Journal: Molecular and Cellular Biology

Article Title: Targeting SQSTM1/p62 Induces Cargo Loading Failure and Converts Autophagy to Apoptosis via NBK/Bik

doi: 10.1128/MCB.01383-13

Figure Lengend Snippet: BH3 mimetics induce autophagy and apoptosis in the presence of Cdk inhibitors. (A) Human myeloma U266 cells were exposed to 500 nM GX-015-070 (GX) with or without 100 nM flavopiridol (FP) or 4 nM bortezomib (btz) as a positive control, followed by immunoblot analysis for LC3 (16 h) and PARP cleavage (24 h). In parallel, U266 cells were stably transfected with pEGFP-LC3, followed by exposure (16 h) to 500 nM GX with or without 100 nM FP, and then analyzed for GFP-LC3 puncta by confocal microscopy (bar = 10 μm). F+G, FP plus GX; CF, cleaved fragment; UT, untreated; DAPI, 4′,6-diamidino-2-phenylindole. (B) U266 cells were exposed (24 h) to 500 nM GX with or without 100 nM FP, followed by TUNEL staining using fluorescence microscopy (bar = 40 μm). (C) U266 cells were treated (16 h) with 500 nM GX with or without 100 nM FP in the presence or absence of 10 nM bafilomycin A1 (Baf A1), followed by analysis of autophagic flux by immunoblotting for LC3 (top). LC3-II was quantified relative to actin levels (bottom). Results represent fold increases over the vehicle-treated control (Veh) (means ± SD for three experiments). (D) Alternatively, U266 cells were transiently transfected with a pBABE-puro mCherry-EGFP-LC3B plasmid. After 6 h, cells were treated with 500 nM GX with or without 100 nM FP for an additional 16 h, followed by analysis of autophagic flux by confocal microscopy (bar = 5 μm).

Article Snippet: Human multiple myeloma U266 and RPMI8226 cells were obtained from the ATCC and maintained as described previously ( 5 ), and both cell lines were authenticated (Basic STR Profiling Service, ATCC 135-X) by the ATCC before this study was completed.

Techniques: Positive Control, Western Blot, Stable Transfection, Transfection, Confocal Microscopy, TUNEL Assay, Staining, Fluorescence, Microscopy, Control, Plasmid Preparation

Journal: Molecular and Cellular Biology

Article Title: Targeting SQSTM1/p62 Induces Cargo Loading Failure and Converts Autophagy to Apoptosis via NBK/Bik

doi: 10.1128/MCB.01383-13

Figure Lengend Snippet: Cotreatment with BH3 mimetics and Cdk inhibitors results in inefficient autophagy. (A) U266 cells were exposed (16 h) to 500 nM GX with or without 100 nM FP and then examined by electron microscopy (bar = 1 μm). Asterisks indicate deformed mitochondria. N, nucleus; M, mitochondrion; L, lysosome; G, Golgi apparatus; C, centrosome; A, autophagosome; AL, autolysosome; AV, autophagic vacuoles with clear content (empty). (B) In parallel, a filter trap assay using dot or slot blots probed with an antiubiquitin antibody (α-ubi) was performed to monitor the intracellular accumulation of SDS-insoluble ubiquitin-positive protein aggregates. (C) U266 cells were treated (16 h) with 500 nM GX with or without 100 nM FP or 5 nM SCH727965, after which immunoblot analysis was performed to monitor the accumulation of polyubiquitinated (polyUbi) proteins using an antiubiquitin antibody. (D and E) Alternatively, cells were stained with antiubiquitin antibody (D) to monitor intracellular ubiquitin-positive protein aggregates (arrows) or with LysoTracker (E) to visualize lysosomes (arrows).

Article Snippet: Human multiple myeloma U266 and RPMI8226 cells were obtained from the ATCC and maintained as described previously ( 5 ), and both cell lines were authenticated (Basic STR Profiling Service, ATCC 135-X) by the ATCC before this study was completed.

Techniques: Electron Microscopy, TRAP Assay, Ubiquitin Proteomics, Western Blot, Staining

Journal: Molecular and Cellular Biology

Article Title: Targeting SQSTM1/p62 Induces Cargo Loading Failure and Converts Autophagy to Apoptosis via NBK/Bik

doi: 10.1128/MCB.01383-13

Figure Lengend Snippet: Cdk inhibition downregulates SQSTM1/p62 but fails to affect LC3 processing during autophagy. (A) U266 cells were treated with 500 nM GX with or without 100 nM FP for 6, 16, 24, and 48 h, after which immunoblot analysis was performed to monitor LC3 processing and p62 expression. (B) Blots of p62 were quantified relative to tubulin (Tub) values (fold increase over the vehicle-treated control) (results represent means ± SD for three experiments). (C) RPMI8226 and U266 cells were treated (16 h) with GX (500 nM) with or without FP (100 nM) or SCH727965 (5 nM), after which LC3-II and p62 levels were determined by immunoblot analysis. (D) U266 cells were treated (6 h) with 500 nM GX (as indicated on the x axis) with or without 100 nM FP, after which qPCR was used to monitor p62 mRNA levels (fold increase over the vehicle-treated control) (means ± SD for three experiments). (E) U266 cells were stably transfected with constructs encoding shRNA targeting Cdk9 (left) or its partner cyclin T1 (right), which form the P-TEFb complex, and a scrambled sequence (scr) as a negative control. The cells were then exposed (16 h) to GX, followed by immunoblot analysis to monitor the expression of Cdk9 (p42 and p55 isoforms) or cyclin T1, CTD phosphorylation (p-CTD) (serine 2) of RNA polymerase II, LC3 processing, and p62 expression. The vertical lines (LC3) indicate where additional sample lanes were removed from the images; the horizontal line (phosphorylated CTD and cyclin T1) indicates the splice site in the composite image derived from a single blot.

Article Snippet: Human multiple myeloma U266 and RPMI8226 cells were obtained from the ATCC and maintained as described previously ( 5 ), and both cell lines were authenticated (Basic STR Profiling Service, ATCC 135-X) by the ATCC before this study was completed.

Techniques: Inhibition, Western Blot, Expressing, Control, Stable Transfection, Transfection, Construct, shRNA, Sequencing, Negative Control, Phospho-proteomics, Derivative Assay

Journal: Molecular and Cellular Biology

Article Title: Targeting SQSTM1/p62 Induces Cargo Loading Failure and Converts Autophagy to Apoptosis via NBK/Bik

doi: 10.1128/MCB.01383-13

Figure Lengend Snippet: SQSTM1/p62 downregulation results in cargo loading failure and inefficient autophagy. (A) U266 cells were exposed (16 h) to 100 nM FP plus 500 nM GX in the presence or absence of 7.5 μM spautin-1 (SPT) or 500 μM 3-methyladenine (3-MA), after which LC3 processing and p62 expression were monitored by immunoblot analysis. The vertical line (p62) indicates where additional sample lanes were removed from the image. Values indicate quantification of p62 relative to values for tubulin (fold increase over the untreated control). (B) U266 cells were stably transfected with constructs encoding shRNA targeting Ulk1 or a scrambled sequence as a negative control. Cells were then exposed (16 h) to 500 nM GX with or without 100 nM FP, followed by immunoblot analysis using the indicated antibodies. The vertical line (LC3) indicates where additional sample lanes were removed from the image. Values indicate quantification of p62 relative to tubulin values (fold increase over the untreated control). (C) U266 cells were stably transfected with constructs encoding shRNA targeting p62 or a scrambled sequence and then treated (16 h) with the indicated concentrations of GX (nM), followed by immunoblot analysis for p62 expression, LC3 processing, and intracellular accumulation of polyubiquitinated proteins. (D) U266 cells stably transfected with shRNA directed against p62, Ulk1, Cdk9, cyclin T1, or the scrambled sequence as a control were exposed (16 h) to 500 nM GX in the presence or absence of 100 nM FP or 5 nM SCH727965, followed by a filter trap assay using an antiubiquitin antibody to monitor ubiquitin-positive protein aggregates. Values indicate quantification of the amount of total ubiquitin-positive proteins in SDS-insoluble aggregates (values represent fold increases over the untreated controls of shNC cells). (E) U266 cells stably transfected with shRNA directed against Cdk9, p62, or the scrambled sequence as a control were exposed (16 h) to 500 nM GX and examined by electron microscopy (bar = 2 μm).

Article Snippet: Human multiple myeloma U266 and RPMI8226 cells were obtained from the ATCC and maintained as described previously ( 5 ), and both cell lines were authenticated (Basic STR Profiling Service, ATCC 135-X) by the ATCC before this study was completed.

Techniques: Expressing, Western Blot, Control, Stable Transfection, Transfection, Construct, shRNA, Sequencing, Negative Control, TRAP Assay, Ubiquitin Proteomics, Electron Microscopy

Journal: Molecular and Cellular Biology

Article Title: Targeting SQSTM1/p62 Induces Cargo Loading Failure and Converts Autophagy to Apoptosis via NBK/Bik

doi: 10.1128/MCB.01383-13

Figure Lengend Snippet: Expression of SQSTM1/p62 diminishes the increased lethality of BH3 mimetics in p62-defective cells. (A and B) U266 cells stably transfected with shRNA directed against Cdk9 or a scrambled sequence (A) or wild-type (p62+/+) and p62 knockout (p62−/−) MEFs (B) were transiently transfected with a pBABE-puro mCherry-EGFP-LC3B plasmid. After 6 h, cells were treated with GX (U266 cells, 500 nM; MEFs, 200 nM) for an additional 16 h, followed by analysis of autophagic flux using confocal microscopy (bar = 5 μm [A] or 10 μm [B]). Values indicate the number of autophagosomes (A) (yellow) and autolysosomes (AL) (red). (C) U266 cells stably transfected with p62 shRNA were transiently transfected with a construct encoding GFP-tagged p62 or GFP. After 6 h, cells were treated (24 h) with 500 nM GX, followed by 7-aminoactinomycin D (7AAD) staining to monitor cell death by confocal microscopy (left). Arrows indicate GFP-positive/7AAD-negative cells. Dead (7AAD-positive) cells in the GFP-positive population were then quantified by using flow cytometry (right). (D) Immunoblotting analysis was performed to validate p62 expression in wild-type and p62 ko MEFs (inset). MEFs (left, wt; right, p62 ko) were then exposed to 200 nM GX with or without 100 nM FP for 24 h, followed by 7AAD staining to determine the percentage of cell death by flow cytometry. (E) p62 ko MEFs were transiently transfected with GFP-tagged p62 or GFP (inset) (bar = 30 μm). After 6 h, cells were treated with 200 nM GX with or without 100 nM FP, after which the percentage of cell death was determined by flow cytometry (left, GFP; right, GFP-p62) as described above.

Article Snippet: Human multiple myeloma U266 and RPMI8226 cells were obtained from the ATCC and maintained as described previously ( 5 ), and both cell lines were authenticated (Basic STR Profiling Service, ATCC 135-X) by the ATCC before this study was completed.

Techniques: Expressing, Stable Transfection, Transfection, shRNA, Sequencing, Knock-Out, Plasmid Preparation, Confocal Microscopy, Construct, Staining, Flow Cytometry, Western Blot

Journal: Molecular and Cellular Biology

Article Title: Targeting SQSTM1/p62 Induces Cargo Loading Failure and Converts Autophagy to Apoptosis via NBK/Bik

doi: 10.1128/MCB.01383-13

Figure Lengend Snippet: Cdk9 inhibition upregulates NBK/Bik in cells exposed to BH3 mimetics. (A and B) U266 and RPMI8226 cells were treated (24 h) with the indicated concentrations of GX with or without FP (A), SCH727965 (5 nM) (B), or bortezomib (btz) (4 nM) as a positive control, after which immunoblot analysis was performed to monitor Bik expression and/or PARP cleavage. *, nonspecific band. (C) U266 cells stably transfected with shRNA directed against Cdk9, cyclin T1, or a scrambled sequence as a control were exposed (24 h) to 500 nM or 750 nM GX, followed by immunoblot analysis to monitor the expression of Bik and cleavage of caspase 3 and PARP. (D) After U266 cells were treated (24 h) with the indicated concentrations of GX (nM) with or without FP (nM), the ER membrane fraction was isolated and subjected to immunoblot analysis to monitor the subcellular localization of Bik. The same membranes were probed by an anticalnexin antibody as a loading control for ER membranes. (E) U266 cells were treated (16 h) with 500 nM GX plus 100 nM FP in the presence or absence of 1 μM CHX (top) or 300 nM MG-132 (bottom), followed by immunoblot analysis to monitor Bik expression.

Article Snippet: Human multiple myeloma U266 and RPMI8226 cells were obtained from the ATCC and maintained as described previously ( 5 ), and both cell lines were authenticated (Basic STR Profiling Service, ATCC 135-X) by the ATCC before this study was completed.

Techniques: Inhibition, Positive Control, Western Blot, Expressing, Stable Transfection, Transfection, shRNA, Sequencing, Control, Membrane, Isolation

Journal: Molecular and Cellular Biology

Article Title: Targeting SQSTM1/p62 Induces Cargo Loading Failure and Converts Autophagy to Apoptosis via NBK/Bik

doi: 10.1128/MCB.01383-13

Figure Lengend Snippet: NBK/Bik upregulation occurs during autophagy in association with loading failure. (A) U266 cells were treated with the indicated concentrations of GX with or without FP (100 nM) for 6, 16, 24, and 48 h, after which Bik expression was monitored by immunoblot analysis. (B) Blots of Bik after treatment with 500 nM GX with or without FP were quantified relative to tubulin values (values represent fold increases over the vehicle-treated controls) (means ± SD for three experiments). (C) U266 cells were cotreated (24 h) with 500 nM GX and 100 nM FP in the presence or absence of 7.5 μM spautin-1 (SPT), 50 μM CQ, or 500 μM 3-MA. After treatment, Bik expression and PARP cleavage were determined by immunoblot analysis. (D and E) U266 cells stably transfected with shRNA directed against Ulk1 (D), beclin-1 and Atg5 (E), or the scrambled sequence as a control were exposed (24 h) to 500 nM GX with or without 100 nM FP, followed by immunoblot analysis to monitor Bik expression and PARP cleavage. Vertical lines indicate where additional sample lanes were removed from the images. (F) U266 cells stably transfected with shRNA of p62 or the scrambled sequence as a control were exposed (24 h) to 500 or 750 nM GX, followed by immunoblot analysis for Bik expression and PARP cleavage. (G) U266 cells were exposed (16 h) to 500 nM GX with or without 100 nM FP or 5 nM SCH727965 (top), and U266 cells stably transfected with shRNA directed against p62, Cdk9, cyclin T1, or the scrambled sequence as a control were treated (16 h) with 500 nM GX with or without 100 nM FP or 5 nM SCH727965 (bottom). After drug treatment, a filter trap assay using anti-Bik antibody (α-Bik) was performed to monitor the amount of Bik in SDS-insoluble protein aggregates. (H) U266 cells were exposed (16 h) to 500 nM GX with or without 100 nM FP or 5 nM SCH727965, followed by immunoprecipitation (IP) using anti-Bik antibody and subsequent immunoblot (IB) analysis using antiubiquitin antibody (α-ubi). Immunoprecipitations without anti-Bik antibody (− Ab) (lane 1) or cell lysate (− lysate) (lane 2) were used as controls. IgG(H), IgG heavy chain.

Article Snippet: Human multiple myeloma U266 and RPMI8226 cells were obtained from the ATCC and maintained as described previously ( 5 ), and both cell lines were authenticated (Basic STR Profiling Service, ATCC 135-X) by the ATCC before this study was completed.

Techniques: Expressing, Western Blot, Stable Transfection, Transfection, shRNA, Sequencing, Control, TRAP Assay, Immunoprecipitation

Journal: Molecular and Cellular Biology

Article Title: Targeting SQSTM1/p62 Induces Cargo Loading Failure and Converts Autophagy to Apoptosis via NBK/Bik

doi: 10.1128/MCB.01383-13

Figure Lengend Snippet: NBK/Bik plays a functional role in triggering apoptosis in vitro and in vivo. (A and B) U266 cells were stably transfected with a construct encoding shRNA directed against Bik or a scrambled sequence as a control and then treated (24 h) with 500 nM GX with or without 100 nM FP or 4 nM bortezomib (btz) as a positive control. Immunoblot analysis (A) and flow cytometry (B) were conducted to monitor the expression of the indicated proteins or to determine percentage of apoptotic (annexin V-positive) cells (values represent means ± SD for three experiments). (C) Athymic NCr-nu/nu mice subcutaneously inoculated in the flank with 5 × 106 RPMI8226 cells were treated with GX (3 mg/kg of body weight intramuscularly) with or without FP (5 mg/kg i.p.). Tumors were excised at day 28 after tumor cell inoculation and then homogenized and subjected to immunoblot analysis using the indicated antibodies. (D) NOD/SCID/gamma (NSG) mice were subcutaneously inoculated in each flank with 1 × 107 U266 cells stably transfected with shRNA directed against Bik (right flank) or a scrambled sequence as a control (left flank). FP (3 mg/kg i.p.) with or without GX (3 mg/kg i.p.) was then administered daily for a total of 11 days. Images of tumors removed from two representative mice were captured at day 49 after tumor cell inoculation (bar = 20 mm). (E) NSG mice were injected i.v. via the tail vein with 5 × 106 U266 cells carrying luciferase, after which FP (3 mg/kg i.p.) with or without GX (3 mg/kg i.p.) was administered daily for a total of 19 days. The bioluminescent images of representative mice were captured at day 35 after inoculation of cells. Lumbar vertebrae removed from mice were immunohistochemically (IHC) stained with anti-human CD138 antibody (bar = 10 μm) or hematoxylin and eosin (H.E.) (bar = 20 μm). m, megakaryocyte; v, blood vessel.

Article Snippet: Human multiple myeloma U266 and RPMI8226 cells were obtained from the ATCC and maintained as described previously ( 5 ), and both cell lines were authenticated (Basic STR Profiling Service, ATCC 135-X) by the ATCC before this study was completed.

Techniques: Functional Assay, In Vitro, In Vivo, Stable Transfection, Transfection, Construct, shRNA, Sequencing, Control, Positive Control, Western Blot, Flow Cytometry, Expressing, Injection, Luciferase, Staining

Journal: ACS Omega

Article Title: Hydrogel-Mediated Release of TRPV1 Modulators to Fine Tune Osteoclastogenesis

doi: 10.1021/acsomega.1c06915

Figure Lengend Snippet: Expression and functional analysis of TRPV1 in BMMs and Osteoclasts. (a) BMMs stained for the macrophage marker CD11b (red) and TRPV1 (green) depict the latter’s expression in these cells, both in the absence (upper panel) and presence (lower panel) of RANKL. (b) Expression of TRPV1 (red) in phalloidin-stained osteoclasts (green, upper panel) is confirmed by a peptide segment against anti-TRPV1 antibody (lower panel) that reduces the specific fluorescence signal intensity of the TRPV1 channel.

Article Snippet: For confirming the specificity of the antibody, TRPV1 epitope-specific peptide (Alomone Labs) was used.

Techniques: Expressing, Functional Assay, Staining, Marker, Fluorescence

Journal: ACS Omega

Article Title: Hydrogel-Mediated Release of TRPV1 Modulators to Fine Tune Osteoclastogenesis

doi: 10.1021/acsomega.1c06915

Figure Lengend Snippet: Functional analysis of TRPV1 in BMMs. (a) BMMs were assessed for intracellular Ca 2+ levels upon TRPV1 modulation. Representative intensity profiles of Fluo4-AM intensity at different frames are indicated. (b) Time series graphs of intracellular Fluo4-AM intensities across 200 frames of live imaging. The arrow at the x-axis signifies the time of addition of the respective drugs (20th frame). Gray traces are of individual cells, and the black trace represents the average of 50 cells. (c) Compiled average of different treatments of BMMs, individual cell traces omitted.

Article Snippet: For confirming the specificity of the antibody, TRPV1 epitope-specific peptide (Alomone Labs) was used.

Techniques: Functional Assay, Imaging

Journal: ACS Omega

Article Title: Hydrogel-Mediated Release of TRPV1 Modulators to Fine Tune Osteoclastogenesis

doi: 10.1021/acsomega.1c06915

Figure Lengend Snippet: Functional analysis of TRPV1 in BMMs grown on the CMT:HEMA hydrogel. (a) BMMs grown on hydrogels to check for the endogenous levels of Ca 2+ using Fluo4-AM Ca 2+ -sensitive dye. TRPV1 activation elevates the intracellular Ca 2+ levels, as is quantified in (b); n = 100 cells; one-way ANOVA; ns: non-significant, **** p < 0.0001. (c) Correlation representation of the area of cells and per unit area intensity of Fluo4-AM depicts strong positive correlations under basal and TRPV1-activated conditions but not upon inhibition of the channel.

Article Snippet: For confirming the specificity of the antibody, TRPV1 epitope-specific peptide (Alomone Labs) was used.

Techniques: Functional Assay, Activation Assay, Inhibition

Journal: ACS Omega

Article Title: Hydrogel-Mediated Release of TRPV1 Modulators to Fine Tune Osteoclastogenesis

doi: 10.1021/acsomega.1c06915

Figure Lengend Snippet: Morphological analysis of BMMs grown on the hydrogel. (a) Representative images of BMMs grown on glass or hydrogel in the presence of RANKL and TRPV1 modulators. Right panels denote marked inset of respective images. Phalloidin intensity (b) and morphometric analyses of BMM’s area (c), perimeter (d), length (e), width (f), and LWR (g). n = 18–51 cells per group; one-way ANOVA; ns: non-significant, * p < 0.05, *** p < 0.001, **** p < 0.0001.

Article Snippet: For confirming the specificity of the antibody, TRPV1 epitope-specific peptide (Alomone Labs) was used.

Techniques:

Journal: ACS Omega

Article Title: Hydrogel-Mediated Release of TRPV1 Modulators to Fine Tune Osteoclastogenesis

doi: 10.1021/acsomega.1c06915

Figure Lengend Snippet: Differentiation propensities of BMMs into osteoclasts grown on hydrogel. (a) Representative TRAP assay of BMMs grown on the hydrogel in the presence of the TRPV1 activator (RTX) and inhibitor (5′-IRTX) under differentiating conditions (MCSF + RANKL). (b,c) Quantitation of TRAP-positive cells and multinucleated cells in the presence of capsaicin (b) and RTX (c) shows elevated osteoclastogenesis as compared to MCSF and CMT:HEMA control groups. n = 5–10; one-way ANOVA; ** p < 0.01, *** p < 0.005, **** p < 0.001.

Article Snippet: For confirming the specificity of the antibody, TRPV1 epitope-specific peptide (Alomone Labs) was used.

Techniques: TRAP Assay, Quantitation Assay