cell confluence  (Thermo Fisher)

Journal: bioRxiv

Article Title: Histidine exchange sustains LAT1 activity and proliferation in glutamine-addicted breast cancers

doi: 10.64898/2026.04.15.716193

Figure Lengend Snippet: ( a) LC-MS of His levels in NMG (n = 7) and MMTV-c- Myc (n = 10) tumours after a 5 min [U 13 C]His bolus. Total abundance (left) and isotopologue distribution (right). Mean ± s.d.; p -values from two-way ANOVA (Šídák’s correction). ( b) Timeline: cells cultured in 2.0 or 0.1 mM Gln (24 h) before 20-min [U 13 C]His administration. ( c) Total relative His abundance (n = 3). Data are mean ± s.d.; p -values from two-way ANOVA (Šídák’s correction). ( d ) RT-qPCR of indicated genes expression in cells cultures in 2.0 mM or 0.1 mM Gln (n = 3), normalized to β-actin and expressed relative to 2.0 mM Gln. Mean ± s.d.; p -values from one-way ANOVA (Šídák’s correction). ( e,f ) WB of cells in 2.0 mM or 0.1 mM Gln (24 h), n = 3; in ( f ) MMTV-c- Myc tumour and liver tissue are used as controls. ( g ) Relative confluency of cells in 0-2.0 mM Gln (96 h), normalized to 2.0 mM Gln; IC50 values from non-linear fit of log[Gln] vs. normalized response (n = 2). ( h ) LC-MS of cells in 2.0 mM or 0.1 mM Gln (24 h), followed by a 10 min [U 13 C]His pulse (n = 3). Labelled His abundance is relative to 2.0 mM Gln. Mean ± s.d.; p -values from one-way ANOVA (Šídák’s correction). See also Figure S2.

Article Snippet: Cells were imaged at regular intervals using the Incucyte ® live-cell imaging system, and confluency was quantified using the Incucyte S3 Software (Sartorius v.2021C).

Techniques: Liquid Chromatography with Mass Spectroscopy, Cell Culture, Quantitative RT-PCR, Expressing

Journal: bioRxiv

Article Title: Histidine exchange sustains LAT1 activity and proliferation in glutamine-addicted breast cancers

doi: 10.64898/2026.04.15.716193

Figure Lengend Snippet: ( a,b ) WB of replenished cells after amino acid starvation with ( a ) 1.6 mM His ( n = 3) or ( b ) 1.6 mM D-His (n = 2) for 48 h. Protein levels are normalized to mTOR/Vinc and expressed relative to the maximum. ( c ) WB of puromycin incorporation in cells starved or preloaded with 0.12 mM or 1.6 mM His (48 h), then replenished with DMEM. Protein levels normalized to Ponceau and expressed relative to the maximum. A representative of 2 independent replicates. ( b) Time-course of relative abundance of intracellular Leu (left) and Met (right) in DMEM-replenished cells after His preloading (1.6 mM, 48 h). 0 min represents pre-replenishment levels. Mean ± s.d.; p -value from two-way ANOVA (Šídák’’s correction). ( e ) Confluency of DMEM-replenished cells preloaded with His, normalized to pre-replenishment confluency (n = 3). Gompertz growth curve applied. Mean ± s.d.; p- values from two -way ANOVA (Dunnett correction). See also Figure S5.

Article Snippet: Cells were imaged at regular intervals using the Incucyte ® live-cell imaging system, and confluency was quantified using the Incucyte S3 Software (Sartorius v.2021C).

Techniques: