratio: Gene expression/(GAPDH or Actin) × Control numerical value (Control fold ratio = 1). The grouping of gels/blots cropped from different gels/blots.

Article Snippet: Membranes were then captured using the primary antibodies SREBP-1 (sc-365513, Lot# D1513), C/EBPα (sc-365318, Lot# H0613), FAS (sc-1024, Lot# G0312), GPAT2 (sc-168448, Lot# A1811), DGAT1 (sc-26173, Lot# C2315), DGAT2 (sc-66859, Lot# A1215), CPT-1 (sc-139482, Lot# H2213), Actin (sc-8432, Lot# C3017) (Santa Cruz Biotechnology Inc., Dallas, TX, USA), and the respective secondary antibody (Santa Cruz Biotechnology Inc.).

Techniques: Staining, Expressing, Gene Expression, Control

Journal: American Journal of Cancer Research

Article Title: SHMT2 promotes tumor growth through VEGF and MAPK signaling pathway in breast cancer

doi:

Figure Lengend Snippet: SHMT2 is highly expressed in breast cancer and associated with a poor prognosis. A. The expression of SHMT2 protein in various Breast cancer cell lines (MCF-1, MDA-MB-231, HCC1806, ZR-75-1, BT549) and a normal breast cell line (MCF-10A) was analyzed by Western blotting. B. The expression of SHMT2 protein was tested by Western blotting in 8 breast cancer tissue and matched para-cancer tissue. C. Representative pictures of SHMT2 by immunohistochemistry were shown in breast cancer tissues and matched para-carcinoma tissues. Original magnification, 200 ×. D. Kaplan-Meier method was used to compute survival analysis comparing patients with high (n = 64) and low (n = 76) expression. E. With CPTAC data, the expression of SHMT2 in breast cancer tissues compared with normal breast tissues. F. Survival analysis was further conducted. ***P < 0.001, Student’s t-test. The data are presented as the mean ± SD of three tests. T, breast cancer tissue; N, matched para-carcinoma tissue.

Article Snippet: Reagents and antibodies Antibodies against SHMT2 (A1215), GAPDH (A19056), β-actin (AC026), VEGF (A12303), PEDF (A3475), were obtained from ABclonal Technology Co., Ltd. (ABclonal, Wuhan, China).

Techniques: Expressing, Western Blot, Immunohistochemistry

Journal: American Journal of Cancer Research

Article Title: SHMT2 promotes tumor growth through VEGF and MAPK signaling pathway in breast cancer

doi:

Figure Lengend Snippet: Up-regulation of SHMT2 promotes cell growth in breast cancer. SHMT2 protein was detected by western blot in HCC1806 with SHMT2 knockdown (A) and ZR-75-1 (E) with SHMT2 over-expression. And the cell lines HCC1806 (B) and ZR-75-1 (F) were photographed. The cell viability of breast cancer cell lines HCC1806 (C) and ZR-75-1 (G) were determined by MTT assay 48 h after transfection. The effect of down-regulation (D) or up-regulation (H) of SHMT2 on tumor cell colony formation was analyzed by plate clone formation assay. The relative colony formation rates in HCC1806 (D) and ZR-75-1 (H) cells were calculated. *P < 0.05, Student’s t-test. The data are presented as the mean ± SD of three tests.

Article Snippet: Reagents and antibodies Antibodies against SHMT2 (A1215), GAPDH (A19056), β-actin (AC026), VEGF (A12303), PEDF (A3475), were obtained from ABclonal Technology Co., Ltd. (ABclonal, Wuhan, China).

Techniques: Western Blot, Knockdown, Over Expression, MTT Assay, Transfection, Tube Formation Assay

Journal: American Journal of Cancer Research

Article Title: SHMT2 promotes tumor growth through VEGF and MAPK signaling pathway in breast cancer

doi:

Figure Lengend Snippet: SHMT2 knockdown activates caspase dependent apoptotic pathway. A. Apoptosis rate was detected by annexin-V-FITC and PI double-stained cells combined with flow cytometry fluorescence separation in an HCC1806 cell line with SHMT2 knockdown. B. The apoptosis rate of each group was quantitatively analyzed. *P < 0.05, Student’s t-test. The data are presented as the mean ± SD of three tests. C. Western blot analysis of caspase-dependent proteins in HCC1806 with SHMT2 knockdown. D. Representative images of immunofluorescence to monitor cytochrome-c in HCC1806 with SHMT2 knockdown. Original magnification, 200 ×.

Article Snippet: Reagents and antibodies Antibodies against SHMT2 (A1215), GAPDH (A19056), β-actin (AC026), VEGF (A12303), PEDF (A3475), were obtained from ABclonal Technology Co., Ltd. (ABclonal, Wuhan, China).

Techniques: Knockdown, Staining, Flow Cytometry, Fluorescence, Western Blot, Immunofluorescence

Journal: American Journal of Cancer Research

Article Title: SHMT2 promotes tumor growth through VEGF and MAPK signaling pathway in breast cancer

doi:

Figure Lengend Snippet: SHMT2 over-expression affects multiple signaling pathways and biological processes in the breast cancer cells. A. Clustering results of differentially expressed genes comparing ZR-75-1 cells with SHMT2 over-expression with the control group cells. B. Volcanic map of differentially expressed mRNAs. C. GO enrichment histogram of differentially expressed mRNA. D. The methodology of KEGG is conducted according to the literature reported by Kanehisa [20]. KEGG pathway enrichment bars of differentially expressed mRNA. E. Common signaling pathways are closely associated with cell proliferation and survival. F. SHMT2 over-expression affects a variety of biochemical metabolic processes in breast cancer cells. G. SHMT2 over-expression affects the MAPK pathway, apoptosis pathway, and VEGF pathway.

Article Snippet: Reagents and antibodies Antibodies against SHMT2 (A1215), GAPDH (A19056), β-actin (AC026), VEGF (A12303), PEDF (A3475), were obtained from ABclonal Technology Co., Ltd. (ABclonal, Wuhan, China).

Techniques: Over Expression, Protein-Protein interactions, Control

Journal: American Journal of Cancer Research

Article Title: SHMT2 promotes tumor growth through VEGF and MAPK signaling pathway in breast cancer

doi:

Figure Lengend Snippet: SHMT2 promotes breast cancer cell proliferation by activating MAPK pathway. Western blotting analysis of proteins with phosphorylation or not related in MAPK signaling pathway in breast cancer cells with SHMT2 knockdown (A) and over-expression (B). Cell viability was analyzed by MTT assay in HCC1806 cells with SHMT2 knockdown treated with an ERK inhibitor FR180204 (C) or a p38 inhibitor SB202190 (D). ZR-75-1 cells with SHMT2 overexpression were treated with an ERK inhibitor FR180204 (E) or a p38 inhibitor SB202190 (F). # represents the comparison between inhibitor plus SHMT2 over-expression group and SHMT2 over-expression group. * represents the comparison between treatment group and control group. #, *P < 0.05. Student’s t-test. The data are presented as the mean ± SD. The error bars show the SD.

Article Snippet: Reagents and antibodies Antibodies against SHMT2 (A1215), GAPDH (A19056), β-actin (AC026), VEGF (A12303), PEDF (A3475), were obtained from ABclonal Technology Co., Ltd. (ABclonal, Wuhan, China).

Techniques: Western Blot, Phospho-proteomics, Knockdown, Over Expression, MTT Assay, Comparison, Control

Journal: American Journal of Cancer Research

Article Title: SHMT2 promotes tumor growth through VEGF and MAPK signaling pathway in breast cancer

doi:

Figure Lengend Snippet: SHMT2 regulates the VEGF signaling pathway in breast cells. Western blotting analysis of VEGF and PEDF proteins in MAPK signaling pathway in breast cancer cells with SHMT2 knockdown (A) and overexpression (B). VEGF and PEDF proteins in supernatant detected by ELISA in breast cancer cells with SHMT2 knockdown (C) and over-expression (D). Subsequently, the ratio of VEGF and PEDF was calculated from ELISA results in breast cells with sh-SHMT2 (E). Finally, an angiogenesis experiment was conducted in breast cells with sh-SHMT2, and vessel area and length were calculated (F). *P < 0.05, Student’s t-test. The data are presented as the mean ± SD of three tests.

Article Snippet: Reagents and antibodies Antibodies against SHMT2 (A1215), GAPDH (A19056), β-actin (AC026), VEGF (A12303), PEDF (A3475), were obtained from ABclonal Technology Co., Ltd. (ABclonal, Wuhan, China).

Techniques: Western Blot, Knockdown, Over Expression, Enzyme-linked Immunosorbent Assay

Journal: American Journal of Cancer Research

Article Title: SHMT2 promotes tumor growth through VEGF and MAPK signaling pathway in breast cancer

doi:

Figure Lengend Snippet: Knockdown of SHMT2 inhibits breast tumor growth in vivo. HCC1806 cells with SHMT2 knockdown (KD-SHMT2) and control cells (KD-NC), while BT549 cells (SHMT2) with SHMT2 over-expression and control cells (Vector) were injected into the left and right back subcutaneously in 100 ul PBS. The average tumor volume reached 100 mm3 and tumor volume was measured. Xenograft tumors were measured with vernier calipers, tumor volumes were calculated and growth curves were plotted (A, E). After 7 measurements, the mice were sacrificed and photographed (B, F). The xenograft was removed and photographed (C) and weighed (D, G). Immunohistochemical staining (H) detected the protein expression levels of P-ERK, P-P38, KI67, CD31, and SHMT2 in tumor tissues, *P < 0.05, Student’s t-test. The data are presented as mean ± SD from two independent experiments. N = 5 or 6/group, magnification 200 ×.

Article Snippet: Reagents and antibodies Antibodies against SHMT2 (A1215), GAPDH (A19056), β-actin (AC026), VEGF (A12303), PEDF (A3475), were obtained from ABclonal Technology Co., Ltd. (ABclonal, Wuhan, China).

Techniques: Knockdown, In Vivo, Control, Over Expression, Plasmid Preparation, Injection, Immunohistochemical staining, Staining, Expressing

Journal: Investigative Ophthalmology & Visual Science

Article Title: Assocation Between Leber's Hereditary Optic Neuropathy and MT-ND1 3460G>A Mutation-Induced Alterations in Mitochondrial Function, Apoptosis, and Mitophagy

doi: 10.1167/iovs.62.9.38

Figure Lengend Snippet: Alterations in the structure and stability of MT-ND1. ( A ) Electrostatic interactions in A52 of MT-ND1. Based on the cryo-EM structure of complex I from Homo sapiens (PDB entry: 5XTD), the hydrophobic residue A52 interacts with 48P, 55L, and 56F. ( B ) Western blot analysis. Cellular proteins (20 µg) from various cell lines were electrophoresed through a denaturing polyacrylamide gel, electroblotted, and hybridized with MT-ND1, MT-ND4, NDUFA3, NDUFA8, NDUFA13, AFG3L2, and CLPP antibodies, with GAPDH as a loading control. ( C ) Quantification of MT-ND1. Average relative values of MT-ND1 were normalized to the average values of VDAC1 in various cell lines. The calculations were based on three independent determinations in each cell line. The error bars indicate 2 SEM. P indicates significance based on Student's t -test of the differences between mutant and control cells.

Article Snippet: Other antibodies included NDUFA3 (17257-1-AP; ProteinTech Group, Rosemont, IL, USA), NDUFA8 (A12118; ABclonal Technology, Woburn, MA, USA), and NDUFA13 (A5412; ABclonal Technology).

Techniques: Cryo-EM Sample Prep, Residue, Western Blot, Control, Mutagenesis