Fig 2: Effect of SB on the levels of p21 (FK506 binding protein like), p53, p27 (cyclin dependent kinase inhibitor 1B), p16 and pFAK in p21 knockdown and control A172 cells. (A) Knockdown of p21 was achieved by transfection with specific siRNA against human p21 for 72 h. The effect of 2 mM SB on the levels of p21 (FK506 binding protein like), p53, p27 (cyclin dependent kinase inhibitor 1B), p16 and FAK phosphorylation in control and p21-knockdown cells was determined using western blot analysis. (B) ß-galactosidase positive staining (n=4; upper panel); and cell motility (lower panel; n=6) were measured in the A172 cells in (A). Results are presented as the mean ± standard deviation. *P<0.01 vs. control. (C) Cell cycle analysis of the A172 cells in (A) using a BD FACScalibur system. SB, sodium butyrate; p-, phosphorylated; FAK, focal adhesion kinase; si, small interfering; C, control.

Fig 3: Eribulin normalizes CAF traits in the CDM system. A Illustration depicting the experimental conditions for CAF-generated CDMs treated with DMSO (blue) or eribulin (red) during matrix production. B Representative images of CAF-generated CDMs that resulted from the assorted experimental conditions used during the 5-day matrix production. Images show fibronectin fibers detected by indirect immunofluorescence and digitally pseudo-colored to depict the orientation angles of ECM fibers. The colored bar on the right represents the angle distributions and was normalized to show mode angle as 0° (cyan). The scale bar in the bottom right image corresponds to 50 µm. Note that CAFs treated with paclitaxel at 3 nM were not quantified due to the lack of fibrillogenesis as indicated by the yellow arrows pointing to ECM gaps and by the low cellularity and ECM thickness, measured below 7 µm, as shown in accompanying Supplemental Fig. 1 A. The graph indicates the levels of fiber alignment, quantified as the percentage of fibers oriented within 15° from the mode angle. C Representative cropped immunoblots (for full matching blots see Supplemental Fig. 2) of lysates collected at the end of CAF-generated CDM production showing representative bands for palladin, phospho-SMAD2/3 (pSMAD2/3), and total SMAD2/3 (SMAD), using GAPDH as an intracellular protein loading control. Accompanying graphs depict levels of palladin and activated SMAD (pSMAD2/3) gauged from immunoblots and normalized to levels attained under DMSO treated conditions. D The total intensities of phospho-FAK (pFAK) and activated a5ß1-integrin, calculated using the MetaMorph software 7.8.0.0 and measuring total intensities with the same inclusive intensity thresholds set for all conditions, were normalized to cell numbers (nuclei counts). Experiments were repeated 4 times while a minimum of 6 images were acquired per sample. For statistical significance, one-way ANOVA was used to compare to DMSO vehicle. * P < 0.05; ** p < 0.01; *** P < 0.001; **** p < 0.0001

Fig 4: LDL enhances FA dynamics in a FAK-dependent manner ASchematic outline of plasma membrane D4H labeling. CM and LPDS indicate complete medium (10% FBS in DMEM) and 5% LPDS in DMEM, respectively.BAfter 24-h LDL depletion in 5% LPDS, cells were loaded with 50 µg/ml LDL for the indicated times. Plasma membrane cholesterol was labeled with D4H and imaged with widefield epifluorescence microscopy.CAnalysis of (B). Mean ± SD, n = 15 fields of cells as in (B) pooled from 2 independent experiments.DRepresentative FA lifetime in cells treated without or with 50 µg/ml LDL (-LDL or +LDL) for 1–2 h after 24 h in 5% LPDS. Time-lapse imaging of GFP-paxillin dynamics in cells was acquired by TIRF microscopy and maximum intensity during FA lifetime was normalized to 1.EAnalysis of FA assembly and disassembly rate of cells incubated +/-LDL as in (D). Where indicated, 10 µM PF-228 was added after 30 min of LDL loading. Assembly rate: n = 290 FAs (-LDL); 410 (+LDL); 192 (+LDL+PF-228) and disassembly rate: n = 397 (-LDL); 502 (+LDL); 214 (+LDL+PF-228) pooled from 4 independent experiments. The Tukey box plots represent the median and 25th and 75th percentiles (interquartile range). The whiskers show the highest and lowest observations. Non-parametric Kruskal–Wallis analysis of variance with Bonferroni error correction.FRepresentative immunoblot and quantification of pFAK levels in 5% LPDS (-LDL) and upon increasing LDL loading times. Mean ± SD, n = 3, paired Student’s t-test (*P < 0.05).GRepresentative confocal images of NPC1, internalized integrin ß1, and pFAK localization. Stably NPC1-GFP-expressing cells were loaded with 2 µg/mL integrin ß1 antibody for 30 min before fixation and stained with pFAK antibody followed by secondary antibodies. Dashed line indicates the cell edge.HQuantification of (G) using Mander’s colocalization for the indicated markers. Mean ± SD, n = 5 cells. Cells were treated with 5% LPDS for 1 day and then without (-) or with (+) 50 µg/ml LDL for 1h. Source data are available online for this figure.

Fig 5: Cholesterol dependent activation of FAK A, BControl and degron-ORP2 cells were co-plated, and after 1-day 5% LPDS, treated with 50 µg/ml LDL and IAA (+IAA; ORP2 depletion) for the indicated times and immunostained for pFAK to quantify its intensity. For rescue experiments, degron-ORP2 cells were plated and transfected with GFP-ORP2 or -ORP2-mHHK or -ORP2-?ELSK for 6 h. Cells expressing GFP-ORP2 constructs at levels similar to endo-GFP-ORP2 were used for quantifying pFAK intensity. Dashed lines indicate cell outlines. See also Fig EV6C. Asterisk indicates cells depleted of endogenous ORP2. Images were acquired by confocal microscopy. Mean ± SD, n = 20–23 cells pooled from 2 independent experiments. Student’s t-test.CQuantification of FAK protein in cytosolic and membrane fractions in degron-ORP2 cells with or without IAA. Cells were starved overnight in LPDS and loaded with 50 µg/mL LDL +/- IAA for 2 h. The proportion of FAK signal in the membrane fraction (of total FAK in cytosol + membranes) is presented ± SD. For no IAA n = 13 and for IAA n = 11, 2 independent experiments. Student’s t-test.D–FLiposome-co-sedimentation of FAK FERM with increasing concentrations of PI(4,5)P2 and cholesterol. “S” in (D) indicates the supernatant and “P” pellet containing the FAK FERM-bound to liposomes. The total lipid concentration was kept at 50 µM, and purified FAK FERM concentration at 1 µM. The initial lipid composition was POPC:POPE:POPS:Rhodamine-DHPE:PI(4,5)P2:cholesterol (70:19:10:1:0:0, mol/mol). The increasing PI(4,5)P2 and cholesterol concentrations were compensated by decreasing the amount of POPC. Mean ± SD, n = 5. Student’s t-test (*P < 0.05; **P < 0.002). Numbers under the blots indicate fraction of FAK FERM bound to liposomes (pellet) of total FAK FERM (supernatant + pellet).G, HBinding of Alexa Fluor 488-labeled FAK FERM domain to GUVs containing PI(4,5)P2 and cholesterol. The lipid compositions were POPC:POPE:POPS:Rhodamine-DHPE:PI(4,5)P2:cholesterol (50:19:10:1:20:0) and POPC:POPE:POPS:Rhodamine-DHPE:PI(4,5)P2:cholesterol (20:19:10:1:20:30, mol/mol). Images were acquired by widefield epifluorescence microscopy. Scale bar, 5 µm. Mean ± SD, n = 68–76 pooled from 2 independent experiments. Student’s t-test. Source data are available online for this figure.