Journal: Science Advances

Article Title: PES1 is a critical component of telomerase assembly and regulates cellular senescence

doi: 10.1126/sciadv.aav1090

Figure Lengend Snippet: ( A ) PES1 WT or KO MCF7 cells (left) and MEFs (right) were transiently transfected with the indicated plasmids and immunoprecipitated with anti-Flag. The precipitates were used for assessment of Flag-h/mTERT, PES1, and β-actin expression by immunoblot. TR levels were determined by RT-PCR. Flag-h/mTERT, Flag-tagged human or mouse TERT. Flag-hTERT was used for MCF7 cells, and Flag-mTERT was used for MEFs. ( B ) siRNA-mediated PES1 KD MCF7 or HepG2 cells were transfected with Flag-hTERT and siRNA-resistant Myc-tagged PES1, PES1 (ΔBRCT), or PES1 (W397R) as indicated and were analyzed as in (A). ( C ) siRNA-mediated PES1 KD MEFs were transfected with Flag-mTERT and siRNA-resistant Myc-tagged PES1, PES1 (ΔBRCT), or PES1 (W397R) as indicated and were analyzed as in (A). Data shown are mean ± SD of three independent experiments (A to C). ** P < 0.01. ( D ) Biotinylated hTR was incubated with purified GST-hTERT and WT or mutant His-PES1. The resulting complexes were subject to RNA pull-down assay. Unlabeled hTR was used as a negative control. Asterisks indicate the positions of the expected full-length fusion proteins.

Article Snippet: Human breast cancer MCF7 cells, human liver cancer HepG2 cells, and HEK293T cells were purchased from the American Type Culture Collection and have been previously tested for mycoplasma contamination.

Techniques: Transfection, Immunoprecipitation, Expressing, Western Blot, Reverse Transcription Polymerase Chain Reaction, Incubation, Purification, Mutagenesis, Pull Down Assay, Negative Control

Journal: Science Advances

Article Title: PES1 is a critical component of telomerase assembly and regulates cellular senescence

doi: 10.1126/sciadv.aav1090

Figure Lengend Snippet: ( A ) Direct telomerase assay in PES1 KO MCF7 cells or MEFs transfected with Flag-hTERT, hTR, and PES1 or its mutants. Transfected cells were immunoprecipitated with anti-Flag, followed by telomerase activity detection. Parental cells without Flag-hTERT transfection were used as a negative control. Values shown are mean ± SD of three independent experiments. * P < 0.05, ** P < 0.01. ( B ) PES1 WT and KO mouse hepatocytes were isolated from liver-specific PES1 KO mice generated by mating conditional PES1 KO mice with Alb-Cre transgenic mice and were analyzed by TRAP assay. ( C ) IP-TRAP analysis of PES1 KO MCF7 cells transfected with Flag-tagged WT or mutant PES1. When the binding of Flag-tagged proteins to anti-Flag agarose beads was complete, the reaction mixtures were centrifuged and the supernatants were collected for TRAP assay. The precipitates were washed, lysed, and used for TRAP assay. Flag-tagged PES1 or endogenous PES1 levels were analyzed by immunoblot with anti-PES1. ( D ) IP-TRAP analysis of siRNA-mediated PES1 KD HepG2 cells transfected with Flag-PES1 or empty vector. ( E ) TRAP assay was performed by mixing in vitro transcribed hTR and in vitro translated hTERT with purified WT and mutated His-PES1. ( F ) Representative MCF7 monoclones stably expressing PES1 shRNA or control shRNA and WT or mutant PES1 were harvested at indicated PDLs. Reexpression of WT or mutant PES1 was performed after 25 PDLs by splitting the same clone. Telomere lengths were measured using terminal restriction fragment (TRF) analysis. Representative immunoblot reveals PES1 expression. PES1-R, shRNA-resistant PES1. PES1 W397R-R, shRNA-resistant PES1 W397R. ( G ) PES1 WT and KO mouse hepatocytes isolated from liver-specific PES1 KO mice at 3 months were subjected to TRF analysis. Left and right panels indicate two nest mice. fl, flox.

Article Snippet: Human breast cancer MCF7 cells, human liver cancer HepG2 cells, and HEK293T cells were purchased from the American Type Culture Collection and have been previously tested for mycoplasma contamination.

Techniques: Telomerase Assay, Transfection, Immunoprecipitation, Activity Assay, Negative Control, Isolation, Generated, Transgenic Assay, TRAP Assay, Mutagenesis, Binding Assay, Western Blot, Plasmid Preparation, In Vitro, Purification, Stable Transfection, Expressing, shRNA, Control

Journal: Bone & Joint Research

Article Title: Influence of ceftriaxone on human bone cell viability and in vitro mineralization potential is concentration- and time-dependent

doi: 10.1302/2046-3758.103.BJR-2020-0412

Figure Lengend Snippet: a) Gene expression analysis of human bone progenitor cells (HBCs) cultivated in proliferation medium (Prolif) or osteogenic differentiation medium (Diff) for ten days, both supplemented with increasing concentrations of ceftriaxone (mean and standard deviation (SD), n = 3). Messenger RNA (mRNA) expression levels of alkaline phosphatase ( ALP ), osteocalcin (OC) , and collagen type I ( Col-I ) were normalized to Prolif with RPL13a as housekeeping gene. Statistically significant differences compared to Diff 0 are indicated with * (p = 0.028 at 1,000 mg/l and p = 0.002 at 1,500 mg/l, one-way analysis of variance). b) Immunohistochemical staining demonstrating the influence of ceftriaxone supplementation on ALP expression. ALP is illustrated in green, actin cytoskeleton in red, and nuclei in blue. HBCs were cultured in osteogenic differentiation (Diff) and proliferation medium (Pro) for ten days (scale bar 100 µm). Rel. Exp., relative expression.

Article Snippet: To assess the expression of osteogenic markers on the protein level, the samples were stained against alkaline phosphatase (ALP; B4-78, Developmental Studies Hybridoma Bank, USA), collagen type I (Col-I; C2456, Sigma-Aldrich), actin cytoskeleton (Alexa Fluor 546 Phalloidin, A22283, Thermo Fisher), and nuclei (DAPI; 4',6-diamidino-2-phenylindole, D9542, Sigma-Aldrich, Buchs, Switzerland) after fixation at day 10.

Techniques: Gene Expression, Standard Deviation, Expressing, Immunohistochemical staining, Staining, Cell Culture

Journal: bioRxiv

Article Title: LRRC23 truncation impairs radial spoke 3 head assembly and sperm motility underlying male infertility

doi: 10.1101/2023.02.25.530050

Figure Lengend Snippet: A A consanguineous pedigree with two infertile males (IV-1 and IV-2). IV-1 was subjected for WES (arrow). Genotypes of the variant (blue) in all family members included in this study (III-1, III-2, IV-1, IV-2, IV-3, and IV-4) are confirmed by Sanger sequencing. +, wild-type allele. An infertile female sibling (IV-4) is marked in black circle. B Papanicolaou-stained sperm from the infertile male (IV-2). C Mapping of the LRRC23 variant. Mutation of G to A at the splicing donor site in the 5 th intron is predicted to prevent LRRC23 mRNA from splicing. D Sequencing chromatograms presenting the LRRC23 variant in the infertile male (IV-1) and his father (III-2). The variant is underlined and normal splicing donor site (GT) is boxed. E, F Minigene assay for testing altered splicing of LRRC23 by the variant. (E) Minigene constructs expressing LRRC23 ORF containing the 5 th intron (sashed) with wild-type (WT) or mutant (Mut, red) splicing donor site were generated. The constructs are tagged with FLAG and HA at N- and C-termini, respectively. (F) RT-PCR of the 293T cells transfected with the minigene constructs reveals the 5 th intron is not spliced out and retained by the variant. Intron-spanning primers, F1 and R1, are used. Repeated three times with biological replications.

Article Snippet: Human LRRC23 ORF clone (HG24717-UT, SinoBiological) and the partial region of the human LRRC23 5 th intron amplified by PCR was subcloned into phCMV3 vector to generate mammalian expression constructs for human LRRC23 ( phCMV3-FLAG-hLRRC23 ORF - HA , phCMV3-FLAG-hLRRC23 WT - HA , and phCMV3-FLAG-hLRRC23 Mut - HA ). cDNA clones of human RSPH3 (616166, Horizon Discovery), RSPH6A (5270908, Horizon Discovery), RSPH9 (5296237, Horizon Discovery), and RSPH22 (OHu31347, GenScript) was subcloned into phCMV3 to express human RSPH proteins tagged with FLAG at C-termini.

Techniques: Variant Assay, Sequencing, Staining, Mutagenesis, Mini Gene Assay, Construct, Expressing, Generated, Reverse Transcription Polymerase Chain Reaction, Transfection

Journal: bioRxiv

Article Title: LRRC23 truncation impairs radial spoke 3 head assembly and sperm motility underlying male infertility

doi: 10.1101/2023.02.25.530050

Figure Lengend Snippet: A, B Immunoblotting of LRRC23 in testis (A) and epididymal sperm (B) from mutant male mice. Truncated LRRC23 (arrowheads) is detected from testis microsome fraction (filled), but not in mature sperm (empty), of heterozygous (+/Δ) and homozygous (Δ/Δ) males. Acetylated tubulin (AcTub) is a loading control. Experiments were performed with three biological replications. C Confocal images of immunostained LRRC23 in Lrrc23 +/Δ and Lrrc23 Δ/Δ epididymal sperm Experiments were repeated with three biological replications. D Epididymal sperm counts. n.s., not significant. E Pregnancy rate of Lrrc23 +/Δ and Lrrc23 Δ/Δ males. F Number of litters from fertile females mated with Lrrc23 +/Δ and Lrrc23 Δ/Δ males. G Swimming trajectory of Lrrc23 +/Δ and Lrrc23 Δ/Δ sperm in viscous media (0.3% methylcellulose). Swimming trajectory for 2 seconds is overlaid. Experiments were performed with three biological replications. See . H Flagellar waveforms of Lrrc23 +/Δ and Lrrc23 Δ/Δ sperm before (0 minute) and after (90 minutes) inducing capacitation. Flagellar movements for two beat cycles are overlaid and color coded in time. Experiments were performed with three biological replications. See . Data information: In (A-C), samples from WT were used for positive or negative control of normal or truncated LRRC23. In (D, F), circles indicate sperm counts from individual males (D) and pup numbers from each litter (F), and data represented as mean ± SEM (D, Mann-whiteny U test; F, Student’s t-test). n.s., non-significant.

Article Snippet: Human LRRC23 ORF clone (HG24717-UT, SinoBiological) and the partial region of the human LRRC23 5 th intron amplified by PCR was subcloned into phCMV3 vector to generate mammalian expression constructs for human LRRC23 ( phCMV3-FLAG-hLRRC23 ORF - HA , phCMV3-FLAG-hLRRC23 WT - HA , and phCMV3-FLAG-hLRRC23 Mut - HA ). cDNA clones of human RSPH3 (616166, Horizon Discovery), RSPH6A (5270908, Horizon Discovery), RSPH9 (5296237, Horizon Discovery), and RSPH22 (OHu31347, GenScript) was subcloned into phCMV3 to express human RSPH proteins tagged with FLAG at C-termini.

Techniques: Western Blot, Mutagenesis, Control, Negative Control

Journal: bioRxiv

Article Title: LRRC23 truncation impairs radial spoke 3 head assembly and sperm motility underlying male infertility

doi: 10.1101/2023.02.25.530050

Figure Lengend Snippet: A Sub-tomogram averaging images of RSs from Chlamydomonas reinhardtii ( red ), Trypanosoma brucei (yellow), mouse sperm (sky blue), and human sperm (blue). RSs at 7 th microtubule doublet (MTD) are shown for human sperm. Original data from Electron Microscopy Data Bank was rendered. B Structure of RS in C. reinhardtii . A schematic cartoon shows the RS1 and 2. The structure of RS2 stalk is shown in inset (PDB Id: 7JRJ). C, D Purification of normal (hLRRC23 WT ) and the mutant human LRRC23 (hLRRC23 Mut ) by the splicing site mutation (c.621+1G>A) in this study. (C) Diagrams for the purified recombinant normal and mutant proteins tagged with tagged with GST and HA at N- and C-termini, respectively. (D) Purified proteins by Coomassie blue staining ( left ) and immunoblotting with α-HA ( middle ) and a-LRRC23 ( right ). Proteins matched to the predicted size were marked with asterisks. E A cartoon of the RSPH-trap approach to test LRRC23 interaction with RS proteins. Individual human RS proteins tagged with FLAG (RSPH-FLAG) are expressed in 293T cells and enriched by α-FLAG resin from cell lysates. The recombinant RSPH proteins were incubated with the purified hLRRC23 WT or hLRRC23 Mut and subjected to immunoblotting. F Interaction of hLRRC23 to a RS head component, RSPH9. The purified hLRRC23 were incubated with the RSPH-Trap (RS head, RSPH6A and RSPH9; stalk, RSPH3 and RSPH22) and subjected to immunoblotting. 5% amount of the hLRRC23s used for the trap assay were loaded as inputs. Yellow lines in individual α-HA blot images indicate marker information (75 kDa, left ; 50 kDa, right ). Experiments were repeated four times. Purified GST was used for negative control . Experiments were repeated three times with biological replications. G A phylogenetic tree constructed by Maximum-likelihood analysis of the protein sequences of the C. reinhardtii RSP15 and the orthologs of LRRC23 and LRRC34. LRR37, the first LRRC23 ortholog identified in Ciona intestinalis is marked in bold. H Comparison of the reported RSP15 from C. reinhardtii and the predicted structure of LRRC23 and LRRC34 from human. Atomic structure of the C. reinhardtii RS2 containing RSP15 are represented by ribbon (RS2) and surface (RSP15) diagram ( left , PDB Id: 7JU4). Ribbon diagrams of C. reinhardtii RSP15 and AlphaFold-predicted human LRRC23 ( middle ) and LRRC34 ( right ) are shown for structural comparison. Secondary structures are color-coded. Different from C. reinhardtii RSP15 and LRRC34, LRRC23 does not display repeated α-helix (magenta) between β-sheets (gold).

Article Snippet: Human LRRC23 ORF clone (HG24717-UT, SinoBiological) and the partial region of the human LRRC23 5 th intron amplified by PCR was subcloned into phCMV3 vector to generate mammalian expression constructs for human LRRC23 ( phCMV3-FLAG-hLRRC23 ORF - HA , phCMV3-FLAG-hLRRC23 WT - HA , and phCMV3-FLAG-hLRRC23 Mut - HA ). cDNA clones of human RSPH3 (616166, Horizon Discovery), RSPH6A (5270908, Horizon Discovery), RSPH9 (5296237, Horizon Discovery), and RSPH22 (OHu31347, GenScript) was subcloned into phCMV3 to express human RSPH proteins tagged with FLAG at C-termini.

Techniques: Electron Microscopy, Purification, Mutagenesis, Recombinant, Staining, Western Blot, Incubation, TRAP Assay, Marker, Negative Control, Construct, Comparison

Journal: bioRxiv

Article Title: LRRC23 truncation impairs radial spoke 3 head assembly and sperm motility underlying male infertility

doi: 10.1101/2023.02.25.530050

Figure Lengend Snippet: A Immunostaining of flagellar proteins in different compartments. Shown are midpiece (TOM20), annulus (SEPT4 and SEPT12), fibrous sheath (AKAP4), outer dense fiber (ODF2), and axoneme (acetylated tubulin, AcTub) in Lrrc23 +/Δ ( top ) and Lrrc23 Δ/Δ ( bottom ) sperm. Magnified insets are represented for annulus proteins (scale bars in insets = 2μm). Fluorescence and corresponding DIC images are merged. Sperm heads were counter stained with Hoechst. Lrrc23 +/Δ sperm were used for positive control. Experiments were performed with three biological replications. B Transmission electron microscopy images of Lrrc23 +/Δ ( left ) and Lrrc23 Δ/Δ ( right ) sperm. Shown are longitudinal section of sperm flagella. M, mitochondria; ODF, outer dense fiber; AX, axoneme; CP, central pair; MT, microtubule; FS, fibrous sheath. Lrrc23 +/Δ sperm were used for positive control. C Cryo-electron tomography (cryo-ET) of WT and Lrrc23 Δ/Δ sperm flagella. Shown are representative tomographic slices from WT ( left ) and Lrrc23 Δ/Δ sperm ( right ). The 9+2 axonemal structure are shown in both WT and Lrrc23 Δ/Δ in cross-sectional view ( left ). Axonemal structures are shown with proximal side of the flagellum on the left in longitudinal view ( right ; see ). Magnified insets ( bottom ) reveal that RS1, 2, and 3 are shown in WT sperm ( left , filled arrowheads) but RS3, especially head part, is not clearly visible ( right , red arrowheads) in Lrrc23 Δ/Δ sperm. RS1, 2, and 3 are distinguished by the interval between each set of RS1, 2, and 3, and the electron dense area corresponding to the barrel (RS1) and bridge (RS2–3) structures. WT sperm were used for positive control.

Article Snippet: Human LRRC23 ORF clone (HG24717-UT, SinoBiological) and the partial region of the human LRRC23 5 th intron amplified by PCR was subcloned into phCMV3 vector to generate mammalian expression constructs for human LRRC23 ( phCMV3-FLAG-hLRRC23 ORF - HA , phCMV3-FLAG-hLRRC23 WT - HA , and phCMV3-FLAG-hLRRC23 Mut - HA ). cDNA clones of human RSPH3 (616166, Horizon Discovery), RSPH6A (5270908, Horizon Discovery), RSPH9 (5296237, Horizon Discovery), and RSPH22 (OHu31347, GenScript) was subcloned into phCMV3 to express human RSPH proteins tagged with FLAG at C-termini.

Techniques: Immunostaining, Fluorescence, Staining, Positive Control, Transmission Assay, Electron Microscopy, Tomography

Journal: bioRxiv

Article Title: LRRC23 truncation impairs radial spoke 3 head assembly and sperm motility underlying male infertility

doi: 10.1101/2023.02.25.530050

Figure Lengend Snippet: A, B Sub-tomogram averaging (STA) to analyze structural defects at radial spoke (RS) of WT (A) and Lrrc23 Δ/Δ sperm (B). Shown are STA images resulted from 96-nm doublet repeats from WT and Lrrc23 Δ/Δ sperm. RS2 and 3 are magnified and density to represent RS3 head and the bridge between RS2 and RS3 (red circle) is missed in Lrrc23 Δ/Δ sperm specifically. C Overwrapped STA images from 96 nm-doublet repeats from WT (gray) and Lrrc23 Δ/Δ (gold) sperm, and Chlamydomonas reinhardtii (cyan). D A proposed model of impaired sperm motility and male infertility by the LRRC23 loss of function.

Article Snippet: Human LRRC23 ORF clone (HG24717-UT, SinoBiological) and the partial region of the human LRRC23 5 th intron amplified by PCR was subcloned into phCMV3 vector to generate mammalian expression constructs for human LRRC23 ( phCMV3-FLAG-hLRRC23 ORF - HA , phCMV3-FLAG-hLRRC23 WT - HA , and phCMV3-FLAG-hLRRC23 Mut - HA ). cDNA clones of human RSPH3 (616166, Horizon Discovery), RSPH6A (5270908, Horizon Discovery), RSPH9 (5296237, Horizon Discovery), and RSPH22 (OHu31347, GenScript) was subcloned into phCMV3 to express human RSPH proteins tagged with FLAG at C-termini.

Techniques:

Journal: Cancer cell

Article Title: Th9 Cells Represent a Unique Subset of CD4 + T Cells Endowed with the Ability to Eradicate Advanced Tumors

doi: 10.1016/j.ccell.2018.05.004

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: HEK 293T cells were transiently transfected with a 1256-bp mouse luciferase reporter vector pEZX-PG04 (mTraf6-PG04) inserted into the Traf6 promoter (Genecopoeia) or control vector (NEG-PG04) along with expression vectors for Stat6, Stat5, Stat3, Pu.1, and NF-κB molecules (p50, p52, RelA, RelB and c-Rel, Addgene) by Lipofectamine 2000 (Invitrogen).

Techniques: Blocking Assay, Staining, Extraction, Enzyme-linked Immunosorbent Assay, Luminescence Assay, Recombinant, shRNA, Negative Control, Binding Assay, Modification, Software, Gene Expression

Journal: Neuro-Oncology

Article Title: Increased activation of HDAC1/2/6 and Sp1 underlies therapeutic resistance and tumor growth in glioblastoma

doi: 10.1093/neuonc/noaa103

Figure Lengend Snippet: Sp1 is deacetylated by HDAC1/2/6 in TMZ-resistant GBM cells. (A) The wild-type (Wt) and TMZ-resistant (TMZ-R) GBM cells,12 as well as GBM spheroids formed in serum-free medium/suspension (S/S) culture and the control attached (Adh) cells13 were used for the immunoprecipitation (IP) assay with rabbit IgG, anti-Sp1 (Sp1, panel a), and anti–acetyl-lysine (ac-K, panels b and c) antibodies, and analyzed using immunoblotting (IB) as indicated. In panel c, the protein level of acetylated Sp1 was normalized to its total protein and quantified. (B) Gene expression profiles of HDACs in brain tumors were analyzed using the Oncomine database. HDAC1/3/6/9, shown by the arrows, were upregulated more in cancer tissues than in normal samples. Red indicates upregulation; blue indicates downregulation. The number in the cell represents the number of datasets that pass the filter criteria (threshold: P < 0.05). (C to F) Cells were harvested and analyzed using IB. The Wt (C and E) and TMZ-R (E) A172 cells were treated with the indicated concentrations of TMZ for 3 days. (D) The protein expression of HDACs in Wt and TMZ-R P11 GBM cells was normalized to the loading control and quantified. (F) The levels of HDAC6 and tubulin acetylation in attached GBM cells and tumorspheres. (G) Wt and TMZ-R P11 cells were used for IP assay with anti-Sp1 antibodies and rabbit IgG, and analyzed using IB as indicated. (H) TMZ-R U87MG cells were transfected with a nontargeting control siRNA or HDAC1/2/6-specific siRNAs as indicated. After knockdown, the cells were used for IP assay. (t-test: *P < 0.05, ***P < 0.001)

Article Snippet: Human GBM cell lines U87MG and A172 (both American Type Culture Collection), 2 patient-derived GBM lines, P3 and P11, as well as their TMZ-resistant cells and tumorspheres, were cultivated in different culture media as described in previous studies.

Techniques: Suspension, Control, Immunoprecipitation, Western Blot, Gene Expression, Expressing, Transfection, Knockdown

Journal: Neuro-Oncology

Article Title: Increased activation of HDAC1/2/6 and Sp1 underlies therapeutic resistance and tumor growth in glioblastoma

doi: 10.1093/neuonc/noaa103

Figure Lengend Snippet: HDAC1/2/6 inhibition significantly reduces the growth rates of TMZ-resistant GBM cells. (A) U87MG cells, as well as primary cultures of neurons and glial cells, were treated with 1 μM SAHA (SA), 1 μM azaindolyl sulfonamide compound 12 (MPT0B291, MP), or dimethyl sulfoxide (DMSO) (DM) for 4 days. After treatment, cell viability was assessed using colorimetric MTT assay. (B) In the focus formation assay, parental and TMZ-resistant (TMZ-R) U87MG cells were seeded at low density onto 60-mm plates, and treated with TMZ or MP alone or in combination at different doses every 3 days. Following a 2-week incubation period, the forming foci were stained using crystal violet. Representative images are shown. (C) TMZ-R GBM cell lines, including U87MG-R, A172-R, and P11-R cells, were treated with DMSO or different doses of MP (1, 3, 6 μM) for various time intervals (1 to 4 days). Cell viability was assessed using the MTT assay. (D) TMZ-R U87MG inoculated orthotopic mice were treated with 25 mg/kg TMZ to maintain a TMZ-resistant phenotype, and co-treated with or without 25 mg/kg MP every 2 days for 3 weeks. The brain tumors were observed using serial histology sections along the tumor using hematoxylin and eosin staining. (E) TMZ-R P3 inoculated orthotopic mice were randomly grouped and treated with DMSO, 10 mg/kg TMZ (T), or TMZ plus 10 mg/kg MP (T+M) every 2 days. Survival was plotted using a Kaplan–Meier curve. (F) Cells were transfected with HDAC1-, HDAC2-, and/or HDAC6-specific siRNAs or a nontargeting control siRNA as indicated for 2 days. After knockdown, cell viability was assessed using the MTT assay. (t-test: *P < 0.05, **P < 0.01, ***P < 0.001)

Article Snippet: Human GBM cell lines U87MG and A172 (both American Type Culture Collection), 2 patient-derived GBM lines, P3 and P11, as well as their TMZ-resistant cells and tumorspheres, were cultivated in different culture media as described in previous studies.

Techniques: Inhibition, MTT Assay, Tube Formation Assay, Incubation, Staining, Transfection, Control, Knockdown

Journal: Neuro-Oncology

Article Title: Increased activation of HDAC1/2/6 and Sp1 underlies therapeutic resistance and tumor growth in glioblastoma

doi: 10.1093/neuonc/noaa103

Figure Lengend Snippet: The HDAC/Sp1 pathway plays an important role in regulating cell cycle progression and proliferation. (A) Venn diagram illustrating overlaps between number of genes that were altered by more than 1.5–fold in TMZ-resistant (TMZ-R) cells and in spheroids (serum-free medium/suspension culture, S/S) following MP treatment and were also targeted by Sp1 in U87MG cells. (B) The IPA software program was applied on 139 potential HDACs/Sp1-regulated genes (the intersection genes of Sp1 ChIP-seq data and gene expression microarray data from MP-treated TMZ-R and S/S in [A]) to identify top 10 scoring canonical pathways. (C) Heat map representing the expression levels of 41 cell cycle–related genes, obtained from IPA analysis in (B), following MP treatment. (D) Relationships between MP-treated microarray data (in horizontal) and TCGA-GBM NGS data (in vertical) using Pearson’s correlation coefficient (PCC, r). Each dot represents the expression value of a cell cycle-related gene. (E) Forest plots showing hazard ratios for risk of death in low-grade and high-grade glioma patients with higher expression of the indicated gene(s). The lines on both sides denote 95% confidence intervals. All the original data (Kaplan–Meier curve) were obtained from PROGgeneV2 (Supplementary Figure 15A). Hazard ratios above 1 indicate a worse outcome. (t-test: *P < 0.05, **P < 0.01, ***P < 0.001)

Article Snippet: Human GBM cell lines U87MG and A172 (both American Type Culture Collection), 2 patient-derived GBM lines, P3 and P11, as well as their TMZ-resistant cells and tumorspheres, were cultivated in different culture media as described in previous studies.

Techniques: Suspension, Software, ChIP-sequencing, Gene Expression, Microarray, Expressing

Journal: Neuro-Oncology

Article Title: Increased activation of HDAC1/2/6 and Sp1 underlies therapeutic resistance and tumor growth in glioblastoma

doi: 10.1093/neuonc/noaa103

Figure Lengend Snippet: MPT0B291 induces senescence and diminished the expression stemness-related markers in both GBM spheroids and TMZ-resistant (TMZ-R) cells. (A) Mitotic P3 cells were released into the cell cycle by removing nocodazole from the culture, and then harvested at different time points as indicated. Cells were then fixed for cell cycle progression assay using flow cytometry. The percentages of cells in G1 phase and G2/M phase are shown in right panel. (B) U87MG spheroids (S/S) were treated with DMSO or MP. At 4 days posttreatment, senescence was examined using SA β-gal staining. Photomicrographs of spheroids were randomly selected in microscopic fields, and SA-β-gal positive cells were counted. (C) Cells dissociated from U87MG spheroids via trypsinization were grown in soft agar, followed by treatment with different doses of MP every 4 days. After 3 weeks of incubation at 37°C, the colonies that arose from these single cells were photographed randomly, and a histogram of average colony numbers was plotted after performing the experiment in triplicate. (D and E) Cells, as indicated, after 2 days of MP treatment were harvested, and the cell lysates were analyzed using IB with the indicated antibodies. (t-test: **P < 0.01)

Article Snippet: Human GBM cell lines U87MG and A172 (both American Type Culture Collection), 2 patient-derived GBM lines, P3 and P11, as well as their TMZ-resistant cells and tumorspheres, were cultivated in different culture media as described in previous studies.

Techniques: Expressing, Flow Cytometry, Staining, Incubation

Journal: Neuro-Oncology

Article Title: Increased activation of HDAC1/2/6 and Sp1 underlies therapeutic resistance and tumor growth in glioblastoma

doi: 10.1093/neuonc/noaa103

Figure Lengend Snippet: MPT0B291 decreases Sp1 binding to BMI1 and hTERT promoters. (A) The Sp1 ChIP-seq reads mapped to the promoter region of BMI1 and hTERT. Forward reads are shown in green and reverse reads are shown in red. The significance of ChIP peaks, generated using the CLC Genomics Workbench 10.1.1 software, indicated Sp1 binding loci. (B and C) U87MG spheroids were treated with DMSO or MP for 6 h, and the level of Sp1 binding to the promoter regions of BMI1 and hTERT was assessed using a ChIP assay with rabbit IgG or anti-Sp1 antibodies. DNA was then extracted from the sample for PCR with the primers as indicated. Rabbit IgG acted as a negative control for nonspecific precipitation, and E-box was used as a negative control for nonspecific binding. (D to F) U87MG spheroids were treated with different doses of MP for 2 days. After treatment, the mRNA (D) and protein (E) levels of hTERT in cells were analyzed using real-time PCR and IB, respectively. Furthermore, relative telomerase activity (F) was also detected using the TRAP assay and normalized to the value of the internal PCR control in each reaction. The cell lysis buffer was used as a negative control. The arrow points to the 36-bp internal control. (t-test: *P < 0.05, **P < 0.01)

Article Snippet: Human GBM cell lines U87MG and A172 (both American Type Culture Collection), 2 patient-derived GBM lines, P3 and P11, as well as their TMZ-resistant cells and tumorspheres, were cultivated in different culture media as described in previous studies.

Techniques: Binding Assay, ChIP-sequencing, Generated, Software, Negative Control, Real-time Polymerase Chain Reaction, Activity Assay, TRAP Assay, Control, Lysis

Journal: Journal of Translational Medicine

Article Title: Development of patient-derived xenograft models from a spontaneously immortal low-grade meningioma cell line, KCI-MENG1

doi: 10.1186/s12967-015-0596-8

Figure Lengend Snippet: Immunostaining of original tumor, low passage, and high passage KCI-MENG1 cells. The original patient-derived tumor ( top row ) showed moderate and patchy immunoreactivity for epithelial membrane antigen (EMA); strong and diffuse immunostaining for progesterone receptor (PR); and a Ki-67 proliferative index of 2–3%. There was also strong immunostaining for N-cadherin and vimentin. KCI-MENG1-LP cells ( middle row ) and KCI-MENG1-HP cells ( bottom row ) maintained expression of EMA, N-cadherin, and vimentin but had significantly reduced PR expression compared to the original tumor. Whereas Ki-67 labeling was found in only a small number of cells in the original tumor and low passage cells, it was positive in virtually all P84 cells. Scale bar 50 µm.

Article Snippet: Primary antibodies used targeted the following proteins: EMA (cat.#247M-94), PR (cat.#323R-14), Ki-67 (cat.#275R-14), vimentin (cat.#347R-14; all from CellMarque, Rocklin, CA, USA), and N-cadherin (cat.#NBP1-48309, Novus Biologicals, Littleton, CO, USA).

Techniques: Immunostaining, Derivative Assay, Membrane, Expressing, Labeling

Journal: Journal of Translational Medicine

Article Title: Development of patient-derived xenograft models from a spontaneously immortal low-grade meningioma cell line, KCI-MENG1

doi: 10.1186/s12967-015-0596-8

Figure Lengend Snippet: Immunostaining of original patient tumor, low and high passage KCI-MENG1 cells, and subcutaneous xenograft tumor. The original patient-derived tumor showed moderate immunoreactivity for E-cadherin which was maintained in all in vitro and in vivo models. Scale bar 50 µm.

Article Snippet: Primary antibodies used targeted the following proteins: EMA (cat.#247M-94), PR (cat.#323R-14), Ki-67 (cat.#275R-14), vimentin (cat.#347R-14; all from CellMarque, Rocklin, CA, USA), and N-cadherin (cat.#NBP1-48309, Novus Biologicals, Littleton, CO, USA).

Techniques: Immunostaining, Derivative Assay, In Vitro, In Vivo

Journal: Journal of Translational Medicine

Article Title: Development of patient-derived xenograft models from a spontaneously immortal low-grade meningioma cell line, KCI-MENG1

doi: 10.1186/s12967-015-0596-8

Figure Lengend Snippet: Meningioma cell lines reported in the literature

Article Snippet: Primary antibodies used targeted the following proteins: EMA (cat.#247M-94), PR (cat.#323R-14), Ki-67 (cat.#275R-14), vimentin (cat.#347R-14; all from CellMarque, Rocklin, CA, USA), and N-cadherin (cat.#NBP1-48309, Novus Biologicals, Littleton, CO, USA).

Techniques: Southern Blot, Injection, Expressing, TRAP Assay, Activity Assay, Real-time Polymerase Chain Reaction

Journal: Journal of Translational Medicine

Article Title: Development of patient-derived xenograft models from a spontaneously immortal low-grade meningioma cell line, KCI-MENG1

doi: 10.1186/s12967-015-0596-8

Figure Lengend Snippet: Human meningioma mouse xenograft model KCI-MENG1-LPSX generated with the spontaneously immortal cell line KCI-MENG1-LP. Tumors from immunocompromised SCID mice were dissected ( a ) and the derivative cell line KCI-MENG1-LPSX CL was generated. The H&E staining of the mouse tumor revealed a pattern of moderately cellular meningothelial cells similar to the original patient tumor ( b ). The KCI-MENG1-LPSX CL cells were composed of the round-shaped cells similar to the high passage parent cell line KCI-MENG1-HP ( c ). The EMA, PR, and N-cadherin IHC of the mouse tumor highly resembled the original patient-derived tumor ( d top row ). The vimentin- and Ki-67-stained cells in the mouse tumor tissue were markedly more abundant and more intensely stained than in the original tumor ( d top row ). KCI-MENG1-LPSX CL cells displayed the same patterns of immunostaining as the high passage parent cell line KCI-MENG1-HP, including the loss of PR staining ( d bottom row ). Scale bar 50 µm.

Article Snippet: Primary antibodies used targeted the following proteins: EMA (cat.#247M-94), PR (cat.#323R-14), Ki-67 (cat.#275R-14), vimentin (cat.#347R-14; all from CellMarque, Rocklin, CA, USA), and N-cadherin (cat.#NBP1-48309, Novus Biologicals, Littleton, CO, USA).

Techniques: Generated, Staining, Derivative Assay, Immunostaining