Journal: Cancers

Article Title: Drug-Induced Resistance and Phenotypic Switch in Triple-Negative Breast Cancer Can Be Controlled via Resolution and Targeting of Individualized Signaling Signatures

doi: 10.3390/cancers13195009

Figure Lengend Snippet: TNBC tissues are represented by different patient-specific signaling signatures, majority of which do not include EGFR. ( A ) Fold changes in expression levels of EGFR and pEGFR in TNBC and non-TNBC tumors are shown. Y1068 and Y1173 are EGFR phosphorylation sites; ( B ) Examples for patient-specific sets of active unbalanced processes are shown. Each sample harbors a set of 1–3 active unbalanced processes (PaSSS), represented schematically by a barcode. In each barcode active unbalanced processes are represented by black or gray squares, inactive white. Negative/positive amplitude denotes how the patients are correlated with respect to a particular process. Suggested PaSSS-based therapies appear below each barcode; ( C ) Heterogeneity index of 3 subgroups, represented by a ratio between the number of distinct PaSSSs and the number of samples in each subset, is shown for the TNBC subset of tissues, the entire set (3467 samples from 11 cancer types) and the subset of non-TNBC samples. (Abbreviations: TNBC—Triple Negative Breast Cancer, PaSSS—Patient-specific signaling signature, EGFR—Epidermal Growth Factor Receptor, VEGFR2—Vascular Endothelial Growth Factor Receptor 2, Her2—Human Epidermal growth factor Receptor 2, Src—Proto-oncogene tyrosine-protein kinase Src).

Article Snippet: The following conjugated antibodies were used: anti-p-EGFR (Y1068) (R&D Systems, Minneapolis, MN, USA, cat. no. IC3570G), anti-p-ERK2 (Thr202/Tyr204) (BioLegend, San Diego, CA, USA, cat. No. 675503), anti-p-S6 (Ser235/236) (BioLegend, cat. no. 608605), and anti-GAPDH (Santa Cruz Biotechnology, Dallas, Texas, USA, cat. no. sc-47724AF594).

Techniques: Expressing, Phospho-proteomics

Journal: Cell Death Discovery

Article Title: Grb2 interacts with necrosome components and is involved in rasfonin-induced necroptosis

doi: 10.1038/s41420-022-01106-1

Figure Lengend Snippet: A The level of reactive oxygen species (ROS) was detected as described in Materials and Methods following ACHN cells were treated with rasfonin (12 μM) or positive control for 12 h. B ACHN cells were treated with rasfonin (12 μM) in the presence or absence of N-acetyl-L-cysteine (NAC, 0.5 mM) for 12 h; cell viability was analyzed by MTS assay as described in Materials and Methods. C ACHN cells were challenged with rasfonin (6 and 12 μM) for 4 h and then, the total RNA was extracted, reversed, and detected by real-time PCR. D , E ACHN cells were treated with rasfonin (0–12 μM) for 4 h, the cells were lysed and subjected to immunoblotting with the antibodies indicated. Actin was used as a loading control. Data are presented as mean ± SD and are representatives of three independent experiments. Each performed in triplicate, and the data was analyzed by t -test. Single asterisk denotes that the group is statistically different from the control groups ( p < 0.05), whereas double asterisk means ( p < 0.01). The ratios between EGFR, RIP1 or Grb2 and Actin (A) were shown below the blots ( D ). Similar experiments were repeated at least three times.

Article Snippet: Small interference RNA (siRNA) of RIP1 (sc-36426 and L-004445-00), EGFR (sc-29301 and L-003114-00) and Grb2 (sc-29334 and L-019220-00) were obtained from Santa Cruz Biotechnology and Dharmacon along with control siRNA.

Techniques: Positive Control, MTS Assay, Real-time Polymerase Chain Reaction, Western Blot, Control

Journal: Cell Death Discovery

Article Title: Grb2 interacts with necrosome components and is involved in rasfonin-induced necroptosis

doi: 10.1038/s41420-022-01106-1

Figure Lengend Snippet: A The STRING network analysis of possible interactions among EGFR, Grb2, RIP1 and p62. B–E ACHN cells were exposed to rasfonin (6 or 12 μM) for 6 h, and equal amounts of cell lysates were immunoprecipitated with the mouse monoclonal antibodies of Grb2, p62, EGFR, RIP1 or IgG. Immunoprecipitates were then immunoblotted for the indicated polyclonal (Grb2, p62 and EGFR) or monoclonal (RIP1) antibodies. Similar experiments were repeated twice.

Article Snippet: Small interference RNA (siRNA) of RIP1 (sc-36426 and L-004445-00), EGFR (sc-29301 and L-003114-00) and Grb2 (sc-29334 and L-019220-00) were obtained from Santa Cruz Biotechnology and Dharmacon along with control siRNA.

Techniques: Immunoprecipitation, Bioprocessing

Journal: Cell Death Discovery

Article Title: Grb2 interacts with necrosome components and is involved in rasfonin-induced necroptosis

doi: 10.1038/s41420-022-01106-1

Figure Lengend Snippet: A ACHN cells were treated with rasfonin (12 μM) with or without gefitinib (5 μM) for 4 h. B After transfection with the control (Mock) or EGFR siRNA (siEGFR) for 48 h, ACHN cells were treated with rasfonin (12 μM) for 4 h. Cell lysates in ( A , B ) were analyzed by immunoblotting with the indicated antibodies. C The efficiency of siRNA interference was determined with sample in ( B ). D Schematic representation of the WT EGFR and K721A EGFR. E , F HEK293T cells were transfected transiently with the WT EGFR or K721A EGFR plasmids for 36 h, and treated with rasfonin (12 μM) for 4 h. Cell lysates were analyzed by immunoblotting with the indicated antibody. Actin was used as a loading control. Similar experiments were repeated at least three times.

Article Snippet: Small interference RNA (siRNA) of RIP1 (sc-36426 and L-004445-00), EGFR (sc-29301 and L-003114-00) and Grb2 (sc-29334 and L-019220-00) were obtained from Santa Cruz Biotechnology and Dharmacon along with control siRNA.

Techniques: Transfection, Control, Western Blot

Journal: Cell Death Discovery

Article Title: Grb2 interacts with necrosome components and is involved in rasfonin-induced necroptosis

doi: 10.1038/s41420-022-01106-1

Figure Lengend Snippet: A–E After transfection with the control (Mock) or Grb2 siRNA (siGrb2) for 48 h, ACHN cells were exposed to 12 μM rasfonin for 6 h, and the total RNA was extracted, reversed, and detected by real-time PCR ( A ); cell lysates were analyzed by immunoblotting with the indicated antibodies, Actin was used as a loading control ( B ); the apoptosis and necrosis induced by rasfonin were determined by flow cytometry ( C and D ). Apoptotic: AV-positive and PI-negative; necrotic: PI-positive. The data are presented as mean ± S.D. from three independent experiments. The double asterisk denotes the group is statistically different from the control groups ( P < 0.01). E Cell lysates in ( B ) were analyzed by immunoblotting with the indicated antibodies, and ratios between cleaved PARP-1 (cPARP-1) and PARP-1, or between cPARP-1 and Actin (A), or between PARP-1 and Actin (A) were shown below the blots. Similar experiments were repeated at least three times. F Schematic mechanism of EGFR/Grb2 in rasfonin-dependent autophagy and necroptosis.

Article Snippet: Small interference RNA (siRNA) of RIP1 (sc-36426 and L-004445-00), EGFR (sc-29301 and L-003114-00) and Grb2 (sc-29334 and L-019220-00) were obtained from Santa Cruz Biotechnology and Dharmacon along with control siRNA.

Techniques: Transfection, Control, Real-time Polymerase Chain Reaction, Western Blot, Flow Cytometry

Journal: JCI Insight

Article Title: Interim report on engineered NK cell trial in lung cancer refractory to immune checkpoint inhibitors

doi: 10.1172/jci.insight.186890

Figure Lengend Snippet: Utilizing the multicolor immunofluorescence histochemical staining, we examined lung tumor tissue from patient 006 on 3 occasions using DAPI (blue) for nuclear staining, pan-CK (gray) for lung parenchyma, CD45 (purple) for lymphocytes, CD57 (cyan) and CD56 (green) for NK cells, CD3 (yellow) for T cells, and EGFR (red) combined with CD56 or CD57 to identify NK cells transduced with the tEGFR-sIL15 construct. ( A ) FFPE tissue from a pretreatment tumor biopsy from patient 006 shows the presence of lung parenchyma (DAPI + pan-CK + ) but the absence of tEGFR + (red) transduced CD57 + or CD56 + NK cells. ( B ) FFPE tissue from the skinny needle tumor biopsy collected 24 hours following the fourth and final infusion of sIL15_TRACK NK cells without observable of tEGFR + (red) transduced CD57 + (cyan) or CD56 + (green) cells. ( C ) FFPE tissue from an excisional tumor biopsy collected 7 days following the fourth and final infusion of sIL15_TRACK NK cells shows sIL15_TRACK NK cells identified in DAPI + CD45 + cells by coexpression of tEGFR (red) with CD57 (cyan), as marked by white arrows. ( D ) Selected individual markers of the boxed region shown in C are shown. Scale bars: 10 μm ( A – C ); 10 μm ( D ).

Article Snippet: For assessment of tumor infiltration of NK cells, primary antibodies were as follows: EGFR (clone 31G7, Novus), CD3 (clone LN10, Leica), CD56 (clone MRQ-42, Cell Marque), CD45 (clone D9M81, Cell Signaling Technologies), CD57 (clone HNK-1, Biolegend), and pan-cytokeratin (clone AE1/AE3, Dako).

Techniques: Immunofluorescence, Staining, Transduction, Construct