Journal: Cellular and molecular bioengineering

Article Title: Mouse Pluripotent Stem Cell Differentiation Under Physiological Oxygen Reduces Residual Teratomas.

doi: 10.1007/s12195-021-00687-8

Figure Lengend Snippet: FIGURE 2. Expression of pluripotency markers decrease substantially with time during differentiation and extended culture under reduced oxygen. Cells were differentiated for 10 days followed by up to 80 days of extended culture (90 days total). (a) Flow cytometric quantification of the fraction and number of Oct4-GFP1 for differentiating mESCs and miPSCs at 142, 36, and 7 mmHg pO2gas (n = 3). (b) Representative en face bright field and Oct4-GFP images of cell aggregates after extended culture at 142 or 7 mmHg pO2gas. (c) Relative expression of Oct4 and Nanog mRNA with time in mESCs, miPSCs, and HIF-1a2/2 mESCs at 142, 36, and 7 mmHg pO2gas (n = 3) measured with qPCR.

Article Snippet: Cells were incubated overnight at 4 C with primary antibodies against Oct4 (sc-9081; Santa Cruz Biotechnology, Santa Cruz, CA, USA) diluted 1:100, Nanog (IHC-00205; Bethyl Laboratories, Montogomery, TX, USA) diluted 1:100, SSEA-1 (MC-480; DSHB) diluted 1:10, Foxa2 (sc-6554; Santa Cruz Biotechnology) diluted 1:200, Nestin diluted 1:50, cardiac troponin T (cTnT; MS-295-P1; NeoMarkers, Fremont, CA, USA) diluted 1:50, or Nkx2.5 (sc-8697; Santa Cruz Biotechnology) in 3% DS, 0.1% Triton X-100 solution, washed once with PBS, and incubated with secondary donkey AlexaFluor 488 (green) or 594 (red) antibodies (Invitrogen) against the appropriate species diluted 1:250 in 3% DS, 0.1% Triton X-100 solution for 2 h in the dark.

Techniques: Expressing

Journal: Oncotarget

Article Title: ARID3B increases ovarian tumor burden and is associated with a cancer stem cell gene signature.

doi: 10.18632/oncotarget.2247

Figure Lengend Snippet: Figure 1: SKOV3IP xenograft tumors recapitulate the expression of ARID3B in ovarian cancer. (A) Immunohistochemistry was performed on tissue microarrays containing 102 cases of ovarian cancer. TMAs were scored for ARID3B expression. Representative images of light staining for ARID3B staining: (clear cell carcinoma, grade 2, stage IIC), moderate (serous, grade 3, stage IIIC), or strong (serous, grade3, stage IV). Black arrow indicates nucleus. Original magnification is 20x. (B) IHC was performed on xenografts in nude mice from SKOV3IP-RFP, SKOV3-ARID3BSH, and SKOV3IP-ARID3BFL cells.

Article Snippet: Anti-ARID3B IHC Antibody (Bethyl Laboratories, Montgomery, TX), anti-KI67 (Novus Biologicals, Littleton, CO), and Cleaved PARP (Asp214) (Cell Signaling Technology, Danvers, MA) were used.

Techniques: Expressing, Immunohistochemistry, Staining

Journal: Oncotarget

Article Title: ARID3B increases ovarian tumor burden and is associated with a cancer stem cell gene signature.

doi: 10.18632/oncotarget.2247

Figure Lengend Snippet: Figure 3: ARID3B induces collagen and cancer stem cell genes. qRT-PCR was performed on ascites cells and peritoneal washes obtained from SKOV3IP-RFP (N=6) and SKOV3IP-ARID3BFL (N=13) mouse ascites. (A) COL1A1, (B) COL1A2, (C) WISP1, (D) LGFR5, (E) SFRP1, and (F) SNAI2 were all significantly increased in ascites derived from SKOV3IP-ARDI3BFL cells compared to SKOV3IP-RFP derived ascites. qRT-PCR was performed on OVCA429-RFP and OVCA429-ARDI3BFL cells for (G) WISP1 and (H) SFRP1. (* P ≤ 0.05) (** P ≤ 0.01) (*** P ≤ 0.001)

Article Snippet: Anti-ARID3B IHC Antibody (Bethyl Laboratories, Montgomery, TX), anti-KI67 (Novus Biologicals, Littleton, CO), and Cleaved PARP (Asp214) (Cell Signaling Technology, Danvers, MA) were used.

Techniques: Quantitative RT-PCR, Derivative Assay

Journal: Oncotarget

Article Title: ARID3B increases ovarian tumor burden and is associated with a cancer stem cell gene signature.

doi: 10.18632/oncotarget.2247

Figure Lengend Snippet: Figure 5: ARID3B enhances cancer stem cell selection in sphere-cultures. (A) SKOV3, SKOV3-RFP, and SKOV3-ARID3BFL cells were enriched for CSCs by selection in cisplatin. qRT-PCR was performed for ARID3B, OCT4, and Prom1 on SKOV3, SKOV3-RFP, and SKOV3-ARID3BFL and CSCs isolated from each respective cell line. (B) Flow cytometry was performed for the stem cell markers CD117 and CD133 on Control Cells (not selected with cisplatin for CSCs) and CSCs (SKOV3 Parental), SKOV3-RFP (RFP), and SKOV3- ARID3BFL (ARID3B)). The percentage of CD117+ cells (Y-axis) and CD133+ cells (X-axis) is indicated for each quadrant. (* P ≤ 0.05) (** P ≤ 0.01) (*** P ≤ 0.001)

Article Snippet: Anti-ARID3B IHC Antibody (Bethyl Laboratories, Montgomery, TX), anti-KI67 (Novus Biologicals, Littleton, CO), and Cleaved PARP (Asp214) (Cell Signaling Technology, Danvers, MA) were used.

Techniques: Selection, Quantitative RT-PCR, Isolation, Flow Cytometry, Control

Journal: Molecular Biology of the Cell

Article Title: Kdm3a lysine demethylase is an Hsp90 client required for cytoskeletal rearrangements during spermatogenesis

doi: 10.1091/mbc.E13-08-0471

Figure Lengend Snippet: Two Kdm3a mouse models present arrested spermatogenesis with globozoospermia. (A) Diagram illustrates Cre-mediated deletion of exons 22–24 containing JmjC catalytic domain of Kdm3a in Kdm3a ΔJC mice ( Tateishi et al. , 2009 ). (a), (b), and (c) indicate the position of primers used to genotype mutant mice. Color code: green oval, C6-type zinc finger (ZF); blue, LXXLL nuclear receptor binding; red, JmjC domain; black, exons surrounding and affected by deletion. pAb, polyclonal antibody. (B) Genomic PCR identifying ΔJC deletion mutants. (C) Immunoblot of total MEF extracts with Kdm3a antibody (N-terminal) shows absence of full-length Kdm3a protein (FL, red arrow) in Kdm3a ΔJC/ΔJC . A shorter Kdm3a protein product result of Cre-induced deletion is indicated (ΔJC, black arrow). A nonspecific band is indicated (*). (D) DAPI staining of Kdm3a ΔJC/ΔJC epididymis reveals few mature sperm with rounded nuclei. (E) DAPI staining of testis cross-sections shows some abnormal tubules in Kdm3a ΔJC/+ (boxed region). Kdm3a ΔJC/ΔJC show arrested spermatogenesis. (F) Higher magnification of indicated regions from (E). es, elongated spermatids; arn, accumulation of round nuclei. Numbers represent the proportion of abnormal to normal tubules in each slide. (G) Diagram illustrates the position of the gene-trap insertion in Kdm3a GT/GT . Arrows indicate primers used for RT-PCR shown in (H). (H) Absence of Kdm3a transcript in Kdm3a GT/GT MEFs. Loading control used was 18S. (I) Immunoblot of total MEF extracts with a Kdm3a antibody directed to the N-terminus shows absence of full-length protein (FL, red arrow) in Kdm3a GT/GT . β-gal antibody detects the gene-trap product Kdm3a GT/GT only. (J) Kdm3a GT/GT testes are smaller than wild-type (WT) and heterozygous littermates. (K) Hematoxylin and eosin stain show disorganized tubules in testis sections of homozygous. (L) Immunohistochemistry of testis sections with a commercial antibody to Kdm3b (green) stained the acrosome of elongating spermatids. Note abnormal acrosome shape in Kdm3a GT/GT sections only. (M) DAPI panel shows absence of mature sperm in homozygous-GT epididymis. (N) Bright-field images show round-headed spermatozoon in Kdm3a GT/GT mice.

Article Snippet: The antibodies used along this study are to KDM3A (12835; Proteintech; and NB100-77282; Novus Biologicals, Littleton, CO); KDM3B (IHC 00189; Bethyl Laboratories, Montgomery, TX); Cct4 (ARP34271; Aviva); anti–mono- and dimethylated lysines (ab23366 and ab76118; Abcam, Cambridge, MA); anti-GFP (sc-8334), monoclonal to GAPDH (5019A-2; Imgenex, San Diego, CA), β-gal (A-11132; Molecular Probes), GST (C83271; LSBio); HP1a (clone 15.1952; Upstate); γ-tubulin (GTU-88; Sigma-Aldrich, St. Louis, MO), β-actin (ab8229; Abcam), Hsp90ab1 (MAB32861; R&D Systems), Hsp90aa1 (10713715; Pierce, Rockford, IL), Actbl2 (ab134977; Abcam).

Techniques: Mutagenesis, Binding Assay, Western Blot, Staining, Reverse Transcription Polymerase Chain Reaction, Control, H&E Stain, Immunohistochemistry

Journal: Human Molecular Genetics

Article Title: BBS mutations modify phenotypic expression of CEP290 -related ciliopathies

doi: 10.1093/hmg/ddt394

Figure Lengend Snippet: Physical interaction between CEP290 and the BBSome. (A) Co-immunoprecipitation of CEP290 in HEK293T cells stably expressing FLAG-BBS4 and FLAG-BBS5. Lysates from stable cell lines and control (parental) cells were subjected to immunoprecipitation (IP) with the anti-FLAG antibody and precipitated proteins were analyzed by immunoblotting with indicated antibodies. Normal mouse IgG pull-down was used as a negative control. (B and C) Interaction of endogenous CEP290 and the BBSome in HEK293T cells (B) and mouse retina (C). Lysates from HEK293T cells and mouse retina were subjected to IP using antibodies against CEP290, and precipitated proteins were analyzed by immunoblotting with indicated antibodies. (D) Schematic representation of the CEP290 deletion mutants. Numbers indicate expressed portions of CEP290 in amino acid positions. Known IQCB1-, CC2D2A- and RAB8A-binding domains and the BBSome-interacting region are also summarized. SMC, structural maintenance of chromosomes; MYO-Tail, myosin-tail homology domain. (E) The BBSome binds to the N-terminal part of CEP290. CEP290 deletion mutants (FLAG-tagged) were transfected into HEK293T cells and lysates were analyzed by IP using anti-FLAG antibodies. Untransfected cells were used as a negative control. (F) BBS4 interacts with CEP290. HA-tagged, individual BBSome components were transiently transfected with FLAG-Cep_1 constructs. Lysates were subjected to IP with anti-HA antibodies. (G) PCM1-independent interaction between the BBSome and CEP290. HEK293T cells were co-transfected with Cep_1 fragment and siRNA against PCM1.

Article Snippet: Other antibodies used were purchased from the following sources: mouse monoclonal antibodies against ABCA4 (3F4; Abcam), acetylated tubulin (6–11B-1; Sigma), γ-tubulin (GTU-88; Sigma), FLAG (M2; Sigma), HA (F-7; Santa Cruz Biotechnology), GFP (3E6; Invitrogen), beta-actin (AC-15; Sigma), rhodopsin (RET-P1; Santa Cruz Biotechnology), and synaptophysin (Santa Cruz Biotechnology; sc-55507); mouse polyclonal antibodies against IQCB1 (NPHP5; Abcam; ab69927); rabbit polyclonal antibodies against BBS8 (Sigma; HPA003310), BBS9 (Sigma; HPA021289), CEP290 (for immunoprecipitation, immunoblotting and RPE1 cell immunofluorescence microscopy; Bethyl Lab; IHC-00365), HSPA5/GRP78/BiP (Cell Signaling; 3177), PCM1 (Sigma; HPA023374), PDI (Sigma; P7372), PRPH2 (Proteintech Group; 18109), STAT3 (Santa Cruz Biotechnology; SC-483), phospho-STAT3 (Y705; Cell Signaling; 9131) and Tgoln2/Tgn46 (Abcam; ab16059).

Techniques: Immunoprecipitation, Stable Transfection, Expressing, Control, Western Blot, Negative Control, Binding Assay, Transfection, Construct

Journal: Human Molecular Genetics

Article Title: BBS mutations modify phenotypic expression of CEP290 -related ciliopathies

doi: 10.1093/hmg/ddt394

Figure Lengend Snippet: Co-localization of CEP290 and the BBSome. (A) Co-localization of GFP-tagged BBS4 and CEP290 in RPE1 cells (yellow arrowheads). Localization of CEP290 (red) was probed with the anti-CEP290 antibody in hTERT-RPE1 after 24 h of serum withdrawal, whereas BBS4-GFP was probed with the anti-GFP antibody. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI, blue). Note the variable localization of BBS4, although these cells were cultured in the same condition (from a single well). Scale bar, 5 μm. (B) Co-localization of Bbs4 and Cep290 in the mouse retina. Antibodies against Cep290 (red) and rhodopsin (green; a marker of the OS) were used in WT photoreceptors in the left panel, whereas in the middle and right panels Bbs4 (red) and rhodopsin (green) localizations were probed in WT and Bbs4−/− (4KO) photoreceptors. Both Cep290 and Bbs4 localize to the connecting cilium. CC, connecting cilium; ONL, outer nuclear layer. Scale bar, 10 μm. (C) Co-fractionation of the BBSome and Cep290 in the photoreceptor OS fraction. The photoreceptor OS was isolated from the mouse retina and probed with antibodies against multiple subcellular marker proteins, Bbs4, Bbs7 and Cep290.

Article Snippet: Other antibodies used were purchased from the following sources: mouse monoclonal antibodies against ABCA4 (3F4; Abcam), acetylated tubulin (6–11B-1; Sigma), γ-tubulin (GTU-88; Sigma), FLAG (M2; Sigma), HA (F-7; Santa Cruz Biotechnology), GFP (3E6; Invitrogen), beta-actin (AC-15; Sigma), rhodopsin (RET-P1; Santa Cruz Biotechnology), and synaptophysin (Santa Cruz Biotechnology; sc-55507); mouse polyclonal antibodies against IQCB1 (NPHP5; Abcam; ab69927); rabbit polyclonal antibodies against BBS8 (Sigma; HPA003310), BBS9 (Sigma; HPA021289), CEP290 (for immunoprecipitation, immunoblotting and RPE1 cell immunofluorescence microscopy; Bethyl Lab; IHC-00365), HSPA5/GRP78/BiP (Cell Signaling; 3177), PCM1 (Sigma; HPA023374), PDI (Sigma; P7372), PRPH2 (Proteintech Group; 18109), STAT3 (Santa Cruz Biotechnology; SC-483), phospho-STAT3 (Y705; Cell Signaling; 9131) and Tgoln2/Tgn46 (Abcam; ab16059).

Techniques: Staining, Cell Culture, Marker, Fractionation, Isolation

Journal: Human Molecular Genetics

Article Title: BBS mutations modify phenotypic expression of CEP290 -related ciliopathies

doi: 10.1093/hmg/ddt394

Figure Lengend Snippet: The CEP290 localization in the centriolar satellite and the connecting cilium is BBSome-dependent. (A) The BBSome is required for centriolar satellite localization of CEP290 (red). RPE1 cells were transfected with siRNAs against CEP290, BBS1, BBS4, BBS9 and PCM1. Antibodies against γ-tubulin and acetylated tubulin (green) were used to mark the basal body and cilia, respectively. (B) Quantification of CEP290 mis-localization in BBSome-depleted RPE1 cells. Cells with concentrated CEP290 staining around the centrosome (within 2 μm from the centrosome) were counted as positive, whereas cells with CEP290 only in the TZ and centrosome were considered negative. At least 120 cells per experiment were counted and graphs represent averages of three independent experiments. Error bars represent SEM. One-way ANOVA followed by Tukey's post-test was used for statistical analysis. **P < 0.01 compared with Ctrl KD cells. (C) Localization of Cep290 (green; left) in WT (top), Bbs1M390R/M390R (middle) and Bbs4−/− (bottom) mouse retinas. OS localization of Prph2 (red) with respect to acetylated tubulin (green) is not affected in BBS mutant retinas (right panels). Scale bars, 10 μm.

Article Snippet: Other antibodies used were purchased from the following sources: mouse monoclonal antibodies against ABCA4 (3F4; Abcam), acetylated tubulin (6–11B-1; Sigma), γ-tubulin (GTU-88; Sigma), FLAG (M2; Sigma), HA (F-7; Santa Cruz Biotechnology), GFP (3E6; Invitrogen), beta-actin (AC-15; Sigma), rhodopsin (RET-P1; Santa Cruz Biotechnology), and synaptophysin (Santa Cruz Biotechnology; sc-55507); mouse polyclonal antibodies against IQCB1 (NPHP5; Abcam; ab69927); rabbit polyclonal antibodies against BBS8 (Sigma; HPA003310), BBS9 (Sigma; HPA021289), CEP290 (for immunoprecipitation, immunoblotting and RPE1 cell immunofluorescence microscopy; Bethyl Lab; IHC-00365), HSPA5/GRP78/BiP (Cell Signaling; 3177), PCM1 (Sigma; HPA023374), PDI (Sigma; P7372), PRPH2 (Proteintech Group; 18109), STAT3 (Santa Cruz Biotechnology; SC-483), phospho-STAT3 (Y705; Cell Signaling; 9131) and Tgoln2/Tgn46 (Abcam; ab16059).

Techniques: Transfection, Staining, Mutagenesis

Journal: Human Molecular Genetics

Article Title: BBS mutations modify phenotypic expression of CEP290 -related ciliopathies

doi: 10.1093/hmg/ddt394

Figure Lengend Snippet: Increased body weight and higher leptin levels in Bbs4+/−; Cep290+/rd16 mice. (A) Weight gains in male animals versus age (minimum of six animals per group). Values are expressed as mean + SEM. By month 3, double-heterozygous mice are significantly heavier than single-heterozygous littermates. *P < 0.05 versus single-heterozygous animals; **P < 0.01 versus single-heterozygous animals. (B) Serum leptin levels of single- and double-heterozygous mice. One-way ANOVA and t-test were used for statistical analysis. *P < 0.05 versus single-heterozygous animals. (C) STAT3 phosphorylation upon leptin administration was reduced in double-heterozygous mice. Hypothalamic protein extracts were analyzed by western blotting. (D) Quantification of STAT3 phosphorylation. Band intensities of phospho-STAT3 (P-STAT3) were normalized with those of total STAT3 and induction ratios were calculated by comparison with vehicle-injected samples. Data represent mean + SEM. n = 5 for vehicle and n = 10 for leptin. *P < 0.05.

Article Snippet: Other antibodies used were purchased from the following sources: mouse monoclonal antibodies against ABCA4 (3F4; Abcam), acetylated tubulin (6–11B-1; Sigma), γ-tubulin (GTU-88; Sigma), FLAG (M2; Sigma), HA (F-7; Santa Cruz Biotechnology), GFP (3E6; Invitrogen), beta-actin (AC-15; Sigma), rhodopsin (RET-P1; Santa Cruz Biotechnology), and synaptophysin (Santa Cruz Biotechnology; sc-55507); mouse polyclonal antibodies against IQCB1 (NPHP5; Abcam; ab69927); rabbit polyclonal antibodies against BBS8 (Sigma; HPA003310), BBS9 (Sigma; HPA021289), CEP290 (for immunoprecipitation, immunoblotting and RPE1 cell immunofluorescence microscopy; Bethyl Lab; IHC-00365), HSPA5/GRP78/BiP (Cell Signaling; 3177), PCM1 (Sigma; HPA023374), PDI (Sigma; P7372), PRPH2 (Proteintech Group; 18109), STAT3 (Santa Cruz Biotechnology; SC-483), phospho-STAT3 (Y705; Cell Signaling; 9131) and Tgoln2/Tgn46 (Abcam; ab16059).

Techniques: Phospho-proteomics, Western Blot, Comparison, Injection

Journal: Human Molecular Genetics

Article Title: BBS mutations modify phenotypic expression of CEP290 -related ciliopathies

doi: 10.1093/hmg/ddt394

Figure Lengend Snippet: Impaired rhodopsin trafficking and diminished ERG responses in mice with combined loss of Cep290 and Bbs4 alleles. (A) Immunohistochemical analysis of WT, Bbs4+/+;Cep290rd/rd, Bbs4+/−;Cep290rd/rd and Bbs4−/−; Cep290rd/rd retinas at P21 with antibodies against rhodopsin. Scale bar, 5 μm. (B) DA-SCR ERG b-wave amplitudes in the indicated mouse genotypes (at the age of 1 month). Removing one or two Bbs4 alleles on a Cep290rd/rd background results in a lower ERG response. **P < 0.01 versus Bbs4+/+;Cep290rd/rd mice. Error bars are SD.

Article Snippet: Other antibodies used were purchased from the following sources: mouse monoclonal antibodies against ABCA4 (3F4; Abcam), acetylated tubulin (6–11B-1; Sigma), γ-tubulin (GTU-88; Sigma), FLAG (M2; Sigma), HA (F-7; Santa Cruz Biotechnology), GFP (3E6; Invitrogen), beta-actin (AC-15; Sigma), rhodopsin (RET-P1; Santa Cruz Biotechnology), and synaptophysin (Santa Cruz Biotechnology; sc-55507); mouse polyclonal antibodies against IQCB1 (NPHP5; Abcam; ab69927); rabbit polyclonal antibodies against BBS8 (Sigma; HPA003310), BBS9 (Sigma; HPA021289), CEP290 (for immunoprecipitation, immunoblotting and RPE1 cell immunofluorescence microscopy; Bethyl Lab; IHC-00365), HSPA5/GRP78/BiP (Cell Signaling; 3177), PCM1 (Sigma; HPA023374), PDI (Sigma; P7372), PRPH2 (Proteintech Group; 18109), STAT3 (Santa Cruz Biotechnology; SC-483), phospho-STAT3 (Y705; Cell Signaling; 9131) and Tgoln2/Tgn46 (Abcam; ab16059).

Techniques: Immunohistochemical staining