pha665752  (Santa Cruz Biotechnology)

Journal: Cell Death & Disease

Article Title: Inhibition of TFF3 synergizes with c-MET inhibitors to decrease the CSC-like phenotype and metastatic burden in ER+HER2+ mammary carcinoma

doi: 10.1038/s41419-025-07387-5

Figure Lengend Snippet: A MDA-MB-361 and BT474 cells were treated with varying concentration (0.01, 0.1, 1, 10, or 100 μM) of AMPC (A) and Cabozantinib (C), SU11274 (S), or PHA-665752 (P) for 6 days. The survival fraction (SF) in response to AMPC (A) was assessed using a total cell number counting assay. Data are represented as mean ± SD ( n = 3). B CI was determined using the Chou-Talalay method, where CI < 1 indicated synergy, CI = 1 indicated additivity, CI > 1 indicated antagonism. C Synergy scores were calculated using bliss synergy analysis ( www.synergyfinder.com ), where synergy score> 0 indicated synergy, synergy score <0 indicated antagonism. D Dose-response analysis of the shift in IC 50 of Cabozantinib (C), SU11274 (S), or PHA-665752 (P) in MDA-MB-361 and BT474 cells after co-treatment with AMPC (2.5 μM) was conducted with total cell number assay. The arrow indicates the fold reduction in IC 50 of the respective c-METis in the presence of AMPC. Data are represented as means ± SD ( n = 3). E Foci formation was performed on MDA-MB-361 and BT474 cells treated with vehicle (V), 5 μM AMPC (A), 1 μM Cabozantinib (C), 1 μM SU11274 (S), 2 μM PHA-665752 (P) and the combination in monolayer culture at low cell density for 14 days. Foci were stained by crystal violet. F MDA-MB-361 and BT474 cells were treated with vehicle (V), 5 μM AMPC (A), 1 μM Cabozantinib (C), 1 μM SU11274 (S), 2 μM PHA-665752 (P), and the combination in medium containing 2% FBS and 4% Matrigel for 12 days after 3 days of pre-culture. Bright-field images showed the colonies, while merged images depicted Live/Dead staining, with red indicating dead colonies and green indicating live colonies. Scale bars, 100 μm.

Article Snippet: Additionally, three c-MET inhibitors, namely Cabozantinib, SU11274, and PHA-665752 were sourced from SelleckChem (Houston, TX, USA).

Techniques: Concentration Assay, Staining

Journal: Cell Death & Disease

Article Title: Inhibition of TFF3 synergizes with c-MET inhibitors to decrease the CSC-like phenotype and metastatic burden in ER+HER2+ mammary carcinoma

doi: 10.1038/s41419-025-07387-5

Figure Lengend Snippet: A Cell migration and invasion assays were conducted on MDA-MB-361 and BT474 cells treated with vehicle (V), 5 μM AMPC (A), 1 μM Cabozantinib (C), 1 μM SU11274 (S), 2 μM PHA-665752 (P) or the combination. Data are expressed as mean ± SD ( n = 3). Statistical significance is indicated as * P < 0.05, ** P < 0.01, and *** P < 0.001. B MDA-MB-361 and BT474 cells were seeded in ultralow attachment plates and cultured in spheroid growth media, and treated with vehicle (V), 5 μM AMPC (A), 1 μM Cabozantinib (C), 1 μM SU11274 (S), 2 μM PHA-665752 (P), or the combination for 12 days. The spheroids with diameters greater than 50 μm in each well were counted. Data are expressed as mean ± SD ( n = 3). Statistical significance is indicated as * P < 0.05, ** P < 0.01, and *** P < 0.001. C ALDEFLUOR activity was measured after 3 days of treatment with vehicle (V), 5 μM AMPC (A), 1 μM Cabozantinib (C), 1 μM SU11274 (S), 2 μM PHA-665752 (P), or the combination. MDA-MB-361 and BT474 cells were then harvested and incubated with ALDEFLUOR substrate to define the ALDH1-positive population, with DEAB used to establish the baseline fluorescence. Data are expressed as mean ± SD ( n = 3). Statistical significance is indicated as * P < 0.05, ** P < 0.01, and *** P < 0.001. D Western blot analysis was conducted to assess the expression and/or phosphorylation of TFF3, c-MET, AKT, p44/42 MAPK and CSC-related proteins in MDA-MB-361 and BT474 cells following treatment with vehicle (V), 5 μM AMPC (A), 1 μM Cabozantinib (C), 1 μM SU11274 (S), 2 μM PHA-665752 (P), or the combination for 3 days. β-ACTIN was used as input control. The sizes of detected proteins in kDa are indicated on the left.

Article Snippet: Additionally, three c-MET inhibitors, namely Cabozantinib, SU11274, and PHA-665752 were sourced from SelleckChem (Houston, TX, USA).

Techniques: Migration, Cell Culture, Activity Assay, Incubation, Fluorescence, Western Blot, Expressing, Phospho-proteomics, Control

Journal: Cell Death & Disease

Article Title: Inhibition of TFF3 synergizes with c-MET inhibitors to decrease the CSC-like phenotype and metastatic burden in ER+HER2+ mammary carcinoma

doi: 10.1038/s41419-025-07387-5

Figure Lengend Snippet: A Western blot analysis was conducted to assess the level of c-MET and c-MET phosphorylation at Y1234/1235 in MDA-MB-361 cells with TFF3 forced expression (V & T) and depletion (sh & shT). β-ACTIN was used as input control. The sizes of detected protein blots in kDa are on the left. B Western blot analysis was conducted to assess the level of c-MET and c-MET phosphorylation at Y1234/1235 in MDA-MB-361 and BT474 cells with AMPC (0, 0.1, 0.5, 1, 5, or 10 μM) treatment for 3 days. β-ACTIN was used as input control. The sizes of detected protein blots in kDa are shown on the left. C Western blot analysis was performed to assess the level of TFF3 in MDA-MB-361 and BT474 cells transfected with scrambled siRNA, siMET#1, or siMET#2 plasmid. β-ACTIN was used as input control. The sizes of detected protein blots in kDa are shown on the left. D Western blot analysis was performed to assess the level of TFF3 in MDA-MB-361 and BT474 cells treated with Cabozantinib (0, 0.02, 0.1, 0.2, 1 or 2 μM), SU11274 (0, 0.02, 0.1, 0.2, 1 or 2 μM) or PHA-665752 (0, 0.04, 0.2, 0.4, 2 or 4 μM) for 3 days. β-ACTIN was used as input control. The sizes of detected protein blots in kDa are shown on the left. E A potential c-MET/TFF3 interaction in MDA-MB-361 cells was investigated by immunoprecipitation (IP) and immunoblotting. F MDA-MB-361 and BT474 cells transfected with scrambled siRNA, siMET#1, or siMET#2 plasmid were harvested and incubated with ALDEFLUOR substrate to define the ALDH1-positive population. DEAB was used to establish the baseline fluorescence. Data are expressed as mean ± SD ( n = 3). Statistical significance is indicated as * P < 0.05, ** P < 0.01, and *** P < 0.001. G Western blot analysis was utilized to examine the level of CSC-related proteins in MDA-MB-361 and BT474 cells transfected with scrambled siRNA, siMET#1, or siMET#2 plasmid. β-ACTIN was used as input control. The sizes of detected protein blots in kDa are shown on the left.

Article Snippet: Additionally, three c-MET inhibitors, namely Cabozantinib, SU11274, and PHA-665752 were sourced from SelleckChem (Houston, TX, USA).

Techniques: Western Blot, Phospho-proteomics, Expressing, Control, Transfection, Plasmid Preparation, Immunoprecipitation, Incubation, Fluorescence

Journal: bioRxiv

Article Title: Hypoxia-induced histone methylation and NF-κB activation in pancreas cancer fibroblasts promote EMT-supportive growth factor secretion

doi: 10.1101/2025.01.30.635486

Figure Lengend Snippet: A) 0082T CAF cells were cultured in 21% or 1% O 2 for 72 hr, and qRT-PCR was performed for the indicated transcripts and normalized to CASC3 . n=3, t-test. B) 0082T CAFs were cultured in 21% or 1% O 2 for 120 hr, and immunofluorescence microscopy was performed for IGF-2. n=3, t-test. C) 430 inhibitors were screened for their ability to counteract hypoxic CAF-induced EMT in HPAF-II cells. HPAF-II cells were treated with hypoxic 0082T CM and inhibitors (1 μM for all compounds) for 120 hr. The ability of each inhibitor to reduce vimentin expression was calculated using positive and negative controls contained on each plate (see Methods). Data are displayed as a plot of the kernel density function (displayed as density) for the percent vimentin inhibition. Inhibitors with >50% reduction in vimentin-positive cells were designated as effective (orange region of the density plot of the inhibitor screen). D) Fraction of vimentin-positive HPAF-II cells treated with CM and MET inhibitors (blue) or MEK inhibitors (orange) compared to untreated and CM-only controls. The percentage of effective inhibitors was calculated for each inhibitor class in the inhibitor library. Representative images are shown of effective (i.e., >50% vimentin reduction) MEKi- or METi-treated samples and controls. E) 0082T cells were cultured in 21% or 1% O 2 for 120 hr and assessed for HGF expression by immunofluorescence microscopy. n=3, t-test. F) Immunofluorescence microscopy was performed on HPAF-II cells treated with hypoxic 0082T CM with linsitinib (IGF1Ri, 2.5 μM), PHA665752 (METi, 2.5 μM), both, or DMSO. G) Immunofluorescence microscopy was performed on KPCY tumor samples to determine fraction of HGF-positive CAFs (PDPN+) in oxygenated (HYP-) or hypoxic (HYP+) tumor regions. n=3, paired t-test. Data are presented as mean ± SEM for bar plots (A, B, E, F) or as a box-and- whisker plot that represent the first quartile (lower box bound), median (center bound), or third quartile (upper box bound) (G). * p <0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001

Article Snippet: PHA665752 (Santa Cruz Biotechnology) and linsitinib (Selleck) were used at 2.5 μM ( , ).

Techniques: Cell Culture, Quantitative RT-PCR, Immunofluorescence, Microscopy, Expressing, Inhibition, Whisker Assay

Journal: bioRxiv

Article Title: Hypoxia-induced histone methylation and NF-κB activation in pancreas cancer fibroblasts promote EMT-supportive growth factor secretion

doi: 10.1101/2025.01.30.635486

Figure Lengend Snippet: A) 0082T CAFs were cultured in 21% or 1% O 2 for 120 hr, and qRT-PCR was performed on extracted mRNA for HGF transcripts. CASC3 was used for normalization. n=3. B) Conditioned medium was collected from 0082T cells cultured in 21% or 1% O 2 and analyzed for HGF by ELISA. n=3, t-test. C) HPAF-II cells were untreated or treated with conditioned medium (CM) from PSCs grown at 1% O 2 +/- PHA665752 (METi, 2.5 μM) for 120 hr. Immunofluorescence microscopy was performed for the indicated proteins. n=3, one-way ANOVA with Tukey’s multiple comparison test. D) HPAF-II cells were treated with IGF-2 (100 ng/mL), HGF (5 ng/mL), or IGF-2+HGF for 120 hr. Immunofluorescence microscopy was performed for the indicated proteins. n=3, one-way ANOVA with Tukey’s multiple comparison test. E) Fluorescence-based immunohistochemistry was performed on sections of KPCY tumors for podoplanin (PDPN) and Hypoxyprobe (HYP) to calculate the percentage of CAFs within hypoxic regions. n=3. F) In sections of subcutaneous tumors formed using KPCY-derived 7160.c2 cells, HGF expression was assessed in Hypoxyprobe-positive and -negative CAFs (PDPN+ cells). n=3, t-test. Data are presented as mean ± SEM for bar plots (A, B, C, D, F) or as a mosaic plot indicating the percentage of PDPN+ cells that are positive or negative for Hypoxyprobe (HYP) across biological replicates. * p <0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001

Article Snippet: PHA665752 (Santa Cruz Biotechnology) and linsitinib (Selleck) were used at 2.5 μM ( , ).

Techniques: Cell Culture, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Immunofluorescence, Microscopy, Comparison, Fluorescence, Immunohistochemistry, Derivative Assay, Expressing