Journal: bioRxiv

Article Title: ErbB2/HER2 expression level determines CDK4-inhibitor sensitivity and cyclin D1 and c-Myc dependency at the G1/S transition

doi: 10.1101/2024.05.09.593450

Figure Lengend Snippet: ( A, B ) Control and ErbB2-overexpressing MCF-7 cells (high levels) were treated with CDK4i or DMSO 12 h after HRG stimulation and then harvested at 24 h post-HRG stimulation. Western blotting results (A) showing p-ErbB2, p-RB (Ser 807/811 ), p-RB (Thr 373 ), RB, p-AKT (Thr 308 ), AKT, p-ERK (Thr 202 /Thr 204 ), ERK, c-Myc, cyclin D1, and p27 expression (in control cells, i.e., DMSO treatment) after normalization with GAPDH (B). ( C ) Control and ErbB2-overexpressing cells (moderate and high levels) were individually treated with 250 nM CDK4i or DMSO (no treatment) 12 h after stimulation with 10 nM HRG, and images were acquired every 20 min until 72 h after HRG stimulation. Scale bar: 100 µm. ( D ) Images in (C) were analyzed to quantitatively determine the proportion (%) of each cell-cycle phase in the entire set of images; n = 3. ( E ) Cells that completed the M/G1 transition within 72 h out of the n = 100 individually tracked cells were used. Control cells; CDK4i n = 92, DMSO n = 94, ErbB2 OE-Moderate cells; CDK4i n = 53, DMSO n = 88, ErbB2 OE-High; CDK4i n = 81, DMSO n = 91, with the time to G1/S transition plotted on the x-axis and the time to M/G1 transition plotted on the y-axis. The slope (s) and intercept (i) obtained from the regression equation are shown. The red and blue colors indicate the CDK4i and DMSO treatments, respectively. The mean ± SE time taken to reach the respective time to G1/S transition is shown at the bottom of the graph. ( F ) Using the same data as in E, the time to reach the G1/S transition (G1), the duration of the S/G2/M phase (SG2M), and the overall cell-cycle length (G1+SG2M) were assessed for differences in mean time between the CDK4i and DMSO conditions. ( G ) The number of nuclei increased from 16 h to 72 h after HRG stimulation; n = 3). In (B) and (G), * P < 0.05 (Tukey test).

Article Snippet: A method for constructing MCF-7 (ATCC, HTB-22) overexpressing full-length human ErbB2 (HER2) is described as follows: A human ErbB2 cDNA in pCMV6-XL5 vector was purchased from OriGene (Catalog No: TC128161, Rockville, MD).

Techniques: Control, Western Blot, Expressing

Journal: bioRxiv

Article Title: ErbB2/HER2 expression level determines CDK4-inhibitor sensitivity and cyclin D1 and c-Myc dependency at the G1/S transition

doi: 10.1101/2024.05.09.593450

Figure Lengend Snippet: ( A–G ) ( A - G ) MCF-7 cells overexpressing ErbB2 (high levels) were used. ( A, B ) The following conditions were used: treatment with 250 nM CDK4i 12 h after HRG stimulation, followed by wash/no-wash treatment 4 h later; cells were harvested 40 h after HRG stimulation. Western blotting results showing the expression of p-RB, c-Myc, cyclin D1, p27, and GAPDH. After individually normalizing the proteins relative to GAPDH, the ratio to the no-wash condition was quantified; n = 3. ( C ) Washing operations were performed under the same conditions as in (A, B); cells were fixed 40 h after HRG stimulation and immunostained with p-c-Myc and cyclin D1 antibodies together with DAPI; n = 3,859. The x-axis shows the fluorescence intensity of DAPI, and the y-axis shows the p-c-Myc/cyclin D1 ratio. A higher value on the y-axis indicates that the amount of p-c-Myc in one cell was greater than that of cyclin D1. The black circle shows the vertex of each histogram, and the red dotted line indicates the G1/S transition time point. ( D, E ) Cells were treated for 30 min with/without 64 nM c-Myc inhibitor (EN4) 11.5 h after HRG stimulation. The cells were then incubated with 250 nM CDK4i for 8 h, washed, and imaged 52 h later (72 h after HRG stimulation; D). ( E ) The percentage distributions of G1 and S/G2/M phases were examined based on the images in (D); n = 3. Scale bar: 100 µm. ( F ) Twelve hours after HRG stimulation, the cells were treated with 250 nM CDK4i or DMSO (control). mRNA levels of p27 up to 20 h after HRG stimulation were examined by qPCR. ( G ) After the cells were treated with or without EN4 under the same conditions as in (D–E), RNA was collected 8 h after treatment with inhibitors, and the mRNA levels of CDKN1B were examined by qPCR; n = 3. In (B), (E), (G), * P < 0.05 ( t -test).

Article Snippet: A method for constructing MCF-7 (ATCC, HTB-22) overexpressing full-length human ErbB2 (HER2) is described as follows: A human ErbB2 cDNA in pCMV6-XL5 vector was purchased from OriGene (Catalog No: TC128161, Rockville, MD).

Techniques: Western Blot, Expressing, Fluorescence, Incubation, Control

Journal: bioRxiv

Article Title: ErbB2/HER2 expression level determines CDK4-inhibitor sensitivity and cyclin D1 and c-Myc dependency at the G1/S transition

doi: 10.1101/2024.05.09.593450

Figure Lengend Snippet: (A) In WT MCF-7 cells, the G1/S transition is facilitated by the regulation of cyclin D1/CDK4 via ERK and AKT activities. Conversely, CDK4 inhibition leads to reduced signaling activity of AKT and ERK due to ErbB2 degradation via Hsp90 dis-colocalization, leading to attenuated transcriptional activity of c-Myc activated by epigenetic changes. Consequently, a subpopulation of cells that is unresponsive to CDK4is exhibits a prolonged G1/S transition time. ( B ) In MCF-7 cells expressing high levels of high ErbB2, increased AKT activity leads to increased c-Myc expression. Subsequently, the expression of p27, which is critical for the stability of cyclin D1/CDK4 during the G1 phase, is suppressed by c-Myc, resulting in a slightly delayed G1/S entry compared to that in wild-type cells. Conversely, following CDK4 inhibition, most cells exhibit a cell-cycle arrest. This arrest is reversibly maintained by the strong transcriptional activity of c-Myc, ultimately sustaining survival signals.

Article Snippet: A method for constructing MCF-7 (ATCC, HTB-22) overexpressing full-length human ErbB2 (HER2) is described as follows: A human ErbB2 cDNA in pCMV6-XL5 vector was purchased from OriGene (Catalog No: TC128161, Rockville, MD).

Techniques: Inhibition, Activity Assay, Expressing