Journal: bioRxiv

Article Title: Reversible Dissociation of Mitochondrial Complex V Balances Anabolic and Energy-Generating Needs in Cancer

doi: 10.1101/2025.08.05.668642

Figure Lengend Snippet: (A). Appearance of free F 1 in NIH3T3 fibroblasts during Rho0 cell generation. Cells were maintained in 30 or 60 ng/ml of EtBr for 2 days (lanes 2 & 3) and then switched to EtBr-free medium for 2 wks to allow for recovery (lanes 4&5). At each point, in situ ATPase assays were performed. (B). mtDNA content of EtBr-treated cells described in (A) and normalized to that of the nuclear-encoded Mlx gene . (C). Immuno-blot showing that mitochondrial-encoded Mt-atp6 but not nuclear-encoded Atp5f1a α subunit is depleted in a dose-dependent manner by EtBr (+EtBr) and then restored following subsequent recovery in EtBr-free medium (-EtBr). (D). Immunoblot showing time-dependent Mt-atp6 depletion in NIH3T3 cells in response to doxycycline or chloramphenicol treatment. (E). In situ ATPase assays performed on NIH3T3 cells exposed to doxycycline (10 μg/ml) or chloramphenicol (25 μg/ml) for 48 hr. (F). Stable expression and mitochondrial localization of Mt-atp6-c in NIH3T3 cells. Mitochondria from control (WT) cells or those stably expressing the Mt-atp6-c fusion protein were purified as described in . Loading control immuno-blots were performed for the mitochondrial-localized proteins Pdha1a and ATP5fa1. (G). Identification of TALED mutations in NIH3T3 cells using the T7 endonuclease assay described in . (H). Enforcing Mt-atp6-c or mutating endogenous Mt-atp6 with TALEDs, respectively, alters NIH3T3 cell tsusceptibility to EtBr-mediated F o -F 1 dissociation. In situ ATPase assays were performed on control, untreated NIH3T3 cells or on cells cultured in EtBr (30 ng/ml) as described in (A). (I) Hypoxia and chloramphenicol reduce ATP t 1/2 in BY3 cells. Cells stably expression Mito-targeted iATPSnFR2HaloTag were exposed to 1% oxygen or 25 mg/ml chloramphenicol for 48 hr. They were then stained with Janelia Fluor JFX650 HaloTag® Ligand and subjected to moving average continuous flow cytometry as described in .

Article Snippet: They were transiently co-transfected into BY3 or NIH3T3 murine fibroblasts (obtained from the ATCC, Manassas, VA) at a 10:1 ratio with the Cyto- and Mito-targeted roGFP or pSypHER plasmids described above followed by FACS purification and G418-selection of the GFP+ population 2 days later.

Techniques: In Situ, Western Blot, Expressing, Control, Stable Transfection, Purification, Cell Culture, Staining, Flow Cytometry

Journal: Life

Article Title: Ubiquitination Regulates Reorganization of the Membrane System During Cytomegalovirus Infection

doi: 10.3390/life15081212

Figure Lengend Snippet: PYR-41-inhibited ubiquitination prevents the establishment of the MCMV infection. ( A ) NIH3T3 fibroblasts were infected with C3X GFP MCMV and treated with DMSO (Ø) or indicated concentrations of PYR-41 at 0, 2 and 4 hpi. GFP expression was determined by flow cytometry after 6 hpi . The upper panel shows GFP intensity normalized to mock-treated cells, and the lower panel shows the percentage of GFP-positive cells. ( B ) pIE1 expression after 6 hpi and ( C ) percentage of pIE1-positive cells (mean ± SD) after infection with MCMV and treatment with DMSO (mock) or different concentrations of PYR-41 at 0, 2 and 4 hpi. Bars—25 μm. ( D ) MCMV-infected cells were treated with 15 μM PYR-41 before (0 hpi →) or 4 h after infection (4 hpi →) and the expression of pIE1 was determined by Western blot analysis. Shown are representative Western blots (complete blots are shown in ) and a quantitative analysis of the signals normalized to β-tubulin. The fold changes represent the signal expression relative to the band with the highest intensity in the mock-treated (Ø) samples, the read bars represent the mean ± SD, and the empty circles are data from an independent experiment. Statistical significance was determined using a two-tailed paired Student t -test (*** p < 0.001; ** p < 0.01; * p < 0.05). The numbers of independent experiments are indicated in parenthesis.

Article Snippet: Murine fibroblast-like cell line NIH3T3 (ATCC CRL-1658) was used for most experiments, and murine embryonic fibroblasts (MEFs), derived from 17-day-old BALB/c mouse embryos, were used for virus production and plaque assays.

Techniques: Ubiquitin Proteomics, Infection, Expressing, Flow Cytometry, Western Blot, Two Tailed Test

Journal: Life

Article Title: Ubiquitination Regulates Reorganization of the Membrane System During Cytomegalovirus Infection

doi: 10.3390/life15081212

Figure Lengend Snippet: pIE1 is ubiquitinated in MCMV-infected cells. Doxycycline-treated (2 μg/mL for 48 h) NIH3T3 HA-Ub cells were infected with MCMV and ( A , B ) were left untreated or ( C , D ) treated with either DMSO (mock) or 15 μM PYR-41 at 4 hpi. At indicated times after infection, the whole cell lysates ( A , C ) and anti-HA immunoprecipitate ( B , D ) were analyzed for pIE1 and β-actin expression by Western blot. Signals were quantified by ImageJ and normalized to β-actin in WCLs. Fold changes represent the signal expression relative to the band with the highest intensity in mock-treated cells. The mean ± SD (red bars) and individual data (empty circles) are shown in the graphs. The number of independent experiments is indicated in parenthesis. The original blots are shown in .

Article Snippet: Murine fibroblast-like cell line NIH3T3 (ATCC CRL-1658) was used for most experiments, and murine embryonic fibroblasts (MEFs), derived from 17-day-old BALB/c mouse embryos, were used for virus production and plaque assays.

Techniques: Infection, Expressing, Western Blot

Journal: Life

Article Title: Ubiquitination Regulates Reorganization of the Membrane System During Cytomegalovirus Infection

doi: 10.3390/life15081212

Figure Lengend Snippet: The effect of PYR-41 on the progression of the MCMV replication cycle. ( A – E ) MCMV-infected NIH3T3 cells were treated with either DMSO (mock) or with 15 μM PYR-41 prior to infection (0 hpi) or 4 hpi. The expression of pE1 ( A , B ), pM57 ( C ) and pM74 ( D , E ) was analyzed by Western blot at the indicated time points after infection. β-tubulin or β-actin were used as loading controls. The original blots are shown in . Signals were normalized to the loading control, and fold changes represent signal expression relative to the band with the highest intensity in the mock-treated samples. The empty circles show the data from independent experiments and the red bars show mean values ± SD. Statistical significance was determined using a two-tailed paired Student t -test (*** p < 0.001; ** p < 0.01; * p < 0.05). The number of independent experiments is indicated in parenthesis. ( F ) Supernatants or cell lysates of PYR-41-treated cells (0 hpi → or 4 hpi →) were harvested at 0, 24, 48, 72 and 96 hpi and the number of infectious units was determined by plaque assay. The mean values ± SD of four independent experiments are shown. Statistical significance was determined using the Mann–Whitney test (* p < 0.05).

Article Snippet: Murine fibroblast-like cell line NIH3T3 (ATCC CRL-1658) was used for most experiments, and murine embryonic fibroblasts (MEFs), derived from 17-day-old BALB/c mouse embryos, were used for virus production and plaque assays.

Techniques: Infection, Expressing, Western Blot, Control, Two Tailed Test, Plaque Assay, MANN-WHITNEY

Journal: Life

Article Title: Ubiquitination Regulates Reorganization of the Membrane System During Cytomegalovirus Infection

doi: 10.3390/life15081212

Figure Lengend Snippet: PYR-41 inhibits the formation of pre-AC. ( A ) NIH3T3 cells were infected with ΔFcR MCMV and analyzed for the expression of GM130 (green), Rab10 (red) and pIE1 (blue) by immunofluorescence after 12 hpi. In the uninfected control, the cell nuclei were stained with DAPI (blue). Arrows indicate extended Golgi, arrowheads indicate condensed Golgi, and asterisks indicate perinuclear Rab10 in pre-AC. The percentage of cells expressing condensed Rab10 and GM130 in pre-AC is shown on the right. Means ± SD (red bars) and individual values (empty circles) are shown. The complete experiment is shown in . ( B ) ΔFcR MCMV-infected cells were treated with 15 μM PYR-41 at 0 and 4 hpi or mock treated. After 12 hpi, the expression of GM130 (green), Rab10 (red) and pIE1 (blue) was analyzed. Arrowheads indicate condensed Golgi and arrows indicate dispersed Golgi. Asterisks indicate Rab10 in pre-AC. ( C – E ) The ratio of pIE1-positive cells ( C ), cells with perinuclear Rab10 ( D ) and condensed/dispersed Golgi patterns ( E ) in PYR-41-treated and mock-treated cells. Shown are the mean ± SD (red bars) and individual values (empty circles). The number of independent experiments is indicated in parenthesis. Statistical significance was determined using a two-tailed paired Student t -test (*** p < 0.001; ** p < 0.01). Bars—10 μm.

Article Snippet: Murine fibroblast-like cell line NIH3T3 (ATCC CRL-1658) was used for most experiments, and murine embryonic fibroblasts (MEFs), derived from 17-day-old BALB/c mouse embryos, were used for virus production and plaque assays.

Techniques: Infection, Expressing, Immunofluorescence, Control, Staining, Two Tailed Test

Journal: Life

Article Title: Ubiquitination Regulates Reorganization of the Membrane System During Cytomegalovirus Infection

doi: 10.3390/life15081212

Figure Lengend Snippet: Treatment with PYR-41 in the late phase of infection inhibits the production of infectious virions. NIH3T3 cells were infected with wt MCMV. After 48 hpi, the cell culture medium was replaced with fresh medium, and cells were treated with 15 μM PYR-41 or mock-treated. The supernatants or cell lysates were harvested after 72 and 96 hpi and infectious virion production was determined using the plaque assay. The mean values of four independent experiments are plotted; the error bars show the standard deviation. Statistical significance was determined using the Mann–Whitney test (* p < 0.05). Ctrl.—control level of virus production in mock-treated cells without changing the cell culture medium 48 hpi.

Article Snippet: Murine fibroblast-like cell line NIH3T3 (ATCC CRL-1658) was used for most experiments, and murine embryonic fibroblasts (MEFs), derived from 17-day-old BALB/c mouse embryos, were used for virus production and plaque assays.

Techniques: Infection, Cell Culture, Plaque Assay, Standard Deviation, MANN-WHITNEY, Control, Virus

Journal: Life

Article Title: Ubiquitination Regulates Reorganization of the Membrane System During Cytomegalovirus Infection

doi: 10.3390/life15081212

Figure Lengend Snippet: PYR-41 disrupts Rab10-associated tubulation in the established AC. NIH3T3 EGFP-Rab10 cells were infected with wt MCMV. At 16 hpi, PYR-41 was injected into the tissue culture medium (15 μM concentration), and cells were imaged live with fluorescence-enhanced DHTM for 1 h. Screenshots at the indicated time points are shown and a full time-lapse in (Mock) and (PYR-41). The arrows indicate expanded Rab10-EGFP-positive tubules in AC.

Article Snippet: Murine fibroblast-like cell line NIH3T3 (ATCC CRL-1658) was used for most experiments, and murine embryonic fibroblasts (MEFs), derived from 17-day-old BALB/c mouse embryos, were used for virus production and plaque assays.

Techniques: Infection, Injection, Concentration Assay, Fluorescence

Journal: Life

Article Title: Ubiquitination Regulates Reorganization of the Membrane System During Cytomegalovirus Infection

doi: 10.3390/life15081212

Figure Lengend Snippet: WASHC1 in MCMV-infected cells. ( A ) NIH 3T3 cells were infected with wt MCMV or left uninfected and analysed 16 h later by immunofluorescence for the expression of pIE1 (green) and WASH1C (red). DAPI (blue) was used to stain the cell nuclei. The percentage of cells with perinuclear WASHC1 (mean ± SD (red bar)) is shown on the right. ( B ) Doxycycline-treated NIH3T3 HA-Ub cells were infected with wt MCMV or left uninfected. After 16 h, 10% of cells were lysed for WCL and 90% of cells were lysed for the immunoprecipitation of ubiquitinated proteins with rabbit anti-HA. The expression of WASHC1, pIE1 and β-actin was visualized with the corresponding primary and secondary antibodies. The fold change represents the signal expression of Ub-WASHC1 relative to the uninfected cells. HC—Heavy chain of anti-Rb IgG pAb. ( C , D ) NIH3T3 cells were transfected with Scr. or WASHC1 siRNA or left untransfected (control). After 48 h, cells were infected with wt MCMV for 16 h or left uninfected and analyzed either by Western blot ( C ) or immunofluorescence ( D ). Signals were normalized to β-actin and fold change represents signal expression relative to uninfected and mock-treated samples. ( D ) Triple staining of Rab10 (red), pIE1 (green) and DAPI (blue) was analyzed by confocal imaging. ( E ) Percentage of MCMV-infected (pIE1-positive) cells with perinuclear accumulation of Rab10 in Scr. and WASH1 siRNA-treated cells at 16 h post-infection, shown as mean ± SD. Ctrl., control the level in non-transfected cells. The number of independent experiments is indicated in parenthesis. ( F ) Cells were treated with Scr., Rab11 or WASHC1 siRNA for 48 h and infected with wt MCMV. Supernatants and cell lysates were harvested at 48 hpi, and the number of infectious units was determined by plaque assay. The number of experiments is indicated in the bars. The error bars show the standard deviation. Statistical significance was determined using the Mann–Whitney test (* p < 0.05, ** p < 0.01, *** p < 0.001). Bars—10 μm.

Article Snippet: Murine fibroblast-like cell line NIH3T3 (ATCC CRL-1658) was used for most experiments, and murine embryonic fibroblasts (MEFs), derived from 17-day-old BALB/c mouse embryos, were used for virus production and plaque assays.

Techniques: Infection, Immunofluorescence, Expressing, Staining, Immunoprecipitation, Transfection, Control, Western Blot, Imaging, Plaque Assay, Standard Deviation, MANN-WHITNEY

Journal: bioRxiv

Article Title: Instant fluorescence lifetime imaging microscopy reveals mechano-metabolic reprogramming of stromal cells in breast cancer peritumoral microenvironments

doi: 10.1101/2025.05.28.656717

Figure Lengend Snippet: Representative fluorescence lifetime images (top) and phasor plots (bottom) of a) NIH3T3 fibroblasts (n = 11 uncompressed, 10 compressed), b) 3T3-L1 undifferentiated adipocytes (n = 12 uncompressed and compressed), and c) differentiated 3T3-L1 (d3T3-L1) adipocytes (n = 11 uncompressed, 10 compressed). Fluorescence lifetime analysis reveals that the applied mechanical stress decreases mean fluorescence lifetime ( ) in d) fibroblasts and f) differentiated adipocytes, but produces the opposite effect in e) undifferentiated adipocytes. The decrease in lifetime indicates that compression metabolically rewires g) NIH3T3 and i) d3T3-L1 cells to a more glycolytic state and h) 3T3-L1s towards a more oxidative state. Error bars represent SEM and asterisks indicate statistical significance (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001) determined using a Student’s t-test. Scale bar is 50 µm.

Article Snippet: Murine NIH3T3 fibroblasts (American Type Culture Collection [ATCC], CRL-1568, Manassas, VA, USA) and 3T3-L1 undifferentiated adipocytes (ATCC, CRL-173) were grown in the following complete media: Dulbecco’s Modified Eagle Medium (DMEM; Corning, 10-03-CV, Corning, NY, USA) supplemented with 10% fetal bovine serum (FBS; Corning, 35-015-CV) and 1X penicillin-streptomycin (P/S; Corning, 30-002-CI).

Techniques: Fluorescence, Metabolic Labelling

Journal: bioRxiv

Article Title: Instant fluorescence lifetime imaging microscopy reveals mechano-metabolic reprogramming of stromal cells in breast cancer peritumoral microenvironments

doi: 10.1101/2025.05.28.656717

Figure Lengend Snippet: Gene expression analysis of markers involved in mechanotransduction via qPCR shows that Trpv4 expression is moderately upregulated in a) compressed NIH3T3 fibroblasts (n = 3), but not b) compressed 3T3-L1 undifferentiated adipocytes (n = 3). On the other hand, Ctnnb1 , Piezo1 , and Yap1 expression in both cell types is not significantly affected by compressive stress. Error bars represent SEM and asterisks indicate statistical significance (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001) determined using a Student’s t-test. RQ: relative quantification.

Article Snippet: Murine NIH3T3 fibroblasts (American Type Culture Collection [ATCC], CRL-1568, Manassas, VA, USA) and 3T3-L1 undifferentiated adipocytes (ATCC, CRL-173) were grown in the following complete media: Dulbecco’s Modified Eagle Medium (DMEM; Corning, 10-03-CV, Corning, NY, USA) supplemented with 10% fetal bovine serum (FBS; Corning, 35-015-CV) and 1X penicillin-streptomycin (P/S; Corning, 30-002-CI).

Techniques: Gene Expression, Expressing, Quantitative Proteomics

Journal: bioRxiv

Article Title: Instant fluorescence lifetime imaging microscopy reveals mechano-metabolic reprogramming of stromal cells in breast cancer peritumoral microenvironments

doi: 10.1101/2025.05.28.656717

Figure Lengend Snippet: Representative immunofluorescent images of murine a) NIH3T3 (n = 29 uncompressed, 44 compressed) and b) 3T3-L1 cells (n = 15 uncompressed and compressed) stained for the mechanoresponsive yes-associated protein (YAP). Analysis of log-transformed nuclear-to-perinuclear YAP intensity ratios (log[nuclear:perinuclear intensity]) reveals that compression significantly increases YAP nuclear localization in c) fibroblasts and d) undifferentiated adipocytes. Error bars represent SEM and asterisks indicate statistical significance (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001) determined using a Student’s t-test. Scale bar is 50 µm.

Article Snippet: Murine NIH3T3 fibroblasts (American Type Culture Collection [ATCC], CRL-1568, Manassas, VA, USA) and 3T3-L1 undifferentiated adipocytes (ATCC, CRL-173) were grown in the following complete media: Dulbecco’s Modified Eagle Medium (DMEM; Corning, 10-03-CV, Corning, NY, USA) supplemented with 10% fetal bovine serum (FBS; Corning, 35-015-CV) and 1X penicillin-streptomycin (P/S; Corning, 30-002-CI).

Techniques: Staining, Transformation Assay

Journal: bioRxiv

Article Title: Instant fluorescence lifetime imaging microscopy reveals mechano-metabolic reprogramming of stromal cells in breast cancer peritumoral microenvironments

doi: 10.1101/2025.05.28.656717

Figure Lengend Snippet: a) Representative plots of oxygen consumption rate (OCR) over time for NIH3T3 (left; n = 3) and 3T3-L1 (right; n = 3) cells. b) Relative contribution to ATP production through glycolysis (glycoATP) and oxidative phosphorylation (mitoATP). c) ATP rate index (proportion of mitoATP:glycoATP) for compressed and uncompressed cells. Higher values indicate a preference for energy production via oxidative phosphorylation while lower values suggest a bias towards glycolysis-driven ATP production. d) Percent activity of glycolysis and oxidative phosphorylation is not significantly different between compressed and uncompressed cells. When considered alongside our FLIM results in , these findings suggest that FLIM offers superior sensitivity in detecting in situ metabolic changes in our system compared to traditional metabolic assays such as Seahorse. Error bars represent SEM and asterisks indicate statistical significance (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001) determined using a Student’s t-test.

Article Snippet: Murine NIH3T3 fibroblasts (American Type Culture Collection [ATCC], CRL-1568, Manassas, VA, USA) and 3T3-L1 undifferentiated adipocytes (ATCC, CRL-173) were grown in the following complete media: Dulbecco’s Modified Eagle Medium (DMEM; Corning, 10-03-CV, Corning, NY, USA) supplemented with 10% fetal bovine serum (FBS; Corning, 35-015-CV) and 1X penicillin-streptomycin (P/S; Corning, 30-002-CI).

Techniques: Phospho-proteomics, Activity Assay, In Situ

Journal: bioRxiv

Article Title: Instant fluorescence lifetime imaging microscopy reveals mechano-metabolic reprogramming of stromal cells in breast cancer peritumoral microenvironments

doi: 10.1101/2025.05.28.656717

Figure Lengend Snippet: Following 24 hours of in vitro compression, NIH3T3 fibroblasts were collected for transcriptomic analysis using bulk RNA-sequencing. a) Gene set enrichment analysis (GSEA) based on gene ontology (GO) terms shows that compressed fibroblasts (n = 3) downregulate oxidative phosphorylation and several related mitochondrial processes, as well as upregulate glycolysis and autophagic processes compared to their uncompressed counterparts (n = 3), confirming our FLIM results in . This highlights the sensitivity of instant FLIM as it can detect subtle metabolic changes that are reflected at the transcriptional level. b) GSEA plots from GO terms showing upregulation of glycolysis (top) and downregulation of oxidative phosphorylation (bottom) in compressed fibroblasts. Statistical significance was determined using permutation testing with Benjamini-Hochberg correction for multiple comparisons (* p.adj < 0.05, ** p.adj < 0.01, *** p.adj < 0.001). NES: normalized enrichment score.

Article Snippet: Murine NIH3T3 fibroblasts (American Type Culture Collection [ATCC], CRL-1568, Manassas, VA, USA) and 3T3-L1 undifferentiated adipocytes (ATCC, CRL-173) were grown in the following complete media: Dulbecco’s Modified Eagle Medium (DMEM; Corning, 10-03-CV, Corning, NY, USA) supplemented with 10% fetal bovine serum (FBS; Corning, 35-015-CV) and 1X penicillin-streptomycin (P/S; Corning, 30-002-CI).

Techniques: In Vitro, RNA Sequencing, Phospho-proteomics

Journal: bioRxiv

Article Title: Instant fluorescence lifetime imaging microscopy reveals mechano-metabolic reprogramming of stromal cells in breast cancer peritumoral microenvironments

doi: 10.1101/2025.05.28.656717

Figure Lengend Snippet: Representative images of a) NIH3T3 fibroblasts and b) 3T3-L1 undifferentiated adipocytes stained with MitoTracker Red CMXRos. Qualitative observations of mitochondrial structure suggest that compression-induced mitochondrial damage occurs in undifferentiated adipocytes, but not fibroblasts, as indicated by greater fragmentation in the former. qPCR analysis of genes encoding proteins involved in mitophagy shows that none are differentially expressed in c) fibroblasts (n = 3), while Bnip3 and Park2 are significantly upregulated in d) compressed undifferentiated adipocytes (n = 3), confirming the qualitative data from a) and b) . e) Representative immunofluorescent images of 3T3-L1 cells stained for MFN1 (fusion marker) and DLP1 (fission marker). Immunofluorescent analysis reveals that the f) expression and g) area fraction (proportion of total cell area occupied by protein signal) of MFN1 is significantly lower in undifferentiated adipocytes subjected to compression (n = 3) compared to those that were not compressed (n = 3). On the other hand, there are no significant changes in DLP1 h) expression and i) area fraction between compressed (n = 3) and uncompressed (n = 3) 3T3-L1 cells. This suggests that the increased presence of mitochondrial fragments in compressed undifferentiated adipocytes likely results from impaired fusion dynamics. Error bars represent SEM and asterisks indicate statistical significance (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001) determined using a Student’s t-test. Scale bar is 10 µm.

Article Snippet: Murine NIH3T3 fibroblasts (American Type Culture Collection [ATCC], CRL-1568, Manassas, VA, USA) and 3T3-L1 undifferentiated adipocytes (ATCC, CRL-173) were grown in the following complete media: Dulbecco’s Modified Eagle Medium (DMEM; Corning, 10-03-CV, Corning, NY, USA) supplemented with 10% fetal bovine serum (FBS; Corning, 35-015-CV) and 1X penicillin-streptomycin (P/S; Corning, 30-002-CI).

Techniques: Staining, Marker, Expressing

Journal: bioRxiv

Article Title: Instant fluorescence lifetime imaging microscopy reveals mechano-metabolic reprogramming of stromal cells in breast cancer peritumoral microenvironments

doi: 10.1101/2025.05.28.656717

Figure Lengend Snippet: The inclusion of 4T1 CM counteracts compression-induced metabolic reprogramming in a) NIH3T3 (data from and ) and b) 3T3-L1 cells (data from and ), but not in c) d3T3-L1 cells (data from and ). Additionally, 4T1 CM significantly increases fluorescence lifetime regardless of compression status in d) NIH3T3 (data from and ), e) 3T3-L1 (data from and ), and f) d3T3-L1 (data from and ) cells. Error bars represent SEM. Statistical significance was determined using a Student’s t-test, with asterisks indicating significance levels (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001).

Article Snippet: Murine NIH3T3 fibroblasts (American Type Culture Collection [ATCC], CRL-1568, Manassas, VA, USA) and 3T3-L1 undifferentiated adipocytes (ATCC, CRL-173) were grown in the following complete media: Dulbecco’s Modified Eagle Medium (DMEM; Corning, 10-03-CV, Corning, NY, USA) supplemented with 10% fetal bovine serum (FBS; Corning, 35-015-CV) and 1X penicillin-streptomycin (P/S; Corning, 30-002-CI).

Techniques: Fluorescence