Journal: Cancers

Article Title: The RAL Small G Proteins Are Clinically Relevant Targets in Triple Negative Breast Cancer

doi: 10.3390/cancers16173043

Figure Lengend Snippet: Depletion of RALA or RALB reduces MDA-MB-468 tumor growth and is associated with changes in the tumor microenvironment. ( A ) Western blots displaying RALA and RALB expression in the MDA-MB-468 shRNA control (shCTRL), shRALA, and shRALB cells. ImageJ was used for quantification. ( B , C ) Effects of stable knockdown of RALA or RALB in MDA-MB-468 cells on 2D growth as measured by MTT (( B ), n = 4), and 3D growth as measured by GILA (( C ), n = 4) *, p < 0.05. ( D ) Stable knockdown of RALA or RALB had no effect on MDA-MB-468 cell migration (left: representative images, right: composite mean values, n = 2). ( E ) Comparison of tumor growth following orthotopic mammary fat pad injection of MDA-MB-468 shCtrl ( n = 20), shRALA ( n = 20), or shRALB ( n = 20). Results were combined from three independent experiments. ( F – H ) MDA-MB-468 shCTRL ( n = 9), shRALA ( n = 10), and shRALB ( n = 10) tumors IHC stained for ( F ) Ki-67, ( G ) CC3, or ( H ) CD31. ROIs were determined on each image separating the tumor from stroma before color deconvolution to extract DAB staining. A signal threshold (equivalent for each image) was then applied to the samples before measurement of the ROIs was performed to measure the % area of target staining in each region. Three representative photos from each sample were separately analyzed, and the mean values were used for comparisons among groups. *, p < 0.05. ( I ) MDA-MB-468 tumors stained with Masson’s Trichrome, denoted by white arrows, to analyze the collagen deposition in shCTRL ( n = 7), shRALA ( n = 8), or shRALB ( n = 9) MDA-MB-468 tumors. *, p < 0.05. ( J , K ) Graphs summarize the relative amounts of secreted proteins detected in the conditioned media from the MDA-MB-468 shCTRL, shRALA, and shRALB cultures.

Article Snippet: MDA-MB-468 parental cells were transduced with the Origene lentiviruses shCTRL (TR30021V), shRALA (TL309957VC, 5′-CTGGTTGGTAACAAATCAGATTTAGAAGA-3′), and shRALB (TL309956VD, 5′-GAACAGATTCTCCGTGTGAAGGCTGAAGA-3′).

Techniques: Western Blot, Expressing, shRNA, Control, Knockdown, Migration, Comparison, Injection, Staining

Journal: Cancers

Article Title: The RAL Small G Proteins Are Clinically Relevant Targets in Triple Negative Breast Cancer

doi: 10.3390/cancers16173043

Figure Lengend Snippet: Loss of RALA or RALB does not negatively impact SKBR3 tumor growth or viability. ( A ) Western blots displaying RALA and RALB expression in the SKBR3 shRNA control (shCTRL), shRALA, and shRALB cells. ImageJ was used for quantification. ( B ) Comparison of tumor growth following orthotopic mammary fat pad injection of SKBR3 shCTRL ( n = 10), shRALA ( n = 9), or shRALB ( n = 10). Results were from a single experiment. ( C – E ) Effects of stable knockdown of RALA or RALB in SKBR3 cells on 2D growth as measured by MTT (( C ), n = 5), 3D growth as measured by GILA (( D ), n = 5), and cell migration (( E ), n = 2). *, p < 0.05.

Article Snippet: MDA-MB-468 parental cells were transduced with the Origene lentiviruses shCTRL (TR30021V), shRALA (TL309957VC, 5′-CTGGTTGGTAACAAATCAGATTTAGAAGA-3′), and shRALB (TL309956VD, 5′-GAACAGATTCTCCGTGTGAAGGCTGAAGA-3′).

Techniques: Western Blot, Expressing, shRNA, Control, Comparison, Injection, Knockdown, Migration

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Hypoxia-autophagy axis induces VEGFA by peritoneal mesothelial cells to promote gastric cancer peritoneal metastasis through an integrin α5-fibronectin pathway

doi: 10.1186/s13046-020-01703-x

Figure Lengend Snippet: Hypoxia-induced autophagy mediated degradation of SIRT1 in PMCs promotes VEGFA secretion through acetylation of HIF-1α. a The levels of p62, LC3I/II, SIRT1, HIF-1α, and VEGFA were observed in HMrSV5 cells cultured in hypoxic conditions for the indicated time. b and c Western blot analysis of p62, LC3I/II, SIRT1, HIF-1α, and VEGFA under hypoxia and in response to treatment with chloroquine (CQ) or knockdown of ATG7 in HMrSV5 cells, or knockout of ATG7 in HEK293. d The levels of LC3I/II, p62, SIRT1, HIF-1α, and VEGFA were analyzed in HMrSV5 cells exposed to normoxia or hypoxia for 24 h with or without overexpression of SIRT1. e ATG7 was knocked out in HEK293 cells and knocked down in HMrSV5 cells, followed by exposure to normoxic or hypoxic conditions for 24 h . Immunoprecipitation was performed with a pan-acetyl antibody followed by immunoblot analysis using antibodies against HIF-1α. f Immunohistochemical analysis of LC3BI/II and VEGFA in benign mouse peritonea and GC metastatic peritonea. G. MGC-803 cells were subjected to normal media or conditioned media (CM1: conditioned media from hypoxic PMCs, CM2: conditioned media of hypoxic shRNA-Atg7 PMCs) and to CM2 with synchronous addition of exogenous VEGFA. Representative photographs of adherent and migratory cells are shown. Scale bars represent 100 μm. Bars represent SD of the mean. * P < 0.05. ** P < 0.01. *** P < 0.001

Article Snippet: For lentiviral production and infection, control shRNA (shCtrl/NC) lentivirus, shRNA against Atg7 (sh-Atg7), sgRNA against VEGFR1 (sgRNA-VEGFR1/FLT1), sgRNA against SIRT1 (sgRNA-SIRT1), sgRNA against Atg7 (sgRNA-Atg7) lentivirus and plasmids for Flag-p62, HA-SIRT1 N terminal domain (NTD, aa 1–234), sirtuin (catalytic) domain (SD, aa 234–510), C terminal domain (CTD, aa 510–747), full length (FL, aa 1–747) were purchased from Shanghai GeneChem Company.

Techniques: Cell Culture, Western Blot, Knockdown, Knock-Out, Over Expression, Immunoprecipitation, Immunohistochemical staining, shRNA