Journal: Cell death & disease

Article Title: Gossypol and an HMT G9a inhibitor act in synergy to induce cell death in pancreatic cancer cells.

doi: 10.1038/cddis.2013.191

Figure Lengend Snippet: Figure 4 BNIP3 mRNA and protein levels are increased upon compound treatment. (a) Fold change in gene expression in PANC-1 cells treated for 3 days with 2.5 mM gossypol alone or the combination of 2.5 mM gossypol and 2.5 mM BRD4770. The 10 gene transcripts that showed the greatest fold induction between the two conditions are shown. The total RNA was extracted, and the 84 key genes involved in autophagy were profiled by a 384-well format human autophagy pathway-focused expression real-time PCR array. Six housekeeping genes were used as internal controls for data normalization (see the Materials and Methods section). Data for BRD4770 treatment were generated by gene expression profiling (see the Materials and Methods section). (b) Protein levels of BNIP3, BNIP3L, beclin1, and BCL-xL were assessed in PANC-1 cells treated for 3 days with the indicated concentrations of BRD4770. BNIP3 was observed as a 30-kDa monomer and a 60-kDa dimer. Tubulin was used as an internal loading control

Article Snippet: Plasmids encoding human BNIP3 (RC205087) and BNIP3L (RC203315) were obtained from Origene (Rockville, MD, USA). siRNAs (25 nM) and plasmid DNA (0.4 mM) were transfected into PANC-1, mCherry-eGFP-LC3 HeLa, and PC-3 cells using Lipofectamine 2000 reagent (Invitrogen).

Techniques: Gene Expression, Expressing, Real-time Polymerase Chain Reaction, Generated, Control

Journal: Cell death & disease

Article Title: Gossypol and an HMT G9a inhibitor act in synergy to induce cell death in pancreatic cancer cells.

doi: 10.1038/cddis.2013.191

Figure Lengend Snippet: Figure 5 BNIP3 overexpression induces LC3-II expression and autophagosome formation in the presence of gossypol. (a) PANC-1 cells were transiently transfected with BNIP3 and BNIP3L, either individually or in combination. The control cells were transfected with an empty vector. After 24 h, the cells were treated with 2.5 mM gossypol for another 24 h. Tubulin served as an internal loading control. (b) Fluorescence microscopy of mCherry-eGFP-LC3 HeLa cells treated as described in panel (a). Scale bar ¼ 20 mm

Article Snippet: Plasmids encoding human BNIP3 (RC205087) and BNIP3L (RC203315) were obtained from Origene (Rockville, MD, USA). siRNAs (25 nM) and plasmid DNA (0.4 mM) were transfected into PANC-1, mCherry-eGFP-LC3 HeLa, and PC-3 cells using Lipofectamine 2000 reagent (Invitrogen).

Techniques: Over Expression, Expressing, Transfection, Control, Plasmid Preparation, Fluorescence, Microscopy

Journal: Cell death & disease

Article Title: Gossypol and an HMT G9a inhibitor act in synergy to induce cell death in pancreatic cancer cells.

doi: 10.1038/cddis.2013.191

Figure Lengend Snippet: Figure 6 Knockdown of G9a and GLP expression induces BNIP3 expression and cell death. PANC-1 cells were transiently transfected with siRNA against G9a and GLP, both individually and in combination. Scrambled siRNA was used as the negative control. (a) Quantification of the level of knockdown for G9a and GLP by real-time PCR. GAPDH and actin were used to normalize the data. (b) Cellular ATP levels were measured after 3-day treatment with gossypol and either 1.25 mM BRD4770 or siRNA knockdown of G9a and GLP. Data represent the mean and standard error of six independent replicates for each combination. (c) Western blot analysis to examine the protein levels of LC3, BNIP3, and BNIP3L 3 days after transfection. Tubulin served as an internal loading control

Article Snippet: Plasmids encoding human BNIP3 (RC205087) and BNIP3L (RC203315) were obtained from Origene (Rockville, MD, USA). siRNAs (25 nM) and plasmid DNA (0.4 mM) were transfected into PANC-1, mCherry-eGFP-LC3 HeLa, and PC-3 cells using Lipofectamine 2000 reagent (Invitrogen).

Techniques: Knockdown, Expressing, Transfection, Negative Control, Real-time Polymerase Chain Reaction, Western Blot, Control

Journal: Autophagy

Article Title: Mutant HTT (huntingtin) impairs mitophagy in a cellular model of Huntington disease

doi: 10.1080/15548627.2020.1728096

Figure Lengend Snippet: PolyQ tract in mutant HTT does not affect the polyUB labeling of mitochondria. (a) Representative immunoblots of total polyUB (total-UB) and phosphorylated ubiquitin (Ser65; p-UB-S65) in total homogenate and isolated mitochondria from ST-Q7 and ST-Q111 control cells (vehicle, CTR) or treated with rotenone (Rot, 1 μM, 4 h) or CCCP (10 μM, 24 h). Protein levels were normalized relative to Ponceau staining and quantification is depicted as fold-change to control ST-Q7 cells of at least three independent experiments. (b) Representative immunoblots of BNIP3 and BNIP3L in isolated mitochondria from ST-Q7 and ST-Q111 control cells (vehicle, CTR) or treated with rotenone (Rot, 1 μM, 4 h) or CCCP (10 μM, 24 h). Protein levels were quantified using all bands present in each lane independently of the molecular weight or oligomerization state (indicated with line and arrows) and were normalized relative to Ponceau staining and quantification is depicted as fold-change to control ST-Q7 cells. Data are presented as mean ± s.e.m of at least 3 independent experiments and no significant changes were observed after statistical analysis

Article Snippet: OPTN (10837-1-AP) from Proteintech Group, HTT (MAB2166) from Chemicon, SQSTM1/p62 (GP62-C) and NBR1 (H00004077-M01) from Abnova, anti-DNA (AC-30-10) from Progen, MTOR (GT649) mouse (GTX630198) from Genetex, total-UB (U5379) and ACTB/β-actin (A5441) from Sigma-Aldrich, BNIP3 (M01469), BNIP3L (M03107) and FUNDC1 (A08688) from Boster.

Techniques: Mutagenesis, Labeling, Western Blot, Ubiquitin Proteomics, Isolation, Control, Staining, Molecular Weight