Journal: Drug Design, Development and Therapy

Article Title: miR-125b acts as a tumor suppressor in chondrosarcoma cells by the sensitization to doxorubicin through direct targeting the ErbB2-regulated glucose metabolism

doi: 10.2147/DDDT.S90530

Figure Lengend Snippet: Overexpression of miR-125b in chondrosarcoma cells inhibits cells growth rate and increases the sensitivity to doxorubicin. Notes: ( A ) Transfection of pre-miR-125b into JJ012, CH-2879, and SW1353 cells; the expressions of miR-125b were measured by qPCR. ( B ) Overexpression of miR-125b inhibits the JJ012, CH-2879, and SW1353 cells growth rates. Cells were transfected with pre-miR-125b for 48 hours, and then were plated into 48-well plates for the cell growth assays. ( C ) JJ012, CH-2879, and SW1353 cells were transfected with negative control and pre-miR-125b for 48 hours; the cells were plated into 48-well plates for the treatments with doxorubicin at indicated concentrations for 48 hours, followed by the cell viability assays. Columns, mean of three independent experiments; bars, SE. * P <0.05; ** P <0.01; *** P <0.001.

Article Snippet: Overexpression vectors containing wild-type ErbB2 (RC212583) and LDHA (RC228293) were purchased from www.origene.com .

Techniques: Over Expression, Transfection, Negative Control

Journal: Drug Design, Development and Therapy

Article Title: miR-125b acts as a tumor suppressor in chondrosarcoma cells by the sensitization to doxorubicin through direct targeting the ErbB2-regulated glucose metabolism

doi: 10.2147/DDDT.S90530

Figure Lengend Snippet: Overexpression of miR-125b in chondrosarcoma cells downregulates glucose metabolism. Notes: ( A ) Glucose uptake and ( B ) lactate product were measured in miR-125b overexpressing JJ012 (left) and CH-2879 (right) cells compared with negative control. ( C ) Western blotting experiments showed that the expressions of HK II, PDK1, and LDHA were downregulated in miR-125b overexpressing JJ012 (left) and CH-2879 (right) cells compared with negative control. β-actin was used as a loading control. Columns, mean of three independent experiments; bars, SE. * P <0.05.

Article Snippet: Overexpression vectors containing wild-type ErbB2 (RC212583) and LDHA (RC228293) were purchased from www.origene.com .

Techniques: Over Expression, Negative Control, Western Blot, Control

Journal: Drug Design, Development and Therapy

Article Title: miR-125b acts as a tumor suppressor in chondrosarcoma cells by the sensitization to doxorubicin through direct targeting the ErbB2-regulated glucose metabolism

doi: 10.2147/DDDT.S90530

Figure Lengend Snippet: Restoration of ErbB2 in miR-125b overexpressing cells recovers the glucose metabolism and doxorubicin sensitivity. Notes: ( A ) JJ012 cells were transfected with 100 nM pre-miR-negative control and pre-miR-125b for 48 hours, followed by transfection with vector control and overexpression vector containing wild-type ErbB2 for 24 hours, and then cells were collected and prepared for Western blotting with antibody against ErbB2, HK II, PDK1, and LDHA. β-actin was used as a loading control. ( B ) JJ012 cells were transfected with pre-miR-125b and ErbB2 as described in ( A ), then the glucose uptake (left) and lactate product (right) were checked. ( C ) JJ012 cells were transfected with pre-miR-125b and ErbB2 as described in ( A ); cells were collected and replaced in 48-well plates for overnight, followed by doxorubicin treatments at the indicated concentrations for 48 hours. Cells were analyzed by cell viability assays. ( D ) The JJ012 cells were transfected with control siRNA or siErbB2 for 48 hours, and then cells were collected for Western blot analysis (middle) and the measurements of glucose uptake and lactate product. Columns, mean of three independent experiments; bars, SE. * P <0.05; ** P <0.01.

Article Snippet: Overexpression vectors containing wild-type ErbB2 (RC212583) and LDHA (RC228293) were purchased from www.origene.com .

Techniques: Transfection, Negative Control, Plasmid Preparation, Control, Over Expression, Western Blot

Journal: Drug Design, Development and Therapy

Article Title: miR-125b acts as a tumor suppressor in chondrosarcoma cells by the sensitization to doxorubicin through direct targeting the ErbB2-regulated glucose metabolism

doi: 10.2147/DDDT.S90530

Figure Lengend Snippet: Overexpression of miR-125b resensitizes doxorubicin resistant chondrosarcoma cells through the inhibition of glucose metabolism. Notes: ( A ) Overexpression of miR-125b resensitized doxorubicin resistant cells. Cells were transfected with pre-miR-125b for 48 hours and then were treated with doxorubicin at indicated concentrations for 48 hours, followed by the measurement of cell viability. ( B ) Western blotting experiments showed the overexpression of miR-125b in JJ012 doxorubicin resistant cells decreased the expressions of ErbB2, HK II, PDK1, and LDHA to the same levels as those of JJ012 parental cells. ( C ) Glucose uptake (left) and lactate product (right) results showed the overexpression of miR-125b in JJ012 doxorubicin resistant cells decreased the glucose metabolism to the same levels as those of JJ012 parental cells. ( D ) Exogenous overexpression of LDHA into miR-125b pretransfected JJ012 parental cells (left) showed resistant to doxorubicin treatments at the indicated concentrations for 48 hours (right). Columns, mean of three independent experiments; bars, SE. * P <0.05; ** P <0.01. Abbreviation: Doxo R, Doxo resistant.

Article Snippet: Overexpression vectors containing wild-type ErbB2 (RC212583) and LDHA (RC228293) were purchased from www.origene.com .

Techniques: Over Expression, Inhibition, Transfection, Western Blot

Journal: Cell Systems

Article Title: Simultaneous Multiplexed Imaging of mRNA and Proteins with Subcellular Resolution in Breast Cancer Tissue Samples by Mass Cytometry

doi: 10.1016/j.cels.2017.12.001

Figure Lengend Snippet:

Article Snippet: ERBB2 overexpression vector , Addgene , Plasmid #23888.

Techniques: Microarray, Recombinant, Labeling, Multiplex Assay, Purification, Expressing, Over Expression, Plasmid Preparation, Software

Journal: The Journal of Biological Chemistry

Article Title: The receptor tyrosine kinase ErbB2/HER2 governs CDK4 inhibitor sensitivity, timing, and irreversibility of the G1/S transition

doi: 10.1016/j.jbc.2025.110865

Figure Lengend Snippet: Time-dependent expression of CDK4 and cyclin D1 in G1/S transition. A – D , dynamic changes in the expression or phosphorylation of individual proteins in MCF-7 cells stably expressing FUCCI, detected by western blotting up to 36 h after treatment with 10 nM HRG. A and B , ErbB2 pathway proteins. C and D , proteins regulating G1/S transition. Protein and phosphorylation levels were normalized relative to those of GAPDH and divided by that of the 0 h sample (0 h = 1); n = 3. The data are displayed as the means ± SEM. FUCCI, fluorescent ubiquitination-based cell cycle indicator; HRG, heregulin.

Article Snippet: MCF-7 (American Type Culture Collection, ATCC, HTB-22) cells overexpressing full-length human ErbB2 (HER2) were established as follows: Human ErbB2 complementary DNA (cDNA) in the pCMV6-XL5 vector was purchased from OriGene (Catalog No: TC128161).

Techniques: Expressing, Phospho-proteomics, Stable Transfection, Western Blot, Ubiquitin Proteomics

Journal: The Journal of Biological Chemistry

Article Title: The receptor tyrosine kinase ErbB2/HER2 governs CDK4 inhibitor sensitivity, timing, and irreversibility of the G1/S transition

doi: 10.1016/j.jbc.2025.110865

Figure Lengend Snippet: High ErbB2 expression induces cell cycle arrest by repressing cyclin D1 expression via c-Myc under CDK4 inhibition. A and B , WT MCF-7 and ErbB2-overexpressing MCF-7 cells (high levels) were treated with CDK4i or DMSO 12 h after HRG stimulation and harvested 24 h after HRG stimulation. Western blotting of ( A ) p-ErbB2, p-RB (Ser807/811), p-RB (Thr373), RB, p-AKT (Thr308), AKT, p-ERK (Thr202/Thr204), ERK, c-Myc, cyclin D1, and p27 expression (in control cells, with DMSO treatment) after normalization with GAPDH expression ( B ). The data are displayed as the means ± SD. C - F , ErbB2-overexpressing cells (moderate) were individually treated with 250 nM CDK4i or DMSO (control) 12 h after stimulation with 10 nM HRG and fixed 16 h after stimulation with 10 nM HRG (CDK4i or DMSO addition 4 h later). Hsp90 and ErbB2 were coimmunostained. The scale bar represents 25 μm ( C ). Colocalization rate of Hsp90 and ErbB2 based on Mander’s tM1 and Pearson’s R values determined using Coloc 2 plugin of Image J; n = 10 ( D ). Hsp90 and PY100 were coimmunostained. The scale bar represents 25 μm ( E ). Colocalization rate of Hsp90 and PY100 based on Mander’s tM2 and Pearson’s R values; n = 10 ( F ). G and H , PLA signals detected in WT MCF-7 cells. Cells were individually treated with 250 nM CDK4i or DMSO (control) 12 h after stimulation with 10 nM HRG and fixed 16 h after stimulation with 10 nM HRG (CDK4i or DMSO addition 4 h later). PLA was performed using ErbB2 and Hsp90 antibodies. G , PLA signals detected in the presence of HER2 and Hsp90 antibodies ( left ). No signals detected after omitting Hsp90 antibody ( right ). The scale bar represents 100 μm. H , mean intensity of PLA signals per image was measured under each condition in ( G ) ( Left ). Number of nuclei per image was counted ( Right ). n = 59. I and J , FUCCI stably expressing MCF-7 cells were treated with CDK4i or DMSO (control) as well as 10 μM MG132, 12 h after stimulation with 10 nM HRG and collected 8 h later. I , ErbB2 and GAPDH expression determined by western blotting. ( J ) ErbB2 expression normalized to GAPDH expression; n = 3. The data are displayed as the means ± SD. K and L , ErbB2 cycloheximide pulse-chase assay was performed with FUCCI stably expressing MCF-7 cells. Cells were treated with CDK4i or DMSO (control) 12 h after stimulation with 10 nM HRG, then 100 μg/ml cycloheximide was added 16 h after stimulation with 10 nM HRG. Cells were harvested at 0, 2, and 4 h after cycloheximide treatment; n = 3. K , ErbB2 and GAPDH expression determined by western blotting. L , ErbB2 expression normalized to GAPDH expression; n = 3. The data are displayed as the means ± SDs. M , coimmunoprecipitation assay in ErbB2-overexpressing MCF-7 cells (moderate). Cells were treated with CDK4i or DMSO (control) as well as 10 μM MG132, 12 h after stimulation with 10 nM HRG, and collected 8 h later. Immunoprecipitates and whole cell lysates were immunoblotted with anti-ubiquitin or anti-ErbB2, or GAPDH antibodies. N , schematic model: In CDK4i-unresponsive WT MCF-7 cells, the loss of ErbB2 protein stability impairs the regulation of c-Myc-mediated G1/S transition, leading to the delayed G1/S transition. O , WT MCF-7 and ErbB2-overexpressing cells (moderate and high levels) were individually treated with 250 nM CDK4i or DMSO (control) 12 h after stimulation with 10 nM HRG, and images were acquired every 20 min until 72 h after stimulation. The scale bar represents 100 μm. P , Images in ( J ) were analyzed to determine the proportion (%) of each cell cycle phase in the entire set of images; n = 3. In ( B ) and ( J ), ∗ p < 0.05 (Tukey’s test). In ( D ), ∗ p < 0.05 (with respect to control and CDK4i; Welch’s t test). In ( F ) and ( H ), ∗ p < 0.05 (with respect to control and CDK4i; two-tailed Student’s t test). In ( L ), the two-way ANOVA revealed significant main effects of both time ( p < 0.0001) and CDK4i treatment (∗ p = 0.02). FUCCI, fluorescent ubiquitination-based cell cycle indicator; HRG, heregulin; PLA, proximity ligation assay; DMSO, dimethyl sulfoxide; RB, retinoblastoma protein.

Article Snippet: MCF-7 (American Type Culture Collection, ATCC, HTB-22) cells overexpressing full-length human ErbB2 (HER2) were established as follows: Human ErbB2 complementary DNA (cDNA) in the pCMV6-XL5 vector was purchased from OriGene (Catalog No: TC128161).

Techniques: Expressing, Inhibition, Western Blot, Control, Stable Transfection, Pulse Chase, Co-Immunoprecipitation Assay, Ubiquitin Proteomics, Two Tailed Test, Proximity Ligation Assay

Journal: The Journal of Biological Chemistry

Article Title: The receptor tyrosine kinase ErbB2/HER2 governs CDK4 inhibitor sensitivity, timing, and irreversibility of the G1/S transition

doi: 10.1016/j.jbc.2025.110865

Figure Lengend Snippet: Reversible cell cycle arrest is maintained under c-Myc transcriptional activity. A - G , MCF-7 cells overexpressing ErbB2 (high levels). A and B , cells were treated with 250 nM CDK4i 12 h after HRG stimulation, subjected to a wash/no-wash treatment 4 h later, and harvested 40 h after HRG stimulation. Western blotting results showing the expression of p-RB, c-Myc, p-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. The data are displayed as the means ± SD. C , washing operations were performed under the same conditions as in ( A and B ); cells were fixed 40 h after HRG stimulation and immunostained with p-c-Myc and cyclin D1 antibodies together with DAPI; n = 3859. 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 circle shows the vertex of each histogram, and the red dotted line indicates the G1/S transition time point. D and E , cells were pretreated 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 , percentage distributions of G1 and S/G2/M phases based on the images in ( D ); n = 4. The scale bar represents 100 μm. The data are displayed as the means ± SD. F , cells were treated with 250 nM CDK4i or DMSO (control) 12 h after HRG stimulation and CDKN1B mRNA levels up to 20 h after HRG stimulation were examined by qPCR; n = 3. 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 CDKN1B mRNA levels were examined by qPCR; n = 3. The data are displayed as the means ± SD. H , WT MCF-7, WT T47D, and ErbB2 ( high ) OE MCF-7 cells were pretreated for 30 min with/without 4 μM c-Myc inhibitor (EN4) 11.5 h after HRG stimulation, incubated with 250 nM CDK4i for 6 days, and SA-β-Gal staining was assessed. I , percentage SA-β-Gal-stained cells based on the images in ( H ); n = 12. J , ErbB2 levels modulate the irreversibility of the G1/S transition under CDK4i treatment. In ( B ), ( E ), ( F ), and ( G ), ∗ p < 0.05 (with respect to control and each inhibitor; two-tailed Student’s t test). In ( I ), ∗ p < 0.05 (with respect to control and each inhibitor; Welch’s t test). DAPI, 4′,6-diamidino-2-phenylindole; DMSO, dimethyl sulfoxide; SA-β-Gal, senescence-associated β-galactosidase; qPCR, quantitative PCR; RB, retinoblastoma protein.

Article Snippet: MCF-7 (American Type Culture Collection, ATCC, HTB-22) cells overexpressing full-length human ErbB2 (HER2) were established as follows: Human ErbB2 complementary DNA (cDNA) in the pCMV6-XL5 vector was purchased from OriGene (Catalog No: TC128161).

Techniques: Activity Assay, Western Blot, Expressing, Incubation, Control, Staining, Two Tailed Test, Real-time Polymerase Chain Reaction

Journal: The Journal of Biological Chemistry

Article Title: The receptor tyrosine kinase ErbB2/HER2 governs CDK4 inhibitor sensitivity, timing, and irreversibility of the G1/S transition

doi: 10.1016/j.jbc.2025.110865

Figure Lengend Snippet: Proposed model the ErbB2 receptor regulation during G1/S transition via cyclin D1 and c-Myc in CDK4i nonresponsive cells. A , in low ErbB2-expressing cells, CDK4 inhibition leads to reduced pathway activity due to ErbB2 degradation via Hsp90 dis-colocalization. This attenuates the transcriptional activity of c-Myc activated by epigenetic changes. Consequently, a subpopulation of cells unresponsive to CDK4i exhibits a delayed G1/S transition. B , in high ErbB2-expressing cells, 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 cell cycle arrest after CDK4 inhibition. This arrest is reversibly maintained by the strong transcriptional activity of c-Myc, ultimately sustaining survival signals.

Article Snippet: MCF-7 (American Type Culture Collection, ATCC, HTB-22) cells overexpressing full-length human ErbB2 (HER2) were established as follows: Human ErbB2 complementary DNA (cDNA) in the pCMV6-XL5 vector was purchased from OriGene (Catalog No: TC128161).

Techniques: Expressing, Inhibition, Activity Assay

Journal: bioRxiv

Article Title: Destabilized 3’UTR ARE therapeutically degrades ERBB2 in drug-resistant ERBB2+ cancer models

doi: 10.1101/2022.08.14.503914

Figure Lengend Snippet: A. Dot blots show the phospho kinase array pattern of the BT474 clone 5 wild type, BT474 clone 5 Vector, BT474 clone 5 desARE3’UTR ERBB2-3 and BT474 clone 5 desARE3’UTR c-MYC 2-3. The dot marked in red box show unique kinases down regulated only in BT474 clone 5 desARE3’UTR ERBB2-3 and the dot marked in green boxes show unique kinases downregulated only in BT474 clone 5 desARE3’UTR c-MYC 2-3. Each kinase is spotted twice. B. The bar chart shows the intensity of the phospho-kinase array dot blot for the BT474 clone 5 wild type (black bar), BT474 clone 5 Vector (blue bar), BT474 clone 5 desARE3’UTR ERBB2-3 (red bar) and BT474 clone 5 desARE3’UTR c-MYC 2-3 (green bar). C. The bar charts show YES1 pT60 and WNK1 pT60 kinases specifically down regulated in BT474 clone 5 desARE3’UTR ERBB2-3 in red bar. Controls BT474 clone 5 wild type (black bar), BT474 clone 5 Vector (blue bar), and BT474 clone 5 desARE3’UTR c-MYC 2-3 (green bar). T-test (*** Pval=0.00413, desARE3’UTRERBB2-3 for YES and WNK1) D. Venn diagram show kinases downregulated in destabilized desARE3’UTR ERBB2-3, 30 NCI-H1975 (p=0.02) blue (from RNA Seq), and in destabilized desARE3’UTR ERBB2-3 BT474 clone 5 (red-kinase array) and the shared kinases found downregulated in destabilized ERBB2 in both resistant cell lines WNK1 and YES1 in pink. E. Western blot show YES1 and GAPDH expression across the NCI-H1975 wildtype cells, vector, cells containing constructs desARE3’UTR ERBB2-1, 3 and 30 and wild type trastuzumab treated NCI-H1975 cells (n=2). F. Bar chart show quantification of the YES1 expression normalized against GAPDH on NCI-H1975 wildtype cells, vector and cells containing constructs desARE3’UTR ERBB2-1, 3 and 30 (n=2). T-test (***Pval=0.0021, desARE3’UTRERBB2-1,3,30) G. Western blot show WNK1 and GAPDH expression across the NCI-H1975 wildtype cells, vector, cells containing constructs desARE3’UTR ERBB2-1, 3 and 30 and wild type trastuzumab treated NCI-H1975 cells. H. Western blot show YES1 and GAPDH expression across the NCI-H1975 destabilized with constructs desARE3’UTR ERBB2-3 and 30 (lane 1 and 3) and over expressed with YES1 in lane 2 and 4. I. Bar chart show the wound healing closing of NCI H1975 wildtype, vector, desARE3’UTRERBB2-1, 3,30 and desARE3’UTR ERBB2-30,30 overexpressed with YES1 from 0hr to 72hrs. At 24hrs *** p=0.00025 desARE3’UTRERBB2-3,30. At 48hrs ****p=0.000018 desARE3’UTR ERBB2-3 and at 72hrs *** p=0.0003 desARE3’UTRERBB2-3 and **p=0.002 desARE3’UTRERBB2-30.

Article Snippet: YES1 Y537F overexpression vector (Addgene: 51299) was transfected into desARE3’UTR ERBB2-3 and 30 cells that have lost ERBB2 and YES1 by introduction of destabilized desARE3’UTR ERBB2.

Techniques: Plasmid Preparation, Dot Blot, RNA Sequencing, Western Blot, Expressing, Construct

Journal: bioRxiv

Article Title: Destabilized 3’UTR ARE therapeutically degrades ERBB2 in drug-resistant ERBB2+ cancer models

doi: 10.1101/2022.08.14.503914

Figure Lengend Snippet: A. Microscope images of wound healing assay of NCI H1975 WT, vector, desARE3’UTRERBB2-1, 3 and 30 and desARE3’UTRERBB2-3 and 30 overexpressed with YES1.Images were taken at 0hr, 24hr, 48hrs and 72hrs at 10x.

Article Snippet: YES1 Y537F overexpression vector (Addgene: 51299) was transfected into desARE3’UTR ERBB2-3 and 30 cells that have lost ERBB2 and YES1 by introduction of destabilized desARE3’UTR ERBB2.

Techniques: Microscopy, Wound Healing Assay, Plasmid Preparation

Journal: Genes & development

Article Title: Stromal-derived NRG1 enables oncogenic KRAS bypass in pancreas cancer.

doi: 10.1101/gad.351037.123

Figure Lengend Snippet: Figure 1. Cancer-associated fibroblasts promote resistance to KRAS∗extinction in a PDAC model. (A) Crystal violet staining of iKPC1 cell colony formation assay, with Kras∗on (on dox), MRTX1133, or Kras∗off (off dox). The presence of 50% conditional medium from pCAF1, pCAF2, or CAF1 significantly increased the capacity of cell growth 4 d after treatment. (B) Measurement of cell viability using Celltiter-Glo in the same settings as in A 4 d after treatment. (C) Measurement of colony diameter of iKPC1 in Matrigel culturing assay in the same settings as in A 7 d after treatment. (D) Luciferase image of mice injected with 1 × 106 iKPC1-luc cells alone, iKPC2-luc cells alone, or iKPC1-luc or iKPC2-luc cells together with 1 × 106 pCAF1 or CAF1 cells 7 d after injection. Coinjection of iKPC and CAFs sig- nificantly increased KRAS∗-independent growth. (E) Quantification of luciferase signal from D. (F) Histology (HE, trichome, GFP, and KRASG12D staining) of tissue mass from iKPC + CAF1 injection from D. (G) Measurement of colony diameter of iKPC1 in Matrigel cul- turing assay 7 d after treatment with the indicated condition and treatment. (DMSO) Dox on with DMSO, (MRTX) dox on with 100 nM MRTX1133, (Kras∗off) dox off with DMSO, (CM) the presence of 50% CAF-CM in culture, (CM-HI) the presence of 50% heat-inactivated CAF-CM in culture. (H) Summary of the strategy for isolating CAF-secreted protein (CAF-pro). (I) Experimental settings and Western blot analysis of iKPC1 cells at the indicated time points after adding CAF-pro. (J) RTK array analysis of two iKPC cell lines after 5 min of CAF- pro or heat-inactivated CAF-pro (HI-CAF-pro) treatment. (K) Quantification of J. (L) Confirming ERBB2, ERBB3, and AKT phosphorylation in four different iKPC cell lines. Data are represented as mean ± SD for B, C, and G, and mean only for E. For B, C, E, and G, Student’s t-test was performed to calculate statistical values.

Article Snippet: Plasmid construction and gene knockdown, knockout, and overexpression LentiORFs were purchased from Origene for mouse Erbb2 (NM_001003817, MR227307), mouse Erbb3 (NM_010153, MR227559), mouse Nrg1 (NM_178591, MR226911L3), and human NRG1 (NM_013956, RC220134).

Techniques: Staining, Colony Assay, Luciferase, Injection, Western Blot, Phospho-proteomics

Journal: Genes & development

Article Title: Stromal-derived NRG1 enables oncogenic KRAS bypass in pancreas cancer.

doi: 10.1101/gad.351037.123

Figure Lengend Snippet: Figure 2. Paracrine Nrg1–Erbb2/3 signaling activation confers KRAS∗resistance. (A) Western blot of NRG1 protein levels in various cell lines. (B) Multiplexed RNAscope immunofluorescence staining of iKPC tumor with NRG1-specific RNA probe (white), GFP (green), PDPN (red), and DAPI (blue). (C) qPCR results of Nrg1 on flow cytometry-sorted GFP+ PDAC cells or PDPN+ CAFs from iKPC GEM tu- mors. Two independent tumors were pooled together for dissociation. (D) Measurement of colony diameter of iKPC1 in 3D Matrigel cul- turing assay with the indicated treatment for 7 d. (on) Kras∗on with dox, (off) Kras∗off without dox, (MRTX) 100 nM MRTX1133 treatment, (CAF) the presence of CAF1-pro in culture, (NRG1) the presence of 10 ng/mL rmNRG1 in culture. Data are represented as mean ± SEM; Student’s t-test. (E) Western blot of the indicated iKPC1 knockout clones Kras∗off for 48 h and serum-starved for 2 h prior to 5-min stimulation of the indicated treatments (CAF1-pro or 10 ng/mL rmNRG1). (F) Western blot of the indicated iKPC1 ERBB3 knock- out or rescued clones K Kras∗off for 48 h and serum-starved for 2 h prior to 5-min stimulation of the indicated treatments (CAF1-pro or 10 ng/mL rmNRG1). (G) Western blot of iKPC1 cells with 48 h of K Kras∗off and 2 h of serum starvation prior to 5-min stimulation of the indicated treatments (CAF1-pro cells were preincubated with 100 μg/mL mouse IgG or the indicated concentration of NRG1 neutraliza- tion antibody YW538.24.71 for 1 h at 37°C). (H) Western blot of AsPC1, DMSO, or 100 nM MRTX1133 treatment for 24 h and serum star- vation for 2 h with the presence of 10 μg/mL control human IgG or 10 μg/mL pertuzumab prior to 5-min stimulation of the indicated treatments (CAF-pro, 10 ng/mL rmNRG1, or CAF-sgNrg1-pro). (I) Cell viability assay with AsPC1 with the indicated treatment for 4 d. (MRTX) Treated with 100 nM MRTX1133 (with pCAF1-pro, CAF1-pro, or 10 ng/mL rmNRG1), (mIgG + hIgG) treated with 10 μg/ mL control mouse IgG and 10 μg/mL control human IgG, (anti-NRG1) the presence of 10 μg/mL YW538.24.71, (pertuzumab) the presence of 10 μg/mL pertuzumab. Data are represented as mean ± SD; two-way ANOVA. (J) Luciferase assay of iKPC1-luc cells coinjected with pCAF3 or CAF1. One day after cell implantation, luciferase signal was measured to acquire the initial value of luciferase activity, and IgG or YW538.24.71 was given at a dose of 25 mg/kg. Luciferase signal was acquired again at 7 d after implantation. (K) Quantification of luciferase signal from J. Paired multiple t-test at day 7.

Article Snippet: Plasmid construction and gene knockdown, knockout, and overexpression LentiORFs were purchased from Origene for mouse Erbb2 (NM_001003817, MR227307), mouse Erbb3 (NM_010153, MR227559), mouse Nrg1 (NM_178591, MR226911L3), and human NRG1 (NM_013956, RC220134).

Techniques: Activation Assay, Western Blot, RNAscope, Immunofluorescence, Staining, Flow Cytometry, Knock-Out, Clone Assay, Concentration Assay, Control, Viability Assay, Luciferase, Activity Assay

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