Journal: bioRxiv

Article Title: Aberrant regulation of serine metabolite drives extracellular vesicle release and cancer progression

doi: 10.1101/2022.05.10.491299

Figure Lengend Snippet: a. Venn diagram showing overlaps among genes downregulated by miR-891b transfection in HCT116 and A549 cells (Fc < -1.5) and target genes of miR-891b predicted by TargetScan. b. Schematic of the miR-891b target gene screening. After siRNAs were transfected into HCT116 or A549 cells, secreted EV amount was measured by ExoScreen (both CD9- and CD63-positive) c. Heat map showing EV secretion levels of cells with gene silencing. d. EV secretion levels after siPSAT1 introduction. EV secretion was measured by the ExoScreen based on the surface CD9 or CD63 levels. The showing the mean ± SE (n = 3). *P < 0.05, the Student’s t test. e. Nanoparticle tracking analysis of EVs collected from siPSAT1-transfected cancer cells. The particle count was normalized by the cell number, when the conditioned medium collected. The showing the mean ± SE. *P < 0.05, the Student’s t test. f. PSAT1 expression levels in miR-891b-transfected cancer cells. The relative expression levels of PSAT1 were normalized to β-actin expression. The values are presented as the means±SEs (n = 3). *, p<0.05. g. Western blot analysis of PSAT1 after miR-891b was transfected. Each lane was loaded with 15 μg protein. h. Summary of miR-891b target sites and mutated sites (shown in bold) in the 3′UTRs of PSAT1. i. The results of the luciferase reporter assay with the wild-type and mutant PSAT1 3’UTR cotransfected with miR-891b. The values are the means ± SE (n = 8, *, p<0.05, Student’s t test)

Article Snippet: MCF-7 and BM022 cells (1 × 10 5 cells) were seeded into 24-well dishes, and the PSAT1 overexpression vector (RC202475, Origene) was transfected by using Lipofectamine LTX Plus reagent (15338-100, Thermo Fisher).

Techniques: Transfection, Expressing, Western Blot, Luciferase, Reporter Assay, Mutagenesis

Journal: bioRxiv

Article Title: Aberrant regulation of serine metabolite drives extracellular vesicle release and cancer progression

doi: 10.1101/2022.05.10.491299

Figure Lengend Snippet: a. Co-staining of the nucleus (blue) with PSAT1 (red) and CD63 (green) in PSAT1-silenced cells. b. Co-staining of Rab7, a late endosome marker (red), with CD63 (green). c. Quantification of sum of the CD63 single-positive area in HCT116 and A549 cells in the PSAT1-silenced and control groups (n = 10, *, p<0.05, Student’s t test)

Article Snippet: MCF-7 and BM022 cells (1 × 10 5 cells) were seeded into 24-well dishes, and the PSAT1 overexpression vector (RC202475, Origene) was transfected by using Lipofectamine LTX Plus reagent (15338-100, Thermo Fisher).

Techniques: Staining, Marker

Journal: bioRxiv

Article Title: Aberrant regulation of serine metabolite drives extracellular vesicle release and cancer progression

doi: 10.1101/2022.05.10.491299

Figure Lengend Snippet: a. qRT–PCR analysis of the expression level of PSAT1 across colorectal and lung cancer cell lines. Expression levels were normalized to β-actin expression. The values are the means ± SE (n = 3). *, p<0.05, the one-way analysis of variance test. b. Positive correlation between PSAT1 expression and EV secretion. c. Expression levels of PSAT1 between cancer and normal tissues in several types of cancer based on the Oncomine database, Student’s t test. d. Kaplan–Meier analysis based on PSAT1 expression in lung cancer (all stages, stage I, II and III). e. The effect of siPSAT1 on EV secretion across cancer cell lines. EV secretion was measured by NTA. The values are the means ± SE (n = 3). *, p<0.05, Student’s t test.

Article Snippet: MCF-7 and BM022 cells (1 × 10 5 cells) were seeded into 24-well dishes, and the PSAT1 overexpression vector (RC202475, Origene) was transfected by using Lipofectamine LTX Plus reagent (15338-100, Thermo Fisher).

Techniques: Quantitative RT-PCR, Expressing

Journal: bioRxiv

Article Title: Aberrant regulation of serine metabolite drives extracellular vesicle release and cancer progression

doi: 10.1101/2022.05.10.491299

Figure Lengend Snippet: a. Western blot analysis of PSAT1 in parental cells (MCF7 and MDA-MB-231) and the corresponding metastatic cells (BM022 and D3H2LN). b. Relative EV secretion levels in parental and metastatic cell lines. The number of EVs secreted was normalized to the cell number. The values are the means ± SE (n = 3). *, p<0.05, Student’s t test. c. Relative EV secretion levels in PSAT1-overexpressing cells. The values are the means ± SE (n = 3). *, p<0.05, Student’s t test. N.S. = not significant. d. Diagram of the strategy for evaluating the effect of BM022-derived EVs on osteoclast differentiation of RAW 264.7 cells in a Boyden chamber system. Osteoclast differentiation was evaluated by TRAP staining. e. Number of TRAP-positive cells among PSAT1-overexpressing cells treated with sRANKL. The values are the means ± SE (n = 10, *, p<0.05, the one-way analysis of variance test). f. Number of TRAP-positive cells after the addition of EVs collected from PSAT1-overexpressing BM022 cells. The values are the means ± SE (n =10, *, p<0.05, the one-way analysis of variance test). g. NTA was performed to compare particle sizes between PSAT1-overexpressing BM022 cells and control cells. N.S. = not significant.

Article Snippet: MCF-7 and BM022 cells (1 × 10 5 cells) were seeded into 24-well dishes, and the PSAT1 overexpression vector (RC202475, Origene) was transfected by using Lipofectamine LTX Plus reagent (15338-100, Thermo Fisher).

Techniques: Western Blot, Derivative Assay, Staining

Journal: bioRxiv

Article Title: Aberrant regulation of serine metabolite drives extracellular vesicle release and cancer progression

doi: 10.1101/2022.05.10.491299

Figure Lengend Snippet: a. Scheme for establishment of the in vivo bone metastasis model via caudal artery injection. The bulk cell populations of PSAT1-overexpressing BM022 and control cells were used for this in vivo study. b. Representative IVIS images of mice injected with PSAT1-overexpressing BM022 and control cells on Days 0, 7, and 28. c. Relative luminescence intensity of BM022 cells, which express a luciferase construct, in mice injected with PSAT1-overexpressing BM022 and control cells. *, p<0.05. d. Computed tomography of bones in mice injected with PSAT1-overexpressing BM022 and control cells. e-g Images of HE staining ( e ), TRAP staining ( f ) and ALP staining ( g ) in bone from mice injected with PSAT1-overexpressing BM022 and control cells. Mice were sacrificed on Day 28 after IVIS imaging.

Article Snippet: MCF-7 and BM022 cells (1 × 10 5 cells) were seeded into 24-well dishes, and the PSAT1 overexpression vector (RC202475, Origene) was transfected by using Lipofectamine LTX Plus reagent (15338-100, Thermo Fisher).

Techniques: In Vivo, Injection, Luciferase, Construct, Computed Tomography, Staining, Imaging

Journal: Photosynthesis Research

Article Title: Photosystem I-LHCII megacomplexes respond to high light and aging in plants

doi: 10.1007/s11120-017-0447-y

Figure Lengend Snippet: High light-inducible bands are Photosystem I/LHCII megacomplexes. a , Western blotting of fractions (i.e., green gel bands 1–4) derived from DM/OG-solubilized spinach thylakoid membranes, probed with antibodies for PsbA, Lhcb1, and PsaD as representative components of Photosystem II (PSII), major light-harvesting antenna (LHCII), and Photosystem I (PSI) complexes, respectively. Bands were excised from gels and proteins were electroeluted so that the components of each band could be compared on a quantitative basis. Normalization was performed on a per chlorophyll basis before samples were denatured and run on standard SDS-PAGE prior to Western blot analysis. b , Silver staining of the same samples shown in a , but also including the PSII-LHCII supercomplex for comparison. Protein identifications were assigned by overlaying stained gels with Western blots and by comparison with published gels (Jarvi et al. ; Scheller et al. ). c A duplication of Band 3, the main PSI band, from panel b , with bands identified by reference to Scheller et al.

Article Snippet: All antibodies (i.e., PsaD, PsbA, Lhcb1, PsaH, and PsaL) were purchased from Agrisera and were used at approximately 1/1000 dilution, followed by anti-rabbit HRP-conjugated secondary antibody.

Techniques: Western Blot, Derivative Assay, SDS Page, Silver Staining, Comparison, Staining

Journal: Photosynthesis Research

Article Title: Photosystem I-LHCII megacomplexes respond to high light and aging in plants

doi: 10.1007/s11120-017-0447-y

Figure Lengend Snippet: Non-stoichiometric changes in photosystem I subunits as leaf ages. a Photosystem I-MCs trend in leaf age series. Leaves of 6-week-old spinach plants were grouped by age from youngest to oldest as young (Y), mature (M), and old (O): yellow unexpanded leaves (Y0), first set of expanding leaves (Y1), second set of expanding leaves (Y2), first set of fully expanded leaves (M1), second set of fully expanded leaves (M2), second-to-last set of fully expanded leaves (O1), and last set of fully expanded leaves (O2). Leaves were sampled midday. b Western blotting for major photosystem components. The same samples that were loaded onto the green gel in panel A were denatured and run on SDS-PAGE, followed by Western blotting. c Simultaneous Western blot of PsaD and PsaH from leaf age series. A leaf age series from a different set of spinach leaves than the one used for panels A and B is shown. The membrane was probed for both PsaD and PsaH simultaneously to clearly show the changing ratios of the two proteins relative to one another

Article Snippet: All antibodies (i.e., PsaD, PsbA, Lhcb1, PsaH, and PsaL) were purchased from Agrisera and were used at approximately 1/1000 dilution, followed by anti-rabbit HRP-conjugated secondary antibody.

Techniques: Western Blot, SDS Page, Membrane

Journal: The Journal of Biological Chemistry

Article Title: De novo synthesis of serine and glycine fuels purine nucleotide biosynthesis in human lung cancer tissues

doi: 10.1074/jbc.RA119.008743

Figure Lengend Snippet: Activation of Ser/Gly synthesis from glucose, their efficient incorporation into purines, and reverse flux of the Ser–Gly one-carbon pathway underlie preference for glucose as substrate for purine synthesis in lung cancer tissues ex vivo. The three schemes depict our key findings on the metabolism of exogenous glucose (a), Ser (b), and Gly (c) into purines in CA lung tissues. These were modified from the mammalian cell literature (4, 32, 40,–43). a, 1) net [13C3]glucose (Glc) uptake, enhanced conversion to [13C]Ser/[13C2]Gly in the cytoplasm (pool 1, light green square), and efficient incorporation (thick red arrow) into purine carbons (red circle) (e.g. adenine of ATP) via the action of cytoplasmic SHMT1 (route 1), MTHFD1 (route 2), and other enzymes (not shown) in the purine nucleotide synthesis pathway; 2) lack of cytoplasmic and mitochondrial exchange for [13C3]Ser/[13C2]Gly. b, 3) net uptake of exogenous D3-Ser into the cytoplasm (pool 2, peach square), which does not readily exchange with pool 1 but exchanges with the mitochondrial pool (lavender square) and interconverts with Gly and one-carbon metabolites (green double-headed arrow); 4) less access of D3-Ser-derived Gly and one carbon metabolites (peach square pools) to purine synthesis machinery (e.g. orange square purinosomes (44)); 5) enhanced mitochondrial Gly to Ser fluxes (uneven green double-headed arrow) possibly driven by activation of SHMT2 and MTHFD2 (thick green double-headed arrow). b and c, 6) loss of D (or gain of H) in one carbon metabolites via mitochondrial MTHFD2/2L and cytoplasmic MTHFD1 exchange reactions (green double-headed arrow); 7) loss of D via direct incorporation of D-Gly into C5,6 of purines (thin arrow); 8) negligible incorporation of Gly-derived one carbon metabolites into purines (gray arrow). c, 9) net Gly efflux (uneven arrow); 9) less favored direct (route 1, thin blue arrow) (c) and indirect (route 2, not depicted) incorporation of [13C2]Gly into purines. PSAT1, phosphoserine aminotransferase; MTHFD1, cytoplasmic NADP+-dependent methylene tetrahydrofolate (CH2-THF) dehydrogenase/methylene THF; cyclohydrolase/formyl THF (CHO-THF) synthetase; MTHFD2, mitochondrial NAD+-dependent methylene THF dehydrogenase/methylene THF cyclohydrolase; MTHFD2L, mitochondrial NADP+-dependent methylene THF dehydrogenase; MTHFD1L, mitochondrial formyl THF synthetase; 3-PG, 3-phosphoglycerate; 3-POHPyr, 3-phosphohydroxypyruvate. Solid and dashed arrows: one- and multistep reactions, respectively.

Article Snippet: The primary antibodies used were obtained from ProteinTech Group with the following catalogue numbers: GAPDH, 60004-1-Ig; PSAT1, 10501-1-AP; MTHFD2, 12270-1-AP; SHMT2, 11099-1-AP; and PHGDH, 14719-1-AP; GLDC, 24827-1-AP.

Techniques: Activation Assay, Ex Vivo, Modification, Derivative Assay

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Central role for ferritin in the day/night regulation of iron homeostasis in marine phytoplankton

doi: 10.1073/pnas.1506074112

Figure Lengend Snippet: Day/night and circadian regulation of ferritin and major iron-containing proteins in the field and in cultures. (A) Relative transcript abundance of ferritin (Ftn) and iron-containing proteins of PSI (PsaC), cytochrome b6/f (PetD), ferredoxin (PetF), and ferredoxin (Fd-GOGAT) during two complete day/night cycles [from an environmental dataset sampled on the Pacific Coast of the United States between September 16 and September 18, 2010 (25)]. The gray areas indicate the night period [photosynthetically active radiation (PAR) = 0]. (B) Day/night changes in the level of the PsaC protein. (Upper) Western blot analysis of PsaC under 12-h/12-h light/dark conditions. (Lower) Density quantitation of PsaC levels normalized to the mean signal. Data are shown as mean ± SD (n = 3). (C) Day/night changes in the level of ferritin inferred from the relative luminescence of the ferritin-luc (FTN-luc) translational reporter normalized to the mean signal. Data are shown as mean ± SD (n = 5). (D) Circadian regulation of ferritin expression. Translational reporters of FTN-luc (solid line) and circadian clock-associated 1 protein (CCA1-Luc, dashed line) were placed in constant light after entrainment under 12-h/12-h light/dark conditions. Normalized luminescence to the mean signal is shown (mean ± SD, n = 5).

Article Snippet: PsaC protein standard (Agrisera) was used as a calibration standard curve.

Techniques: Western Blot, Quantitation Assay, Expressing