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

Journal: Biomedicines

Article Title: The hTERT and iCasp9 Transgenes Affect EOMES and T-BET Levels in NK Cells and the Introduction of Both Genes Improves NK Cell Proliferation in Response to IL2 and IL15 Stimulation

doi: 10.3390/biomedicines12030650

Figure Lengend Snippet: Retroviral transduction of NK cells with hTERT or/and iCasp9 genes. ( A ) The ACTB normalized expression levels of hTERT transgene evaluated by qPCR analysis ( n = 7). * p < 0.05. ( B ) The ACTB normalized expression levels of iCasp9 transgene evaluated by qPCR analysis ( n = 7). Friedman test with Dunn’s multiple comparison test, median, p -value: * p < 0.05; ** p < 0.01. ( C ) Telomerase activity in modified NK cells determined by TRAP assay for qPCR method ( n = 4). The K562 cell line was used as positive control. For comparison, data were divided into 2 groups (1st with hTERT transgenes: hTERT + with hTERT + iCasp9 + ; 2nd without hTERT transgenes: untransduced with iCasp9 + ). Mann–Whitney test, median, p -value: * p < 0.05. ( D ) Proportion of dead NK cells treated for 24 h with 100 nM chemical inductor of dimerization (CID) ( n = 6). NK cells cultured without CID were used as a control. Dead cells were determined as positive for AnnexinV and/or SYTOX staining. The increase in percentage of dead NK cells was calculated by the following formula: Δ% = 100 nM CID-treated dead NK cells %—dead NK cells in control %. Ordinary one-way ANOVA with Tukey’s multiple comparison test, mean, p -value: * p < 0.05; ** p < 0.01.

Article Snippet: The plasmid xlox TERT PGK iCasp9 IRES GFP consisting of both genes was assembled from Addgene #69809 and Addgene #15567.

Techniques: Retroviral, Transduction, Expressing, Comparison, Activity Assay, Modification, TRAP Assay, Positive Control, MANN-WHITNEY, Cell Culture, Control, Staining

Journal: Biomedicines

Article Title: The hTERT and iCasp9 Transgenes Affect EOMES and T-BET Levels in NK Cells and the Introduction of Both Genes Improves NK Cell Proliferation in Response to IL2 and IL15 Stimulation

doi: 10.3390/biomedicines12030650

Figure Lengend Snippet: Proliferation assay started 1 month after ex vivo isolation of NK cells modified with hTERT and/or iCasp9 transgenes. Stimulation with IL2, IL2+K562-mbIL21 and IL2+IL15 was performed. ( A ) Numbers of NK cells cultured for one month with IL2+IL15. Mixed effects analysis, Sidak’s multiple comparisons test, n = 5, median, p -value: * p < 0.05. For IL2, IL2+K562-mbIL21 and IL2+IL15 types of stimulations: ( B ) the proportion of live NK cells and ( C ) the weekly increase n (week+1) /N week are presented. Vertical dashed line stands for a time point of 2 months after isolation. Friedman test with Dunn’s multiple comparisons, n = 7, median, p -value: * p < 0.05; ** p < 0.01; *** p < 0.001. ( D ) The proportions of live NK cells for each type of stimulation in 2 months of ex vivo isolation. ( E ) The comparison of expansion coefficient between IL2, IL2+K562-mbIL21 and IL2+IL15 stimulations in 2 months of ex vivo isolation. Friedman test with Dunn’s multiple comparisons, median, n = 5, p -value: * p < 0.05; ** p < 0.01.

Article Snippet: The plasmid xlox TERT PGK iCasp9 IRES GFP consisting of both genes was assembled from Addgene #69809 and Addgene #15567.

Techniques: Proliferation Assay, Ex Vivo, Isolation, Modification, Cell Culture, Comparison

Journal: Biomedicines

Article Title: The hTERT and iCasp9 Transgenes Affect EOMES and T-BET Levels in NK Cells and the Introduction of Both Genes Improves NK Cell Proliferation in Response to IL2 and IL15 Stimulation

doi: 10.3390/biomedicines12030650

Figure Lengend Snippet: The expression of EOMES and T-BET transcription factors in NK cells modified with hTERT and/or iCasp9 genes. ( A ) NK cells cultivated with IL2+K562-mbIL21 are represented. A change in the proportion of EOMES- and T-BET-expressing cells in 1st and to 2nd month after ex vivo isolation (N ex vivo = 8, N 1 month = 5, N 2 months = 2, mean). ( B ) The distribution of EOMES +/− T-BET +/− subsets of NK cells cultured for 1 month. EOMES − T-BET − , EOMES + T-BET + (for comparison, data were divided into 2 groups (1st: hTERT + with hTERT + iCasp9 + ; 2nd: untransduced with iCasp9 + )), EOMES + T-BET − (for comparison, data were divided into 2 groups (1st: untransduced with hTERT + ; 2nd: hTERT + iCasp9 + with iCasp9 + )), EOMES − T-BET + hTERT and/or iCasp9 modified NK cells a month after ex vivo isolation ( n = 5). Unpaired t -test, mean, p -value: * p < 0.05. ( C ) Representative density plots for EOMES and T-BET distributions in NK cells ex vivo, 1 month and 2 months after isolation.

Article Snippet: The plasmid xlox TERT PGK iCasp9 IRES GFP consisting of both genes was assembled from Addgene #69809 and Addgene #15567.

Techniques: Expressing, Modification, Ex Vivo, Isolation, Cell Culture, Comparison

Journal: Biomedicines

Article Title: The hTERT and iCasp9 Transgenes Affect EOMES and T-BET Levels in NK Cells and the Introduction of Both Genes Improves NK Cell Proliferation in Response to IL2 and IL15 Stimulation

doi: 10.3390/biomedicines12030650

Figure Lengend Snippet: The proportions of NK cells modified with hTERT and/or iCasp9 genes 2 months after ex vivo isolation. Data for IL2, IL2+K562-mbIL21 and IL2+IL15 stimulated NK cells are represented. ( A ) The expression levels of immune checkpoints TIM-3, TIGIT and KLGR-1. Dotted boxes stand to determine the groups compared. Two-way ANOVA with Tukey’s multiple comparisons test, n = 3, mean, p -value: * p < 0.05; ** p < 0.01; *** p < 0.001. ( B ) Surface expression (means) of CD16, KIR (KIR2DL2/DL3), PD-1, NKG2C, CD57, CD56, NKG2A, HLA-DR, NKp30, NKp44 and NKp46 for NK cells stimulated by IL2+K562-mbIL21.

Article Snippet: The plasmid xlox TERT PGK iCasp9 IRES GFP consisting of both genes was assembled from Addgene #69809 and Addgene #15567.

Techniques: Modification, Ex Vivo, Isolation, Expressing

Journal: Biomedicines

Article Title: The hTERT and iCasp9 Transgenes Affect EOMES and T-BET Levels in NK Cells and the Introduction of Both Genes Improves NK Cell Proliferation in Response to IL2 and IL15 Stimulation

doi: 10.3390/biomedicines12030650

Figure Lengend Snippet: The dynamics of normalized mRNA expression levels of the EOMES and TBX21 genes encoding transcription factors EOMES and T-BET along with expression levels of pro-survival BCL2 , MCL1 , BCL2L1 (BCL-X L ) and BIRC5 , pro-apoptotic BAX , BAD , BAK , DIABLO and BBC3 (PUMA), immune-checkpoint-encoding genes TIM3 , TIGIT and LAG3 and exhaustion-associated SOCS1-3 and CISH . Data obtained at time points of 1 month (1 m) and 2 months (2 m) after ex vivo isolation of hTERT and/or iCasp9 modified NK cells cultured with IL2+K562-mbIL21. Dotted boxes stand to highlight groups compared. Mann–Whitney, N1m = 10, N2m = 3, median, p -value: * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: The plasmid xlox TERT PGK iCasp9 IRES GFP consisting of both genes was assembled from Addgene #69809 and Addgene #15567.

Techniques: Expressing, Ex Vivo, Isolation, Modification, Cell Culture, MANN-WHITNEY

Journal: Biomedicines

Article Title: The hTERT and iCasp9 Transgenes Affect EOMES and T-BET Levels in NK Cells and the Introduction of Both Genes Improves NK Cell Proliferation in Response to IL2 and IL15 Stimulation

doi: 10.3390/biomedicines12030650

Figure Lengend Snippet: The functional activity of NK cells modified with hTERT and/or iCasp9 genes a month after their ex vivo isolation. ( A ) The proportion of NK cells accumulating IFNγ in response to IL2, IL12 and IL18 cytokines (Top). The intensity of cytokine-dependent IFNγ production per cell measured by mean fluorescence intensity normalized to FMO control (bottom). ( B ) The fraction of NK cells capable of degranulation measured by LAMP-1 surface exposure in response to the recognition of K562 target cells (Top). The intensity of degranulation in response to K562 cells production per cell is measured by mean fluorescence intensity normalized to basal degranulation (bottom). The cells were also divided into groups I (IFNγ > 50% and MFI LAMP-1 < 5) and II (IFNγ < 50% and MFI LAMP-1 > 5). ( C ) The donor dependency of IFNγ production. Ordinary one-way ANOVA, n = 7 donors.

Article Snippet: The plasmid xlox TERT PGK iCasp9 IRES GFP consisting of both genes was assembled from Addgene #69809 and Addgene #15567.

Techniques: Functional Assay, Activity Assay, Modification, Ex Vivo, Isolation, Fluorescence, Control

Journal: Human molecular genetics

Article Title: IRE1 plays an essential role in ER stress-mediated aggregation of mutant huntingtin via the inhibition of autophagy flux.

doi: 10.1093/hmg/ddr445

Figure Lengend Snippet: Figure 2. ER stress promotes the aggregation of mtHTT via IRE1 activation. (A) ER stress enhances mtHTT aggregation. SH-SY5Y cells were co-transfected with pHTTex120Q-GFP and either pcDNA or pIRE1, and then exposed to DMSO, 1 mm thapsigargin (Tg) or 2 mg/ml tunicamycin (Tuni.) for the indicated times. Cells were then observed under fluorescence microscope (left panel) or cell lysates were examined with western blot analysis using the indi- cated antibodies (right panel). Bars represent mean values+SD from at least three independent experiments. P-values were calculated using t-test and were versus control (∗P , 0.05; ∗∗P , 0.01; ∗∗∗P , 0.001). (B) Down-regulation of IRE1 reduces ER stress-induced mtHTT aggregation. SH-SY5Y/pSuper-Neo (SH/Neo) and SH-SY5Y/pSuper-shIRE1 (SH/shIRE1 #5 and #8) cells were transfected with pHTTex120Q-GFP and incubated with DMSO or 1 mm thapsi- gargin (Tg) for 24 h. Percentages of mtHTT aggregation were determined under fluorescence microscope as in (A) (left panel). Cell extracts were prepared and examined by western blot analysis using the indicated antibodies (right panel). (C) Inhibitory effect of dominant-negative IRE1 on ER stress-induced mtHTT aggregation. After co-transfection with pHTTex120Q-GFP and either pcDNA or each dominant-negative mutant of ER sensor proteins (IRE1 DN, ATF6 DN and PERK DN), SH-SY5Y cells were left untreated or exposed to 1 mm thapsigargin (Tg). The aggregation of mtHTT was examined under fluorescence microscope (left panel) and cell extracts were analyzed with western blotting using anti-HA, anti-GRP78 and anti-b-actin antibodies (right panel). Arrowheads indicate the expression of each construct.

Article Snippet: The following antibodies were used for western blot analyses: p-IRE1 (Abcam), CHOP (Santa Cruz Biotechnology), GRP78 (Santa Cruz Biotechnology), p62 (Abnova), LC3 (Novus Biologicals), ATG5 (Novus Biologicals), Beclin-1 (Novus Biologicals), cleaved caspase-3 (Cell Signaling), caspase-12 (Santa Cruz Biotechnology), a-tubulin (Sigma-Aldrich), b-actin (Sigma-Aldrich), Flag (Sigma-Aldrich) and GFP (Santa Cruz Biotechnology) antibodies.

Techniques: Activation Assay, Transfection, Microscopy, Western Blot, Control, Incubation, Dominant Negative Mutation, Cotransfection, Expressing, Construct

Journal: Human molecular genetics

Article Title: IRE1 plays an essential role in ER stress-mediated aggregation of mutant huntingtin via the inhibition of autophagy flux.

doi: 10.1093/hmg/ddr445

Figure Lengend Snippet: Figure 1. Ectopic expression of IRE1 accumulates mtHTT aggregation. (A) Increased aggregation of mtHTT by IRE1 over-expression. AF5 cells were co-transfected with pHTTex120Q-GFP (mtHTT) and either pcDNA (PCD), pArfaptin2 (Arfaptin2) or pIRE1 (IRE1) for the indicated times and examined for the aggregation under fluorescence microscope. Percentages of aggregation were determined by counting cells showing mtHTT aggregates among total GFP-positive cells. Bars represent mean values+SD from at least three inde- pendent experiments. P-values were calculated using t-test and were versus control (∗∗∗P , 0.001). (B) Detection of insoluble aggregates induced by IRE1. HEK293F cells were co-transfected with pHTTex120Q-GFP and either pcDNA, pArfaptin2 or pIRE1 for 36 h. Cell lysates were prepared, sepa- rated into soluble and insoluble fractions and analyzed by western blotting using anti-GFP or anti-a-tubulin antibody (upper panels). Cells were resus- pended in PBS and a filter trap assay was performed as described in Materials and Methods. The amounts of insoluble mtHTT-GFP aggregates on the blot were examined with western blot analysis using anti-GFP antibody (lower panel). (C) Stimulatory effect of IRE1 on mtHTT aggregation in primary neur- onal cells. Rat primary striatal and cortical neurons were cultured from embry- onic day 16 and maintained for 3 days in vitro. The striatal and cortical neurons were then co-transfected with pHTTex120Q-GFP and either pcDNA, pArfaptin2 or pIRE1 for 24 h. Bars represent mean values+SD (n ¼ 3). (D) Detergent-resistant aggregates of mtHTT formed by IRE1. SH-SY5Y cells were co-transfected with pHTTex120Q-GFP (green) and either pcDNA or pIRE1 for 24 h. Detergent-resistant aggregation assay was then performed using Triton X-100 and SDS (T/S) as described in Materials and Methods. Arrows indicate detergent-resistant aggregates of mtHTT.

Article Snippet: The following antibodies were used for western blot analyses: p-IRE1 (Abcam), CHOP (Santa Cruz Biotechnology), GRP78 (Santa Cruz Biotechnology), p62 (Abnova), LC3 (Novus Biologicals), ATG5 (Novus Biologicals), Beclin-1 (Novus Biologicals), cleaved caspase-3 (Cell Signaling), caspase-12 (Santa Cruz Biotechnology), a-tubulin (Sigma-Aldrich), b-actin (Sigma-Aldrich), Flag (Sigma-Aldrich) and GFP (Santa Cruz Biotechnology) antibodies.

Techniques: Expressing, Over Expression, Transfection, Microscopy, Control, Western Blot, TRAP Assay, Cell Culture, In Vitro

Journal: Human molecular genetics

Article Title: IRE1 plays an essential role in ER stress-mediated aggregation of mutant huntingtin via the inhibition of autophagy flux.

doi: 10.1093/hmg/ddr445

Figure Lengend Snippet: Figure 3. Kinase activity of IRE1 is required for mtHTT aggregation. (A) Schematic diagram of functional domains and various mutants of IRE1. Asterisks indicate amino acid substitution of Lys599 with Ala in serine/threonine kinase domain and of Gly923 with Ala in endoribonuclease domain (left panel). HEK293F cells were transfected with pcDNA (PCD), pIRE1-HA or its mutant for 36 h and cell lysates were analyzed by western blotting using anti-p-IRE1, anti-HA and anti-a-tubulin antibodies. Arrowheads indicate the expression of IRE1 and its mutant and asterisk shows non-specific band, respectively. (B and C) Effects of IRE1 kinase activity on mtHTT aggregation. AF5 cells were co-transfected with pHTTex120Q-GFP and either pcDNA, pIRE1-HA or its mutant for the indicated times and then examined for the aggregation under fluorescence microscope. Bars represent mean values+SD from at least three independent experiments. P-values were calculated using t-test and were versus control (∗P , 0.05; ∗∗∗P , 0.001) (B). HEK293F cells were co-transfected with pHTTex120Q-GFP and either pcDNA, pIRE1-HA or its mutant for 36 h. Cell lysates were separated into soluble and insoluble fractions and the fractions were subjected to western blotting using anti-GFP and anti-a-tubulin antibodies (C).

Article Snippet: The following antibodies were used for western blot analyses: p-IRE1 (Abcam), CHOP (Santa Cruz Biotechnology), GRP78 (Santa Cruz Biotechnology), p62 (Abnova), LC3 (Novus Biologicals), ATG5 (Novus Biologicals), Beclin-1 (Novus Biologicals), cleaved caspase-3 (Cell Signaling), caspase-12 (Santa Cruz Biotechnology), a-tubulin (Sigma-Aldrich), b-actin (Sigma-Aldrich), Flag (Sigma-Aldrich) and GFP (Santa Cruz Biotechnology) antibodies.

Techniques: Activity Assay, Functional Assay, Transfection, Mutagenesis, Western Blot, Expressing, Microscopy, Control

Journal: Human molecular genetics

Article Title: IRE1 plays an essential role in ER stress-mediated aggregation of mutant huntingtin via the inhibition of autophagy flux.

doi: 10.1093/hmg/ddr445

Figure Lengend Snippet: Figure 5. IRE1 links ER stress-induced inhibition of autophagy flux. (A) ER stress impairs autophagy flux. SH-SY5Y cells were treated with DMSO or 1 mm thapsigargin (Tg) for 18 h and then exposed to 20 nM bafilomycin A1 (Baf.A1) for 6 h. Cells were then harvested and cell extracts were subjected to western blotting using the indicated antibodies. (B) Down-regulated IRE1 reduces ER stress-induced p62 accumulation. SH-SY5Y/pSuper-Neo (SH/Neo) and SH-SY5Y/ pSuper-shIRE1 (SH/shIRE1) stable cells were incubated with DMSO or 1 mm thapsigargin (Tg) for the indicated times and cell extracts were then analyzed with western blotting using anti-p62, anti-LC3, anti-p-IRE1, anti-GRP78 and anti-b-actin antibodies. (C) Down-regulation of IRE1 rescues thapsigargin-induced autophagy inhibition. SH-SY5Y/pSuper-Neo (SH/Neo) and SH-SY5Y/pSuper-shIRE1 (SH/shIRE1) cells were transfected with pmCherry-GFP-LC3 and incu- bated with DMSO or 1 mm thapsigargin (Tg) for 24 h. LC3 dots with GFP or mCherry signals were examined under confocal microscope (left panel). The cell numbers showing co-localization of both mCherry and GFP signals were counted and summarized with mean values+SD from at least three independent experiments (right panel). (D) Kinase activity of IRE1 contributes to autophagy inhibition. SH-SY5Y cells were transfected with either pcDNA, pIRE1-HA, pIRE1 DC-HA or pIRE1 DR-HA for 48 h and then harvested for western blot analysis using anti-p62, anti-LC3, anti-HA, anti-GRP78 and anti-b-actin anti- bodies. Cells exposed to 1 mm thapsigargin (Tg) for 24 h were used as a positive control. (E). Dominant-negative TRAF2 suppresses thapsigargin-induced autop- hagy inhibition. SH-SY5Y cells were transfected with pcDNA, pIRE1 DC-HA or pTRAF2 DN for 48 h and then incubated with DMSO or 1 mm thapsigargin (Tg) for 24 h. Cells were collected and analyzed with western blotting using the indicated antibodies.

Article Snippet: The following antibodies were used for western blot analyses: p-IRE1 (Abcam), CHOP (Santa Cruz Biotechnology), GRP78 (Santa Cruz Biotechnology), p62 (Abnova), LC3 (Novus Biologicals), ATG5 (Novus Biologicals), Beclin-1 (Novus Biologicals), cleaved caspase-3 (Cell Signaling), caspase-12 (Santa Cruz Biotechnology), a-tubulin (Sigma-Aldrich), b-actin (Sigma-Aldrich), Flag (Sigma-Aldrich) and GFP (Santa Cruz Biotechnology) antibodies.

Techniques: Inhibition, Western Blot, Incubation, Transfection, Microscopy, Activity Assay, Positive Control, Dominant Negative Mutation

Journal: Human molecular genetics

Article Title: IRE1 plays an essential role in ER stress-mediated aggregation of mutant huntingtin via the inhibition of autophagy flux.

doi: 10.1093/hmg/ddr445

Figure Lengend Snippet: Figure 6. Inhibition of autophagy flux by ER stress regulates mtHTT aggregation. (A and B) An increase in mtHTT aggregation by ER stress or autophagy inhibition was alleviated in IRE1 knock-down cells. SH-SY5Y/pSuper-Neo (SH/Neo) and SH-SY5Y/pSuper-shIRE1 (SH/shIRE1) cells were transfected with pHTTex120Q-GFP and exposed to DMSO or 1 mm thapsigargin (Tg) for 24 h in the presence or absence of 20 nM bafilomycin A1 (Baf.A1). Percentages of mtHTT aggregation were determined by counting cells showing aggregated pHTTex120Q-GFP under fluorescence microscope (A). Cell extracts were prepared and analyzed with western blotting using anti-p62, anti-LC3, anti-p-IRE1, anti-GRP78 and anti-b-actin antibodies (B). Bars represent mean values+SD from at least three independent experiments. P-values were calculated using t-test and were versus control (∗P , 0.05; ∗∗∗P , 0.001). (C and D) ER stress-induced mtHTT aggregation is mediated via ATG5-dependent autophagy. M5-7 cells were incubated in the presence or absence of 10 ng/ml doxycycline (Dox.) for 4 days. After transfection with pHTTex120Q-GFP for 6 h, cells were exposed to DMSO or 30 nM thapsigargin (Tg) for additional 18 h. The aggregation of mtHTT was examined as in A (C). Cells extracts were subjected to western blotting (D). Bars represent mean values+SD (n ¼ 3).

Article Snippet: The following antibodies were used for western blot analyses: p-IRE1 (Abcam), CHOP (Santa Cruz Biotechnology), GRP78 (Santa Cruz Biotechnology), p62 (Abnova), LC3 (Novus Biologicals), ATG5 (Novus Biologicals), Beclin-1 (Novus Biologicals), cleaved caspase-3 (Cell Signaling), caspase-12 (Santa Cruz Biotechnology), a-tubulin (Sigma-Aldrich), b-actin (Sigma-Aldrich), Flag (Sigma-Aldrich) and GFP (Santa Cruz Biotechnology) antibodies.

Techniques: Inhibition, Knockdown, Transfection, Microscopy, Western Blot, Control, Incubation

Journal: Human molecular genetics

Article Title: IRE1 plays an essential role in ER stress-mediated aggregation of mutant huntingtin via the inhibition of autophagy flux.

doi: 10.1093/hmg/ddr445

Figure Lengend Snippet: Figure 7. IRE1 enhances mtHTT-mediated neuronal cell death via its kinase activity. (A) IRE1 knock-down reduces ER stress-induced mtHTT toxicity. SH-SY5Y/pSuper-Neo (SH/Neo) and SH-SY5Y/pSuper-shIRE1 (SH/shIRE1 #5 and #8) cells were transfected with pHTTex120Q-GFP and incubated with DMSO or 1 mm thapsigargin (Tg) for 24 h. Percentages of cell death were determined under fluorescence microscope after staining with ethidium homodimer. (B) IRE1 increases the neurotoxicity of mtHTT. SH-SY5Y cells were co-transfected with pIRE1 and either pEGFP, pHTTex18Q-GFP (wtHTT) or pHTTex120Q-GFP (mtHTT) for 24 h. Percentages of cell death were determined under fluorescence microscope after staining with ethidium homodimer as in (A). Bars represent mean values+SD (n ¼ 3). (C and D) Kinase activity of IRE1 contributes to death of STHdhQ111/111 cells. STHdhQ7/7 (Q7/7) and STHdhQ111/111 (Q111/111) cells were co-transfected with pEGFP and either pcDNA, pIRE1-HA, pIRE1 DC-HA or pIRE1 DR-HA for 72 h. Cells death was analyzed as in A (C) or cell extracts were prepared and subjected to western blotting using anti-caspase-12, anti-cleaved caspase-3, anti-p-IRE1, anti-HA and anti-b-actin antibodies (D). The arrowheads indicate specific signals detected by each antibody and the asterisks show non-specific signals. Bars represent mean values+SD (n ¼ 3). (E) STHdhQ111/111 striatal cells are more sensitive to ER stress-induced cell death than STHdhQ7/7 cells. STHdhQ7/7 (Q7/7) and STHdhQ111/111 (Q111/111) cells were co-transfected with pEGFP and either pcDNA or pIRE1 DC-HA for 48 h and then left untreated or exposed to DMSO or 100 nM thapsigargin (Tg) for additional 24 h. Cells death was analyzed as in (A). (F) Impaired autophagy activity increases mtHTT-mediated cell death under ER stress. M5-7 cells were cultured in the presence or absence of 10 ng/ml doxycycline (Dox.) for 4 days. After transfection with pEGFP or pHTTex120Q-GFP for 6 h, cells were exposed to DMSO or 30 nM thapsigargin (Tg) for additional 18 h. Cells death was analyzed as in (A). Bars represent mean values+SD from at least three independent experiments.

Article Snippet: The following antibodies were used for western blot analyses: p-IRE1 (Abcam), CHOP (Santa Cruz Biotechnology), GRP78 (Santa Cruz Biotechnology), p62 (Abnova), LC3 (Novus Biologicals), ATG5 (Novus Biologicals), Beclin-1 (Novus Biologicals), cleaved caspase-3 (Cell Signaling), caspase-12 (Santa Cruz Biotechnology), a-tubulin (Sigma-Aldrich), b-actin (Sigma-Aldrich), Flag (Sigma-Aldrich) and GFP (Santa Cruz Biotechnology) antibodies.

Techniques: Activity Assay, Knockdown, Transfection, Incubation, Microscopy, Staining, Western Blot, Cell Culture

Journal: Journal of cell science

Article Title: PML-IV functions as a negative regulator of telomerase by interacting with TERT.

doi: 10.1242/jcs.048066

Figure Lengend Snippet: Fig. 1. PML induced by IFNα suppresses intrinsic telomerase activities in H1299 cells. (A) Ablation of endogenous PML under IFNα treatment. H1299 cell lines were treated with 1000 U/ml IFNα in the presence of control or 200 nM PML siRNA. The levels of PML and actin in cell extracts harvested at indicated times were subjected to immunoblotting using anti-PML and anti-actin antibodies. The cell extracts (30 ng) were added for TRAP analyses. NC and PC denote negative control and positive control, respectively. TRAP analysis for NC was performed in the absence of cell extracts. The 36 bp represents the internal TRAP assay standards. The ImageJ (NIH) program was used to measure the relative amounts of bands in the TRAP assays after normalizing to 36 bp band, and are shown in the panel on the right. (B) Detection of the TERT mRNA levels. The mRNA of the cells treated with control or PML siRNA in the presence or absence of IFNα was extracted and tested for the levels of TERT mRNA using RT-PCR. The mRNA levels of GAPDH were analyzed as a control. (C) Co-localization of TERT-HA with PML-NBs. H1299 cells were transfected with plasmid expressing TERT-HA with or without IFNα. Cells were fixed after 24 hours and analyzed by immunofluorescence with anti-HA and anti-PML antibodies followed by Alexa Fluor 594 rabbit (red) and Alexa Fluor 488 mouse (green) secondary antibodies. A fluorescent microscope was used to detect the proteins, with a total of 200 cells counted for each experiment. DAPI was used to visualize the nuclei. Cells displaying colocalization of TERT and PML-NBs were counted and shown as graph on right. Cells were captured with the same exposure times (200 milliseconds for TERT, 800 milliseconds for PML). (D) Immunoprecipitation of TERT-HA with endogenous PML. Plasmid expressing TERT-HA was transfected into untreated or IFNα-treated H1299 cells. Whole cell lysates (WCLs) were immunoprecipitated with anti-HA antibodies and immunoblotted with anti-PML, anti-HA and anti- actin antibodies. Cell lysates (5% or 20%) used for immunoprecipitation were subjected to Western blot as an input.

Article Snippet: Anti-PML (PG-M3), anti-HA mouse (F-7), anti-Myc (9E10) and anti-HA rabbit (Y11) antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Control, Western Blot, Negative Control, Positive Control, TRAP Assay, Reverse Transcription Polymerase Chain Reaction, Transfection, Plasmid Preparation, Expressing, Immunofluorescence, Microscopy, Immunoprecipitation

Journal: Journal of cell science

Article Title: PML-IV functions as a negative regulator of telomerase by interacting with TERT.

doi: 10.1242/jcs.048066

Figure Lengend Snippet: Fig. 2. PML-IV interacts specifically with TERT. (A) Schematic view of the six PML isoforms. (B) The interaction between TERT and PML isoforms. Six FLAG- PML isoforms (I-IV) were transfected into HEK293T cells along with TERT-HA. Whole cell lysates were immunoprecipitated with anti-FLAG antibodies. FLAG immunoprecipitates and 20% input were loaded and immunoblotting was performed using anti-HA and anti-FLAG. (C) Co- localization of TERT with PML-IV. H1299 cells were co-transfected with plasmids expressing TERT-HA and FLAG-PML isoforms. Cells were fixed after 24 hours and analyzed by immunofluorescence using anti-HA and anti-FLAG antibodies followed by Alexa Fluor 594 rabbit (red) and Alexa Fluor 488 mouse (green) secondary antibodies. Nuclei were visualized by DAPI staining.

Article Snippet: Anti-PML (PG-M3), anti-HA mouse (F-7), anti-Myc (9E10) and anti-HA rabbit (Y11) antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Transfection, Immunoprecipitation, Western Blot, Expressing, Immunofluorescence, Staining

Journal: Journal of cell science

Article Title: PML-IV functions as a negative regulator of telomerase by interacting with TERT.

doi: 10.1242/jcs.048066

Figure Lengend Snippet: Fig. 3. The C-terminal region of PML-IV is required for TERT recruitment. (A) Schematic representation of various PML mutants showing their functional domains. (B) Interaction between TERT and C-terminal deletion PML mutants. Expression plasmids for TERT-HA and/or FLAG-tagged C-terminal deletion mutants of PML-IV were transfected into HEK293T cells. 24 hours after transfection, whole cell lysates were immunoprecipitated with anti-FLAG antibodies and assayed by immunoblotting with anti-HA and anti-FLAG antibodies. (C) Interaction between TERT and PML mutants. Plasmids expressing wild-type or truncated PML mutants and/or TERT-HA were transfected into HEK293T cells. Whole cell lysates were immunoprecipitated with anti-FLAG antibodies. Immunoblotting was performed with anti-HA and anti-FLAG antibodies. (D) Interaction between PML3M and TERT. Plasmids expressing TERT-HA, FLAG-PML-IV, or FLAG- PML3M were transfected into HEK293T cells. Whole cell lysates were subjected to immunoprecipitation using anti-FLAG antibodies followed by western blotting using anti-HA and anti-FLAG antibodies. (E) The interaction between TERT and the C-terminal fragment of PML-IV. Plasmids expressing TERT-HA, Myc-PML- IV, Myc-553-633 or Myc-571-633 were transfected into HEK293T cells. Whole cell lysates were subjected to immunoprecipitation using anti-Myc antibodies followed by western blotting using anti-HA and anti-Myc antibodies. The asterisks indicate heavy and light chains. pCS3-MT-BX empty vector produces 25 kDa polypeptide consisting of 6Myc epitope (lane 2). In the immunoprecipitation analysis (B-E), 20% of lysates were used as an input.

Article Snippet: Anti-PML (PG-M3), anti-HA mouse (F-7), anti-Myc (9E10) and anti-HA rabbit (Y11) antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Functional Assay, Expressing, Transfection, Immunoprecipitation, Western Blot, Plasmid Preparation

Journal: Journal of cell science

Article Title: PML-IV functions as a negative regulator of telomerase by interacting with TERT.

doi: 10.1242/jcs.048066

Figure Lengend Snippet: Fig. 4. Deletion fragments of TERT localize to the nucleoplasm in speckles. (A) Schematic representation of the TERT deletion constructs used in this study (Autexier and Lue, 2006; Xia et al., 2000). (B) Subcellular localization of TERT and its deletion constructs. H1299 cells were transfected with plasmids expressing TERT or the TERT fragments. 24 hours after transfection, cells were lysed and immunoblotted using anti-HA-rabbit antibodies (right panel). H1299 cells were transfected with plasmids expressing TERT-HA or its deletion mutants. The expressed proteins were detected with anti-HA antibodies followed by Alexa Fluor 594 rabbit (red) secondary antibodies. (C) Colocalization of TERT or its deletion mutants with PML-NBs. H1299 cells were co-transfected with expression vector for TERT-HA (panels 1-4) or its mutants (panels 5-20). Cells were fixed and detected using anti-HA and anti-PML antibodies followed by Alexa Fluor 594 rabbit (red) and Alexa Fluor 488 mouse (green) secondary antibodies. Transfected cells were analyzed using confocal microscopy. Endo PML, endogenous PML; PC, phase contrast. A total of 100 cells were counted for each experiment.

Article Snippet: Anti-PML (PG-M3), anti-HA mouse (F-7), anti-Myc (9E10) and anti-HA rabbit (Y11) antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Construct, Transfection, Expressing, Plasmid Preparation, Confocal Microscopy

Journal: Journal of cell science

Article Title: PML-IV functions as a negative regulator of telomerase by interacting with TERT.

doi: 10.1242/jcs.048066

Figure Lengend Snippet: Fig. 5. Two regions of TERT, 1-350 and 595-946, are required for TERT interaction with PML-IV. (A) Interaction between PML-IV and TERT. To detect the interaction between TERT and PML-IV, HEK293T cells were transfected with expression plasmids for TERT-HA and/or FLAG-PML-IV. Immunoprecipitation analyses were carried out as indicated in Fig. 2B. (B) The interaction between TERT fragments and PML-IV. To analyze the binding domain of TERT, HEK293T cells were transfected with expression plasmids for FLAG-PML-IV and/or HA-TERT mutants. Cell lysates were immunoprecipitated as described above using anti-HA antibodies and immunoblotted using anti-HA and anti-FLAG antibodies. (C) Interaction between TERT fragments and PML-IV 553-633 fragment. To analyze the binding domain between TERT and PML-IV, HEK293T cells were transfected with expression plasmids for FLAG-PML-IV (553-633) and/or HA- TERT mutants. Immunoprecipitations were carried out as described above. Immunoblotting was performed using anti-Myc and anti-HA antibodies. (D) Co- localization of TERT or its deletion mutants with PML-IV. H1299 cells were cotransfected with expression plasmids for TERT-HA (panels 1-4) or its mutants (panels 5-20) with FLAG-PML-IV. Cells were fixed and detected using anti-HA and anti-FLAG antibodies followed by Alexa Fluor 594 rabbit (Red) and Alexa Fluor 488 mouse (Green) secondary antibodies. Transfected cells were analyzed as described n Fig. 2C. A total of 100 cells were counted for each experiment. Cells were captured with the same exposure times (200 milliseconds for TERT, 800 milliseconds for PML).

Article Snippet: Anti-PML (PG-M3), anti-HA mouse (F-7), anti-Myc (9E10) and anti-HA rabbit (Y11) antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Transfection, Expressing, Immunoprecipitation, Binding Assay, Western Blot

Journal: Journal of cell science

Article Title: PML-IV functions as a negative regulator of telomerase by interacting with TERT.

doi: 10.1242/jcs.048066

Figure Lengend Snippet: Fig. 6. PML-IV negatively regulates telomerase activity in H1299 cells. (A) The effect of PML-IV on telomerase activity. H1299 cells were transfected with either empty vector or the plasmids expressing FLAG-PML-IV. Increasing concentrations of cell extracts (10, 30 and 50 ng) were added for TRAP analyses as described in Fig. 1A. (B) The effects of PML isoforms on telomerase activities. H1299 cells were transfected with the empty plasmid or the vectors expressing PML I-IV (upper panel). The extracts from the transfected cells (30 ng) were tested for telomerase activities and subjected to immunoblotting using anti-FLAG and anti-actin antibodies. (C) The effects of PML C-terminal fragment on telomerase activities. H1299 cells were transfected with the vectors expressing 6Myc epitope (control vector), Myc-PML-IV, Myc-553-633 or Myc-571-633 (upper panel). The extracts from the transfected cells (30 ng) were tested for telomerase activity and subjected to immunoblotting using anti-Myc and anti-actin antibodies. (D) Detection of the TERT mRNA levels. H1299 cells transfected with expression vectors for Myc-PML-IV, Myc-553-633 or Myc-571-633. After RNA extraction from transfected cells, mRNA levels of TERT were measured by RT-PCR. The mRNA levels of GAPDH were analyzed as a control. (E) Telomerase activity of the immunoprecipitated TERT by PML-IV and its 553-633 deletion mutant. Plasmids expressing FLAG-Ku70, FLAG-PML-IV, Myc-PML-IV, Myc-553-633, or empty vectors were transfected into HEK293T cells with expression vector for TERT- HA. Whole cell lysates were subjected to immunoblotting with anti-HA, anti-FLAG and anti-Myc antibodies (lanes 1-6). The lysates were immunoprecipitated with anti-FLAG or anti-Myc antibodies and assayed by immunoblotting with anti-HA antibodies (lanes 7-12) to detect TERT. 5% of the immunoprecipitated sample was analyzed for telomerase activity using a TRAP assay.

Article Snippet: Anti-PML (PG-M3), anti-HA mouse (F-7), anti-Myc (9E10) and anti-HA rabbit (Y11) antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Activity Assay, Transfection, Plasmid Preparation, Expressing, Western Blot, Control, RNA Extraction, Reverse Transcription Polymerase Chain Reaction, Immunoprecipitation, Mutagenesis, TRAP Assay

Journal: Journal of cell science

Article Title: PML-IV functions as a negative regulator of telomerase by interacting with TERT.

doi: 10.1242/jcs.048066

Figure Lengend Snippet: Fig. 7. H1299 stable cell lines overexpressing PML-IV suppress TERT activity. (A) FLAG-PML-IV expression in PML stable cell lines. PML- IV, expressed in PML-IV-stable cell lines, was detected using anti-PML or anti-HA antibodies. H1299 cell lines stably transfected with pCMV- Tag2B (mock) were also tested using the same antibodies. The asterisk indicates stably overexpressed FLAG-PML-IV. (B) Detection of the TERT mRNA and hTERC levels in the stable cell lines. The RNA of the stable cell lines was extracted and tested for the TERT mRNA or hTERC using RT-PCR as described in Fig. 1. The mRNA levels of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were tested as a control. (C) Telomerase activity of the PML-IV-stable cell lines. The extracts of H1299, mock, or stable cell lines were tested for telomerase activity as described in Fig. 5. (D) Detection of telomere length in stable cell lines. The telomere length of the cell lines used above was determined by TRF analyses. The mock cell line was used as a control.

Article Snippet: Anti-PML (PG-M3), anti-HA mouse (F-7), anti-Myc (9E10) and anti-HA rabbit (Y11) antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Stable Transfection, Activity Assay, Expressing, Transfection, Reverse Transcription Polymerase Chain Reaction, Control

Journal: Genes & Development

Article Title: ZCCHC8 , the nuclear exosome targeting component, is mutated in familial pulmonary fibrosis and is required for telomerase RNA maturation

doi: 10.1101/gad.326785.119

Figure Lengend Snippet: Linkage analysis and whole-genome sequencing identify novel disease gene ZCCHC8 in familial pulmonary fibrosis with low telomerase RNA ( TR ). ( A ) Pedigree with pulmonary fibrosis proband (arrow) with affected relatives are indicated by the shaded symbols (key). The clinical history below each of the four shaded pedigree symbols refers to the age of onset of lung disease including idiopathic pulmonary fibrosis (IPF). (?) Asymptomatic individuals who had unknown affected status at the time of clinical assessment; (gray shading) unknown cause of death; (*) individuals with DNA who were included in the linkage analysis. ( B ) TR levels measured by quantitative real time PCR (qRT-PCR) in lymphoblastoid cell lines (LCLs). Arrow refers to proband (red) and pedigree identifiers refer to A . TR level from a DKC1 mutation carrier is a positive control. The data represent a mean of three experiments, each from independent RNA isolations. ( C ) Telogram shows age-adjusted lymphocyte telomere length by flow cytometry and fluorescence in situ hybridization (flowFISH) in the proband (arrow) and family (pedigree designations as in A ). The validated telogram is based on 192 controls. ( D ) Phenotype assignments used in linkage (key) and genotype below each individual refers to ZCCHC8 SNP. Italicized genotypes refer to obligate carriers. ( E ) Log of the odds (LOD) ratio across autosomal chromosomes calculated from SNP data from 14 individuals, with arrow on chromosome 12 pointing to maximum LOD. ( F ) p.P186L conservation across eight vertebrate ZCCHC8 species with darker shading denoting more conserved residues. CCHC refers to Zinc-knuckle domain; PSP refers to proline-rich domain.

Article Snippet: Myc-tagged mouse ZCCHC8 cDNA (NM_028151) was purchased in a pCMV3 expression vector (MG51487-NM, Sino Biological).

Techniques: Sequencing, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Mutagenesis, Positive Control, Flow Cytometry, Fluorescence, In Situ Hybridization

Journal: Genes & Development

Article Title: ZCCHC8 , the nuclear exosome targeting component, is mutated in familial pulmonary fibrosis and is required for telomerase RNA maturation

doi: 10.1101/gad.326785.119

Figure Lengend Snippet: ZCCHC8 loss of function is sufficient to cause low TR levels. ( A ) Immunoblot of ZCCHC8 in lymphoblastoid cell lines (LCLs) from healthy controls (C1 and C2) and unaffected relatives and mutation carriers labeled with pedigree identifiers from A. Quantification from one blot and result replicated twice from independently harvested protein lysates. ( B ) Immunoblot of ZCCHC8, SKIV2L2, and RBM7 levels in proband's primary skin fibroblasts. ( C ) Immunoblot of transfected Myc-tagged (293FT cells) and endogenous ZCCHC8. ( D ) Mean ZCCHC8 mRNA levels ± SEM from LCLs in unaffected family members ( n = 4) and ZCCHC8 p.P186L mutation carriers ( n = 3). ( E ) Chromatogram showing that ZCCHC8 p.P186L mutation is expressed in LCL mRNA from proband (also verified in two other mutation carriers). ( F ) Immunoblot showing efficiency of shRNA knockdown of Luciferase (Luc), ZCCHC8, and NAF1 in HeLa cells. ( G ) Total TR levels measured by qRT-PCR (mean ± SEM from three independent knockdowns and RNA isolations). ( H ) Northern blot of TR after stable knockdown of ZCCHC8 and NAF1 (replicated twice with independent RNA isolations). (**) P < 0.01; (***) P < 0.001 (Student's t -test, two-sided).

Article Snippet: Myc-tagged mouse ZCCHC8 cDNA (NM_028151) was purchased in a pCMV3 expression vector (MG51487-NM, Sino Biological).

Techniques: Western Blot, Mutagenesis, Labeling, Transfection, shRNA, Luciferase, Quantitative RT-PCR, Northern Blot

Journal: Genes & Development

Article Title: ZCCHC8 , the nuclear exosome targeting component, is mutated in familial pulmonary fibrosis and is required for telomerase RNA maturation

doi: 10.1101/gad.326785.119

Figure Lengend Snippet: ZCCHC8 is required for its 3′ end maturation and telomerase function. ( A ) Compound heterozygous frameshift (fs) mutations introduced using CRISPR/Cas9 in HCT116 pseudodiploid cells. ( B ) Immunoblot for ZCCHC8 in HCT116-edited cells. ( C ) Scheme summarizing TR 3′ rapid amplification of cDNA ends sequencing (3′RACE-seq). TR 3′ ends were generally divided into mature (451 bp) and extended (>451 bp) where extensions are denoted by gray N's. ( D ) Summary of TR 3′RACE-seq fractions in isogenic ZCCHC8 +/+ and ZCCHC8 −/− cells. Color-coded key shows four categories of TR forms: mature (451 nt), adenylated (A)n, short genomically extended (g)n (<465 nt), and long genomically extended (>465 nt). Data are mean of three independent 3′RACE-seq analyses from three RNA isolations each from a different aliquot of a single clone. ( E ) qRT-PCR of extended TR forms beyond the 451 mature end (>20, >51, >784 nt). Data are mean of three independent RNA isolations similar to D . ( F ) TR Northern blot of edited ZCCHC8 +/+ and ZCCHC8 −/− cells. ( G ) Total TR levels by Northern bot (six blots from three RNA isolations). ( H ) Telomerase activity measured by telomere repeat amplification protocol (TRAP) assay in ZCCHC8 +/+ , ZCCHC8 −/− , and NAF1 S329/S329 HCT116 cell extracts. Activity was quantified on serially diluted extracts (1, 1/5, 1/25, and 1/125) against a PCR-amplified internal control (IC). RNase-treated wild-type extract and no template PCR reaction are included as negative controls. ( I ) Mean TRAP activity of 1/5× diluted extracts (three independent TRAP assays, each from a different lysate). ( J ) Summary of 3′RACE-seq of TR forms from control and proband's primary skin fibroblasts with speciation as in D . ( K ) qRT-PCR values of extended TR forms in primary skin fibroblasts as in E , mean of three technical replicates). ( L ) Amplified TR from input and Myc-ZCCHC8 immunprecipitated fractions (293FT cells) using primers falling within the mature TR sequence. ( M ) qRT-PCR of extended TR (>51 nt extended beyond the 3′ mature TR end) after transfection of tagged ZCCHC8, DIS3, EXOSC10/RRP6, and PARN into HCT116 ZCCHC8 −/− cells (three to four independent transfections/experiment). Data are expressed as mean ± SEM (*) P < 0.05; (**) P < 0.01 (Student's t -test, two-sided).

Article Snippet: Myc-tagged mouse ZCCHC8 cDNA (NM_028151) was purchased in a pCMV3 expression vector (MG51487-NM, Sino Biological).

Techniques: CRISPR, Western Blot, Rapid Amplification of cDNA Ends, Sequencing, Quantitative RT-PCR, Northern Blot, Activity Assay, Amplification, TRAP Assay, Transfection

Journal: Genes & Development

Article Title: ZCCHC8 , the nuclear exosome targeting component, is mutated in familial pulmonary fibrosis and is required for telomerase RNA maturation

doi: 10.1101/gad.326785.119

Figure Lengend Snippet: Zcchc8- null mice have TR insufficiency. ( A – C ) Immunoblot for ZCCHC8, SKIV2L2, and RBM7, respectively, on lysates from mouse ear fibroblasts. ( D , E ) Northern blot for mouse TR and quantification. For E , mean reflects mice Zcchc8 +/+ ( n = 4, 2M/2F), Zcchc8 +/− ( n = 4, 2M/2F), Zcchc8 −/− ( n = 3M), mTR +/− ( n = 2, sex unknown) and mTR −/− ( n = 2, sex unknown). ( F ) TR 3′ extended levels (>20 bp) relative to Hprt as measured by qRT-PCR. Mouse numbers and M/F designations as in E . Data are expressed as mean ± SEM. (*) P < 0.05; (**) P < 0.01 (Student's t -test, two-sided).

Article Snippet: Myc-tagged mouse ZCCHC8 cDNA (NM_028151) was purchased in a pCMV3 expression vector (MG51487-NM, Sino Biological).

Techniques: Western Blot, Northern Blot, Quantitative RT-PCR

Journal: Genes & Development

Article Title: ZCCHC8 , the nuclear exosome targeting component, is mutated in familial pulmonary fibrosis and is required for telomerase RNA maturation

doi: 10.1101/gad.326785.119

Figure Lengend Snippet: ZCCHC8 complete loss causes progressive and fatal neurodevelopmental phenotype. ( A , top row) Images showing head profile of Zcchc8 wild-type, heterozygous, and homozygous null mice (41–46 d-old). The labels show the genotype with a male ( left ) and a female ( right ) for each genotype. Zcchc8 −/− mice have abnormal head profiles with domed crania, as outlined by the dashed line. ( Middle row ) CT head mid-sagittal images show Zcchc8 −/− mice have dome-shaped crania. ( Bottom row). Volume-rendered (VR) CT images of mouse calvaria show widened cranial sutures in Zcchc8 −/− mice (seen in three of six imaged). None of Zcchc8 +/+ or Zcchc8 +/− mice (four mice imaged/genotype) had this feature. Each vertical image group is from the same mouse except the last column (two different females). ( B ) Representative H&E coronal sections from 8-wk-old heads (all male) show no differences in Zcchc8 +/− mice (11 examined) compared with Zcchc8 +/+ mice (10 examined). In contrast, Zcchc8 −/− mice had severe ventricular dilation (nine of 11 examined). ( C ) E12.5 brain sections show Zcchc8 −/− have microcephaly but intact brain structures with no ventriculomegaly in utero. ( D ) Image of E12.5 embryos from a single dam showing expected Mendelian ratios but Zcchc8 −/− embryos have small crania. ( E ) Cranial area of newborn (P0) pups measured on VR CT images and corrected to left femur length on the same images ( Zcchc8 +/+ n = 7, 3M/4F; Zcchc8 +/− n = 10, 3M/7F; Zcchc8 −/− n = 4, 3M/1F). ( F ) Mean cranial area ± SEM relative to age for all three genotypes showing that Zcchc8 −/− mice develop macrocephy after birth. Newborn mice include those listed in E and older mice ( Zcchc8 +/+ , n = 4, 3M/1F; Zcchc8 +/− , n = 4, 3M/1F; Zcchc8 −/− , n = 5, 3M/2F). Dashed lines denote 95% confidence intervals. (**) P < 0.01 (Student's t -test, two-sided).

Article Snippet: Myc-tagged mouse ZCCHC8 cDNA (NM_028151) was purchased in a pCMV3 expression vector (MG51487-NM, Sino Biological).

Techniques: In Utero

Journal: Genes & Development

Article Title: ZCCHC8 , the nuclear exosome targeting component, is mutated in familial pulmonary fibrosis and is required for telomerase RNA maturation

doi: 10.1101/gad.326785.119

Figure Lengend Snippet: The Zcchc8 −/− transcriptome shows up-regulation and misprocessing of low-abundance intronless RNAs other than TR . ( A ) Heat map with dendrogram of gene expression showing unsupervised analysis of 9788 high-quality genes from brain RNA-seq analysis. Colors denote mean-subtracted FPKM expression values on a log 2 scale ( Zcchc8 +/+ , n = 5; Zcchc8 +/− , n = 3; Zcchc8 −/− , n = 6 embryonic brains sequenced). Each column is labeled below by WT, HET, and KO followed by the embryo number (1, 2, 3, etc.), referring to respective Zcchc8 genotypes. The log 2 expression value was subtracted from the mean log 2 expression value of the entire cohort. The dendrogram showing relatedness of the samples is above , and relatedness of the gene transcripts is at the left . The differential change in RNA expression is shown as positive and negative change on color scale in the key above the top right corner. ( B , C ) Volcano plots depicting the log 2 -fold changes ( X -axis) versus −log 10 P -values calculated by two-tailed one-way ANOVA ( Y -axis) for the Zcchc8 +/− and Zcchc8 −/− versus Zcchc8 +/+ comparisons, respectively. Each dot represents a single transcript. ( D ) Histogram of number of genes at each expression value denoted on the x -axis by the mean log 2 FPKM values obtained from Zcchc8 wild-type embryos ( n = 5). RNAs that have more than two SD higher levels in the Zcchc8 +/+ versus Zcchc8 −/− comparison are shown in red ( n = 197) and fall on the low end of the histogram with TR and its mean FPKM in wild-type embryos shown. Down-regulated RNAs, defined as less than two SD ( n = 43), are shown in blue appear uniformly distributed on the distribution. ( E ) Histogram of the most up-regulated (>2SD) transcripts in the Zcchc8 −/− versus Zcchc8 +/+ by exon number shows the largest subset is intronless RNAs (42 of 188 with known gene structure, 22%). The pie chart divides the intronless RNAs by functional category. ( F ) Annotation of 28 up-regulated intronless RNAs ( TR , histones and cilia) shows a majority of the histones represented are replication-dependent histones (RDH) (23 of 24, 96%). The majority have an annotated transcript size in the range of TR between 400 and 560 (22 of 28). Columns referring to 5′ end and 3′ end refer to visualized additional reads beyond annotated gene boundaries with 5′ end reads referring to upstream reads that are not necessarily contiguous (manually identified in the Integrative Genome Viewer [IGV]). ( G , H ) Genome browser read coverage plots from IGV viewer showing extended 3′ ends as labeled above from two histone genes in each of Zcchc8 +/+ , Zcchc8 +/− , Zcchc8 −/− transcriptomes. ( I , J) Coverage plots for two coiled-coil domain containing cilia genes Ccdc89 and Ccdc182 , respectively, by genotype. For Ccdc89 , there is also an increase in discontinuous upstream of gene 5′end reads that resemble so-called promoter upstream transcripts (PROMPTs).

Article Snippet: Myc-tagged mouse ZCCHC8 cDNA (NM_028151) was purchased in a pCMV3 expression vector (MG51487-NM, Sino Biological).

Techniques: Expressing, RNA Sequencing Assay, Labeling, RNA Expression, Two Tailed Test, Functional Assay

Journal: Molecular Cancer

Article Title: A new KSRP-binding compound suppresses distant metastasis of colorectal cancer by targeting the oncogenic KITENIN complex

doi: 10.1186/s12943-021-01368-w

Figure Lengend Snippet: DKC1125 inhibits the function of Dvl by interfering with the interaction of KSRP with Dvl2. a Association of KSRP with Dvl2 was inhibited after treatment of DKC1125. Co-IP experiments were performed in Caco2 cells stably expressing EV or KITENIN-V5 using cytoplasmic–nuclear fractionation after treatment with vehicle (V) or DKC1125 treatment (0.5 μM). Precipitates were analyzed by immunoblotting to detect KSRP-Dvl binding. Whole-cell lysate (WCL) of the same pool of cells was co-analyzed as a control. b The amount of Dvl2 protein adhering to KSRP was markedly reduced by treatment with DKC1125. Dvl2-ΔDIX-GFP transfected HCT116 cells were collected and lysed, and the supernatants were mixed with purified KSRP-His by Ni-NTA after in vitro bacterial expression and subjected to GFP-Trap. GFP-Trap, consisting of an anti-GFP Nanobody/VHH coupled to agarose beads, was used for effective pulldown of GFP-fusion proteins. Interaction of ΔDIX-DVL2-GFP with bacterially expressed KSRP-His was verified by immunoblotting with anti-His antibody. c Effect of DKC1125 on Dvl binding of WT-KSRP or ΔKH34-KSRP. Co-IP analysis was performed using cytoplasmic–nuclear fractions obtained from WT-KSRP or ΔKH34-KSRP transfected Caco2 cells stably expressing KITENIN-V5 or EV. Binding was verified by immunoblotting. d Effect of DKC1125 on activation of WNT/β-catenin by expression of KITENIN and/or KSRP. 293 T cells were transfected with the TOP-flash reporter gene and KITENIN-V5, WT-KSRP-myc, or ΔKH34-KSRP-myc, in parallel, and treated with vehicle or DKC1125 (0.5 μM). Luciferase activity was measured at 48 h after transfection and normalized against the activity of TK-Renilla. Data are shown relative to the corresponding TOP-flash value in control cells. Differences in transcriptional activity of TCF/LEF by expression of KITENIN, WT-KSRP, and ΔKH34-KSRP were compared between the presence and absence of DKC1125. Error bars indicate SEM. The asterisk indicates a significant difference between groups (NS, not significant; ** P < 0.01)

Article Snippet: Antibodies against the following proteins were obtained from the indicated suppliers: Dvl1, Dvl3, Lamin A/C, GFP, and tubulin (Santa Cruz Biotechnology); DVL2, Myc, GAPDH, and c-Jun (Cell Signaling Technol); KSRP (Novus); V5 (MBL); HA and Actin (Sigma); KITENIN (Atlas); His and RACK1 (Abcam); and myc-Trap and GFP-Trap (Chromotek).

Techniques: Co-Immunoprecipitation Assay, Stable Transfection, Expressing, Fractionation, Western Blot, Binding Assay, Control, Transfection, Purification, In Vitro, Activation Assay, Luciferase, Activity Assay

Journal: Molecular Cancer

Article Title: A new KSRP-binding compound suppresses distant metastasis of colorectal cancer by targeting the oncogenic KITENIN complex

doi: 10.1186/s12943-021-01368-w

Figure Lengend Snippet: Treatment with DKC1125 results in autophagic degradation of Dvl2 and KITENIN through increased binding to RACK1. a RACK1 directly binds to DKC1125. Proteins pulled down by chemical probe using HCT116 lysates were verified by immunoblot analysis using antibodies against RACK1 and Dvl2. b Interactions of RACK1 with KSRP or Dvl2 within the functional KITENIN complex following DKC1125. Caco2 cells were transfected with empty vector (EV) or KITENIN-V5 (KIT-V5), and treated with vehicle (V) or DKC1125 (DKC) (0.5 μM). The cell lysates were immunoprecipitated with anti- RACK1 antibody and immunoblotted with anti-KSRP or anti-Dvl2 antibody. c Levels of Dvl2 and KITENIN are reduced more after DKC1125 treatment under RACK1 expression. Caco2 cells were transfected with empty vector (EV) or KITENIN-V5, or co-transfected with KITENIN-V5 and RACK1-GFP, and then treated with vehicle or DKC1125 (0.5 μM). The protein levels of Dvl2 and KITENIN were checked after treatment with cycloheximide at the indicated times. d RACK1 affects the activation of canonical WNT signaling by Dvl2. 293 T cells were transfected with the TOP-flash reporter gene and Dvl2, RACK1, or si-RACK1, in parallel, and treated with vehicle or DKC1125 (0.5 μM). Differences in transcriptional activity of TCF/LEF by Dvl2, alone or in combination with RACK1 overexpression or knockdown, were compared between the presence or absence of DKC1125. e Autophagic degradation of Dvl2 and KITENIN by DKC1125. Caco2 cells were initially pretreated with vehicle, the proteasome inhibitor MG132 (MG, 10 μM), the lysosomal degradation inhibitors bafilomycin A1 (A1, 100 nM) and chloroquine (CQ, 10 μM), or the autophagosome blocker type III phosphatidylinositol 3-kinase inhibitor (3-MA, 1 mM), and later treated with a high concentration of DKC1125 (5 μM). Levels of Dvl2 and KITENIN were examined by immunoblot analyses. f Staining of autophagosomes in DKC1125-treated cells. Autophagosomes were stained in stably KITENIN-expressing Caco2 cells after 24 h treatment with DKC1125 using CYTO-ID autophagy detection dye. Rapamycin was included as a positive control for autophagic induction

Article Snippet: Antibodies against the following proteins were obtained from the indicated suppliers: Dvl1, Dvl3, Lamin A/C, GFP, and tubulin (Santa Cruz Biotechnology); DVL2, Myc, GAPDH, and c-Jun (Cell Signaling Technol); KSRP (Novus); V5 (MBL); HA and Actin (Sigma); KITENIN (Atlas); His and RACK1 (Abcam); and myc-Trap and GFP-Trap (Chromotek).

Techniques: Binding Assay, Western Blot, Functional Assay, Transfection, Plasmid Preparation, Immunoprecipitation, Expressing, Activation Assay, Activity Assay, Over Expression, Knockdown, Concentration Assay, Staining, Stable Transfection, Positive Control

Journal: Molecular Cancer

Article Title: A new KSRP-binding compound suppresses distant metastasis of colorectal cancer by targeting the oncogenic KITENIN complex

doi: 10.1186/s12943-021-01368-w

Figure Lengend Snippet: RACK1 and miR-124 are required for the suppressive effects of DKC1125 on cell invasiveness. a RACK1 in the KITENIN complex plays a major role in the inhibition of cell invasion by DKC1125. Cell invasion was examined in empty vector (EV)- or KITENIN-transfected Caco2 cells after knockdown of RACK1 via siRNA transfection (left panel), or under ectopic expression of RACK1 (right panel) after treatment with vehicle (V) or DKC1125 (D) (0.5 μM). Data are expressed as in Fig. a . b Dvl2 downregulation by DKC1125 was associated with elevated binding of Dvl2 to RACK1. Several deletion mutants within the binding site of KSRP to DKC1125 were designed and co-expressed in Caco2 cells with RACK1-GFP. Dvl2–RACK1 binding was examined using GFP-Trap and immunoblot analysis after treatment with DKC1125 (0.5 μM), and compared with that of empty vector (EV) or wild-type (WT) KSRP expression. An increase in Dvl2–RACK1 interaction was observed after DKC1125 treatment in cells expressing the Q417A-KSRP or N467A-KSRP mutant, but not in cells expressing the R411A-KSRP or R415A-KSRP mutant. c Modulation of miR-124 is also involved in increased cellular invasiveness by the functional KITENIN complex. Detection of transcript of miR-124-3p in stably miR-null- or miR-124-transfected Caco2 cells (left panel). Cell invasion was examined in Caco2 cells stably expressing the miR-null vector or miR-124 that were transfected with the empty vector (EV)-, KITENIN-, WT-KSRP-, or Δ34KH-KSRP, and treated with vehicle or DKC1125 (0.5 μM) (right panel). Data are expressed as in Fig. a . d Inhibitor of miR-124-3p significantly restored the inhibitory effect of DKC1125 on the KITENIN-mediated increase in cell invasion. Cell invasion was compared in empty vector (EV)- or KITENIN-transfected Caco2 cells treated with vehicle or DKC1125, or co-treated with DKC1125 (0.5 μM) and a synthetic-oligo inhibitor of miR-124-3p (50 nM). The asterisk indicates a significant difference in Caco2/KITENIN-V5 cells after treatment with DKC1125, and a significant difference in DKC1125-treated Caco2/KITENIN-V5 cells after treatment with a synthetic-oligo inhibitor of miR-124-3p (** P < 0.01). Data are expressed as in Fig. a

Article Snippet: Antibodies against the following proteins were obtained from the indicated suppliers: Dvl1, Dvl3, Lamin A/C, GFP, and tubulin (Santa Cruz Biotechnology); DVL2, Myc, GAPDH, and c-Jun (Cell Signaling Technol); KSRP (Novus); V5 (MBL); HA and Actin (Sigma); KITENIN (Atlas); His and RACK1 (Abcam); and myc-Trap and GFP-Trap (Chromotek).

Techniques: Inhibition, Plasmid Preparation, Transfection, Knockdown, Expressing, Binding Assay, Western Blot, Mutagenesis, Functional Assay, Stable Transfection

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: Assay identification numbers for NBK/Bik and SQSTM1/p62 were Hs00154189_m1 and Hs01061917_g1, respectively.

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: Assay identification numbers for NBK/Bik and SQSTM1/p62 were Hs00154189_m1 and Hs01061917_g1, respectively.

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: Assay identification numbers for NBK/Bik and SQSTM1/p62 were Hs00154189_m1 and Hs01061917_g1, respectively.

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: 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: Mechanistic model of SQSTM1/p62 and NBK/Bik acting as novel molecular switches converting autophagy to apoptosis. During autophagy (e.g., induced by BH3 mimetics), the adaptor protein SQSTM1/p62 is responsible for recognition and loading of cargo, including malfolded (unfolded or misfolded) proteins and damaged organelles, into autophagosomes for removal. Targeting p62 by blocking its resynthesis through inhibition of transcription (e.g., by Cdk9 inhibition) disrupts this process, resulting in a failure of the cargo loading process and dysfunctional removal of malfolded proteins and damaged organelles, defined here as inefficient autophagy. The latter event promotes the accumulation of the BH3-only protein NBK/Bik, a substrate of ubiquitination and degradation via autophagy, which in turn triggers the activation of the apoptotic signaling cascade. Therefore, whereas targeting p62 converts cytoprotective autophagy into an inefficient form, NBK/Bik switches inefficient autophagy to apoptosis. Thus, SQSTM1/p62 and NBK/Bik cooperate to convert autophagy to apoptosis. This autophagy-targeting strategy (A) may provide theoretical advantages over alternatives, e.g., inhibition of autophagy initiation (B) or disruption of autophagosome maturation/fusion with lysosomes (C), including (i) upregulation of prodeath Bik, which requires autophagy initiation and does not occur in the case of stage B, and (ii) downregulation of oncogenic and prosurvival p62 at the transcriptional level by Cdk9 inhibition, in contrast to disruption of autophagy at either early (B) (e.g., initiation) or late (C) (e.g., maturation or lysosome biogenesis) stages, which results in p62 upregulation due to interference with its autophagic degradation. L, lysosome; c, cytochrome c.

Article Snippet: Assay identification numbers for NBK/Bik and SQSTM1/p62 were Hs00154189_m1 and Hs01061917_g1, respectively.

Techniques: Blocking Assay, Inhibition, Ubiquitin Proteomics, Activation Assay, Disruption

Journal: Scientific Reports

Article Title: The lethal response to Cdk1 inhibition depends on sister chromatid alignment errors generated by KIF4 and isoform 1 of PRC1

doi: 10.1038/srep14798

Figure Lengend Snippet: ( A ) Schematic outline of the RO-3306 barcode screen in U2OS cells. ( B ) Reconstitution of the RNAi-resistant human PRC1-1 cDNA in PRC1 knockdown cells. The pRS vector (shCTRL) was used as a control. ( C ) Western blot analysis of protein lysates corresponding to the cell lines shown in ( B ). shPRC1 Lib#1 targets only endogenous PRC1, while shPRC1 Lib#2 is able to target both endogenous and Venus-NT-PRC1 #1. ( D ) Western blot analysis of U2OS cells stably expressing individual Venus-PRC1 isoforms after transfection of siRNA duplexes specifically targeting the indicated splice variants. CTRL refers to cells lacking ectopic expression of Venus-PRC1. ( E ) Depletion of PRC1-1 rescues cell proliferation when Cdk1 activity is compromised. The functional phenotypes of the individual shRNAs targeting the PRC1 variants are indicated by the colony formation assay. The pRS vector (shCTRL) was used as a control. The knockdown efficiency of each individual shRNA was measured by examining the mRNA level of the PRC1-1 and PRC1-2 target genes by qRT-PCR. (Mean ± s.d. n = 3). ( F ) KIF4 is a PRC1 binding partner. Immunoprecipitations (IPs) with GFP-Trap_A beads were performed on extracts of U2OS WT (CTRL) or U2OS cells stably expressing Venus-PRC1-1 (PRC1-1). Aliquots of the IPs were analysed by SDS-PAGE. ( G ) Compromised Cdk1 activity in mitosis results in premature binding of PRC1 to KIF4. U2OS cells were synchronised in mitosis using thymidine and nocodazole. Cells arrested in mitosis were obtained by mitotic shake-off, released from the nocodazole and harvested after 60 minutes (G1) or, alternatively, treated with 5 μM MG132 with or without 3 μM RO-3306 and harvested after 30 minutes (M). Cell extracts were subjected to immunoprecipitation using a rabbit anti-PRC1. Aliquots of the immunoprecipitates were analysed by Western blotting. The signal intensity of the KIF4 band in the PRC1 IP was quantified and normalized to 1 for the G1 sample. * p < 0.05, Student’s t test. Western blots in panels ( C , D , F , G ) have been cropped and full-length gels can be viewed in .

Article Snippet: Immunoprecipitations of GFP for mass spectrometry were performed with GFP-Trap_A beads (Chromotek), according to the manufacturer’s protocol.

Techniques: Plasmid Preparation, Western Blot, Stable Transfection, Expressing, Transfection, Activity Assay, Functional Assay, Colony Assay, shRNA, Quantitative RT-PCR, Binding Assay, SDS Page, Immunoprecipitation

Journal: bioRxiv

Article Title: Bi-allelic MCM10 mutations cause telomere shortening with immune dysfunction and cardiomyopathy

doi: 10.1101/844498

Figure Lengend Snippet: a) Schematic of human MCM10 indicating NK- and RCM-associated patient mutations (annotated based on MCM10 transcript NM_018518.5) and exons targeted using CRISPR-Cas9 (exon 3) or rAAV (exon14) to generate MCM10 +/- cell lines. b) Pedigree and segregation of MCM10 mutations in RCM family. Blue shading indicates fetal RCM. Individuals that underwent exome (§) or genome (*) sequencing are indicated. Clinically unaffected children that are not carriers of both pathogenic mutations (†) are indicated (carrier status of minors thereby not disclosed). c) Structural model of the human Mcm10-ID with a bound single-stranded DNA, based on Xenopus laevis Mcm10-ID (Protein Data Bank accession codes 3EBE and 3H15 ). The zinc ion is shown as a gray sphere. The locations of the R426C and <ι>Δ N20 mutations are indicated. d) (Left) Coomassie blue-stained SDS-PAGE gel of purified WT and <ι>Δ N20 Mcm10-ID. (Right) Size exclusion chromatography profiles comparing elution of WT and <ι>Δ N20 Mcm10-ID. The molecular weight standard (gray) included thyroglobulin (670 kDa), <ι>γ -globulin (158 kDa), ovalbumin (44 kDa), myoglobin (17 kDa), and vitamin B12 (1.3 kDa). e) Western blot for Mcm10 with GAPDH as a loading control. Quantification of Mcm10 levels normalized to loading control, relative to wild type is indicated. f) Average proliferation rate in MCM10 +/- cells normalized to wild type. For each cell line n = 6 wells across three biological replicates. g) Comparison of clonogenic survival of HCT116 wild type (top) and MCM10 +/- cells (middle/bottom). Cells plated per well are noted. h) Percentage clonogenic survival in HCT116 wild type (blue) and MCM10 +/- cells (red), n = 15 wells across ten biological replicates. i) Average percentage of each population represented by early apoptotic, late apoptotic or dead cells. HCT116 wild type (blue) and clonal MCM10 +/- cell lines (red) are shown. Error is indicated in f, h and i as standard deviation and significance was calculated using two-tailed student’s t-test with *<.05; **<.01, ***<.001.

Article Snippet: Primary antibodies were incubated in 5% BLOT-QuickBlocker (G-Biosciences 786-011) as follows: rabbit anti-Mcm10 (Bethyl A300-131A; 1:500), rabbit anti-Mcm10 (Novus, H00055388-D01P, 1:500), mouse anti-Cdc45 (Santa Cruz G12, SC55568; 1:500), mouse anti-Mcm4 (Santa Cruz G7, SC28317; 1:500), mouse anti-Mus81 (Abcam ab14387; 1:500), mouse anti-PCNA (Abcam Ab29; 1:3,000), rabbit anti-RPA32 (S4/8) (Bethyl A300-245A; 1:2000), mouse anti-GAPDH (GeneTex GTX627408; 1:5,000), mouse anti- <ι>α -Tubulin (Millipore T9026, clone DM1A; 1:10,000).

Techniques: CRISPR, Sequencing, Staining, SDS Page, Purification, Size-exclusion Chromatography, Molecular Weight, Western Blot, Standard Deviation, Two Tailed Test

Journal: bioRxiv

Article Title: Bi-allelic MCM10 mutations cause telomere shortening with immune dysfunction and cardiomyopathy

doi: 10.1101/844498

Figure Lengend Snippet: a) Sequencing analysis from the proband validating the c.236delG mutation in MCM10 exon 3 that introduces a premature stop codon. Sequencing base calls and corresponding translation is shown below the trace image. On the 3’ end the base calls the wild type allele is shown on top and the mutant allele with a single G deletion is shown on the bottom. The translation of each codon is shown with the single letter abbreviations for each amino acid and their position in the Mcm10 protein. Annotation is in reference to MCM10 transcript NM_018518.5. (Right) Cartoon depiction of the wild type and mutant c.236delG exon 3 MCM10 alleles. b) Sequencing analysis from the mother showing the wild type MCM10 exon5/6 junction and from the father validating the c.764+5G>A mutation that results in loss of exon 6 from the mature mRNA. Sequencing base calls and corresponding translation is shown below each trace image. The translation of each codon is shown with the single letter abbreviations for each amino acid and their position in the Mcm10 protein. Below each translation is a bar indicating whether that region is coded for by MCM10 exon 5 (red), exon 6 (green) or exon 7 (blue). Annotation is in reference to MCM10 transcript NM_018518.5. (Right) Cartoon depiction of the wild type and mutant c.764+5G>A alleles. c) Modified cartoon of human Mcm10 including the N-terminal domain that harbors a coiled-coil (CC, orange) motif. The internal domain contains a PCNA-interacting peptide (PIP) box (red), Hsp10-like domain (purple), an oligonucleotide/oligosaccharide binding (OB)-fold (gray) and zinc-finger motif 1 (ZnF1, green). The C-terminal domain contains ZnF2 (green), the zinc ribbon (ZnR, blue) and winged helix motif (WH, light gray). Mcm10 structural domains are connected by two flexible linker regions (yellow). The location of the RCM-associated G79EfsTer6 and D198GfsTer10 ( <ι>Δ 198-255) and NKD- associated R426C and R582X mutations are indicated. Annotation is in reference to MCM10 transcript NM_018518.5. d) CD spectra comparing WT and <ι>Δ N20 Mcm10-ID at 25°C and 60°C. e) Melt curves for WT and <ι>Δ N20 Mcm10-ID obtained by plotting the normalized CD signal (ellipticity) at 205 nm from 40°C to 90°C.

Article Snippet: Primary antibodies were incubated in 5% BLOT-QuickBlocker (G-Biosciences 786-011) as follows: rabbit anti-Mcm10 (Bethyl A300-131A; 1:500), rabbit anti-Mcm10 (Novus, H00055388-D01P, 1:500), mouse anti-Cdc45 (Santa Cruz G12, SC55568; 1:500), mouse anti-Mcm4 (Santa Cruz G7, SC28317; 1:500), mouse anti-Mus81 (Abcam ab14387; 1:500), mouse anti-PCNA (Abcam Ab29; 1:3,000), rabbit anti-RPA32 (S4/8) (Bethyl A300-245A; 1:2000), mouse anti-GAPDH (GeneTex GTX627408; 1:5,000), mouse anti- <ι>α -Tubulin (Millipore T9026, clone DM1A; 1:10,000).

Techniques: Sequencing, Mutagenesis, Modification, Binding Assay

Journal: bioRxiv

Article Title: Bi-allelic MCM10 mutations cause telomere shortening with immune dysfunction and cardiomyopathy

doi: 10.1101/844498

Figure Lengend Snippet: a) Cell cycle distribution of HCT116 wild type and MCM10 +/- cells. Percentage of each population in G1-(green), S-(purple) or G2/M-phase (gray) is shown. b) Cell cycle distribution of HCT116 wild type and MCM10 +/- cell lines from three biological replicates. Percentage of each population in G1-(green), S-(purple) and G2/M-phase (gray) is shown. c) Schematic of flow cytometry analysis. Cell-cycle phase is defined by DNA content, EdU incorporation and chromatin loaded MCM2. d) Analytical flow cytometry plots for HCT116 wild type and MCM10 +/- cells. G1-phase/MCM positive cells (blue), S-phase/MCM positive cells (orange) and G1- or G2/M-phase/MCM negative cells (gray) are indicated. e) Comparison of origin licensing (left) and quantification of G1 loaded MCM2 (n = 3) in HCT116 wild type (gray) and MCM10 +/- cells (blue/green). f) Comparison of S-phase DNA synthesis (left) and mean EdU intensity (n = 3) in HCT116 wild type (gray) and MCM10 +/- cells (red/orange). Error bars in b, e and f indicate standard deviation and significance was calculated using two-tailed student’s t-test with *<.05; **<.01, ***<.001. g) Inter-origin distance (IOD) quantification from three technical replicates across two biological replicates in wild type (blue) and MCM10 +/- cells (red). Average IOD and number (n) quantified is listed. h) Fork speed from three technical replicates across two biological replicates in wild type (blue) and MCM10 +/- cells (red). Average fork speed (kb/minute) and number (n) quantified is listed. i) Fork stability from three technical replicates across two biological replicates in wild type (blue) and MCM10 +/- cells (red). Average fork stability and number (n) quantified is listed. Statistical significance for g-i was calculated using Mann-Whitney Ranked Sum Test with *<.05; **<.01, ***<.001. j) Chromatin associated PCNA, PCNA-Ub and phospho-RPA32, which binds to ssDNA exposed during replication stress, with and without 40 J UV treatment. Quantification of PCNA-Ub levels normalized to unmodified PCNA, relative to the first lane wild type sample is indicated.

Article Snippet: Primary antibodies were incubated in 5% BLOT-QuickBlocker (G-Biosciences 786-011) as follows: rabbit anti-Mcm10 (Bethyl A300-131A; 1:500), rabbit anti-Mcm10 (Novus, H00055388-D01P, 1:500), mouse anti-Cdc45 (Santa Cruz G12, SC55568; 1:500), mouse anti-Mcm4 (Santa Cruz G7, SC28317; 1:500), mouse anti-Mus81 (Abcam ab14387; 1:500), mouse anti-PCNA (Abcam Ab29; 1:3,000), rabbit anti-RPA32 (S4/8) (Bethyl A300-245A; 1:2000), mouse anti-GAPDH (GeneTex GTX627408; 1:5,000), mouse anti- <ι>α -Tubulin (Millipore T9026, clone DM1A; 1:10,000).

Techniques: Flow Cytometry, DNA Synthesis, Standard Deviation, Two Tailed Test, MANN-WHITNEY

Journal: bioRxiv

Article Title: Bi-allelic MCM10 mutations cause telomere shortening with immune dysfunction and cardiomyopathy

doi: 10.1101/844498

Figure Lengend Snippet: a) Representative phase contrast images of early, middle and late passage MCM10 +/- cell populations. b) Example karyotypes from late passage HCT116 wild type (top) and mid-passage (middle) or late passage (bottom) MCM10 +/- cells. Blue arrows indicate expected HCT116 genomic aberrations. Red arrows indicate non-clonal genomic aberrations. c) TRF analysis comparing early passage HCT116 wild type cells with clonal Mcm10- deficient populations carrying inactivating mutations in one copy of MCM10 exon 3. Yellow dots indicate the location of peak intensity. d) Quantification of <ι>β -gal activity expressed as arbitrary fluorescence units normalized to total protein for HCT116 wild type (blue) and clonal MCM10 +/- cell lines (red). Error bars indicate standard deviation and statistical significance was calculated using two tailed student’s t-test with *<.05; **<.01, ***<.001; n=3 replicate wells for all data points. g) Quantification of signal free-ends (left) and fragile telomeres (right) in late passage HCT116 wild type (blue) and MCM10 +/- cells (red). Error bars indicate standard deviation and statistical significance was calculated using two-tailed student’s t-test with ***<.001; n = number of metaphases quantified per cell line. h) Flow chart for experiment to generate and analyze PURO-marked MCM10 +/- cell lines. i) Analysis of a PURO-marked HCT116 MCM10 +/- het #14 cell population tracking average telomere length by TRF (top) and reversion of the exon 14 locus by PCR (bottom). PDs at each time point is indicated. Yellow dots indicate the location of peak intensity.

Article Snippet: Primary antibodies were incubated in 5% BLOT-QuickBlocker (G-Biosciences 786-011) as follows: rabbit anti-Mcm10 (Bethyl A300-131A; 1:500), rabbit anti-Mcm10 (Novus, H00055388-D01P, 1:500), mouse anti-Cdc45 (Santa Cruz G12, SC55568; 1:500), mouse anti-Mcm4 (Santa Cruz G7, SC28317; 1:500), mouse anti-Mus81 (Abcam ab14387; 1:500), mouse anti-PCNA (Abcam Ab29; 1:3,000), rabbit anti-RPA32 (S4/8) (Bethyl A300-245A; 1:2000), mouse anti-GAPDH (GeneTex GTX627408; 1:5,000), mouse anti- <ι>α -Tubulin (Millipore T9026, clone DM1A; 1:10,000).

Techniques: Activity Assay, Fluorescence, Standard Deviation, Two Tailed Test

Journal: bioRxiv

Article Title: Bi-allelic MCM10 mutations cause telomere shortening with immune dysfunction and cardiomyopathy

doi: 10.1101/844498

Figure Lengend Snippet: a) TRF analysis of HCT116 wild type (left) and MCM10 +/- cells (right). Estimated PDs is indicated. Yellow dots indicate the location of peak intensity. b) Signal free-ends (left) and fragile telomeres (right) in HCT116 wild type (blue) and MCM10 +/- cells (red). Statistical significance was calculated using two-tailed student’s t-test with ***<.001; n = metaphases analyzed per cell line. Scale bars are 1 µm. c) TRAP assay from HCT116 wild type (left) and MCM10 +/- cells (right). The internal PCR control at 36 bp and telomerase above 50 bp are noted. d) TRF analysis of HCT116 wild type (left) and independent late passage MCM10 +/- cell populations from two parental MCM10 +/- cell lines (right). Yellow dots indicate the location of peak intensity. e) MCM10 +/- exon 14 genotyping of alleles carrying a loxP site 3’ of exon 14 (upper band) or a loxP scar (lower band), in comparison to the wild type locus (middle band). A faint non-specific band is noted (asterisk). f) MCM10 +/- exon 14 genotyping PCR in late passage MCM10 +/- populations showing alleles that have one loxP site 3’ of exon 14 (upper band) or a loxP scar (lower band), as well as exon 14 reverted alleles that have retained or lost the 3’ loxP site. A faint non-specific band can also be detected (asterisk). g) TRF analysis in HCT116 wild type and MCM10 +/- cells (top). PDs for each population are shown. Yellow dots indicate the location of peak intensity. Genotyping PCR for each TRF sample is shown (bottom). h) Western blot analyses for Mcm10 with tubulin as a loading control. Quantification of Mcm10 levels normalized to loading control, relative to the first lane wild type sample is indicated. PDs for each cell line are noted. i) Proliferation rate in HCT116 wild type, MCM10 +/- and reverted cells normalized to early passage wild type cells, for each cell line n = 6 replicate wells across two biological replicates. j) Cell cycle distribution of HCT116 wild type, MCM10 +/- and reverted cell lines, n = 4 across two biological replicates. Percentage of each population in G1-(green), S-(purple) and G2/M-phase (gray) is shown. Error bars in h and i indicate standard deviation and significance was calculated using two-tailed student’s t-test with *<.05; **<.01, ***<.001.

Article Snippet: Primary antibodies were incubated in 5% BLOT-QuickBlocker (G-Biosciences 786-011) as follows: rabbit anti-Mcm10 (Bethyl A300-131A; 1:500), rabbit anti-Mcm10 (Novus, H00055388-D01P, 1:500), mouse anti-Cdc45 (Santa Cruz G12, SC55568; 1:500), mouse anti-Mcm4 (Santa Cruz G7, SC28317; 1:500), mouse anti-Mus81 (Abcam ab14387; 1:500), mouse anti-PCNA (Abcam Ab29; 1:3,000), rabbit anti-RPA32 (S4/8) (Bethyl A300-245A; 1:2000), mouse anti-GAPDH (GeneTex GTX627408; 1:5,000), mouse anti- <ι>α -Tubulin (Millipore T9026, clone DM1A; 1:10,000).

Techniques: Two Tailed Test, TRAP Assay, Western Blot, Standard Deviation

Journal: bioRxiv

Article Title: Bi-allelic MCM10 mutations cause telomere shortening with immune dysfunction and cardiomyopathy

doi: 10.1101/844498

Figure Lengend Snippet: a) Western blot analyses for Mcm10, Cdc45 and Mcm4, with GAPDH loading controls. Quantification of Mcm10, Cdc45 or Mcm4 levels normalized to loading control, relative to the first lane wild-type sample is indicated. b) Western blot analyses for Cdc45 or Mcm4 with GAPDH as a loading control. Quantification of Cdc45 or Mcm4 levels normalized to loading control, relative to the first lane wild-type sample is indicated. c) Quantification of growth rate in CDC45 +/- (red) and MCM4 +/- (orange) cell lines normalized to HCT116 wild type cells. For each cell line n=6 replicate wells across three biological replicates; error bars indicate standard deviation and statistical significance was calculated using two-tailed student’s t-test with *<.05; **<.01, ***<.001. d) TRF analysis in HCT116 wild type and CDC45 +/- cell lines. Estimated PDs is indicated. Yellow dots indicate the location of peak intensity. e) TRF analysis in HCT116 wild type and MCM4 +/- cell lines. Estimated PDs is indicated. Yellow dots indicate the location of peak intensity.

Article Snippet: Primary antibodies were incubated in 5% BLOT-QuickBlocker (G-Biosciences 786-011) as follows: rabbit anti-Mcm10 (Bethyl A300-131A; 1:500), rabbit anti-Mcm10 (Novus, H00055388-D01P, 1:500), mouse anti-Cdc45 (Santa Cruz G12, SC55568; 1:500), mouse anti-Mcm4 (Santa Cruz G7, SC28317; 1:500), mouse anti-Mus81 (Abcam ab14387; 1:500), mouse anti-PCNA (Abcam Ab29; 1:3,000), rabbit anti-RPA32 (S4/8) (Bethyl A300-245A; 1:2000), mouse anti-GAPDH (GeneTex GTX627408; 1:5,000), mouse anti- <ι>α -Tubulin (Millipore T9026, clone DM1A; 1:10,000).

Techniques: Western Blot, Standard Deviation, Two Tailed Test

Journal: bioRxiv

Article Title: Bi-allelic MCM10 mutations cause telomere shortening with immune dysfunction and cardiomyopathy

doi: 10.1101/844498

Figure Lengend Snippet: a) Western blot for Mcm10 with tubulin as a loading control. Quantification of Mcm10 levels normalized to loading control, relative to wild type is indicated. b) Average proliferation rate in MCM10 +/- cells normalized to wild type. For each cell line n = 6 wells across three biological replicates. c) Average percentage of each population represented by early apoptotic, late apoptotic or dead cells. RPE-1 wild type (blue) and clonal MCM10 +/- cell lines (red) are shown. Error bars indicate standard deviation and statistical significance was calculated using student’s t-test with *<.05; **<.01, ***<.001; n = 4 for all data points. d) Cell cycle distribution of RPE-1 wild type and MCM10 +/- cells. Percentage of each population in G1-(green), S-(purple) and G2/M-phase (gray) is shown. e) Cell cycle distribution of RPE-1 wild type and MCM10 +/- cells from three biological replicates. Percentage of each population in G1-(green), S-(purple) and G2/M-phase (gray) is shown. f) Flow cytometry plots for RPE-1 wild type and MCM10 +/- cells. G1-phase/MCM positive cells (blue), S-phase/MCM positive cells (orange) and G1- or G2/M-phase/MCM negative cells (gray) are indicated. g) Comparison of origin licensing (left) and G1 loaded MCM2 (n = 3) in RPE-1 wild type (gray) and MCM10 +/- cells (blue). h) Comparison of S-phase DNA synthesis (left) and mean EdU intensity (n = 3) in RPE-1 wild type (gray) and MCM10 +/- cell lines (red). i) G1 loaded Mcm2 (n = 3) in wild type HCT116 (gray) and RPE-1 (blue) cells. j) Mean EdU intensity (n = 3) in wild type HCT116 (red) and RPE-1 (gray) cells. k) Quantification of <ι>β -gal activity expressed as arbitrary fluorescence units normalized to total protein for RPE-1 wild type (blue) and clonal MCM10 +/- cell lines (red). Error bars in b, c, e, and g-k indicate standard deviation and significance was calculated using two-tailed student’s t-test with *<.05; **<.01, ***<.001. l) TRF analysis in RPE-1 wild type and MCM10 +/- clone #20. PDs for each cell line are noted. Yellow dots indicate the location of peak intensity. m) TRF analysis RPE-1 wild type and MCM10 +/- clone #20 in the presence of telomerase inhibitor. PDs for each cell line are noted. Yellow dots indicate the location of peak intensity. n) TRF analysis RPE-1 wild type and MCM10 mutant cell lines carrying NK-associated patient mutations. Yellow dots indicate the location of peak intensity.

Article Snippet: Primary antibodies were incubated in 5% BLOT-QuickBlocker (G-Biosciences 786-011) as follows: rabbit anti-Mcm10 (Bethyl A300-131A; 1:500), rabbit anti-Mcm10 (Novus, H00055388-D01P, 1:500), mouse anti-Cdc45 (Santa Cruz G12, SC55568; 1:500), mouse anti-Mcm4 (Santa Cruz G7, SC28317; 1:500), mouse anti-Mus81 (Abcam ab14387; 1:500), mouse anti-PCNA (Abcam Ab29; 1:3,000), rabbit anti-RPA32 (S4/8) (Bethyl A300-245A; 1:2000), mouse anti-GAPDH (GeneTex GTX627408; 1:5,000), mouse anti- <ι>α -Tubulin (Millipore T9026, clone DM1A; 1:10,000).

Techniques: Western Blot, Standard Deviation, Flow Cytometry, DNA Synthesis, Activity Assay, Fluorescence, Two Tailed Test, Mutagenesis

Journal: bioRxiv

Article Title: Bi-allelic MCM10 mutations cause telomere shortening with immune dysfunction and cardiomyopathy

doi: 10.1101/844498

Figure Lengend Snippet: a) TRF analysis in HCT116 wild type and MCM10 +/- cells (top) in the presence of 10 µM telomerase inhibitor BIBR1532. PDs for each population are shown. Yellow dots indicate the location of peak intensity. Genotyping PCR for each time point is shown (bottom). b) TRF analyses in MCM10 +/- clone #8 reverted cells in the presence or absence of telomerase inhibitor. PDs for each population are indicated. Yellow dots indicate the location of peak intensity. c) TRF analysis in HCT116 wild type, MCM10 +/- cell lines and ST derivatives of each parental cell line. d) Representative TRAP assay comparing telomerase activity in whole cell extracts from HCT116 wild type (left) and MCM10 +/- ST cell lines (right). The internal PCR control at 36 bp and telomerase products beginning at 50 bp are noted. For each cell line, two concentrations of cell extract were used representing a 10-fold dilution. e) Western blot analyses of whole cell extracts from HCT116 ST wild type and MCM10 +/- cell lines for Mcm10 with tubulin as a loading control. Quantification of Mcm10 levels normalized to loading control, relative to the first lane wild-type ST3 sample is indicated. f) Average proliferation rate in HCT116 wild type, MCM10 +/- and ST cell lines normalized to HCT116 wild type cells. For each cell line n = 4 replicate wells across two biological replicates; error bars indicate standard deviation and statistical significance was calculated using two-tailed student’s t-test with *<.05; **<.01, ***<.001. g) Comparison of clonogenic survival in HCT116 wild type, MCM10 +/- and ST cell lines. The number of cells plated per well is indicated.

Article Snippet: Primary antibodies were incubated in 5% BLOT-QuickBlocker (G-Biosciences 786-011) as follows: rabbit anti-Mcm10 (Bethyl A300-131A; 1:500), rabbit anti-Mcm10 (Novus, H00055388-D01P, 1:500), mouse anti-Cdc45 (Santa Cruz G12, SC55568; 1:500), mouse anti-Mcm4 (Santa Cruz G7, SC28317; 1:500), mouse anti-Mus81 (Abcam ab14387; 1:500), mouse anti-PCNA (Abcam Ab29; 1:3,000), rabbit anti-RPA32 (S4/8) (Bethyl A300-245A; 1:2000), mouse anti-GAPDH (GeneTex GTX627408; 1:5,000), mouse anti- <ι>α -Tubulin (Millipore T9026, clone DM1A; 1:10,000).

Techniques: TRAP Assay, Activity Assay, Western Blot, Standard Deviation, Two Tailed Test

Journal: bioRxiv

Article Title: Bi-allelic MCM10 mutations cause telomere shortening with immune dysfunction and cardiomyopathy

doi: 10.1101/844498

Figure Lengend Snippet: a) Telomere combing images with telomeric DNA (green), nascent DNA (red) and merged images with examples of unreplicated telomeres (left), partially replicated (middle) and completely replicated telomeres (right). Telomeric and sub-telomeric regions are indicated. Scale bars are 3 µm. b) Average percentage of unreplicated telomeres in wild type (blue) and MCM10 +/- cell lines (red) ST cell lines, n= total number of telomeres quantified including two or more replicate experiments. c) Average percentage of completely replicated (yellow) versus partially/stalled telomeres (green) in ST cell lines, n= total number of replicated telomeres quantified including two or more replicate experiments. Error bars in b and c indicate standard deviation and significance was calculated using two-tailed student’s t-test with *<.05; **<.01, ***<.001. d) (Left) Cartoon of t-complex DNA as formerly depicted . (Middle) Diagram of double-stranded telomere restriction fragment (ds-TRF), telomere circle (t-circle) and telomere complex (t-complex) DNA species from 2D gel electrophoresis. (Right) Comparison of 2D gels from ST cell lines. e) Comparison of DNA species from 2D gel electrophoresis in HCT116 wild type ST cells with (bottom) and without (top) 4-day HU treatment. f) Comparison of DNA species from 2D gels in HCT116 wild type and MCM10 +/- ST cell lines with (bottom) and without (top) S1 nuclease digestion. g) (Left) Image of t-SCE staining in ST cell lines. Examples of a t-SCE event and chromosomes without t-SCE are highlighted. (Right) Percentage t-SCE per chromosome ends in ST cell lines. Bars represent median percentage t-SCE per chromosome ends; n = >14 metaphases for each cell line. Significance was calculated using two-tailed student’s t-test with *<.05; **<.01, ***<.001. Scale bar is 10 µm.

Article Snippet: Primary antibodies were incubated in 5% BLOT-QuickBlocker (G-Biosciences 786-011) as follows: rabbit anti-Mcm10 (Bethyl A300-131A; 1:500), rabbit anti-Mcm10 (Novus, H00055388-D01P, 1:500), mouse anti-Cdc45 (Santa Cruz G12, SC55568; 1:500), mouse anti-Mcm4 (Santa Cruz G7, SC28317; 1:500), mouse anti-Mus81 (Abcam ab14387; 1:500), mouse anti-PCNA (Abcam Ab29; 1:3,000), rabbit anti-RPA32 (S4/8) (Bethyl A300-245A; 1:2000), mouse anti-GAPDH (GeneTex GTX627408; 1:5,000), mouse anti- <ι>α -Tubulin (Millipore T9026, clone DM1A; 1:10,000).

Techniques: Standard Deviation, Two Tailed Test, Two-Dimensional Gel Electrophoresis, Electrophoresis, Staining

Journal: bioRxiv

Article Title: Bi-allelic MCM10 mutations cause telomere shortening with immune dysfunction and cardiomyopathy

doi: 10.1101/844498

Figure Lengend Snippet: a) (Top left) Diagram of double-stranded telomere restriction fragment (ds-TRF), telomere circle (t-circle) and telomere complex (t-complex) DNA species from 2D TRF gel electrophoresis. (Right) Representative comparison of 2D TRF gel electrophoresis in HCT116 wild type, MCM10 +/- and reverted ST cell lines. (Bottom left) Genotyping PCR for exon 14 in cell populations showing alleles that have one loxP site 3’ of exon 14 (upper band) or a loxP scar (lower band), as well as exon 14 reverted alleles that have retained or lost the 3’ loxP site. A faint non-specific band can also be detected (asterisk). b) Average proliferation rate in HCT116 wild type, MCM10 +/- and reverted ST cell lines normalized to HCT116 wild type ST3. For each cell line n = 5 replicate wells across two biological replicates; error bars indicate standard deviation and statistical significance was calculated using two-tailed student’s t-test with *<.05; **<.01, ***<.001.

Article Snippet: Primary antibodies were incubated in 5% BLOT-QuickBlocker (G-Biosciences 786-011) as follows: rabbit anti-Mcm10 (Bethyl A300-131A; 1:500), rabbit anti-Mcm10 (Novus, H00055388-D01P, 1:500), mouse anti-Cdc45 (Santa Cruz G12, SC55568; 1:500), mouse anti-Mcm4 (Santa Cruz G7, SC28317; 1:500), mouse anti-Mus81 (Abcam ab14387; 1:500), mouse anti-PCNA (Abcam Ab29; 1:3,000), rabbit anti-RPA32 (S4/8) (Bethyl A300-245A; 1:2000), mouse anti-GAPDH (GeneTex GTX627408; 1:5,000), mouse anti- <ι>α -Tubulin (Millipore T9026, clone DM1A; 1:10,000).

Techniques: Nucleic Acid Electrophoresis, Standard Deviation, Two Tailed Test

Journal: bioRxiv

Article Title: Bi-allelic MCM10 mutations cause telomere shortening with immune dysfunction and cardiomyopathy

doi: 10.1101/844498

Figure Lengend Snippet: a) Western blot analyses for Mus81 and Mcm10 with GAPDH (left) as a loading control. Quantification of Mcm10 levels normalized to tubulin loading control, relative to the first lane wild type sample is indicated. b) Proliferation rate in HCT116 wild type, MUS81 -/- , MCM10 +/- and double mutant cell lines normalized to HCT116 wild type, n = 6 replicate wells across three biological replicates. c) Comparison of clonogenic survival of HCT116 wild type, MUS81 -/- , MCM10 +/- and double mutant cell lines. d) Average percentage of each population represented by early apoptotic, late apoptotic or dead cells in HCT116 wild-type, MUS81 -/- , MCM10 +/- and double mutant cell lines, n = 2 replicates for HCT116 wild type and MCM10 +/- single mutants; n = 4 replicates across two biological replicates for all MUS81 -/- cell lines. e) TRF analysis of early passage HCT116 wild type, MUS81 -/- , MCM10 +/- and double mutant cell lines. Yellow dots indicate the location of peak intensity. f) <ι>β -gal activity expressed as arbitrary fluorescence units normalized to total protein for MUS81 -/- (black) and MUS81 -/- , MCM10 +/- mutant cell lines (gray). Average levels for HCT116 wild type and MCM10 +/- cell lines from are indicated with dashed lines, n = 3 replicate wells for all data points. Error bars in b, d and f indicate standard deviation and significance was calculated using two-tailed students t-test with *<.05; **<.01, ***<.001. g) Model of MCM10 -associated telomeropathies. Different cell lineages have an inherent developmental threshold Mcm10-level to achieve complete development and differentiation. As mono- or bi-allelic mutations decrease the amount of functional Mcm10, telomere erosion is accelerated. When Mcm10 function is reduced below the required threshold, eroded telomeres cause replicative senescence and prevent complete development.

Article Snippet: Primary antibodies were incubated in 5% BLOT-QuickBlocker (G-Biosciences 786-011) as follows: rabbit anti-Mcm10 (Bethyl A300-131A; 1:500), rabbit anti-Mcm10 (Novus, H00055388-D01P, 1:500), mouse anti-Cdc45 (Santa Cruz G12, SC55568; 1:500), mouse anti-Mcm4 (Santa Cruz G7, SC28317; 1:500), mouse anti-Mus81 (Abcam ab14387; 1:500), mouse anti-PCNA (Abcam Ab29; 1:3,000), rabbit anti-RPA32 (S4/8) (Bethyl A300-245A; 1:2000), mouse anti-GAPDH (GeneTex GTX627408; 1:5,000), mouse anti- <ι>α -Tubulin (Millipore T9026, clone DM1A; 1:10,000).

Techniques: Western Blot, Mutagenesis, Activity Assay, Fluorescence, Standard Deviation, Two Tailed Test, Functional Assay