Fig 2: Domains of LASP1 differentially associate with Snail1 and its stabilizing proteins. (A) Top panel–Affinity purified full length LASP1 and its domains fused to GST were separated by 10% SDS-PAGE. Proteins were visualized by staining the gel with Imperial Blue Protein Stain; Bottom panel—A schematic depiction of the constructs of recombinant full length of LASP1 and its various domains that were bacterially expressed (B) Association of LASP1 and its domains with Snail1 upon stimulation of stably expressed CXCR4 by CXCL12. Notably, 1.5 nmol of each of the GST and LASP1 and its domains fused to GST were incubated with 200 µg of nuclear lysate derived from 293-CXCR4 cells that were transiently expressing Flag-Snail1-WT and stimulated with 20 nM CXCL12. The association of Flag-Snail1 with full length LASP1 and its domains were analyzed by 10% SDS-PAGE, followed by immunoblotting; n = 3 (C) and (D) Association of LASP1 and its domains with proteins that contribute to the stabilization of the Snail1. Nuclear lysates were prepared from MDA-Bone-Un cells that were serum-starved and stimulated with 20 nM CXCL12. 1.5 nmol of each of the GST and LASP1 and its domains fused to GST were incubated with 250 µg of nuclear lysate derived from CXCL12-stimulated MDA-Bone-Un cells. The association of endogenous A20, LSD1, AKT and GSK-3β with full length LASP1 and its domains were analyzed by 10% SDS-PAGE followed by immunoblotting; n = 3.

Fig 3: Phospho-Mutants of LASP1 Differentially Interact with Ago2. (A) Schematic diagram of designed LASP1 phosphorylation mutants. (B) Representative profile of purified GST-LASP1 single phospho-mutants: 1.5 nmol of each purified mutant was resolved by 10% SDS-PAGE and visualized by staining with Imperial Blue stain. (C) LASP1 single-mutant pulldown for Ago2: 1 mg of Bone-Un LASP1 KO total lysate was incubated with 1.5 nmol of each LASP1 single-mutant protein. Associated Ago2 was detected by immunoblot analysis and loading of GST-fusion proteins was visualized by Ponceau S staining. (D) Direct binding of LASP1 single-mutants to recombinant Ago2: 100 ng of purified recombinant Ago2 was incubated with 1.5 nmol of each purified single-mutant protein. Directly bound Ago2 was detected by immunoblot analysis and loading of GST-fusion proteins was visualized by Ponceau S staining. (E) Representative profile of purified GST-LASP1 double phospho-mutants: 1.5 nmol of each of the purified proteins were resolved by 10% SDS-PAGE and proteins were visualized by staining with Imperial Blue stain. (F) LASP1 double-mutant pulldown for Ago2: 1 mg of Bone-Un LASP1-KO total lysate was incubated with 1.5 nmol of each of the double-mutants. Associated Ago2 was detected by immunoblot analysis and loading of GST-fusion proteins was visualized by Ponceau S staining. (G) Direct binding of LASP1 double-mutants to recombinant Ago2: 100 ng of purified recombinant Ago2 was incubated with 1.5 nmol of purified LASP1 double mutants. Directly bound Ago2 was detected by immunoblot analysis and loading of GST-fusion proteins was visualized by Ponceau S staining. Uncropped version of the Western Blots is available in Figure S6.

Fig 4: LASP1 Alters Let-7a Guided Ago2 Activity. (A) Stable ectopic expression of LASP1 WT and its mutants SAYD and SDYF in the LASP1-KO background. Total lysates from Bone-Un CRISPR control (CC) and LASP1-KO (LASP1 KO) as well as the LASP1-KO cells with expression of re-introduced LASP1 WT and its doublemutants: pcDNA 3.0 empty vector (EV) control, LASP1 wild-type rescue (WT), dominant active phospho-mutant LASP1 (SAYD), and dominant-negative phospho-mutant LASP1 (SDYF). 20 µg of total was resolved by 10% SDS-PAGE and immunoblotted for LASP1. (B–D) Luciferase assays in LASP1 rescue cell lines with dual-luciferase reporters. LASP1 rescue cell lines WT, SAYD, SDYF, and EV were incubated overnight with 4 µg of each psiCHECK2 dual-luciferase vector containing an internal firefly luciferase control and experimental Renilla luciferase including no 3'-UTR (EV Luc), artificial 3'-UTR containing Let-7a target sites (Let-7a), artificial 3'-UTR containing miR-100 target sites (miR-100 Tar), artificial 3'-UTR containing miR-100 target sites with mis-matched seed sequences (miR-100 Not-Tar), and the inserted 3'-UTR of the mTOR mRNA (mTOR). Cell lysates were harvested from each condition and relative luciferase units (RLUs) measured by luminometer. Final readings for each condition was expressed as the ratio of Renilla to firefly RLUs with Let-7a compared against EV Luc (B), miR-100 Tar vs. miR-100 Not-Tar (C), and mTOR vs. miR-100 Not-Tar (D). All experiments performed n = 3 with eight technical replicates per experiment. Uncropped version of the Western Blots is available in Figure S7. ** p < 0.01.

Fig 5: LASP1 interacts with the eIF4F complex in a CXCL12-dependent manner. (A) Co-immunoprecipitation assay of eIF4A and LASP1 in 231S cells following stimulation with 20 nM CXCL12 (n = 2). (B) Co-immunoprecipitation assay of eIF4B and LASP1 in 231S cells following stimulation with 10 nM CXCL12 (n = 3). (C) Co-immunoprecipitation assay of LASP1 and eIF4A/B following stimulation with 20 nM CXCL12 in 231S cells (n = 2). Fold change was calculated based off the densitometry ratio of co-immunoprecipitated/immunoprecipitated protein signal with 0 min. set to 1. (D–E) m7GTP pulldown assay in 231S cells following stimulation with 20 nM CXCL12 and 100 nM AMD3465 examining the interaction between: (D) LASP1-eIF4E (n = 3) and (E) eIF4G-eIF4E (n = 3). Fold change was calculated based off the densitometry ratio of co-precipitate (LASP1 or eIF4G)/precipitate (eIF4E) protein signal with 0 min. set to 1.