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Additional file 2: Table S1. Primer sequences for qRT-PCR. Table S2. Antibodies. Table S3. Primer sequences for mutagenesisof eIF2α.

Additional file 1: Figure S1. The ArgS treatment leads to a reduction in global O-GlcNAcylation levels and affects the transcription of HBP and O-GlcNAc recycling enzyme genes.Immunoblot analysis of OGA and OGT in the whole cell extracts from BT-549 cells cultured in the -Gln or -Glc m4dium for 24 h. One representative immunoblotis shown. The relative OGA and OGT protein levels are determined after normalizing against the densitometric signal intensity of the OGA or OGT in the control group, which was set as 1 after normalization with actin or H3 signal.A schematic overview of HBP. The HBP enzymesand metabolitesare depicted.Gene expression of the indicated genes in BT-549 cells after incubation in full medium for 48 h or -Arg medium for 24 and 48 h, respectively. The results of the qRT-PCR analysis of the mRNA encoding HBP enzymes shown in the left panel in BT-549 cells after ArgS treatment for 24 and 48 h, respectively. The qRT-PCR analyses were performed using gene-specific primer pairs, and the 2–∆∆Ct method was used to analyze the results. ΔΔCT = ΔCT− ΔCT.The Staudinger ligation was performed in protein lysates where O-GlcNAz-modified proteins were conjugated with phosphine-PEG3-biotin. The O-GlcNAz-modified proteins were then pulled-down using streptavidin.A heat map shows the abundance ratioof 2054 quantified O-GlcNAz-modified proteins. The scale of the heat map is limited to; n=4.GlcNAz-labeled proteins in BT-549 cells were pulled down and subjected to immunoblot analyses. BT-549 cells were maintained in -Gln and -Glc medium supplemented with GlcNAzfor 48 h prior to cell harvest. O-GlcNaz-modified proteins were collected, followed by immunoblot analysis using an anti-eIF2α antibody to determine the endogenous eIF2α O-GlcNAcylation levels.Data are shown as mean ± s.e.m.; *: p5 normal samples were presented as indicated. Standard boxplots were applied to visualize the log2-transformed HMOX1 expression levelsand the number of samples was labeled at the bottom.HMOX1 expressionin breast tumors across different molecular subtypes and adjacent normal tissues. The number of samples was labeled at the bottom.*: p0.05; Wilcoxon tests; p-values were adjusted for multiple comparison using Bonferroni method with the normal samples set as the reference group.Kaplan-Meier survival curves comparing the HO-1 protein levels and overall survivalrates of breast cancer patients. 58 breast tumor samples expressing detectable/no detectable HO-1 in the Tang database were applied to the comparison using log-rank test. Figure S3. HO-1 expression enhances cell recovery from ArgS.Immunoblot analysis of HO1 in different breast cancer cell lines. The whole cell lysates were collected from the different cell lines grown in complete medium.Immunoblot analysis of HO-1 in BT-549 cellstransfected with siHMOX1or treated with ZnPPIX, and in MDA-MB-231 cellsstably overexpressing HO-1 or treated with COPPIX.Measurement of ROS levels in BT-549 and MDA-MB-231 cells subjected to ArgS for 48 h. ROS levels were assessed using DCF-DA oxidation; n=3.Comparison of relative cell viability in BT-549 and MDA-MB-231 cells subjected to ArgS for 24 and 48 h. Assessment of relative cell recovery of MDA-MB-231 cells after treatment with ADI-PEG20for 24 h. The ADIPEG20 treatment was terminated after 24 h; n=3. Comparison of cell recovery in MDA-MB-231 cells overexpressing HO-1 after 24-h treatment with ADI-PEG20; n=3. The ADI-PEG20 treatmentwas terminated by replacing the medium with complete medium. Cell recovery was monitored daily from day 0 to day 3using ACP assays.Assessment of cell migration of MDA-MB-231 cells subjected to ArgS for 24 h; n=3. Cell migration was determined as the percentage of confluence within the gap area.Data are shown as mean ± s.e.m.; *: p

Herein, we evaluated the anti-cancer effect and molecular mechanisms of a novel betulinic acid (BA) derivative, SYK023, by using two mouse models of lung cancer driven by KrasG12D or EGFRL858R. We found that SYK023 inhibits lung tumor proliferation, without side effects in vivo or cytotoxicity in primary lung cells in vitro. SYK023 triggered endoplasmic reticulum (ER) stress. Blockage of ER stress in SYK023-treated cells inhibited SYK023-induced apoptosis. In addition, we found that the expression of cell cycle-related genes, including cyclin A2, B1, D3, CDC25a, and CDC25b decreased but, while those of p15INK4b, p16INK4a, and p21CIP1 increased following SYK023 treatment. Finally, low doses of SYK023 significantly decreased lung cancer metastasis in vitro and in vivo. Expression of several genes related to cell migration, including synaptopodin, were downregulated by SYK023, thereby impairing F-actin polymerization and metastasis. Therefore, SYK023 may be a potentially therapeutic treatment for metastatic lung cancer.

Renewing liquid medium induced radicle development in up to 60% of asexual embryos of cacao (Theobroma cacao L.). In combination with medium renewal, radicle development was promoted further by eliminating casein hydrolysate and by reducing the concentration of Murashige and Skoog salts in liquid medium.

Herein, we evaluated the anti-cancer effect and molecular mechanisms of a novel betulinic acid (BA) derivative, SYK023, by using two mouse models of lung cancer driven by KrasG12D or EGFRL858R. We found that SYK023 inhibits lung tumor proliferation, without side effects in vivo or cytotoxicity in primary lung cells in vitro. SYK023 triggered endoplasmic reticulum (ER) stress. Blockage of ER stress in SYK023-treated cells inhibited SYK023-induced apoptosis. In addition, we found that the expression of cell cycle-related genes, including cyclin A2, B1, D3, CDC25a, and CDC25b decreased but, while those of p15INK4b, p16INK4a, and p21CIP1 increased following SYK023 treatment. Finally, low doses of SYK023 significantly decreased lung cancer metastasis in vitro and in vivo. Expression of several genes related to cell migration, including synaptopodin, were downregulated by SYK023, thereby impairing F-actin polymerization and metastasis. Therefore, SYK023 may be a potentially therapeutic treatment for metastatic lung cancer.