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