Fig 2: Design and verification of sample preparation for projection electrophoresis of single mammalian cells.(a) High-density endogenous protein bands (ii) correspond to single-cell settling in microwells (i). Scale bars represent 1 mm (left full-gel images) and 200 µm (right zoom images). (b) Illustration of top-view and side-view geometries shown in protein dilution studies (c, d). (c) Modeling and experimental quantification of diffusional dilution during lysis. Simulated and experimental top-view images of diffusional protein dilution during lysis, and side-view simulated results are shown. The simulated initial TurboGFP concentration was 2 µM. Experimental image is representative of 12 monitored cells across 3 independent lysis experiments. Scale bars represent 50 µm. (d) Modeling the impact of diffusion during electrophoresis on detectable in-gel protein concentration. Side and top view TurboGFP concentration profiles are shown at different times during electrophoresis. Simulated initial TurboGFP concentration (before lysis and electrophoresis) was 2 µM. Scale bars represent 30 µm. (e) Quantification of the percent of protein remaining in the microwell region during lysis (experiment plots mean and standard deviation of n = 12 cells across 3 lysis experiments). (f) Quantification of the change in the spatial maximum protein concentration as a function of time after protein solubilization (experiment plots mean and standard deviation of n = 12 cells across 3 lysis experiments). (g) Simulated maximum protein concentration vs. electrophoresis time, for 3 model proteins. (h) Representative ß-tubulin separations from U251 glioblastoma cells lysed with different buffer formulations (2× RIPA lysis buffer and 2× RIPA including 8 M urea), both after 10 s electrophoresis. Lysis/EP buffer requires 8 M urea for fast protein solubilization and electromigration. (i) Maximum intensity projection 3D renderings and z-intensity profiles of probed GAPDH bands from single BT474 breast tumor cells. (j) Microwell packing density (impacting assay throughput) is dependent on protein band diffusion before photocapture. Protein diffusion profiles confirm that a microwell pitch of 200 µm is sufficient to resolve bands from neighboring microwells. After 10 s EP, the mean peak width (sxy) of the xy GAPDH spots is 32 ± 11 µm (mean ± standard deviation of n = 47 single-cell separation lanes across 5 replicate separation gels). The depicted confocal slice micrograph is representative of 20 confocal image stacks, across different regions of 5 replicate separation gels. At a microwell pitch of 192 µm (6sxy), <0.3% of the signal should bleed into the neighboring lane. Scale bar represents 50 µm. Source data are provided as a Source Data file.

Fig 3: Projection electrophoresis simultaneously lyses and separates both nuclear and cytosolic proteins from hundreds of single cells.(a) Projection electrophoresis device photograph and workflow schematic. A 9 × 9 mm projection electrophoresis PAG contains > 1000 microwells that each serves as a separation lane. Cells in PAG microwells are lysed in situ using a lysis buffer-soaked hydrogel delivery matrix, and the lysate is then electrophoretically injected into the polyacrylamide and proteins are separated by size through the depth of the gel. After separation, proteins are covalently linked to the gel matrix using a UV-initiated capture. All cells are lysed and analyzed simultaneously, the active assay time (from cell lysis to photocapture) is less than 90 s, and in situ analysis reduces potential losses from transfer steps. (b) In contrast, capillary electrophoresis analyzes cells in series, with each cell lysed and proteins separated at a different time. (c–f) Different visualizations of z-directional protein separation in a single separation lane. (c) xz (y-summed) contour plots of background-subtracted protein signal within the separation lane for nuclear and cytosolic example proteins. Protein signal is visualized as peak height and false-colored. (d) z-directional intensity profiles (summed fluorescence intensity in arbitrary fluorescence units, AFU, vs. z) for PTBP1 (nuclear, blue) and GAPDH (cytosolic, green) in the same lane, revolved around the z-axis to generate a 3D rendering of fluorescence distribution. (e, f) Representative confocal xy slice images (representative of 9 confocal stacks acquired of different regions of 2 independent separation gels) for separated PTBP1 (blue) and GAPDH (green) at two z-depths into the gel ((e) at the 185 µm depth of the PTBP1 band; (f) at the 335 µm depth of the GAPDH band). Each slice image shows 4 separation lanes, 3 of which appear to have been occupied by BT474 cells prior to analysis. Overlaid squares depict xy regions of interest for the lane plotted in (c) and (d), over which fluorescence intensities were summed to yield z-directional intensity profiles. Scale bar represents 50 µm. (g) Quantified z-migration distances for PTBP1 (57 kDa) and GAPDH (37 kDa) from n = 15 (PTBP1) and n = 17 (GAPDH) separation lanes. Source data are provided as a Source Data file.

Fig 4: SREBP1 mediates induction of 6PGD downstream of the androgen receptor (AR).(A) ChIP-seq data showing AR DNA binding near the 6PGD gene in non-malignant and prostate tumour samples (Pomerantz et al., 2015) and the LNCaP (Barfeld et al., 2017) and VCaP (Asangani et al., 2014) cell line models. The grey box indicates a region ±50 kb of the 6PGD transcriptional start site. (B) ChIP-seq data showing SREBP1 DNA binding at the 6PGD promoter in HEPG2 and MCF7 cells. Data is from ENCODE (ENCODE Project Consortium, 2012; HEPG2: ENCFF000XXR; MCF7: ENCFF911YFI). (C) Effect of siSREBP1 on 6PGD protein. LNCaP cells were transfected with siRNA (siSREBP1; 12.5 nM) or control (siCon) for 72 hr after which SREBP1 and 6PGD protein levels were evaluated by immunoblotting. GAPDH was used as loading control. (D) Effect of siSREBP1 on 6PGD induction by DHT. LNCaP (left) or VCaP (right) cells were transfected with siRNA (siSREBP1; 12.5 nM) or control (siCon) in charcoal-stripped FBS media for 72 hr and then treated with 10 nM DHT for another 24 hr. SREBP1 and 6PGD protein levels were evaluated by immunoblotting. GAPDH was used as loading control. (E) RT-qPCR of 6PGD expression in response to DHT and fatostatin in LNCaP cells. Cells were serum starved in charcoal-stripped FBS media for 72 hr and then treated with Veh or 10 nM DHT ±10 µM fatostatin for another 24 hr. Gene expression was normalised to GUSB and L19 and represents the mean + SEM of three biological replicates. Treatment effects were evaluated using ANOVA and Dunnett’s multiple comparison tests (*p<0.05; **p<0.01; ****p<0.0001; NS, not significant).

Fig 5: Western blot of CSB. (A,C) α-CSB rabbit polyclonal Abcam. (B,D) α-CSB rabbit polyclonal Bethyl. (A,B): the full-length form of the CSB protein is absent from CS10 and CS14 fibroblasts whereas it is present in a healthy control. (C,D): larger-sized Western blots at a lower exposure than in (A,B); arrows indicate the expected position of the CSB-piggyBac fusion protein (which is more abundant than the full-length CSB); hatched rectangles correspond to the part of the blot shown in (A,B). Lower panels: immunoblot of the loading control GAPDH.