Fig 2: Alchemilla monticola Opiz. (ALM), astragalin (AST) and quercitrin (QUE) diminished the protein levels of key adipogenic factors in human adipocytes. Representative images from the Western blot analysis (A). Relative protein levels of phosphoinositide 3-kinase (PI3K; B), protein kinase B (AKT; C), peroxisome proliferator-activated receptor gamma (PPARγ; D), CCAAT/enhancer-binding protein alpha (C/EBPα; E), and adiponectin (F). Tubulin or GAPDH were used as housekeeping proteins. Results are expressed as mean ± SEM and are representative of three independent experiments. *p <0.05 compared to the vehicle control group.

Fig 3: Hepcidin activity post-transcriptionally represses NCOA4 in J774 macrophages. J774 cells were treated with PR73, a minihepcidin analog, to mimic the biological effects of hepcidin in vitro. In experiments involving erythrophagocytosis, J774 cells were treated with opsonized red cells 4 h before PR73 treatment. For RNAi, cells were treated with NCOA4 siRNA 24 h prior to the red blood cell treatment. (a) Responses of NCOA4, ferritin, and IRP2 to PR73 (1 μM) in erythrocyte-free macrophages. Relative protein abundance was normalized to GAPDH (n = 3 independent experiments). (b) Lack of changes in Ncoa4, Fth1, and Tfrc mRNA abundance by PR73, normalized to Tbp (n = 3 independent experiments). (c) NCOA4 RNAi negates the increase in ferritin by hepcidin activity (1 μM). Ferritin quantification was normalized to GAPDH (n = 3 independent experiments). (d) PR73 (4 μM) repressed NCOA4 and prevented the facilitated turnover of ferritin between 12 and 24 h of erythrophagocytosis (n = 3 independent experiments). (e) Deficiency of NCOA4 represses the magnitude of change in ferritin by hepcidin activity on erythrocyte-ingested macrophages (n = 3 independent experiments). All data presented as mean ± SD. *, p < 0.05. n.s., not significant. x, non-specific band. ◄, NCOA4 band.

Fig 4: NCOA4 facilitates ferritin turnover and cellular iron release at later stage of erythrophagocytosis. J774 cells were treated with opsonized red blood cells at 0 h and washed at 2 h to remove non-ingested red cells. For NCOA4 depletion, cells were transfected with NCOA4 siRNA 24 h prior red cell treatment. (a) Expression of NCOA4 and native ferritin in erythrocyte-treated cells (n = 3 independent experiments). (b) Lack of changes in ferritin (Fth1, Ftl1) and Ncoa4 transcript abundance by erythrophagocytosis, normalized to Tbp (n = 3 biological replicates). (c) NCOA4 knockdown confirmed by qPCR (n = 3 independent experiments) (d) Steady ferritin expression after erythrophagocytosis in J774 cells deficient of NCOA4. Protein abundance was normalized to GAPDH (n = 4 independent experiments). (e) Higher abundance of ferritin oligomers after 24 h of erythrophagocytosis in NCOA4-deficient cells, detected by western blot analysis of equal amounts of protein separated by native PAGE (n = 3 biological replicates). (f) Cellular mineral contents of erythrocyte-ingested J774 macrophages with or without NCOA4 deficiency, normalized to cell counts (n = 5 biological replicates). (g) Schematic illustration of the flux of iron and regulated ferritinophagy at different iron-recycling steps after erythrophagocytosis. All data presented as mean ± SD. *, p < 0.05. n.s., not significant. x, non-specific band. ◄, NCOA4 band.

Fig 5: 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.