Journal: Communications Biology

Article Title: β-cell-specific deletion of PFKFB3 restores cell fitness competition and physiological replication under diabetogenic stress

doi: 10.1038/s42003-022-03209-y

Figure Lengend Snippet: a Representative immunofluorescence images of islets from WT, PFKFB3 WT DS and PFKFB3 βKO DS at 13 weeks of HFD immunostained for MCM2 (red), insulin (green) and nuclei (blue). b Quantification of images shown under ( a ) (** p = 0.006, * p = 0.029). c Quantification of the total of all medium and strong expressing MCM2 positive β-cells in the islets from WT, PFKFB3 WT DS and PFKFB3 βKO DS at 8 weeks of HFD and PFKFB3 βKO DS chow (4 weeks under 16 h restricted chow diet following 4 weeks of HFD). d Representative immunofluorescence images of islets from PFKFB3 WT DS and PFKFB3 βKO DS at 13 weeks immunostained for c-Myc (red), insulin (green) and nuclei (blue). e Quantification of cytoplasmic c-Myc indicates cells undergoing hIAPP-induced calpain activation (damage) as revealed by immunostaining in ( d ) (* p = 0.021; * p = 0.0182, n = 3, n = 4 for PFKFB3 βKO DS-independent animals, SEM).

Article Snippet: The following antibodies were used: rabbit anti-PFKFB3 (Abcam ab181861, Cambridge, MA, USA, 1:100); mouse anti-minichromosome maintenance-2 protein (MCM2) (BD Transduction Laboratories 610700, San Diego, CA, USA, 1:100); rabbit anti-cleaved caspase-3 (Cell Signalling Technology 9664 S, Danvers, MA, USA, 1:400); guinea pig anti-insulin (Abcam ab195956, Cambridge, MA, USA, 1:400); mouse anti-glucagon (Sigma-Aldrich G2654, St. Louis, MO, USA, 1:1000); mouse anti-c-Myc (Santa Cruz Biotechnology Inc 9E10 sc-40, Dallas, Texas, USA, 1:100); mouse anti-HIF1α (Novus Biologicals NB100-105, Centennial, CO, USA, 1:50); polyclonal rabbit anti-Mcm2 (Abcam ab441) and polyclonal rabbit anti-GADD 153 (Santa Cruz Biotechnology F-168 sc-575).

Techniques: Immunofluorescence, Expressing, Activation Assay, Immunostaining

Journal: Communications Biology

Article Title: β-cell-specific deletion of PFKFB3 restores cell fitness competition and physiological replication under diabetogenic stress

doi: 10.1038/s42003-022-03209-y

Figure Lengend Snippet: An initial insult or injury can change the fitness level of some β-cells within β-cell populations and trigger cell competition. a After metabolic stress such as intake of an HFD in non-diabetic (ND) or wild-type (WT) mice, suboptimal cells (blue) are eliminated from the tissue by competition with healthy β -cells (green), which replicate to regenerate the lost tissue. b In a cell competition in which injury is sustained (T2D) or in PFKFB3 WT DS mice, injured β-cells (pink) survive inspite of reduced fitness and cannot be purged from the tissue because of metabolic remodelling by the HIF1α–PFKFB3 pathway. These injured β-cells may impede healthy β-cell replenishment (replication). c Under conditions described in ( b ), the targeting of the pro-survival PFKFB3 leads to activation of cell competition and elimination of suboptimal (damaged) β-cells (light pink). Elimination of suboptimal (damaged) β-cells leads to replication of the remaining healthy β-cells (MCM2-positive cells, dark green).

Article Snippet: The following antibodies were used: rabbit anti-PFKFB3 (Abcam ab181861, Cambridge, MA, USA, 1:100); mouse anti-minichromosome maintenance-2 protein (MCM2) (BD Transduction Laboratories 610700, San Diego, CA, USA, 1:100); rabbit anti-cleaved caspase-3 (Cell Signalling Technology 9664 S, Danvers, MA, USA, 1:400); guinea pig anti-insulin (Abcam ab195956, Cambridge, MA, USA, 1:400); mouse anti-glucagon (Sigma-Aldrich G2654, St. Louis, MO, USA, 1:1000); mouse anti-c-Myc (Santa Cruz Biotechnology Inc 9E10 sc-40, Dallas, Texas, USA, 1:100); mouse anti-HIF1α (Novus Biologicals NB100-105, Centennial, CO, USA, 1:50); polyclonal rabbit anti-Mcm2 (Abcam ab441) and polyclonal rabbit anti-GADD 153 (Santa Cruz Biotechnology F-168 sc-575).

Techniques: Activation Assay

Journal: Translational Oncology

Article Title: Minichromosome Maintenance Expression Defines Slow-Growing Gastroenteropancreatic Neuroendocrine Neoplasms 1

doi: 10.1016/j.tranon.2016.07.006

Figure Lengend Snippet: Protein expression of MCM2 and MCM3 in small intestinal mucosa and SI-NENs (primaries and metastases). Immunohistochemistry (A): MCM2 and MCM3 were identified in crypts associated with the proliferative zone (PZ) (inset) which is the site of cells involved in renewing mucosa where the highest mitosis rates are expected. Additional cells were MCM3 positive at the base of crypts; these cells were not stained by MCM2 (red arrows). Dual staining with chromogranin A identified only a few (<2%) normal neuroendocrine cells to be MCM2 or MCM3 positive (yellow arrows). Both primary tumor and metastasis exhibited nuclear expression of MCM2 and MCM3, and expression of both proteins tended to be higher in metastasis in comparison to the primary tumor. This overexpression was also identified by Western blot (B), especially for MCM3, but this was not statistically significant (Kruskal-Wallis, MCM3: P = .06, MCM2: P = .15). DAPI: nuclei (blue), FITC: target marker (green), Cy5: chromogranin A (red-white arrows), dual-stained cells (yellow). PZ = proliferative zone. Mean ± SD.

Article Snippet: CNV was measured using PCR-Taqman Copy Number Reference Assay from Life Technologies according to the manufacturer's instructions using the primer sets Hs00575269_cn and Hs02422238_cn (MCM2) and Hs00378664_cn (MCM3).

Techniques: Expressing, Immunohistochemistry, Staining, Comparison, Over Expression, Western Blot, Marker

Journal: Translational Oncology

Article Title: Minichromosome Maintenance Expression Defines Slow-Growing Gastroenteropancreatic Neuroendocrine Neoplasms 1

doi: 10.1016/j.tranon.2016.07.006

Figure Lengend Snippet: Clinical relevance of MCM and Ki67 expression in SI-NENs. (A) MCM expression in normal mucosa tended to be higher than in neoplastic small intestinal tumors, whereas Ki67 was not significantly different between any tissue type (* P < .05 versus normal mucosa). (B) Ki67 and MCM2 expression was significantly increased (* P < .05) in grade 2 compared to grade 1 tumors. MCM3 was also increased, but this did not achieve statistical significance. (C) Correlation between MCM mRNA expression and Ki67 staining identified a relationship for MCM2 ( r 2 = 0.44, P < .05) but not for MCM3. Expression of either MCM significantly correlated with Ki67 mRNA expression (MCM2: r = 0.6, P = .0027, MCM3: r = 0.53, P = .0092) and with each other ( r = 0.69, P = .004). (D) High copy number variance was noted for all three markers in both primary tumors and in metastases; this was not seen in normal mucosa. Mean ± SD.

Article Snippet: CNV was measured using PCR-Taqman Copy Number Reference Assay from Life Technologies according to the manufacturer's instructions using the primer sets Hs00575269_cn and Hs02422238_cn (MCM2) and Hs00378664_cn (MCM3).

Techniques: Expressing, Staining

Journal: Translational Oncology

Article Title: Minichromosome Maintenance Expression Defines Slow-Growing Gastroenteropancreatic Neuroendocrine Neoplasms 1

doi: 10.1016/j.tranon.2016.07.006

Figure Lengend Snippet: Survival curves for AQUA analysis of MCM2 and MCM3 in SI-NENs. Low expressions of MCM2 (A) and MCM3 (B) were both associated with notably longer OS. Using a combination of proliferative markers, patients with low–proliferation score tumors (0-1; n = 21) had prolonged survival (median OS 12 years) compared to those with high–proliferation scores tumors (2-3; n = 24) (median OS years 6.5) (C, P = .06, HR 0.46). A high MCM3 expression was correlated with worst prognosis, whereas after more than 25-years of follow-up, the median OS of patients with low MCM3 expression was not reached in the subset of tumors that were only Ki67% positive (D, P = .019, HR 0.13).

Article Snippet: CNV was measured using PCR-Taqman Copy Number Reference Assay from Life Technologies according to the manufacturer's instructions using the primer sets Hs00575269_cn and Hs02422238_cn (MCM2) and Hs00378664_cn (MCM3).

Techniques: Expressing

Journal: Translational Oncology

Article Title: Minichromosome Maintenance Expression Defines Slow-Growing Gastroenteropancreatic Neuroendocrine Neoplasms 1

doi: 10.1016/j.tranon.2016.07.006

Figure Lengend Snippet: mRNA and protein expression of MCM2 and MCM3 in normal pancreas and pancreatic NENs (primaries and metastases). (A) MCM2 and MCM3 mRNAs were elevated in metastases in comparison to normal pancreas and to primaries, whereas Ki67 was elevated in metastases compared to normal pancreas (Kruskal-Wallis P < 0.0001, * P < .05 versus normal, # P < .05 versus primaries). (B) Ki67 and MCM mRNA expression as a function of grade identified no significant differences in pancreatic NENs. (C) Using Western blot, an elevation of MCM protein expression in pNEN metastases was confirmed (Kruskal-Wallis, MCM2: P = .24, MCM3: P = .44). Mean ± SD.

Article Snippet: CNV was measured using PCR-Taqman Copy Number Reference Assay from Life Technologies according to the manufacturer's instructions using the primer sets Hs00575269_cn and Hs02422238_cn (MCM2) and Hs00378664_cn (MCM3).

Techniques: Expressing, Comparison, Western Blot

Journal: Cancer medicine

Article Title: MCM8 promotes gastric cancer progression through RPS15A and predicts poor prognosis.

doi: 10.1002/cam4.7424

Figure Lengend Snippet: FIGURE 1 MCM8 is significantly elevated in GC and predicts poor prognosis. (A) MCM8 expression in GC (n = 408) and normal tissues (n = 211) from TCGA and GTEx. (B) MCM8 expression in GC and GES-1 cells. (C, D) IHC staining of MCM8 in GC and normal tissues. Scale bar: 100 μm. (E) Kaplan–Meier plots of the overall survival (OS) rate of GC patients. *p < 0.05, **p < 0.01, and ***p < 0.001.

Article Snippet: MCM8 (1:500, Proteintech, Chicago, USA), RPS15A (1:2000, Proteintech, Chicago, USA), CDK4 (1:1000, Proteintech, Chicago, USA), MAPK14 (1:2000, Proteintech, Chicago, USA), CCND1 (1:750, CST, Boston, USA), SMAD3 (1:1000, Abcam, Cambridge, UK), SMAD4 (1:500, Santa Cruz, California, USA), ARAF (1:1000, CST, Boston, USA), RPS6KA1 (1:500, Proteintech, Chicago, USA), GAPDH (1:30000, Proteintech, Chicago, USA), P53 (1:3000, Proteintech, Chicago, USA), p- P53 (1:2000, Proteintech, Chicago, USA), STAT3 (1:1500, CST, Boston, USA), p- STAT3 (1:500, CST, Boston, USA), cJun (1:1000, Proteintech, Chicago, USA), p- c- Jun (1:2000, CST, Boston, USA), p70S6K (1:1000, Affinity, Cincinnati, USA), p- p70S6K (1:1000, Affinity, Cincinnati, USA), P38α(1:2000, Abcam, Cambridge, UK), p- P38α(1:1000, Abcam, Cambridge, UK), LYN (1:1000, CST, Boston, USA), p- LYN (1:1000, CST, Boston, USA), PYK2 (1:2000, Abcam, Cambridge, UK), p- PYK2 (1:1000, CST, Boston, USA), STAT1 (1:1000, Proteintech, Chicago, USA), p- STAT1 (1:2000, Abcam, Cambridge, UK).

Techniques: Expressing, Immunohistochemistry

Journal: Cancer medicine

Article Title: MCM8 promotes gastric cancer progression through RPS15A and predicts poor prognosis.

doi: 10.1002/cam4.7424

Figure Lengend Snippet: FIGURE 2 MCM8 knockdown suppresses the phenotypic functions of GC. (A) Celigo cell counting assay was used to analyze proliferation of MGC-803 and AGS cells after MCM8 knockdown. (B, C) Flow cytometry was performed to detect (B) cell apoptosis and (C) cell cycle of MGC-803 and AGS cells. (D–F) MGC-803 and AGS cells migration and invasion ability was accessed by (D) Wound-healing assay (scale bar: 1 μm) and (E, F) Transwell assay (200×). *p < 0.05, **p < 0.01, and ***p < 0.001.

Article Snippet: MCM8 (1:500, Proteintech, Chicago, USA), RPS15A (1:2000, Proteintech, Chicago, USA), CDK4 (1:1000, Proteintech, Chicago, USA), MAPK14 (1:2000, Proteintech, Chicago, USA), CCND1 (1:750, CST, Boston, USA), SMAD3 (1:1000, Abcam, Cambridge, UK), SMAD4 (1:500, Santa Cruz, California, USA), ARAF (1:1000, CST, Boston, USA), RPS6KA1 (1:500, Proteintech, Chicago, USA), GAPDH (1:30000, Proteintech, Chicago, USA), P53 (1:3000, Proteintech, Chicago, USA), p- P53 (1:2000, Proteintech, Chicago, USA), STAT3 (1:1500, CST, Boston, USA), p- STAT3 (1:500, CST, Boston, USA), cJun (1:1000, Proteintech, Chicago, USA), p- c- Jun (1:2000, CST, Boston, USA), p70S6K (1:1000, Affinity, Cincinnati, USA), p- p70S6K (1:1000, Affinity, Cincinnati, USA), P38α(1:2000, Abcam, Cambridge, UK), p- P38α(1:1000, Abcam, Cambridge, UK), LYN (1:1000, CST, Boston, USA), p- LYN (1:1000, CST, Boston, USA), PYK2 (1:2000, Abcam, Cambridge, UK), p- PYK2 (1:1000, CST, Boston, USA), STAT1 (1:1000, Proteintech, Chicago, USA), p- STAT1 (1:2000, Abcam, Cambridge, UK).

Techniques: Knockdown, Cell Counting, Flow Cytometry, Migration, Wound Healing Assay, Transwell Assay

Journal: Cancer medicine

Article Title: MCM8 promotes gastric cancer progression through RPS15A and predicts poor prognosis.

doi: 10.1002/cam4.7424

Figure Lengend Snippet: FIGURE 5 Overexpression of RPS15A reverses the changes of MCM8 knockdown in GC cells. (A) The transfection efficiency was detected by WB. (B–E) sh-Ctrl+oe-Ctrl, sh-Ctrl+oe-RPS15A, sh-MCM8+oe-Ctrl, and sh-MCM8+oe-RPS15A groups were established in AGS cells. The loss or gain of function experiments showed that RPS15A reversed the changes of MCM8 knockdown in GC cells, including (B, C) proliferation, (D) apoptosis, and (E) migration (100×). *p < 0.05, **p < 0.01, and ***p < 0.001.

Article Snippet: MCM8 (1:500, Proteintech, Chicago, USA), RPS15A (1:2000, Proteintech, Chicago, USA), CDK4 (1:1000, Proteintech, Chicago, USA), MAPK14 (1:2000, Proteintech, Chicago, USA), CCND1 (1:750, CST, Boston, USA), SMAD3 (1:1000, Abcam, Cambridge, UK), SMAD4 (1:500, Santa Cruz, California, USA), ARAF (1:1000, CST, Boston, USA), RPS6KA1 (1:500, Proteintech, Chicago, USA), GAPDH (1:30000, Proteintech, Chicago, USA), P53 (1:3000, Proteintech, Chicago, USA), p- P53 (1:2000, Proteintech, Chicago, USA), STAT3 (1:1500, CST, Boston, USA), p- STAT3 (1:500, CST, Boston, USA), cJun (1:1000, Proteintech, Chicago, USA), p- c- Jun (1:2000, CST, Boston, USA), p70S6K (1:1000, Affinity, Cincinnati, USA), p- p70S6K (1:1000, Affinity, Cincinnati, USA), P38α(1:2000, Abcam, Cambridge, UK), p- P38α(1:1000, Abcam, Cambridge, UK), LYN (1:1000, CST, Boston, USA), p- LYN (1:1000, CST, Boston, USA), PYK2 (1:2000, Abcam, Cambridge, UK), p- PYK2 (1:1000, CST, Boston, USA), STAT1 (1:1000, Proteintech, Chicago, USA), p- STAT1 (1:2000, Abcam, Cambridge, UK).

Techniques: Over Expression, Knockdown, Transfection, Migration

Journal: Cancer medicine

Article Title: MCM8 promotes gastric cancer progression through RPS15A and predicts poor prognosis.

doi: 10.1002/cam4.7424

Figure Lengend Snippet: FIGURE 6 MCM8/RPS15A axis promotes P38α, LYN, and p70S6K phosphorylation in GC. (A, B) Human Phospho-Kinase Array Kit (ARY003C) was used to detect changes in phosphorylation of key proteins after RPS15A knockdown. (C, D) WB was applied to detect RPS15A, P53, LYN, P38α, c-Jun, STAT1, STAT3, PYK2, p70S6K and their phosphorylated forms. *p < 0.05, **p < 0.01, and ***p < 0.001.

Article Snippet: MCM8 (1:500, Proteintech, Chicago, USA), RPS15A (1:2000, Proteintech, Chicago, USA), CDK4 (1:1000, Proteintech, Chicago, USA), MAPK14 (1:2000, Proteintech, Chicago, USA), CCND1 (1:750, CST, Boston, USA), SMAD3 (1:1000, Abcam, Cambridge, UK), SMAD4 (1:500, Santa Cruz, California, USA), ARAF (1:1000, CST, Boston, USA), RPS6KA1 (1:500, Proteintech, Chicago, USA), GAPDH (1:30000, Proteintech, Chicago, USA), P53 (1:3000, Proteintech, Chicago, USA), p- P53 (1:2000, Proteintech, Chicago, USA), STAT3 (1:1500, CST, Boston, USA), p- STAT3 (1:500, CST, Boston, USA), cJun (1:1000, Proteintech, Chicago, USA), p- c- Jun (1:2000, CST, Boston, USA), p70S6K (1:1000, Affinity, Cincinnati, USA), p- p70S6K (1:1000, Affinity, Cincinnati, USA), P38α(1:2000, Abcam, Cambridge, UK), p- P38α(1:1000, Abcam, Cambridge, UK), LYN (1:1000, CST, Boston, USA), p- LYN (1:1000, CST, Boston, USA), PYK2 (1:2000, Abcam, Cambridge, UK), p- PYK2 (1:1000, CST, Boston, USA), STAT1 (1:1000, Proteintech, Chicago, USA), p- STAT1 (1:2000, Abcam, Cambridge, UK).

Techniques: Phospho-proteomics, Knockdown

Journal: Cancer medicine

Article Title: MCM8 promotes gastric cancer progression through RPS15A and predicts poor prognosis.

doi: 10.1002/cam4.7424

Figure Lengend Snippet: FIGURE 7 Knockdown of MCM8 attenuates GC growth in vivo. (A) The left picture is the subcutaneous tumor mice. The right is the mice with the tumor removed. (B) Tumor volume and (C) weight in sh-MCM8 and sh-Ctrl groups. (D, E) The fluorescence images displayed the tumor burden. (F) Ki67 and (G) HE staining were detected in tissue sections. Scale bars: 100 μm. (H) IHC staining of MCM8, RPS15A, P38α, p-38α, LYN, p-LYN, p70S6K, and p-p70S6K in sh-MCM8 and sh-Ctrl tissue sections. Scale bars: 100 μm. *p < 0.05, and ***p < 0.001.

Article Snippet: MCM8 (1:500, Proteintech, Chicago, USA), RPS15A (1:2000, Proteintech, Chicago, USA), CDK4 (1:1000, Proteintech, Chicago, USA), MAPK14 (1:2000, Proteintech, Chicago, USA), CCND1 (1:750, CST, Boston, USA), SMAD3 (1:1000, Abcam, Cambridge, UK), SMAD4 (1:500, Santa Cruz, California, USA), ARAF (1:1000, CST, Boston, USA), RPS6KA1 (1:500, Proteintech, Chicago, USA), GAPDH (1:30000, Proteintech, Chicago, USA), P53 (1:3000, Proteintech, Chicago, USA), p- P53 (1:2000, Proteintech, Chicago, USA), STAT3 (1:1500, CST, Boston, USA), p- STAT3 (1:500, CST, Boston, USA), cJun (1:1000, Proteintech, Chicago, USA), p- c- Jun (1:2000, CST, Boston, USA), p70S6K (1:1000, Affinity, Cincinnati, USA), p- p70S6K (1:1000, Affinity, Cincinnati, USA), P38α(1:2000, Abcam, Cambridge, UK), p- P38α(1:1000, Abcam, Cambridge, UK), LYN (1:1000, CST, Boston, USA), p- LYN (1:1000, CST, Boston, USA), PYK2 (1:2000, Abcam, Cambridge, UK), p- PYK2 (1:1000, CST, Boston, USA), STAT1 (1:1000, Proteintech, Chicago, USA), p- STAT1 (1:2000, Abcam, Cambridge, UK).

Techniques: Knockdown, In Vivo, Fluorescence, Staining, Immunohistochemistry

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Glucagon-Like Peptide-2 Stimulates S-Phase Entry of Intestinal Lgr5+ Stem Cells

doi: 10.1016/j.jcmgh.2022.02.011

Figure Lengend Snippet: Acute hGly2–GLP-2 treatment increases the expression of transcripts involved in G1/S-phase transition. Jejunal mucosal isolates from the mice described in Figure 1 were analyzed by reverse-transcription quantitative polymerase chain reaction for the expression of the following: ( A ) Mcm2 , ( B ) Mcm3 , ( C ) Mcm4 , ( D ) Mcm5 , ( E ) Mcm6 , ( F ) Mcm7 , ( G ) Cdk2 , ( H ) Ccnd1 , ( I ) s100a6 , and ( J ) Igf1 . N = 6–7 for all panels. ∗ P < .05. mRNA, messenger RNA; rRNA, ribosomal RNA.

Article Snippet: Mcm7 , Mm04207570_g1.

Techniques: Expressing, Sublimation, Reverse Transcription, Real-time Polymerase Chain Reaction

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Glucagon-Like Peptide-2 Stimulates S-Phase Entry of Intestinal Lgr5+ Stem Cells

doi: 10.1016/j.jcmgh.2022.02.011

Figure Lengend Snippet: Acute GLP-2 3–33 treatment decreases the expression of transcripts involved in G1/S-phase transition. Jejunal mucosal isolates from the mice described in Figure 5 were analyzed by reverse-transcription quantitative polymerase chain reaction for the expression of the following: ( A ) Mcm2 , ( B ) Mcm3 , ( C ) Mcm4 , ( D ) Mcm5 , ( E ) Mcm6 , ( F ) Mcm7 , ( G ) Cdk2 , ( H ) Ccnd1 , ( I ) s100a6 , and ( J ) Igf1 . N = 5 for all panels. ∗ P < .05. mRNA, messenger RNA; rRNA, ribosomal RNA.

Article Snippet: Mcm7 , Mm04207570_g1.

Techniques: Expressing, Sublimation, Reverse Transcription, Real-time Polymerase Chain Reaction

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Glucagon-Like Peptide-2 Stimulates S-Phase Entry of Intestinal Lgr5+ Stem Cells

doi: 10.1016/j.jcmgh.2022.02.011

Figure Lengend Snippet: Quantitative Polymerase Chain Reaction TaqMan Primers (ThermoFisher Scientific, Mississauga, Canada)

Article Snippet: Mcm7 , Mm04207570_g1.

Techniques: Real-time Polymerase Chain Reaction

Journal: Glia

Article Title: AXON CONTACT-DRIVEN SCHWANN CELL DEDIFFERENTIATION

doi: 10.1002/glia.23131

Figure Lengend Snippet: DRG neurons express a component that prevents cAMP-induced SC differentiation. A–C. Blockage of SC differentiation by treatment with a preparation of disrupted DRG neurons. Isolated SCs were left untreated (control) or treated with CPT-cAMP in the absence or presence of a mitogenically active DRG neuron homogenate (indicated as ‘disrupted DRG neurons’ in this and subsequent figures). Cells were analyzed for O1 expression (green) and BrDU incorporation (red) 3 days after treatment initiation. Representative images and quantification of results are provided in A and B, respectively. The lower panels in A are a close up view of the sections denoted by the white frames (upper panels). In C, cultures subjected to identical experimental conditions were pre-labelled with O1 antibodies prior to Western blot analysis of the indicated markers of differentiation (Krox-20, c-Jun, O1 and GFAP) and proliferation (MCM2). D–F. Mitogenic and differentiating activity of the disrupted DRG neurons. SCs (undifferentiated) were left untreated (Control, C) or treated for the indicated time points with disrupted DRG neurons, soluble neuregulin (10 nM, positive control for proliferation) or 250 μM CPT-cAMP (positive control for SC differentiation), as indicated. The phosphorylation of ErbB3 (Tyr-1289), Akt (Thr-473), and ERK1/2 (Tyr-204) (D) are shown along with the incorporation of [3H]-thymidine (E) to confirm the mitogenic activity of the disrupted neurons. The expression Krox-20 and c-Jun (Western blots) is shown to confirm the differentiating activity of CPT-cAMP (F). Disrupted DRG neurons promoted ErbB activation (D and F) and proliferation (E) without changing the basal levels of Krox-20 and c-Jun (C, right lanes, and F).

Article Snippet: Minichromosome maintenance protein-2 (MCM2) antibodies were from Bethyl Lab (Montgomery, TX).

Techniques: Isolation, Control, Expressing, BrdU Incorporation Assay, Western Blot, Activity Assay, Positive Control, Phospho-proteomics, Activation Assay

Journal: Glia

Article Title: AXON CONTACT-DRIVEN SCHWANN CELL DEDIFFERENTIATION

doi: 10.1002/glia.23131

Figure Lengend Snippet: Contact with live DRG axons drives SC dedifferentiation by counteracting the effect of cAMP. Undifferentiated SCs (SCs) or SCs induced to differentiate with CPT-cAMP (dSCs) were trypsin-dissociated and re-plated as a single cell suspension onto cultures of DRG neurons (SC-neuron) or laminin-coated dishes (SC-only). Experiments were carried out in the absence or presence of CPT-cAMP (A–B) or other cAMP-stimulating agents (D–E) and analyzed by immunofluorescence microscopy (A–B and D–E) and Western blot (C) using the indicated antibodies. In A–B, cultures were analyzed 40 h after co-culture initiation to reveal early changes in cell morphology and differentiation. In D–E, cultures were analyzed 72 h post-plating. Note that differentiated SCs lost the expression of O1 and re-acquired an elongated shape only when plated on DRG neurons. A quantification of O1 positive cells (B and E) is provided. Arrowheads in A (middle panel) point out to a representative S100 positive mitotic figure, indicative of cell division. Note that reduced levels of O1 and P0 (along with increased levels of MCM2) occur without concurrent changes in O4 and ErbB3 in differentiated SCs co-cultured with DRG neurons (C, right lanes). O4 was highly expressed in cAMP-differentiated SCs, as expected based on its well-known sensitivity to cAMP stimulation.

Article Snippet: Minichromosome maintenance protein-2 (MCM2) antibodies were from Bethyl Lab (Montgomery, TX).

Techniques: Suspension, Immunofluorescence, Microscopy, Western Blot, Co-Culture Assay, Expressing, Cell Culture

Journal: Glia

Article Title: AXON CONTACT-DRIVEN SCHWANN CELL DEDIFFERENTIATION

doi: 10.1002/glia.23131

Figure Lengend Snippet: Stimulation with live or disrupted DRG neurons induces SC dedifferentiation. cAMP-differentiated SCs (A, dSCs) or undifferentiated SCs (B, SCs) were stimulated with live DRG neurons (re-plated as a single cell suspension onto the SC cultures) or a preparation of disrupted neurons in medium containing CPT-cAMP and BrDU. Detection of O1, BrDU and neurofilament (NF) was done 3 days after treatment. An enlarged section (a) is provided in A (bottom panels) to show the axonal engagement of proliferating (BrDU positive) SCs. The low levels of O1 expression in some of these cells (arrowhead) was interpreted as indication of dedifferentiation. In C, SCs and dSCs were treated for the indicated time points with disrupted neurons and the levels of MCM2 expression and ErbB3 phosphorylation (P-ErbB3) were determined by Western blot. Note that MCM2 expression was increased to similar levels regardless of the state of differentiation and CPT-cAMP stimulation. In D–E, dSCs were incubated for 3 days in medium containing (middle panel) or lacking (left panel) disrupted DRG neurons and CPT-cAMP. Cultures were analyzed by immunofluorescence microscopy (D, O1/BrDU co-staining) or Western blot (E, O1/ErbB3). As expected, removal of the cAMP stimulus (-cAMP) induces O1 loss (D–E) in the absence of concomitant proliferation (D).

Article Snippet: Minichromosome maintenance protein-2 (MCM2) antibodies were from Bethyl Lab (Montgomery, TX).

Techniques: Suspension, Expressing, Phospho-proteomics, Western Blot, Incubation, Immunofluorescence, Microscopy, Staining

Journal: Glia

Article Title: AXON CONTACT-DRIVEN SCHWANN CELL DEDIFFERENTIATION

doi: 10.1002/glia.23131

Figure Lengend Snippet: An activity of DRG neurons induces SC dedifferentiation without reducing the expression of Krox-20 or increasing the expression of c-Jun, GFAP or p75NGFR. Differentiated SCs (dSCs) were stimulated with disrupted DRG neurons (A–B) or DRG membranes collected by centrifugation (C–E) in the presence of CPT-cAMP for 3 days. A condition in which CPT-cAMP was removed from the culture medium (- cAMP) was also included in the analysis to re-establish p75NGFR, GFAP and c-Jun expression (A–B) and concomitantly reduce that of Krox-20 (A–C). The cultures were analyzed by immunofluorescence microscopy and Western blot using the markers indicated in each panel. Undifferentiated cells (SCs) were used as controls in panels B–D. In E, Krox-20 immunolabeling (green) was performed together with BrDU (red, left panels) and MCM2 (red, right panels). A subset of Krox-20 positive SCs labelled positive for nuclear BrDU and MCM2 (arrows) indicative of cell cycle re-entry.

Article Snippet: Minichromosome maintenance protein-2 (MCM2) antibodies were from Bethyl Lab (Montgomery, TX).

Techniques: Activity Assay, Expressing, Centrifugation, Immunofluorescence, Microscopy, Western Blot, Immunolabeling

Journal: BMC Cancer

Article Title: Prediction and diagnosis of bladder cancer recurrence based on urinary content of hTERT , SENP1 , PPP1CA , and MCM5 transcripts

doi: 10.1186/1471-2407-10-646

Figure Lengend Snippet: hTERT and MCM5 ROC curves . A : ROC curve for hTERT in combination with cytology grade 2 or grade 3 atypical cells and follow-up information included in the classification. The area under the curve is 81%. B : ROC curve for MCM5 in combination with cytology grade 2 or grade 3 atypical cells and follow-up information included in the classification.

Article Snippet: QPCR was performed using an ABI 7500 with Taqman probes for MCM5 (Hs00198823_m1).

Techniques:

Journal: BMC Cancer

Article Title: Prediction and diagnosis of bladder cancer recurrence based on urinary content of hTERT , SENP1 , PPP1CA , and MCM5 transcripts

doi: 10.1186/1471-2407-10-646

Figure Lengend Snippet: Sensitivity and specificity of the urine markers when using cystoscopy as gold standard for detection of bladder cancer

Article Snippet: QPCR was performed using an ABI 7500 with Taqman probes for MCM5 (Hs00198823_m1).

Techniques:

Journal: BMC Cancer

Article Title: Prediction and diagnosis of bladder cancer recurrence based on urinary content of hTERT , SENP1 , PPP1CA , and MCM5 transcripts

doi: 10.1186/1471-2407-10-646

Figure Lengend Snippet: Recurrence-free survival estimates . Kaplan-Meier plot of recurrence-free survival for hTERT (N = 65) ( A ) and MCM5 (N = 47) ( B ) alone and in combination with cytology ( C and D ). E : Kaplan-Meier plot of recurrence-free survival based on cytology results. Only urine samples from patients with no cystoscopy detectable tumors are included.

Article Snippet: QPCR was performed using an ABI 7500 with Taqman probes for MCM5 (Hs00198823_m1).

Techniques:

Journal: BMC Cancer

Article Title: Prediction and diagnosis of bladder cancer recurrence based on urinary content of hTERT , SENP1 , PPP1CA , and MCM5 transcripts

doi: 10.1186/1471-2407-10-646

Figure Lengend Snippet: Sensitivity and specificity of urine markers when including follow-up data for detection of bladder cancer

Article Snippet: QPCR was performed using an ABI 7500 with Taqman probes for MCM5 (Hs00198823_m1).

Techniques:

Journal: Brain Structure & Function

Article Title: Developmental, tract-tracing and immunohistochemical study of the peripheral olfactory system in a basal vertebrate: insights on Pax6 neurons migrating along the olfactory nerve

doi: 10.1007/s00429-012-0486-2

Figure Lengend Snippet: Primary and secondary antibodies

Article Snippet: The specificity of the immunoreaction of both Pax6 antibodies in the retina and brain of S. canicula was previously tested in our laboratory by pre-adsorbing the primary antibodies with the antigenic Pax6 peptide (NB100-2913PEP; Novus Biologicals, Littleton, CO) used for generation of the NB100-2913 antiserum.

Techniques:

Journal: Brain Structure & Function

Article Title: Developmental, tract-tracing and immunohistochemical study of the peripheral olfactory system in a basal vertebrate: insights on Pax6 neurons migrating along the olfactory nerve

doi: 10.1007/s00429-012-0486-2

Figure Lengend Snippet: Pax6 expression in relation to the components of the olfactory system during development. Sagittal sections through the head of embryos at stages 21 (first period, a ), 25–30 (second period, b – j ) and 31 (third period) ( k – m ). a In stage embryos, Pax6 expression was observed throughout the olfactory placode, being faint in the neurogenic region from which HuC/D-ir pioneer cells delaminated. b Section of the olfactory pit at stage 25 to show high levels of Pax6 expression in a subset of HuC/D-ir neurons in the neurogenic region ( arrows ) and the mesenchyme ( arrowheads ). This section is parallel to that of Fig. c. c , d Adjacent sections of the olfactory epithelium of a stage-28 embryo to show high numbers of Pax6-ir cells neighboring DCX-ir fibers that invaded the mesenchyme. e , f Adjacent sections of a stage-28 embryo after application of neurobiotin into the olfactory epithelium, to show anterogradely labeled cells ( arrowheads ) and their outgrowing axons ( open arrowheads ). Inset in e : detail to show that neurobiotin-labeled fibers were DCX immunoreactive. The inset in f shows that some of these neurobiotin-labeled cells (maturing ORNs) were Pax6-ir ( arrowhead ). Note in f Pax6/HuC/D-ir cells (immature neurons) along unlabeled olfactory axons ( arrow ). g Section of the olfactory epithelium of a stage-29 embryo to show the increased number of Pax6-ir cells within the olfactory epithelium and some Pax6-ir cells in apposition to the DCX-ir olfactory fibers. h Detail of the olfactory nerve of a stage-29 embryo double labeled for GFAP and Pax6 to show that there were no colocalization between these markers. i Section across the olfactory nerve-olfactory bulb junction at stage 29 (compare with Fig. k). Pax6 (HuC/D-ir) neurons seemed to accumulate at this point as they never were detected along the terminal primordium. j Section of the head showing the string of terminal HuC/D-ir cells apposed to the ventral part of the telencephalic hemisphere. Inset : detail of the squared area to show Pax6 immunonegativity of primordial terminal ganglion cells. k Section of a stage-31 embryo to show the increased number of Pax6-ir cells within the olfactory epithelium and the scarce number of Pax6-ir cells within the olfactory nerve, identified by the DCX immunoreactivity, as detailed in the inset . l Section of the olfactory bulb of a stage-31 embryo to show that Pax6-ir cells formed corridors along the entrance of bundles of neurobiotin-labeled olfactory axons in the olfactory bulb ( arrows ). m Section of the olfactory bulb of a stage-31 embryo to show apoptotic (TUNEL positive) cells at the entrance of the olfactory bulb, which mirrored the position of Pax6 cells at the same stage. Scale bars 100 μm ( a , i , m ); 150 μm ( c , d , k , l ); 50 μm ( b , h ); 75 μm ( e – g , j )

Article Snippet: The specificity of the immunoreaction of both Pax6 antibodies in the retina and brain of S. canicula was previously tested in our laboratory by pre-adsorbing the primary antibodies with the antigenic Pax6 peptide (NB100-2913PEP; Novus Biologicals, Littleton, CO) used for generation of the NB100-2913 antiserum.

Techniques: Expressing, Labeling, TUNEL Assay

Journal: Brain Structure & Function

Article Title: Developmental, tract-tracing and immunohistochemical study of the peripheral olfactory system in a basal vertebrate: insights on Pax6 neurons migrating along the olfactory nerve

doi: 10.1007/s00429-012-0486-2

Figure Lengend Snippet: Pax6 expression during late third period (stages 32–34) ( a , b , d , e ) and juvenile ( c ). a – c Sections of the olfactory epithelium to show the changes in distribution of Pax6-ir cells as the epithelium became stratified. Note that Pax6 cells progressively adopted a more basal position as development proceeded (compare a and b ). The inset in a shows that Pax6 immunoreactivity was absent from retrogradely labeled cells (mature ORNs) after application of neurobiotin to the olfactory bulb. Mature ORNs were also identified by its intense Gα 0 immunoreactivity ( large arrows in b ; note also the absence of Pax6 expression in these cells). Arrowheads in b and c indicate cells at intermediate olfactory epithelium levels with weak immunoreactivity to Pax6 and Gα 0 , probably representing early maturing ORNs. Open arrows in c point to basal cells with weak immunoreactivity to Pax6 and PCNA, probably early postmitotic cells; short arrows in c indicate PCNA-negative basal cells with intense immunoreactivity to Pax6 and DCX, possible immature/differentiating ORNs. d , e Sections across olfactory axons to show the scarcity of Pax6-ir cells along the olfactory nerve during stage 32 ( d ), and their absence in stage 33 ( e ). Asterisks indicate the apical surface of the olfactory epithelium; see list for abbreviations. Scale bars 100 μm ( a ); 75 μm ( b ); 40 μm ( c ); 300 μm ( d , e )

Article Snippet: The specificity of the immunoreaction of both Pax6 antibodies in the retina and brain of S. canicula was previously tested in our laboratory by pre-adsorbing the primary antibodies with the antigenic Pax6 peptide (NB100-2913PEP; Novus Biologicals, Littleton, CO) used for generation of the NB100-2913 antiserum.

Techniques: Expressing, Labeling