Journal: The Journal of Cell Biology

Article Title: Adhesion to fibronectin regulates Hippo signaling via the FAK–Src–PI3K pathway

doi: 10.1083/jcb.201501025

Figure Lengend Snippet: PI3K, PDK1, and Src regulation of nuclear YAP via Lats in serum-starved, subconfluent cells. (A) PI3K and PDK1 inhibitors relative to Lats. MCF-10A cells transfected with control, Nf2, or Lats1/2 siRNAs were serum starved and treated with DMSO (solvent control), 10 µM wortmannin (PI3K inhibitor), or 5 µM BX-795 (PDK1 inhibitor) for 30 min. On-target plus nontargeting pool was used as a control siRNA. Localization of endogenous YAP was identified by immunofluorescence staining. (B) SFK inhibitors and YAP localization. Serum-starved, low cell density MCF-10A cells were incubated with SFK inhibitors (10 µM each of PP2, dasatinib, SKI-1, and SU6656) for 30 min. 10 µM each of PP3 and imatinib were used as controls. YAP subcellular localization was determined by immunofluorescence staining. Alexa Fluor 594 secondary antibody was used for SU6656, which has high background green fluorescence. (C) Biochemical effects of Src inhibition. PP3- or PP2-treated MCF-10A cells were analyzed by Western blot using anti-YAP and anti–phospho-YAP (S127) antibodies. Phosphorylated YAP was detected by mobility shift on Phos-tag SDS-PAGE. (D) SFK inhibitors relative to Lats. MCF-10A cells transfected with control, Nf2, or Lats1/2 siRNA were serum starved and treated with 10 µM PP3 or PP2. After 30 min, cells were fixed for immunofluorescence staining with anti-YAP antibody. (E) Depletion of individual SFK. MCF-10A cells were transfected with control, Src, Fyn, or Yes siRNA. After serum starvation, subcellular localization of endogenous YAP was identified by immunofluorescence staining and quantified based on the criteria shown under the graph. More than 120 cells from four random views were quantified. (F) Src knockdown relative to Lats. MCF-10A cells were transfected with control, Src, Lats1/2, or combined siRNA of Src and Lats1/2. Cells were serum starved for 24 h before fixation and stained with anti-YAP antibody. (A, B, and D–F) One of three independent results is presented. Bars, 25 µm.

Article Snippet: The following inhibitors were used: SFK inhibitors SKI-1 and SU6656, Src/Abl inhibitor dasatinib monohydrate, Abl inhibitor imatinib mesylate, PDK1 inhibitor BX-795, FAK inhibitors PF-573228 and PF-562271 (Santa Cruz Biotechnology, Inc.), SFK inhibitor PP2 and its inactive analogue PP3 (EMD Millipore), and PI3K inhibitor wortmannin (Sigma-Aldrich).

Techniques: Transfection, Control, Solvent, Immunofluorescence, Staining, Incubation, Fluorescence, Inhibition, Western Blot, Mobility Shift, SDS Page, Knockdown

Journal: International Journal of Medical Sciences

Article Title: The combination of PF-429242 and chloroquine triggers pH-dependent cell death in hepatocellular carcinoma cells

doi: 10.7150/ijms.109069

Figure Lengend Snippet: Involvement of SREBP signaling in PF-429242/chloroquine-induced cell death. (A) PLC5 and HepG2 cells were transfected with siRNAs targeting MBTPS1 or MBTPS2 for 48 h. Following transfection, mRNA levels of MBTPS1 and MBTPS2 were measured using a real-time qPCR. *, **, and *** represent statistically significant differences ( p < 0.05, p < 0.01, and p < 0.001, respectively) compared to control siRNA-transfected cells. (B) Cells transfected with MBTPS1 or MBTPS2 siRNAs were replated into 96-well plates. After 72-h treatment with 20 µM chloroquine, cell viability was assessed using alamarBlue staining. (C) PLC5 and HepG2 cells transfected with MBTPS1 or MBTPS2 siRNAs for 24 h were subsequently treated with 20 µM chloroquine for 48 h. Plasma membrane integrity was evaluated using 7-AAD staining, with red fluorescence indicating 7-AAD-positive cells. (D) Cells were transfected with plasmids encoding SREBP1a/c or SREBP2 for 24 h and then treated with 20 µM PF-429242 for an additional 24 h. Protein expression levels were analyzed via Western blotting. (E) SREBP1a/c- or SREBP2-transfected PLC5 and HepG2 cells were replated into 96-well plates and co-treated with 10 µM PF-429242 and 20 µM chloroquine (PF/CQ) for 72 h. Cell viability was measured using alamarBlue staining.

Article Snippet: After transfection for 24-48 h, cells were used for further experiments. siRNA specific for the LC3B (#sc-43390), ATP6V0D1 (#sc-63207), MBTPS1 (#sc-36496), and MBTPS2 (#sc-41652) genes were purchased from Santa Cruz Technology. pEGFP-STAT3 (#111934), pcDNA3-SREBP1a (#26801), pcDNA3-SREBP1c (#26802), and pcDNA3-SREBP2 (#26807) plasmids were purchased from Addgene.

Techniques: Transfection, Control, Staining, Clinical Proteomics, Membrane, Fluorescence, Expressing, Western Blot

Journal: Cell

Article Title: ER Stress Drives Lipogenesis and Steatohepatitis via Caspase-2 Activation of S1P

doi: 10.1016/j.cell.2018.08.020

Figure Lengend Snippet: A) Indicated expression vectors were transfected into HEK293 cells. After 48 hr, cells were incubated with 25 μg/ml N-acetyl-leucine-leucine-norleucinal (ALLN) for 3 hr before harvest. WCL were IB’d with antibodies to SREBP1 and 2. B) Indicated expression vectors were transfected into SCAP-ablated HEK293 cells (293ΔSCAP). After 5 hr, cells were incubated with 1% lipoprotein-deficient serum (LPDS) for 16 hr, followed by ethanol or 50 μM mevalonate + 5 μg/ml cholesterol for 12 hr and 3 hr with ALLN before harvest. WCL, membranes, and NE were IB’d. Red stars: N-terminal Myc epitope-containing S1P polypeptides. C) Intracellular localization of Casp2-HA, S1P-Myc and Flag-SREBP2 in S1P-ablated HEK293 cells (293ΔS1P). Transiently expressed Casp2, S1P, and SREBP2 were visualized by immunofluorescence with HA, Myc, and FLAG antibodies, respectively. Nuclei were counterstained with DAPI. Scale bar: 10 μm. D) Indicated proteins were expressed in HEK293 cells. After 48 hr, WCL and culture supernatants (CS) were IB’d with Myc and S1P antibodies (2 lanes per condition). E) Indicated proteins were expressed in 293ΔSCAP cells. 5 hr after transfection, the cells were incubated with 1% LPDS for 16 hr followed by 3 hr with ALLN. WCL, membranes, and NE were prepared and Casp2 and S1P were IB’d with HA and Myc antibodies. CS were IB’d for S1P. Arrow: cleaved Casp2, red stars: N-terminal S1P fragments that retained the Myc epitope. F) ER and Golgi fractions were isolated by differential centrifugation from livers of LFD-fed 7-week-old MUP-uPA and Casp2−/−/MUP-uPA mice. Proteins in each fraction were de-glycosylated with PNGase F and IB’d as indicated. Star: nonspecific band present in the Golgi fraction of Casp2-null liver, F.L: full-length, C2-Cl.: Casp2-cleaved, A-Cl.: autocleaved. G) BL6 hepatocytes introduced with Adv carrying GFP or uPA. After 24 hr, ER, Golgi and nuclear fractions were isolated by differential centrifugation and IB’d for indicated proteins. F.L: full-length, Cl.: cleaved, L.E: long-exposure, S.E: short-exposure. H) IB of S1P in sera of HFD-fed (12 weeks) mice (n = 3) of indicated genotypes. I) Sera from normal individuals, NAFLD and NASH patients (n = 3 per group) with different liver fibrosis scores were IB’d with S1P antibody.

Article Snippet: Mouse S1P (MC204593) and mouse Casp2 cDNA clone (MC206011) were purchased from Origene Technologies, Inc. Flag-tagged SREBP2 (#32018) cDNA clone was from Addgene.

Techniques: Expressing, Transfection, Incubation, Immunofluorescence, Isolation, Centrifugation

Journal: JCI Insight

Article Title: Site-1 protease–mediated cholesterol metabolism is essential for lymphatic development in mice

doi: 10.1172/jci.insight.188637

Figure Lengend Snippet: ( A ) The diagram on the left depicts the tamoxifen-induced deletion of S1P to generate iEC Mbtps1 –/– mice. Gross images on the right show embryos at E14.5 ( N > 25 embryos in each genotype). Arrow shows lymphedema. ( B ) Left: Diagrams showing the collection of fetal anterior dorsal skin for whole-mount immunostaining and the migration of LECs to the midline of the dorsal skin at different developmental stages. Right: Images of immunostained anterior dorsal skin of WT and iEC Mbtps1 –/– mice at E14.5. Data represent at least 3 experiments. SMA, smooth muscle actin. ( C ) Quantification of the distance to the midline covered by lymphatic vessels (left) and the vessels’ diameters (right). Based on the blood vascular patterning, comparable regions among samples ( n > 5 images in each genotype) were selected as regions of interest for quantification. Midline closure and vessel diameter were measured using ImageJ (NIH). Littermates, WT. Data represent mean ± SD; unpaired t test with Welch’s correction was performed for the statistical analysis. **** P < 0.0001. ( D ) Immunostaining of dermal lymphatic vessels (green), arterioles (red), and blood capillaries (blue) in WT and LEC Mbtps1 –/– mice at E15.5 (top). Quantification of VEGFR3 intensity normalized to the vessel area (bottom, n > 5 images in each genotype) using ImageJ. Data represent mean ± SD. One-way ANOVA was performed for the statistical analysis. **** P < 0.0001. ( E ) Chylous ascites in LEC Mbtps1 –/– mice ( N > 10 mice). Littermates, WT.

Article Snippet: For generation of mice with constitutive and inducible deletion of S1P in endothelial cells ( Mbtps1 fl/fl Tie2Cre + and Mbtps1 fl/fl Cdh5CreER T2+ ), female Mbtps1 fl/fl mice (provided by Jay Horton, University of Texas Southwestern Medical Center, Dallas, Texas, USA) were crossed with the male Mbtps1 fl/wt Tie2Cre transgenic mice (008863, Jackson Laboratory) or male Mbtps1 fl/wt Cdh5CreER T2 transgenic mice.

Techniques: Immunostaining, Migration

Journal: JCI Insight

Article Title: Site-1 protease–mediated cholesterol metabolism is essential for lymphatic development in mice

doi: 10.1172/jci.insight.188637

Figure Lengend Snippet: ( A ) Representative whole-mount IF images of endothelial cells (green) and lymphatic vessels (red) in sections of jejunum from littermate control and LEC Mbtps1 –/– mice (20 days of age). ( B ) Quantification of the length and width of the lacteal of littermate controls and LEC Mbtps1 –/– mice. ( C ) Whole-mount IF images of VEGFR3 of lacteals of jejunum from WT littermates and LEC Mbtps1 –/– or LEC Srebf2 –/– mice (20 days of age). Fluorescence intensity of VEGFR3 was quantified using ImageJ and normalized to the average intensity of control lacteals. ( D ) Representative images of Oil Red O staining of jejunum section of LEC Srebf2 –/– mice and littermates (20 days of age). Arrow marks Oil Red O–positive lacteals. N = 3 for control littermates; N = 4 for each mutant genotype respectively. A total of 12 lacteals from each animal were analyzed. Each dot represents 1 animal on the graph. The graphs were plotted as mean ± SD. An unpaired t test was performed for the statistical analysis. * P < 0.05; ** P < 0.01.

Article Snippet: For generation of mice with constitutive and inducible deletion of S1P in endothelial cells ( Mbtps1 fl/fl Tie2Cre + and Mbtps1 fl/fl Cdh5CreER T2+ ), female Mbtps1 fl/fl mice (provided by Jay Horton, University of Texas Southwestern Medical Center, Dallas, Texas, USA) were crossed with the male Mbtps1 fl/wt Tie2Cre transgenic mice (008863, Jackson Laboratory) or male Mbtps1 fl/wt Cdh5CreER T2 transgenic mice.

Techniques: Control, Fluorescence, Staining, Mutagenesis

Journal: Cell Death & Disease

Article Title: SREBP1 site 1 protease inhibitor PF-429242 suppresses renal cell carcinoma cell growth

doi: 10.1038/s41419-021-03999-9

Figure Lengend Snippet: Sequences utilized in this study.

Article Snippet: SREBP1 shRNA lentiviral particles (sc-44327-V, Santa Cruz Biotech, Santa Cruz, CA) or S1P shRNA lentiviral particles (sc-36496-V, Santa Cruz Biotech) were added to RCC cells for 48 h. Puromycin was added to the medium.

Techniques: Sequencing

Journal: Cell Death & Disease

Article Title: SREBP1 site 1 protease inhibitor PF-429242 suppresses renal cell carcinoma cell growth

doi: 10.1038/s41419-021-03999-9

Figure Lengend Snippet: Stable RCC1 cells expressing S1P lentiviral shRNA (“sh-S1P” cells) or the CRSPR/Cas9-S1P-KO-GFP construct (“ko-S1P” cells) were established, control cells were transduced with scrambled control shRNA plus CRSPR/Cas9 empty vector (“sh-C + Cas9-C”); Expression of listed genes was shown ( A and B ); Cells were then treated with or without PF-429242 (10 μM), and cultured for applied time periods, total cholesterol levels were tested ( C ); Viable cell number, cell death, proliferation, and migration were tested by CCK-8 ( D ), LDH release ( D ), nuclear EdU staining ( E ) and “Transwell” ( F ) assays, respectively, and results were quantified.RCC2, RCC3, and A498 cells, expressing S1P lentiviral shRNA (“sh-S1P” cells) or scramble control shRNA (“sh-C”), were established and S1P mRNA expression was shown ( G ); Cells were further cultured for applied time periods, total cholesterol levels were tested ( H ); Cell proliferation, migration, and cell death were tested by nuclear EdU staining ( I ), “Transwell” ( J ) and medium LDH release ( K ) assays, respectively. For each assay, n = 5. Data were expressed as the mean ± standard deviation (SD).* P < 0.05 vs. “sh-C + Cas9-C”/“sh-C” group. “n.s.” stands for no statistical difference ( C – F ). In this figure, experiments were repeated three times, and similar results were obtained each time. Scale bar = 100 μm ( E and F ).

Article Snippet: SREBP1 shRNA lentiviral particles (sc-44327-V, Santa Cruz Biotech, Santa Cruz, CA) or S1P shRNA lentiviral particles (sc-36496-V, Santa Cruz Biotech) were added to RCC cells for 48 h. Puromycin was added to the medium.

Techniques: Expressing, shRNA, Construct, Control, Transduction, Plasmid Preparation, Cell Culture, Migration, CCK-8 Assay, Staining, Standard Deviation

Journal: Cell Death & Disease

Article Title: SREBP1 site 1 protease inhibitor PF-429242 suppresses renal cell carcinoma cell growth

doi: 10.1038/s41419-021-03999-9

Figure Lengend Snippet: Primary RCC cells (“RCC1/RCC2/RCC3”) or A498 cells, bearing the lentiviral S1P expression construct (“OE-S1P”) or empty vector (“Vec”), were established, expression of listed genes in these cells and parental control cells (“Pare”) was shown ( A – C and G ). Cells were further cultured for applied time periods, CCK-8 OD ( H ), proliferation (by recording nuclear EdU ratio, D and I ), migration, and invasion (“Transwell” assay, E , F , and J ) were tested. RCC1 cells bearing SREBP1 shRNA (“sh-SREBP1”) or SREBP1-expressing construct (OE-SREBP1) were established, control cells were transduced with scrambled control shRNA plus empty vector (“Vec+shC”), expression of SREBP1 mRNA and protein was shown ( K ). Cells were further cultured for applied time periods, viable cell number ( L ), proliferation ( M ), and migration ( N ) were tested similarly. For each assay, n = 5. Data were expressed as the mean ± standard deviation (SD). * P < 0.05 vs. “Vec”/“Vec+shC” group. In this figure, experiments were repeated three times, and similar results were obtained each time.

Article Snippet: SREBP1 shRNA lentiviral particles (sc-44327-V, Santa Cruz Biotech, Santa Cruz, CA) or S1P shRNA lentiviral particles (sc-36496-V, Santa Cruz Biotech) were added to RCC cells for 48 h. Puromycin was added to the medium.

Techniques: Expressing, Construct, Plasmid Preparation, Control, Cell Culture, CCK-8 Assay, Migration, Transwell Assay, shRNA, Transduction, Standard Deviation

Journal: Cell Death & Disease

Article Title: SREBP1 site 1 protease inhibitor PF-429242 suppresses renal cell carcinoma cell growth

doi: 10.1038/s41419-021-03999-9

Figure Lengend Snippet: RCC1 xenograft-bearing SCID mice were subjected to i.v . injection of PF-429242 (“PF”, at 10 mg/kg body weight, daily, for 21days) or vehicle control (“Veh”), tumor volumes ( A ) and mice body weights ( D ) were recorded every seven days; The estimated daily tumor growth, in mm 3 per day, was calculated using the described formula ( B ). On Day-35 tumors of the two groups were separated through surgery and individually weighted ( C ). On Day-7 and Day-14, one tumor of each group was isolated and homogenized in tissue lysis buffer, expression of listed genes was shown ( E and F ). RCC1 xenograft-bearing SCID mice were subjected to intratumoral injection of SREBP1 shRNA lentivirus (“shSREBP1”), S1P shRNA lentivirus (“shS1P”) or scramble control shRNA lentivirus (“shC”), daily for five days, tumor volumes ( G ) and mice body weights ( H ) were recorded every seven days; At Day-14, one tumor of each group was isolated and homogenized in tissue lysis buffer, expression of listed proteins was shown ( I ). Data were expressed as the mean ± standard deviation (SD).* P < 0.05 vs. “Veh”/ “shC” group.

Article Snippet: SREBP1 shRNA lentiviral particles (sc-44327-V, Santa Cruz Biotech, Santa Cruz, CA) or S1P shRNA lentiviral particles (sc-36496-V, Santa Cruz Biotech) were added to RCC cells for 48 h. Puromycin was added to the medium.

Techniques: Injection, Control, Isolation, Lysis, Expressing, shRNA, Standard Deviation