Journal: American Journal of Physiology - Renal Physiology

Article Title: The mechanosensitive BKα/β1 channel localizes to cilia of principal cells in rabbit cortical collecting duct (CCD)

doi: 10.1152/ajprenal.00256.2016

Figure Lengend Snippet: Antibodies (Abs) used for immunoperfusion

Article Snippet: 3D reconstructions were generated using Leica Application Suite (LAS AF) software. table ft1 table-wrap mode="anchored" t5 Table 1. caption a7 Primary Abs Dilution Provider Secondary Abs Dilution Provider Goat IgG anti-maxi-Kβ1 (N-15) 1:100 Santa Cruz Biotechnology (sc-14749) A488-rabbit IgG anti-goat IgG 1:500 Molecular Probes ( {"type":"entrez-nucleotide","attrs":{"text":"A11078","term_id":"490929","term_text":"A11078"}} A11078 ) Mouse IgG2a anti-maxi-Kβ4 1:100 Stress Marq Biosciences (SMC-38D) A488-goat IgG anti-mouse IgG2a 1:500 Molecular Probes ( {"type":"entrez-nucleotide","attrs":{"text":"A21131","term_id":"514092","term_text":"A21131"}} A21131 ) Mouse IgG2b anti-acetylated α-tubulin (6-11B-1) 1:1,000 Abcam (ab24610) A488-goat IgG anti-mouse IgG 1:1,000 Molecular Probes ( {"type":"entrez-nucleotide","attrs":{"text":"A11029","term_id":"492395","term_text":"A11029"}} A11029 ) Goat IgG anti-aquaporin-2 (C-17) 1:100 Santa Cruz Biotechnology (sc-9882) A488-rabbit IgG anti-goat IgG 1:500 Molecular Probes ( {"type":"entrez-nucleotide","attrs":{"text":"A11078","term_id":"490929","term_text":"A11078"}} A11078 ) Mouse IgG2a anti-polycystin-2 (YCE2) 1:100 Santa Cruz Biotechnology (sc-47734) A647-goat IgG anti-mouse IgG 1:500 Molecular Probes ( {"type":"entrez-nucleotide","attrs":{"text":"A21236","term_id":"583506","term_text":"A21236"}} A21236 ) Rabbit anti-pendrin 1:2,000 Gift from Dr. S. Wall, Emory University School of Medicine A488-goat IgG anti-rabbit IgG 1:500 Molecular Probes ( {"type":"entrez-nucleotide","attrs":{"text":"A11008","term_id":"492390","term_text":"A11008"}} A11008 ) Open in a separate window Antibodies (Abs) used for immunoperfusion The effect of K + intake on relative BKα expression in the cilia was determined by analysis of CCDs isolated from HK ( n = 4)- or LK ( n = 4)-fed rabbits and immunoperfused simultaneously with Abs directed against BKα and acetylated α-tubulin (Ac α-tubulin), followed by appropriate secondary Abs ( ).

Techniques:

Journal: Cancer Communications

Article Title: Snail acetylation by autophagy‐derived acetyl‐coenzyme A promotes invasion and metastasis of KRAS ‐ LKB1 co‐mutated lung cancer cells

doi: 10.1002/cac2.12332

Figure Lengend Snippet: LKB1 loss impairs antioxidant defenses, resulting in increased reactive oxygen species (ROS) levels and Snail stabilization in KL cells. (A) ROS levels detected using the CM‐H2DCFDA probe in hb30‐KP and hb30‐KPL cells. (B) Mitochondrial ROS levels in four K and four KL cell lines. The bar plots show the mean ± SD of four independent experiments. (C) Real‐time PCR analyses of the relative expression of the indicated genes in hb30‐KP and hb30‐KPL cells exposed to 150 μmol/L H 2 O 2 for 6 h (2 RNA samples from each cell line). All data are presented as the fold change ± SD compared to control cells without H 2 O 2 exposure. (D) Steady‐state accumulation of the p62, phospho‐p62 (Ser351) and NRF2 proteins was assessed by Western blotting analysis of whole‐cell lysates from hb30‐KP and hb30‐KPL cells. Beta‐actin was used as the loading control (left). Detection of steady‐state NRF2 protein in hb30‐KP and hb30‐KPL cells by immunofluorescence (scale bar, 50 μm) (middle). Analyses were performed with anti‐NRF2 antibody (green). DAPI was used to visualize nuclei. The bar plots show the nuclear to cytosolic ratio of NRF2 protein intensity, quantified by ImageJ software (three independent experiments). (E) Steady‐state accumulation of the phospho‐AMPK protein was assessed by Western blotting analysis of whole‐cell lysates from hb30‐KP and hb30‐KPL cells. HSP90 was used as the loading control. (F) The effects of AMPK knockdown in hb30‐KPL cells on the accumulation of Snail and 2D invasion were evaluated by Western blotting (left) and Matrigel invasion assays (right), respectively. (G and H) Proteins from hb30‐KPL cells that were untreated or treated with H 2 O 2 or NAC for 6 h were analyzed by Western blotting for phospho‐AMPK, total AMPK (G), and Snail (H). Beta‐actin was used as the loading control. (I) Effects of treatment with the NRF2 activator CDDO‐ME for 24 h on the expression of the indicated proteins in hb30‐KPL cells, as determined by Western blotting. Beta‐actin was used as the loading control. (J) The effects of ectopic p62 expression on the Snail protein levels in hb30‐KPL cells were determined by Western blotting 24 h post‐transfection. Beta‐actin was used as the loading control. (K) Steady‐state accumulation of phospho‐CAMKII protein was assessed by Western blotting analysis of whole‐cell lysates from hb30‐KP and hb30‐KPL cells. HSP90 was used as the loading control. (L) Effects of CAMKK2 knockdown by shRNA on the expression of the indicated proteins in hb30‐KPL cells, as determined by Western blotting. HSP90 was used as the loading control. (M and N) The effects of treatment with the CAMKK2 inhibitor STO‐609 for 24 h on Snail accumulation and 2D invasion in hb30‐KPL cells were evaluated by Western blotting (M) and Matrigel invasion assays (N). Beta‐actin was used as the loading control. (N) The bar plots on the bottom left show the mean relative invaded cell number ± SD, while bar plots on the bottom right show the mean relative live cell count ± SD of three independent experiments. (O) Effects of STO‐609 (25 μmol/L) for 7 days on the invasion of spheroids generated from the indicated KL cell line under 3D culture conditions. The bars indicate the mean ± SD of six invasion lengths in two spheroids per group. DMSO was used as a vehicle control. All experiments were performed in triplicate unless otherwise indicated. The data are presented as mean ± SD, unless otherwise indicated. Significant differences between groups were determined by a two‐sided unpaired Student's t ‐test. * P < 0.05, ** P < 0.01, *** P < 0.001, NS : not significant. Abbreviations: ROS, reactive oxygen species; CM‐H2DCFDA, chloromethyl dichlorodihydrofluorescein diacetate, acetyl ester; PCR, polymerase chain reaction; CTG, CellTiter Glo; NRF2, NF‐E2‐related factor 2; DAPI, 4′,6‐diamidino‐2‐phenylindole; NAC, N‐acetyl cysteine; AMPK, AMP‐activated protein kinase, CDDO‐ME, bardoxolone methyl; CAMKII, Calcium/Calmodulin‐dependent Protein Kinase II; CAMKK2, calcium/calmodulin‐dependent protein kinase kinase 2; 2D, two‐dimensional; 3D, three‐dimensional; HSP90, heat shock protein 90; DMSO, dimethyl sulfoxide

Article Snippet: Rabbit monoclonal anti‐heat shock protein 90 (HSP90) (#4877, Cell Signaling Technology, RRID: AB_2233307), mouse monoclonal anti‐β‐actin (#sc‐47778, Santa Cruz, RRID: AB_2714189), rabbit monoclonal anti‐microtubule‐associated protein 1 light chain 3 alpha (LC3) (#3868, Cell Signaling Technology, RRID: AB_2137707), rabbit monoclonal anti‐Vimentin (#5741, Cell Signaling Technology, RRID: AB_10695459) were used at a 1:3000 dilution.

Techniques: Real-time Polymerase Chain Reaction, Expressing, Control, Western Blot, Immunofluorescence, Software, Knockdown, Transfection, shRNA, Cell Counting, Generated, Polymerase Chain Reaction

Journal: Cancer Communications

Article Title: Snail acetylation by autophagy‐derived acetyl‐coenzyme A promotes invasion and metastasis of KRAS ‐ LKB1 co‐mutated lung cancer cells

doi: 10.1002/cac2.12332

Figure Lengend Snippet: CAMKK2‐AMPK‐dependent activation of autolysosomes drives invasion through GSK3β‐independent Snail stabilization. (A) Detection of steady‐state lysosomes and autophagosomes in hb30‐KPL cells by immunofluorescence (scale bar, 50 μm). LC3 (green dots) represents autophagosomes, and LysoTracker (red dots) represents lysosomes. DAPI was used to visualize nuclei. Arrows indicate cells with co‐localized LC3 and LysoTracker puncta. (B) Western blotting of LC3 proteins in hb30‐KP and hb30‐KPL cells in response to treatment with 50 μmol/L CQ for 6 h. Beta‐actin was used as the loading control. Values obtained from a quantitative densitometric analysis of LC3‐II relative to beta‐actin are shown underneath the LC3 blots. (C) Steady‐state relative luciferase activity in hb30, hb30‐KP and hb30‐KPL cells transiently transfected with a CLEAR motif‐driven luciferase reporter construct. Relative luciferase intensities are expressed as the fold change relative to the hb30 control cells. (D‐E) Effects of treatment with NAC (D) or 50 μmol/L STO‐609 (E) for 24 h on the relative luciferase activity in hb30‐KPL cells stably transfected with a TFEB‐luciferase reporter construct. (F‐G) The effects of genetic suppression of autophagy with the indicated siRNAs (F) or inhibition by the indicated concentrations of CQ for 12‐48 h (G) on Snail protein accumulation were determined by Western blotting analysis of whole‐cell lysates from hb30‐KPL cells. Beta‐actin and HSP90 were used as the loading control. (H and I) Effects of treatment with CQ (50 μmol/L for H) or STO‐609 at the indicated concentrations for 12 h in the presence or absence of 20 nmol/L bortezomib for the last 4 h on Snail protein accumulation in hb30‐KPL cells. Beta‐actin was used as the loading control. Values obtained from a quantitative densitometric analysis of Snail relative to beta‐actin are shown underneath the Snail blots. (J) Steady‐state accumulation of the indicated proteins was assessed by Western blotting of whole‐cell lysates from hb30‐KP and hb30‐KPL cells (left). Beta‐actin was used as the loading control. (K) Effect of treatment with the GSK3β inhibitor CHIR‐99021 (3 μmol/L for 5 h) in the presence or absence of 50 μmol/L CQ on Snail protein accumulation in hb30‐KPL cells. HSP90 was used as the loading control. Activated beta‐catenin was used as the positive control GSK3β substrate. (L,M,O) Effects of the indicated drugs: CQ (lysomotropic weak base) 25 μmol/L, bafilomycin A1 (BafA1: V‐ATPase inhibitor) 10 nmol/L, wortmannin (Wort: PI3K inhibitor) 1 μmol/L, saliphenylhalamide (Sali: V‐ATPase inhibitor) 10 nmol/L and rapamycin (mTORC1 inhibitor) 2 μmol/L for 24 h (L, O) or knockdown with the indicated siRNAs (M) on Matrigel invasion of hb30‐KPL cells (L‐M) or hb30‐KP cells (O). Quantified data of cells that invaded through Matrigel‐coated Transwell membranes are shown. (N) Effects of ATG5 knockdown on the invasion of spheroids generated from the indicated KL cell line under 3D culture conditions. The bars indicate the mean ± SD of nine invasion lengths in three spheroids per group. All experiments were performed in duplicate unless otherwise indicated. The data are presented as mean ± SD, unless otherwise indicated. Significant differences between groups were determined by a two‐sided unpaired Student's t ‐test. * P < 0.05, ** P < 0.01, *** P < 0.001. Abbreviations: LC3, microtubule‐associated protein 1A/1B light chain 3; DAPI, 4′,6‐diamidino‐2‐phenylindole; CQ, chloroquine; CLEAR, coordinated lysosomal expression and regulation; NAC, N‐acetyl‐l‐cysteine; NC, negative control; TFEB, transcription factor EB; TFEB RE, TFEB reporter; ULK1, Unc‐51‐like kinase; ATG5, autophagy related 5; GSK3β, glycogen synthase kinase 3 beta; HSP90, heat shock protein 90, 3D, three‐dimensional; DMSO, dimethyl sulfoxide

Article Snippet: Rabbit monoclonal anti‐heat shock protein 90 (HSP90) (#4877, Cell Signaling Technology, RRID: AB_2233307), mouse monoclonal anti‐β‐actin (#sc‐47778, Santa Cruz, RRID: AB_2714189), rabbit monoclonal anti‐microtubule‐associated protein 1 light chain 3 alpha (LC3) (#3868, Cell Signaling Technology, RRID: AB_2137707), rabbit monoclonal anti‐Vimentin (#5741, Cell Signaling Technology, RRID: AB_10695459) were used at a 1:3000 dilution.

Techniques: Activation Assay, Immunofluorescence, Western Blot, Control, Luciferase, Activity Assay, Transfection, Construct, Stable Transfection, Inhibition, Positive Control, Knockdown, Generated, Expressing, Negative Control

Journal: Cancer Communications

Article Title: Snail acetylation by autophagy‐derived acetyl‐coenzyme A promotes invasion and metastasis of KRAS ‐ LKB1 co‐mutated lung cancer cells

doi: 10.1002/cac2.12332

Figure Lengend Snippet: Acetyl‐CoA supplied by autophagy underlies the enhanced invasion of KL cells. (A) Volcano plot of 225 metabolites compared between 14 KL and 31 K lung cancer cell lines under steady‐state growth conditions based on the CCLE metabolomic data (left). Metabolites with statistically significant differences between the two sets of cells ( P < 0.05, two‐sided Wilcoxon rank sum test) are shown as colored dots. The red dots in the box enclosed by dashed lines represent statistically significantly upregulated metabolites in KL cells (delta>0), whereas the blue dots represent downregulated metabolites (delta<0) with a P value < 0.05. Delta values were obtained by subtracting the mean levels of a metabolite in K cell lines from the mean levels in KL cell lines. The black highlighting indicates metabolites downregulated upon ATG5 knockdown in hb30‐KPL cells, as shown in Supplementary Figure . The heatmap depicts the cell line‐specific levels of 11 metabolites significantly upregulated in KL cells (right) (B) Steady‐state levels of citrate in hb30 cells, hb30‐KP cells, and hb30‐KPL cells. Relative absorbance is expressed as the fold change relative to the hb30 control cells (left). Steady‐state acetyl‐CoA levels in hb30‐KP and hb30‐KPL cells. Relative absorbance is expressed as the fold change relative to the hb30‐KP (right). The data are shown as the mean ± SD of two and four independent experiments, respectively. (C) Acetyl‐CoA level in hb30‐KPL cells with the indicated genetic knockdown as a fold change relative to the control cells. (D) Levels of citrate in hb30‐KPL cells treated with 8 mmol/L MP, 3 mmol/L MOG or 200 μmol/L rosiglitazone for 24 h in the presence or absence of 50 μmol/L CQ. (E) Western blotting of the indicated proteins in hb30‐KP cells treated with 2 μmol/L rapamycin for 5 h. Beta‐actin was used as the loading control (left). Increased conversion of LC3‐I (upper band) to LC3‐II (lower band) indicates autophagic activation. The levels of citrate and acetyl‐CoA under the same conditions (right). (F) Schematic of acetyl‐CoA metabolism in mammalian cells. Citrate is synthesized by two independent pathways via mitochondrial citrate synthase (CS) and cytosolic isocitrate dehydrogenase 1 (IDH1). Acetyl‐CoA is synthesized from citrate by ATP citrate lyase (ACLY) or from acetate by acetyl‐CoA synthetase 2 (ACSS2). Acetyl‐CoA is then used as a precursor for synthesizing lipid molecule synthesis or as an acetyl group donor for protein acetylation. Blue text indicates enzymes. (G‐H) Effects of ACLY or ACSS2 knockdown on the acetyl‐CoA level (measured by fluorescence intensity) (G) and Matrigel invasion (H) in hb30‐KPL cells. (I) Effect of 10 mmol/L acetate treatment for 24 h on the Matrigel invasion of hb30‐KPL cells (left). Matrigel invasion of hb30‐KPL cells with or without ACSS2 knockdown and 10 mmol/L acetate treatment for the last 24 h (right). (J) Effects of CS or IDH1 knockdown on citrate in hb30‐KPL cells. (K) Effects of CS or ACLY knockdown in the presence or absence of 8 mmol/L MP (left) or CS , IDH1 , or ACLY knockdown in the presence or absence of 3 mmol/L MOG (right) on Snail accumulation in hb30‐KPL cells, as determined by Western blotting. HSP90 was used as the loading control in both blots. Values obtained from a quantitative densitometric analysis of Snail relative to HSP90 are shown underneath the Snail blots. (L) Effect of IDH1 knockdown and treatment with 50 μmol/L CQ in the presence or absence of 3 mmol/L MOG for 24 h on Snail accumulation in hb30‐KPL cells. HSP90 was used as the loading control. (M) Effects of ACACA knockdown on the Matrigel invasion and migration of hb30‐KPL cells (left) and indicated KL cell lines (right). (N) Effect of CREBBP knockdown on Matrigel invasion in hb30‐KPL cells (left) and the indicated KL cell lines (right). (O) Effect of treatment with the CBP inhibitor C646 (25 μmol/L) for 24 h in the presence or absence of 10 mmol/L acetate on Matrigel invasion. All experiments were performed in duplicate unless otherwise indicated. The data are presented as mean ± SD, unless otherwise indicated. Significant differences between groups were determined by a two‐sided unpaired Student's t ‐test. * P < 0.05, ** P < 0.01, *** P < 0.001, NS : not significant. Abbreviations: CCLE, Cancer Cell Line Encyclopedia; NC, negative control; ATG5, autophagy related 5; TFEB, transcription factor EB; CAMKK2, calcium/calmodulin dependent protein kinase kinase 2; MP, methyl pyruvate; MOG, dimethyl‐2‐oxoglutarate; Rosi, rosiglitazone; CQ, chloroquine;TCA, tricarboxylic acid cycle; Cyto, cytoplasm; Mito, mitochondria; Rapa, rapamycin; LC3, microtubule associated protein 1 light chain 3 alpha; ACLY, ATP citrate lyase; ACSS2, acetyl‐CoA synthase short chain family member 2, CS, citrate synthase; IDH1, isocitrate dehydrogenase 1; CREBBP, CREB‐binding protein; ACACA, Acetyl‐CoA carboxylase alpha; HSP90, heat shock protein 90

Article Snippet: Rabbit monoclonal anti‐heat shock protein 90 (HSP90) (#4877, Cell Signaling Technology, RRID: AB_2233307), mouse monoclonal anti‐β‐actin (#sc‐47778, Santa Cruz, RRID: AB_2714189), rabbit monoclonal anti‐microtubule‐associated protein 1 light chain 3 alpha (LC3) (#3868, Cell Signaling Technology, RRID: AB_2137707), rabbit monoclonal anti‐Vimentin (#5741, Cell Signaling Technology, RRID: AB_10695459) were used at a 1:3000 dilution.

Techniques: Knockdown, Control, Western Blot, Activation Assay, Synthesized, Fluorescence, Migration, Negative Control, Binding Assay

Journal: Cancer Communications

Article Title: Snail acetylation by autophagy‐derived acetyl‐coenzyme A promotes invasion and metastasis of KRAS ‐ LKB1 co‐mutated lung cancer cells

doi: 10.1002/cac2.12332

Figure Lengend Snippet: Autophagy‐derived acetyl‐CoA enhances KL cell invasion via CBP‐mediated Snail acetylation. (A) Effect of exposure to BMS‐303141, NDI‐091143 and SB‐204990 (ACLY inhibitor) at the indicated concentrations for 24 h, 24 h, and 2 h in the presence or absence of 20 nmol/L bortezomib for the last 3 hours (BMS‐303141) on Snail protein accumulation in hb30‐KPL cells, as determined by Western blotting. Beta‐actin and HSP90 were used as the loading controls (left panels). Acetyl histone H3 was used as a control to represent intracellular acetyl‐CoA levels. The effect of exposure of hb30‐KPL cells to 50 μmol/L BMS‐303141 for 24 h, 1 μmol/L NDI‐091143 for 24 h and 20 μmol/L SB‐204990 for 2 h on Matrigel invasion. Quantified data of cells that invaded through Matrigel‐coated Transwell membranes are shown in the right panels. The bar plots show the mean ± SD of three independent experiments. (B) Steady‐state levels of total protein acetylation in hb30‐KP and hb30‐KPL cells (left) and immunoprecipitation‐Western blotting analysis of acetylated Snail levels in hb30‐KP and hb30‐KPL cells (right). Beta‐actin was used as the loading control. se, short exposure; le, long exposure. (C‐D) Immunoprecipitation‐Western blotting analysis of acetylated Snail levels upon treatment with 50 μmol/L CQ for 24 h (C) or ATG5 or TFEB knockdown (D) in hb30‐KPL cells. Values obtained from a quantitative densitometric analysis of acetylated Snail relative to beta‐actin are shown underneath the Snail blots. (E) The effect of 2 μmol/L rapamycin (for 5 h) with or without ACLY knockdown on Snail protein accumulation was assessed by Western blotting analysis. Values obtained from a quantitative densitometric analysis of acetylated Snail relative to beta‐actin are shown underneath the Snail blots. (F) Effect of the CBP inhibitor C646 (25 μmol/L) for 2 h on the levels of the indicated proteins in hb30‐KPL (left) and KL cell lines (right), as determined by Western blotting. Real‐time PCR analyses of relative SNAI1 gene expression in hb30‐KPL cells after exposure to 25 μmol/L C646 for 2 h (middle; 2 RNA samples). The schematic shows the modes of action of the tested compounds. (G) The effects of hb30‐KPL (left) and KL cell line (right) treatment with CREBBP ‐specific siRNA for 72 h on Snail protein accumulation were assessed by Western blotting analysis. HSP90 was used as the loading control. (H) Effect of the 3 μmol/L HDAC inhibitor SAHA for 15 h on the levels of the indicated proteins in hb30‐KPL cells (left). Immunoprecipitation‐Western blotting analysis of acetylated Snail levels in hb30‐KPL cells upon treatment with 3 μmol/L SAHA for 15 h (right). Beta‐actin and HSP90 were used as the loading controls. Values obtained from a quantitative densitometric analysis of acetylated Snail relative to HSP90 are shown underneath the Snail blots. (I) Matrigel invasion assay of hb30‐KPL cells with SNAI1 knockdown with or without 3 μmol/L SAHA treatment for the last 15 h. Representative bright‐field micrographs (20×) of cells that invaded through Matrigel‐coated Transwell membranes are shown in the left panel, and quantification data of invaded cell numbers are shown in the right panel. (J) The effect of exposure of hb30‐KPL cells to a combination of 3 μmol/L SAHA and 50 μmol/L CQ for 24 h on Snail protein accumulation was assessed by Western blotting. Beta‐actin was used as the loading control. (K) The effects of ectopic expression of wild‐type or acetylation‐deficient Snail (K146R or K187R) on Snail protein levels in the indicated K cell lines were determined by Western blotting analysis at 24 h post‐transfection. HSP90 was used as the loading control (left). Representative images of Matrigel invasion under the above conditions are shown in the middle panels, and quantified data are shown in the right panels. (L) Immunoprecipitation‐Western blotting analysis of phosphorylated/acetylated Snail in A549 cells transfected with wild‐type Snail (pFLAG‐Snail WT) or an acetylation‐deficient Snail mutant (pFLAG‐SnailK146R) with or without 25 μmol/L C646 treatment for 2 h (left) or 3 μmol/L SAHA treatment for 5 h (right). Flag was immunoprecipitated from 1‐μg samples of cell lysates with 40 μL of anti‐Flag M2 Affinity gel and Western blotted with the indicated antibodies. HSP90 was used as the loading control. Phospho‐Ser/Thr, phosphorylated serine/threonine. (M) Immunoprecipitation‐Western blotting for analysis of polyubiquitinated Snail in A549 cells co‐transfected with HA‐ubiquitin and wild‐type Snail (pFLAG‐Snail WT) or an acetylation‐deficient Snail mutant (pFLAG‐SnailK146R) with or without 25 μmol/L C646 treatment for 2 h (left) or 3 μmol/L SAHA treatment for 5 h (right). Flag was immunoprecipitated from 1‐μg samples of cell lysates with 40 μL of anti‐Flag M2 Affinity gel and Western blotted with the indicated antibodies. HSP90 was used as the loading control. All experiments were performed in duplicate unless otherwise indicated. The data are presented as mean ± SD, unless otherwise indicated. Significant differences between groups were determined by a two‐sided unpaired Student's t ‐test. * P < 0.05, ** P < 0.01, *** P < 0.001, NS : not significant. Abbreviations: ACLY, ATP citrate lyase; HSP90, heat shock protein 90; DMSO, dimethyl sulfoxide; CQ, chloroquine; NC, negative control; ATG5, autophagy related 5; TFEB, transcription factor EB;; HDAC, histone deacetylase; SAHA, suberoylanilide hydroxamic acid; PCR, polymerase chain reaction; CREBBP, CREB Binding Protein; IB, immunoblot; IP, immunoprecipitation; HA, hemagglutinin; UB, ubiquitin

Article Snippet: Rabbit monoclonal anti‐heat shock protein 90 (HSP90) (#4877, Cell Signaling Technology, RRID: AB_2233307), mouse monoclonal anti‐β‐actin (#sc‐47778, Santa Cruz, RRID: AB_2714189), rabbit monoclonal anti‐microtubule‐associated protein 1 light chain 3 alpha (LC3) (#3868, Cell Signaling Technology, RRID: AB_2137707), rabbit monoclonal anti‐Vimentin (#5741, Cell Signaling Technology, RRID: AB_10695459) were used at a 1:3000 dilution.

Techniques: Derivative Assay, Western Blot, Control, Immunoprecipitation, Knockdown, Real-time Polymerase Chain Reaction, Gene Expression, Invasion Assay, Expressing, Transfection, Mutagenesis, Ubiquitin Proteomics, Negative Control, Histone Deacetylase Assay, Polymerase Chain Reaction, Binding Assay

Journal: Cancer Communications

Article Title: Snail acetylation by autophagy‐derived acetyl‐coenzyme A promotes invasion and metastasis of KRAS ‐ LKB1 co‐mutated lung cancer cells

doi: 10.1002/cac2.12332

Figure Lengend Snippet: Elevated TFEB acetylation triggers a positive feedback loop to activate autophagy. (A) Effect of ectopic expression of wild‐type TFEB or acetylation‐deficient TFEB[4KR] for 24 h on the relative luciferase activity in hb30‐KP cells transiently co‐transfected with a CLEAR motif‐driven luciferase reporter construct (left). Relative luciferase activity after the addition of 3 μmol/L SAHA for the last 5 h under identical conditions (right). (B) Steady‐state levels of TFEB and phospho‐TFEB (Ser211) in hb30‐KP and hb30‐KPL cells, as determined by Western blotting (left) and immunoprecipitation‐Western blotting analysis of acetylated TFEB in hb30‐KP and hb30‐KPL cells (right). Beta‐actin was used as the loading control. (C) Detection of steady‐state TFEB expression in hb30‐KP and hb30‐KPL cells by immunofluorescence (scale bar, 50 μm). Analyses were performed with anti‐TFEB antibody (green). DAPI was used to visualize nuclei (left). The bar plots show the numbers of hb30‐KP and hb30‐KPL cells with nuclear TFEB (four independent experiments, right). (D) Detection of TFEB expression in hb30‐KPL cells upon treatment with 10 mmol/L acetate for 24 h or 3 μmol/L SAHA for 5 h by immunofluorescence (scale bar, 50 μm). Analyses were performed with anti‐TFEB antibody (green). DAPI was used to visualize nuclei. Arrows indicate cells with nuclear‐localized TFEB. (left). The bar plots show the number of hb30‐KPL cells with nuclear TFEB (four independent experiments, right). (E) Effect of treatment with 10 mmol/L acetate for 24 h or 3 μmol/L SAHA for 5 h on relative luciferase activity in hb30‐KPL cells stably transfected with a CLEAR motif‐driven luciferase reporter construct. The bar plots show the mean ± SD of three (left panel) and two (right panel) independent experiments. (F) The influence of treatment with 10 mmol/L acetate for 24 h or 3 μmol/L SAHA for 5 h with or without 50 μmol/L CQ for the last 3 h on LC3‐II accumulation in hb30‐KPL cells was assessed by Western blotting. Beta‐actin was used as the loading control. Values obtained from a quantitative densitometric analysis of LC3‐II relative to beta‐actin are shown underneath the LC3 blots. se, short exposure; le, long exposure. (G) mRFP‐GFP‐LC3 lysosome delivery assay in hb30‐KPL cells in response to 10 mmol/L acetate for 24 h (left) or 3 μmol/L SAHA treatment for 15 h (right). Representative GFP‐LC3, RFP‐LC3 and overlay images are shown. The number of red puncta (mRFP+/GFP−) and yellow puncta (mRFP+/GFP+) per cell was quantified (15–30 cells/treatment). (H) The effect of exposure of hb30‐KPL cells to 10 mmol/L acetate for 24 h on the accumulation of the indicated proteins was determined by Western blotting. Beta‐actin was used as the loading control (left). Immunoprecipitation‐Western blotting analysis of acetylated TFEB under the same conditions in hb30‐KPL cells (right). (I) The effect of exposure of hb30‐KPL cells to 3 μmol/L SAHA for 18 h on the accumulation of the indicated proteins was determined by Western blotting. Beta‐actin was used as the loading control (left). Immunoprecipitation‐Western blotting analysis of acetylated TFEB under identical conditions in hb30‐KPL cells (right). (J) Real‐time PCR analyses of relative TFEB gene expression in hb30‐KPL cells exposed to 10 mmol/L acetate (2 RNA samples) for 24 h or 3 μmol/L SAHA (3 RNA samples) for 5 h. (K) Immunoprecipitation‐Western blotting analysis of acetylated TFEB upon ACLY knockdown in hb30‐KPL cells (lower left). The effect of 2 μmol/L rapamycin (for 5 h) with or without ACLY knockdown on relative luciferase activity in hb30‐KPL cells stably transfected with a CLEAR motif‐driven luciferase reporter construct (lower right). The schematic in the upper panel shows the mode of action of the tested compounds. (L) The effect of exposure of hb30‐KPL cells to 1 μmol/L FK506 (last 1 h) with or without 10 mmol/L acetate and 3 μmol/L SAHA for 15 h on the accumulation of the indicated proteins was determined by Western blotting (left). Beta‐actin was used as the loading control. The relative luciferase activity in hb30‐KPL cells stably transfected with a CLEAR motif‐driven luciferase reporter construct was measured under the same conditions (right). (M) Immunoprecipitation‐Western blotting analysis of acetylated TFEB in hb30‐KPL cells upon treatment with 50 μmol/L STO‐609 for 24 h. Beta‐actin was used as the loading control. (N) The effect of exposure of hb30‐KPL cells to a combination of 10 mmol/L acetate or 3 μmol/L SAHA and 50 μmol/L CQ for 24 h on TFEB protein accumulation was determined by Western blotting. Beta‐actin was used as the loading control. All experiments were performed in duplicate unless otherwise indicated. The data are presented as mean ± SD, unless otherwise indicated. Significant differences between groups were determined by a two‐sided unpaired Student's t ‐test. * P < 0.05, ** P < 0.01, *** P < 0.001, NS : not significant. Abbreviations: TFEB, transcription factor EB; CLEAR, coordinated lysosomal expression and regulation; TFEB RE, TFEB reporter; DAPI, 4′,6‐diamidino‐2‐phenylindole; SAHA, suberoylanilide hydroxamic acid; CQ, chloroquine; mRFP, monomeric red fluoresecnet protein; GFP, green fluorescent protein; LC3B, microtubule‐associated protein 1A/1B‐light chain 3B; PCR. polymerase chain reaction; IP, immunoprecipitation; IB, immunoblot; Rapa, Rapamycin; ACLY, ATP citrate lyase; HDAC, histone deacetylase; HSP90, heat shock protein 90; DMSO, dimethyl sulfoxide

Article Snippet: Rabbit monoclonal anti‐heat shock protein 90 (HSP90) (#4877, Cell Signaling Technology, RRID: AB_2233307), mouse monoclonal anti‐β‐actin (#sc‐47778, Santa Cruz, RRID: AB_2714189), rabbit monoclonal anti‐microtubule‐associated protein 1 light chain 3 alpha (LC3) (#3868, Cell Signaling Technology, RRID: AB_2137707), rabbit monoclonal anti‐Vimentin (#5741, Cell Signaling Technology, RRID: AB_10695459) were used at a 1:3000 dilution.

Techniques: Expressing, Luciferase, Activity Assay, Transfection, Construct, Western Blot, Immunoprecipitation, Control, Immunofluorescence, Stable Transfection, Real-time Polymerase Chain Reaction, Gene Expression, Knockdown, Polymerase Chain Reaction, Histone Deacetylase Assay

Journal: Life Science Alliance

Article Title: Oxidised metabolites of the omega-6 fatty acid linoleic acid activate dFOXO

doi: 10.26508/lsa.201900356

Figure Lengend Snippet: (A) Tumour incidence in heterozygous hop Tum females flies was decreased by reduction in dFOXO ( Foxo Δ94 ) or Bsk ( bsk 1 ) levels but increased by removal of Puc ( puc E69 ). (B) JNK activation was confirmed by Western analysis of human HeLa and Drosophila S2 cells 24 h after 9-(S)-HODE treatment using anti-pJNK antibodies. MAb E7 anti-tubulin is used as a loading control. Expected pJNK (p54 and p56) species in S2 extracts are indicated. (C) Quantitation of pJNK levels in 9-(S)-HODE–treated cells relative to mock-treated control. (D) Confocal immunofluorescence microscopy of mock and 9-(S)-HODE–treated S2 cells using anti-pJNK anti-dFOXO antibodies confirms JNK activation after 9-(S)-HODE treatment. (E) Quantitation of pJNK signals in S2 cells. (F) Fat body–specific over-expression of dominant-negative Bsk variants or RNAi-mediated inhibition bsk reduces 9-(S)-HODE stimulated dFOXO-mCherry nuclear entry. (G) Quantitation of dFOXO-mCherry nuclear signal in fat body tissue. (H) Pretreatment with JNK inhibitor SP600125 prevents nuclear dFOXO localisation after 9-(S)-HODE treatment. (I) Quantitation of dFOXO nuclear signal in S2 cells after treatment with 9-(S) HODE alone or with the JNK inhibitor SP600125. (J) 9-(S) HODE treatment of S2 cells does not increase reactive oxygen species as revealed by CellROX staining. (K) Reactive oxygen species are not required for 9-(S)-HODE–triggered dFOXO nuclear localisation as simultaneous treatment with N-acetyl-L-cysteine (NAC) does not prevent nuclear entry. (L) Proposed model indicating how 9-HODE activation of FOXO could confer insulin resistance. Data information: in (A), 10 replicate crosses were used for each assay point. Box and whiskers plots were generated using R. * indicates values statistically significantly different from unsupplemented, P -value < 0.001. In (E, G, I, K), 50 determinations were used for each assay point. Box and whiskers plots were generated using R. * indicates values statistically significantly different from unsupplemented, P -value < 0.001 determined using t test.

Article Snippet: Primary antibodies used were rabbit anti-dFOXO ( ) (1:1,000) and mouse anti-pJNK (phospho-SAPK/JNK (Thr183Tyr185) MAb G9, CST, 1:100).

Techniques: Activation Assay, Western Blot, Quantitation Assay, Immunofluorescence, Microscopy, Over Expression, Dominant Negative Mutation, Inhibition, Staining, Generated

Journal: eLife

Article Title: Nanchangmycin regulates FYN, PTK2, and MAPK1/3 to control the fibrotic activity of human hepatic stellate cells

doi: 10.7554/eLife.74513

Figure Lengend Snippet: ( A ) Kinase array analysis of HSCs treated with DMSO or 1 µM NCMC for 1 or 18 hr. Blue bars indicate mean fold change (n=2) in phosphorylation at specified sites in NCMC-treated cells compared to DMSO-treated cells at 1 hr (light blue) or 18 hr (dark blue). Red triangles indicate the mean RPKM of each corresponding kinase mRNA based on RNA sequencing of HSCs . The three dotted lines represent 20% increase/decrease or no change in phosphorylation. Kinases highlighted in red were chosen for further investigation. ( B ) The expression of each candidate kinase gene was depleted using pooled siRNAs, and after 72 hr, COL1A1 level was determined by qRT-PCR in HSCs isolated from human donor 1 (top), 3 (middle), or 4 (bottom). A non-targeting siRNA is used as a control (NTC). Error bars represent mean ± SEM (n≥4, as indicated by the number of dots). ns indicates not significant (p>0.05), * indicates p<0.05, ** indicates p<0.01, *** indicates p<0.001, and **** indicates p<0.0001 (one-way ANOVA test). ( C ) Knockdown efficiency of each siRNA pool in HSCs from human donor 1. Error bars represent mean ± SEM (n≥4). ns indicates not significant (p>0.05), * indicates p<0.05, ** indicates p<0.01, *** indicates p<0.001, and **** indicates p<0.0001 (Student’s t-test performed for FYN , HSPB1 , and PTK2 depletion, and one-way ANOVA test performed for MAPK1 , MAPK3 , STAT5A, and STAT5B depletion). One experiment was performed independently for each HSC line shown. This figure has one supplement.

Article Snippet: Human HSPB1/HSP27 , Hs00356629_g1.

Techniques: Phospho-proteomics, RNA Sequencing, Expressing, Quantitative RT-PCR, Isolation, Control, Knockdown

Journal: eLife

Article Title: Nanchangmycin regulates FYN, PTK2, and MAPK1/3 to control the fibrotic activity of human hepatic stellate cells

doi: 10.7554/eLife.74513

Figure Lengend Snippet:

Article Snippet: Human HSPB1/HSP27 , Hs00356629_g1.

Techniques: Dominant Negative Mutation, Mutagenesis, Amplification, Plasmid Preparation, Isolation, Recombinant, Generated, Expressing, Sequencing, Cloning, Control, Real-time Polymerase Chain Reaction, Software

Journal: eLife

Article Title: Nanchangmycin regulates FYN, PTK2, and MAPK1/3 to control the fibrotic activity of human hepatic stellate cells

doi: 10.7554/eLife.74513

Figure Lengend Snippet:

Article Snippet: Human HSPB1/HSP27 , Hs00356629_g1.

Techniques: