Journal: British journal of pharmacology

Article Title: A novel compound, denosomin, ameliorates spinal cord injury via axonal growth associated with astrocyte-secreted vimentin.

doi: 10.1111/j.1476-5381.2012.02211.x

Figure Lengend Snippet: Figure 7 Denosomin-mediated axonal outgrowth in primary cultures is mediated by vimentin secretion from astrocytes. (A–C) Isolated astrocytes were cultured in the presence or absence of 1 mM denosomin (Pre-Deno) for 6 days. Rat spinal cord cells were cocultured on the astrocyte layer without denosomin for 7 days after the astrocytes had been treated with denosomin. (D–F) Isolated astrocytes were cultured in the presence or absence of 1 mM denosomin (Pre-Deno) for 6 days. The culture medium was then replaced with fresh medium without denosomin, and the medium was collected 24 h later for use as astrocyte-conditioned medium (ACM). Rat primary cultured spinal cord cells were cultured in normal medium or ACM for 6 days (F). (G and H) Cultured astrocytes that were treated with or without denosomin (Deno) (G) and ACM collected from the astrocyte culture (H) were used for the ELISA assay. (I–K) Isolated mouse cortical cells were cultured with or without 1 or 10 ng·mL-1 vimentin for 6 days. The cells were cultured on slides with (J and K) or without (I–J) a CSPG coating. The cells were immunostained for pNF-H and MAP2, and the densities of pNF-H-positive axons on each neuron were quantified (B, D, I and K). Cellular distributions of neurons and non-neuronal cells per 0.1 mm2 were evaluated by the quantification of MAP2- and DAPI-positive cells (E). In (B, G and H): *P < 0.05, Student’s unpaired t-test (two-tailed); #P < 0.05; one-way ANOVA followed by the post hoc Bonferroni test, versus the control ACM treatment (D), versus the control cells (I) or versus CSPG-coated control cells (K). The numbers of photos (B, D, E, I, and K) or measurements (G and H) are shown in parentheses in the columns. The scale bar indicates 100 mm.

Article Snippet: The vimentin concentrations in these samples were quantified using a rat vimentin ELISA kit according to the manufacturer’s protocol (Cusabio Biotech, Wuhan, Hubei, China).

Techniques: Isolation, Cell Culture, Enzyme-linked Immunosorbent Assay, Two Tailed Test, Control

Journal: bioRxiv

Article Title: Cells in the Polyaneuploid Cancer Cell State are Pro-Metastatic

doi: 10.1101/2024.07.12.603285

Figure Lengend Snippet: Western Blot Antibodies

Article Snippet: VIM (Vimentin) , Rabbit IgG Monoclonal: D21H3 (Cell Signaling Technologies 5741) , 1:5000 , IRDye 800CW Goat anti Rabbit IgG (Licor).

Techniques: Western Blot, Concentration Assay

Journal: Oncotarget

Article Title: Post-translational modifications of vimentin reflect different pathological processes associated with non-small cell lung cancer and chronic obstructive pulmonary disease

doi: 10.18632/oncotarget.27332

Figure Lengend Snippet: ( A ) The reactivity of the VIM antibody was tested against the selection peptide (RLRSSVPGVR), elongated peptide (RLRSSVPGVRL), truncated peptide (RLRSSVPGV), nonsense peptide (LLARDFEKNY), nonsense biotinylated peptide (LLARDFEKNY-biotin) and the VICM peptide (RLRSSVPGV-Citrulline). %B/B0: B equals the OD at x ng/ml peptide and B0 equals the OD at 0 ng/ml peptide. ( B ) The reactivity of the VIM antibody was tested against recombinant full length vimentin, recombinant full length vimentin cleaved with MMP2 and recombinant full length vimentin cleaved with MMP8.

Article Snippet: To test the specificity of the VIM antibody towards vimentin cleaved by MMP2 and MMP8, recombinant human vimentin (Novus Biologicals, CO, USA) was reconstituted to a final concentration of 1000 ug/mL in 4 mM HCl.

Techniques: Selection, Recombinant

Journal: Oncotarget

Article Title: Post-translational modifications of vimentin reflect different pathological processes associated with non-small cell lung cancer and chronic obstructive pulmonary disease

doi: 10.18632/oncotarget.27332

Figure Lengend Snippet: Technical validation of the MMP-degraded non-citrullinated vimentin (VIM) assay

Article Snippet: To test the specificity of the VIM antibody towards vimentin cleaved by MMP2 and MMP8, recombinant human vimentin (Novus Biologicals, CO, USA) was reconstituted to a final concentration of 1000 ug/mL in 4 mM HCl.

Techniques: Biomarker Discovery, Intra Assay, Inter Assay

Journal: Oncotarget

Article Title: Post-translational modifications of vimentin reflect different pathological processes associated with non-small cell lung cancer and chronic obstructive pulmonary disease

doi: 10.18632/oncotarget.27332

Figure Lengend Snippet: Levels of VIM and VICM in serum were evaluated in two independent cohorts of patients with NSCLC and healthy controls. VIM and VICM levels were compared to each other using the nonparametric Mann-Whitney test. Significance level: p < 0.05. ( A ) Evaluation of VIM in cohort 2. ( B ) Evaluation of VICM in cohort 2. ( C ) Evaluation of VIM in cohort 3. ( D ) Evaluation of VICM in cohort 3.

Article Snippet: To test the specificity of the VIM antibody towards vimentin cleaved by MMP2 and MMP8, recombinant human vimentin (Novus Biologicals, CO, USA) was reconstituted to a final concentration of 1000 ug/mL in 4 mM HCl.

Techniques: MANN-WHITNEY

Journal: Oncotarget

Article Title: Post-translational modifications of vimentin reflect different pathological processes associated with non-small cell lung cancer and chronic obstructive pulmonary disease

doi: 10.18632/oncotarget.27332

Figure Lengend Snippet: Levels of VIM and VICM in serum were evaluated in patients with COPD and healthy controls. VIM and VICM levels were compared to each other using the nonparametric Mann-Whitney test. Significance level: p < 0.05. ( A ) Evaluation of VIM in COPD. ( B ) Evaluation of VICM in COPD.

Article Snippet: To test the specificity of the VIM antibody towards vimentin cleaved by MMP2 and MMP8, recombinant human vimentin (Novus Biologicals, CO, USA) was reconstituted to a final concentration of 1000 ug/mL in 4 mM HCl.

Techniques: MANN-WHITNEY

Journal: Oncotarget

Article Title: Post-translational modifications of vimentin reflect different pathological processes associated with non-small cell lung cancer and chronic obstructive pulmonary disease

doi: 10.18632/oncotarget.27332

Figure Lengend Snippet: Levels of VIM and VICM in serum were evaluated in the two groups of patients with NSCLC stratified according to TNM disease stage I, II, III, IV ( A and B ) and I–II vs. III–IV ( C and D ). VIM and VICM levels were compared to each other using a one-way ANOVA with Kruskal-Wallis test (A and B) and the nonparametric Mann-Whitney test (C and D). Significance level: p < 0.05.

Article Snippet: To test the specificity of the VIM antibody towards vimentin cleaved by MMP2 and MMP8, recombinant human vimentin (Novus Biologicals, CO, USA) was reconstituted to a final concentration of 1000 ug/mL in 4 mM HCl.

Techniques: MANN-WHITNEY

Journal: Oncotarget

Article Title: Post-translational modifications of vimentin reflect different pathological processes associated with non-small cell lung cancer and chronic obstructive pulmonary disease

doi: 10.18632/oncotarget.27332

Figure Lengend Snippet: Levels of VIM and VICM in serum from patients with non-small cell lung cancer, chronic obstructive pulmonary disease and healthy controls where compared pairwise and evaluated by the Pearson correlation coefficient (r).

Article Snippet: To test the specificity of the VIM antibody towards vimentin cleaved by MMP2 and MMP8, recombinant human vimentin (Novus Biologicals, CO, USA) was reconstituted to a final concentration of 1000 ug/mL in 4 mM HCl.

Techniques:

Journal: Oncotarget

Article Title: Post-translational modifications of vimentin reflect different pathological processes associated with non-small cell lung cancer and chronic obstructive pulmonary disease

doi: 10.18632/oncotarget.27332

Figure Lengend Snippet: The diagnostic power of VIM and VICM to discriminate between NSCLC vs. healthy controls ( A–B ), COPD vs. controls ( C–D ) and NSCLC vs. COPD ( E–F ) were calculated using the area under the receiver operating characteristics curve (AUROC).

Article Snippet: To test the specificity of the VIM antibody towards vimentin cleaved by MMP2 and MMP8, recombinant human vimentin (Novus Biologicals, CO, USA) was reconstituted to a final concentration of 1000 ug/mL in 4 mM HCl.

Techniques: Diagnostic Assay

Journal: Breast Cancer Research : BCR

Article Title: A hybrid epithelial-mesenchymal transition program enables basal epithelial cells to bypass stress-induced stasis and contributes to a metaplastic breast cancer progenitor state

doi: 10.1186/s13058-024-01920-8

Figure Lengend Snippet: Transient activation of EMT in stasis HMECs precedes variant emergence. A Human breast tissue samples from three donors were used to generate HMEC cultures in MEGM. The first passage after partial trypsinization was considered passage 1 (P1). At each passage, the cells were counted, and the cumulative population doubling was calculated. The resulting growth curves illustrate three distinct phases: the initial logarithmic growth phase ( a ), the stasis growth plateau ( b ), and the second logarithmic growth phase initiated by the expansion of variants ( c ). Protein, RNA, and conditioned media (CM) were collected from passage 2 (P2) HMECs, stasis HMECs (S), and variant HMECs at two passages beyond stasis (S + 2). Crystal violet-stained flasks depict the pattern of variant emergence from the stasis cultures. The graphic to the right illustrates a mixed population of cultured HMECs encountering the stasis barrier; while most cells are eliminated from the cultures, a small population of variants bypass this ‘stress-induced’ barrier and reinitiates growth. B Cell proliferation was measured in HMEC cultures using the Click-iT EdU cell proliferation assay and quantified by flow cytometry using cells collected from P2, S, and S + 2 cultures, which were re-plated overnight and pulsed with 10 µM EdU for one hour. C Gene expression analysis of p21 and p16 was performed on cDNA produced from cells collected from P2, S, and S + 2 cultures by qPCR. Representative images of P2 and S HMECs immunostained with antibodies against p16 and p21, and counterstained with DAPI are shown. D CM collected directly from P2 and S HMEC cultures (incubated with cells for 24 h) was mixed with an equal amount of fresh MEGM and used to culture freshly isolated HMECs (P1). E Sandwich ELISA kits were used to determine concentrations of TGF-β, Activin A, and PAI-1. An equal number of cells was harvested from P2, S, and S + 2 cultures and cultured in basal media (supplements excluded) for 24 h. Concentrations were normalized to the final cell number. F MMP-2 and FN were analyzed by qPCR. G E-cadherin, N-cadherin, P-cadherin, Slug, Snail, Twist, and Zeb were analyzed by qPCR. H Western blot analysis of lysates from P2, S, and S + 2 cells using antibodies against Snail, Zeb, Slug, E-cadherin (E-Cad.), N-cadherin (N-Cad.), Vimentin, and Actin (loading control)

Article Snippet: Pre-made TaqMan Gene Expression Assays (Thermo Fisher Scientific) were used: ZEB1: Hs00232783_m1, SNAI1: Hs00195591_m1, SNAI2: Hs00161904_m1, TWIST1: Hs01675818_s1, CDH3: Hs00999915_m1, VIM: Hs00958111_m1, CDH1: Hs01023895_m1, CDH2: Hs00983056_m1, CDKN1A: Hs00355782_m1, MMP-2: Hs01548727_m1, FN1: Hs01549976_m1, CHRDL2: Hs01060234_m1, GREM1: Hs01879841_s1, and WNT5a: Hs00998537_m1.

Techniques: Activation Assay, Variant Assay, Staining, Cell Culture, Proliferation Assay, Flow Cytometry, Gene Expression, Produced, Incubation, Isolation, Sandwich ELISA, Western Blot, Control

Journal: Breast Cancer Research : BCR

Article Title: A hybrid epithelial-mesenchymal transition program enables basal epithelial cells to bypass stress-induced stasis and contributes to a metaplastic breast cancer progenitor state

doi: 10.1186/s13058-024-01920-8

Figure Lengend Snippet: Basal epithelial cells exhibit enhanced TGF-β signaling and EMT activation compared to luminal epithelial cells. A Human breast tissues from the same three donors utilized in Fig. A were further processed into single cells. The cell suspension was stained with conjugated antibodies against CD10, EpCAM, CD49f, CD2, CD3, CD4, CD16, CD64, CD31, and CD45 then DAPI and conjugated streptavidin. Dead cells (DAPI + ), doublets, and immune/stromal cell types (CD2 + , CD3 + , CD4 + , CD16 + , CD64 + , CD31 + , and/or CD45 + ) were removed. The breast epithelium was separated into luminal (EpCAM + ) and basal (EpCAM − CD49f + CD10 + ) enriched populations by FACS and expanded separately in MEGM in 6-well plates. Notably, these luminal and basal populations contain both mature cell type and stem and/or progenitors. Representative wells were immunostained prior to passaging (P0) with the luminal markers cytokeratin 19 (CK19) and mucin 1 (MUC1) or the basal markers p63 and cytokeratin 14 (CK14). B Basal and luminal epithelial cells were further expanded in MEGM, cells were counted at each passage, and the growth curve of the cumulative population doubling over time was plotted. Cells and CM were collected from basal epithelial cells at passages B(P2), B(P5), and B(P7) and from luminal cells at passages L(P2) and L(P3). Crystal violet-stained flasks depict the pattern of variant cell emergence from basal epithelial cell cultures after stasis. C Sandwich ELISA kits were used to determine the TGF-β and Activin A concentrations. An equal number of cells was harvested from the B(P2), B(P5), L(P2), and L(P3) cultures and then cultured in basal media (supplements excluded) for 24 h. Concentrations were normalized to the final cell number. D Representative images of P2 basal and luminal epithelial cells from two donor tissues immunostained with antibodies against pan-cytokeratin and vimentin and then counterstained with DAPI. E E-cadherin, P-cadherin, N-cadherin, Slug, Snail, Twist, and Zeb were analyzed by qPCR using cDNA produced from B(P2), B(P5), L(P2), and L(P3) cells. F Western blot analysis of lysates from B(P2), B(P5), and B(P7) cultures, using antibodies against N-cadherin (N-Cad.), Snail, Zeb, Slug, and Actin (as loading controls). G Same as C, using basal epithelial cells from the B(P3), B(P5), and B(P8) cultures

Article Snippet: Pre-made TaqMan Gene Expression Assays (Thermo Fisher Scientific) were used: ZEB1: Hs00232783_m1, SNAI1: Hs00195591_m1, SNAI2: Hs00161904_m1, TWIST1: Hs01675818_s1, CDH3: Hs00999915_m1, VIM: Hs00958111_m1, CDH1: Hs01023895_m1, CDH2: Hs00983056_m1, CDKN1A: Hs00355782_m1, MMP-2: Hs01548727_m1, FN1: Hs01549976_m1, CHRDL2: Hs01060234_m1, GREM1: Hs01879841_s1, and WNT5a: Hs00998537_m1.

Techniques: Activation Assay, Suspension, Staining, Passaging, Variant Assay, Sandwich ELISA, Cell Culture, Produced, Western Blot

Journal: Breast Cancer Research : BCR

Article Title: A hybrid epithelial-mesenchymal transition program enables basal epithelial cells to bypass stress-induced stasis and contributes to a metaplastic breast cancer progenitor state

doi: 10.1186/s13058-024-01920-8

Figure Lengend Snippet: Inhibition of the TGF-β pathway during cellular transformation reduces metaplastic phenotypes in basal epithelial-derived tumors. A Activation of the TGF-β pathway and EMT collaborate to drive cellular dysfunction and growth arrest. Adapted variants that emerged from stasis cultures exhibit notable changes in gene expression associated with growth and differentiation and are stabilized in a hybrid EMT state. Inhibition of the TGF-β pathway eliminates these selective pressures during the expansion of basal epithelial cells. B Basal epithelial cell populations: (1) P3, cultured in MEGM + DMSO for three passages, (2) variant, cultured in MEGM + DMSO for eight passages, and (3) A83-01, cultured in MEGM + 500 nM A83-01 for three passages, were oncogenically transformed through sequential transduction with TERT and SV40, followed by selection (puromycin and hygromycin) and lentiviral transduction of oncogenic KRAS G12V followed by a second round of selection (blasticidin and IRES-RFP). Western blot analysis was performed on lysates from these oncogenically transformed basal epithelial cells using antibodies against RAS and Actin (as a loading control). C At each passage, Oncogenically transformed basal epithelial cells were counted. A growth curve of the cumulative population doubling over time was plotted. D Transformed basal epithelial populations were suspended in soft agar in each well of a 6-well plate at a concentration of 20,000 cells/well and cultured in DMEM/F12 + 20% FBS. After 4 weeks, the resulting colonies were stained with crystal violet, imaged, and counted using the ImageJ software. E P-cadherin, N-cadherin, Slug, Snail, Twist, and Zeb were analyzed qPCR using cDNA produced from oncogenically transformed basal epithelial cell populations. F Variant and A83-01-treated oncogenically transformed basal epithelial cells were immunostained for P-cadherin and N-cadherin, then visualized by flow cytometry. G Variants and A83-01-treated oncogenically transformed basal epithelial cells (4 × 10 6 ) were injected subcutaneously with 50% Matrigel into 8-to 12-week-old female NSG mice. Tumors formed over a period of 3–6 months. FFPE sections from five tumors were subjected to multiplexed immunohistochemical analysis for p63, cytokeratin 13 (CK13), Slug + Snail, and cytokeratin 5 + vimentin. Human-specific lamin A/C was used to identify the tumor cells

Article Snippet: Pre-made TaqMan Gene Expression Assays (Thermo Fisher Scientific) were used: ZEB1: Hs00232783_m1, SNAI1: Hs00195591_m1, SNAI2: Hs00161904_m1, TWIST1: Hs01675818_s1, CDH3: Hs00999915_m1, VIM: Hs00958111_m1, CDH1: Hs01023895_m1, CDH2: Hs00983056_m1, CDKN1A: Hs00355782_m1, MMP-2: Hs01548727_m1, FN1: Hs01549976_m1, CHRDL2: Hs01060234_m1, GREM1: Hs01879841_s1, and WNT5a: Hs00998537_m1.

Techniques: Inhibition, Transformation Assay, Derivative Assay, Activation Assay, Gene Expression, Cell Culture, Variant Assay, Transduction, Selection, Western Blot, Control, Concentration Assay, Staining, Software, Produced, Flow Cytometry, Injection, Immunohistochemical staining

Journal: OncoTargets and therapy

Article Title: A c-Met Inhibitor Suppresses Osteosarcoma Progression via the ERK1/2 Pathway in Human Osteosarcoma Cells

doi: 10.2147/OTT.S317122

Figure Lengend Snippet: Silencing Met inhibits the proliferation, migration and invasion, while promotes apoptosis in OS cells. ( A ) The expression of Met after transfection of three different Met siRNAs in 143B and U2OS detected by qRT-PCR. (n=3) *p<0.05, **p<0.01, ***p<0.001. ( B ) Colony-formation assay of 143B and U2OS after stable transfection of Met Sh Met or N.C. (vector plasmids). (n=3) *p<0.05, **p<0.01. ( C ) Transwell migration and invasion assay of 143B and U2OS after stable transfection of Met Sh Met or N.C. (vector plasmids). (n=3) **p<0.01, ***p<0.001. ( D ) Wound-healing assay of 143B and U2OS after stable transfection of Met Sh Met or N.C. (vector plasmids). (n=3) *p<0.05, **p<0.01. ( E ) The expression of N-cadherin and vimentin in 143B and U2OS detected by Western blot analysis after stable transfection of Met Sh Met or N.C. (vector plasmids). ( F ) Cell apoptosis rate of 143B and U2OS after stable transfection of Met Sh Met or N.C. (vector plasmids). (n=3) ***p<0.001.

Article Snippet: Anti-GAPDH, anti-N-cadherin and anti-vimentin antibodies were purchased from Proteintech (Chicago, USA); Anti-Cyclin D1, anti-CDK4, anti-ERK1/2 and anti-pERK1/2 (Thy202/Tyr204) antibodies were purchased from Abcam (Cambridge, UK).

Techniques: Migration, Expressing, Transfection, Quantitative RT-PCR, Colony Assay, Stable Transfection, Plasmid Preparation, Invasion Assay, Wound Healing Assay, Western Blot

Journal: OncoTargets and therapy

Article Title: A c-Met Inhibitor Suppresses Osteosarcoma Progression via the ERK1/2 Pathway in Human Osteosarcoma Cells

doi: 10.2147/OTT.S317122

Figure Lengend Snippet: PHA-665752 inhibits proliferation, migration and invasion of OS cells. ( A ) The molecular formula of PHA-665752. ( B ) The anti-proliferative effect of PHA-665752 on 143B and U2OS was detected by CCK-8 assay. (n=5) Cells were treated with different concentration of PHA-665752. Control group contained 0.1% DMSO. *p<0.05, **p<0.01, ***p<0.001. ( C ) Colony-formation assay of 143B and U2OS with control or PHA-665752. (n=3) *p<0.05, **p<0.01. ( D ) Transwell migration and invasion assay of 143B and U2OS with control or PHA-665752. (n=3) *p<0.05, **p<0.01, ***p<0.001. ( E ) Wound-healing assay of 143B and U2OS with control or PHA-665752. (n=3) *p<0.05, **p<0.01. ( F ) The expression of N-cadherin and vimentin in 143B and U2OS with control or PHA-665752 detected by Western blot analysis.

Article Snippet: Anti-GAPDH, anti-N-cadherin and anti-vimentin antibodies were purchased from Proteintech (Chicago, USA); Anti-Cyclin D1, anti-CDK4, anti-ERK1/2 and anti-pERK1/2 (Thy202/Tyr204) antibodies were purchased from Abcam (Cambridge, UK).

Techniques: Migration, CCK-8 Assay, Concentration Assay, Control, Colony Assay, Invasion Assay, Wound Healing Assay, Expressing, Western Blot

Journal: OncoTargets and therapy

Article Title: A c-Met Inhibitor Suppresses Osteosarcoma Progression via the ERK1/2 Pathway in Human Osteosarcoma Cells

doi: 10.2147/OTT.S317122

Figure Lengend Snippet: The ERK1/2 pathway mediates the role of PHA-665752 in proliferation, migration and invasion in OS cells. ( A ) The expression of ERK1/2 and pERK1/2 (Thy202/Tyr204) in 143B and U2OS was detected by Western blot analysis. Cells were treated with different concentration of PHA-665752. ( B ) The molecular formula of LY3214996. ( C ) The effects of PHA-665752 and LY3214996 on the expression of pERK1/2 (Thy202/Tyr204), N-cadherin and vimentin in OS cells as demonstrated by Western blot assay. ( D ) Proliferation of OS cells treated with PHA-665752 (3nM/mL) and various concentrations of LY3214996 was evaluated by CCK-8 assay. (n=5) *p<0.05, **p<0.01, ***p<0.001. ( E ) The effects of PHA-665752 and LY3214996 on the colony-formation ability in OS cells as demonstrated by colony-formation assay. (n=3) *p<0.05, **p<0.01. ( F ) Transwell migration and invasion assay demonstrated the effects of PHA-665752 and LY3214996 on the migration and invasion ability in OS cells. (n=3) *p<0.05, **p<0.01. Wound-healing assay of demonstrated the effects of PHA-665752 and LY3214996 on the migration ability in OS cells. (n=3) *p<0.05, **p<0.01. ( G ) The effects of PHA-665752 and LY3214996 on the migration ability in OS cells as demonstrated by wound-healing assay. (n=3) *p<0.05, **p<0.01.

Article Snippet: Anti-GAPDH, anti-N-cadherin and anti-vimentin antibodies were purchased from Proteintech (Chicago, USA); Anti-Cyclin D1, anti-CDK4, anti-ERK1/2 and anti-pERK1/2 (Thy202/Tyr204) antibodies were purchased from Abcam (Cambridge, UK).

Techniques: Migration, Expressing, Western Blot, Concentration Assay, CCK-8 Assay, Colony Assay, Invasion Assay, Wound Healing Assay

Journal: Cells

Article Title: Nuclear Magnetic Resonance Treatment Induces ßNGF Release from Schwann Cells and Enhances the Neurite Growth of Dorsal Root Ganglion Neurons In Vitro

doi: 10.3390/cells13181544

Figure Lengend Snippet: Characterization of SC cultures and analysis of ßNFG concentration in response to NMRT. ( A ) Representative fluorescence images of the primary SC culture used for NMRT treatment and conditioned media harvest stained for intermediate filament vimentin (VIME, white), S100 (magenta), SOX10 (green) and DAPI (blue); filled arrowheads indicate a SOX10-/S100-/VIME+/DAPI+ fibroblast. Scale bars 50 µm. ( B ) SC culture purity was calculated as percentage of SOX10+/DAPI+ cells (SCs) from DAPI+ cells (all cells). ( C ) Representative phase contrast images illustrating the similar morphology of control SC cultures and NMRT-treated SC cultures after a 10 h treatment cycle. Scale bars 50 µm. ( D ) ßNGF concentration measured in CM CTRL and CM NMRT; values that correspond to the same donor are connected by a line; data are depicted as average values of measured duplicates for each donor ( n = 3); paired t -test; ** p < 0.01.

Article Snippet: Immunostaining was performed using SC markers S100 protein (Dako, Auckland, New Zealand), the glial cell-associated transcription factor SOX10 (Santa Cruz Biotechnology, Dallas, TX, USA) and the intermediate filament vimentin (VIME) (ThermoFisher) to determine the purity of the SC cultures.

Techniques: Concentration Assay, Fluorescence, Staining, Control

Journal: The Open Microbiology Journal

Article Title: Molecular Characterization of Multidrug Resistant Uropathogenic E. Coli Isolates from Jordanian Patients

doi: 10.2174/1874285801812010001

Figure Lengend Snippet: Distribution of 150 MDR E. coli isolates to each antibiotic resistant classes and types of antibiotics.

Article Snippet: E. coli ATCC 25922 and E. coli ATCC 35218 (A & B-lactamase producer) were used as control strains.

Techniques:

Journal: The Open Microbiology Journal

Article Title: Molecular Characterization of Multidrug Resistant Uropathogenic E. Coli Isolates from Jordanian Patients

doi: 10.2174/1874285801812010001

Figure Lengend Snippet: Antimicrobial susceptibility pattern of total 262 E. coli isolates from urine specimens of both hospitalized and community patients.*

Article Snippet: E. coli ATCC 25922 and E. coli ATCC 35218 (A & B-lactamase producer) were used as control strains.

Techniques: Concentration Assay

Journal: The Open Microbiology Journal

Article Title: Molecular Characterization of Multidrug Resistant Uropathogenic E. Coli Isolates from Jordanian Patients

doi: 10.2174/1874285801812010001

Figure Lengend Snippet: Distribution of antimicrobial susceptibility of 262 E. coli isolates from community and hospitalized patients according to their age groups.

Article Snippet: E. coli ATCC 25922 and E. coli ATCC 35218 (A & B-lactamase producer) were used as control strains.

Techniques:

Journal: The Open Microbiology Journal

Article Title: Molecular Characterization of Multidrug Resistant Uropathogenic E. Coli Isolates from Jordanian Patients

doi: 10.2174/1874285801812010001

Figure Lengend Snippet: Distribution of carbapenemase resistance genes, fluoroquinolones-resistance genes, CTX-M-type genes and ST131 clone among 50 representative MDR E. coli isolates.

Article Snippet: E. coli ATCC 25922 and E. coli ATCC 35218 (A & B-lactamase producer) were used as control strains.

Techniques: Bla VIM Assay

Journal: Scientific Reports

Article Title: Metformin exerts anti-cancerogenic effects and reverses epithelial-to-mesenchymal transition trait in primary human intrahepatic cholangiocarcinoma cells

doi: 10.1038/s41598-021-81172-0

Figure Lengend Snippet: Phenotypic characterization of iCCA primary cell cultures and effect of Metformin on cell proliferation of iCCA primary cultures. ( A ) Immunofluorescence for Vimentin, Cytokeratin-19 and E-Cadherin in Large duct-type iCCA (up) and Small duct-type iCCA (down) iCCA primary cell cultures; nuclei were stained with DAPI. The bar graph indicates the percentage of positive cells. More than 70% of cells stained positive for Vimentin while express very low level of Cytokeratin-19 (< 6% of positive cells) and were negative for E-Cadherin. (See also Supplementary Figure A). Figures are representative of N = 5 different cell cultures. Data in the bar graph represent mean ± SD of N = 5 independent experiments. ( B ) Large duct-type iCCA (up) and Small duct-type iCCA (down) primary iCCA cell cultures were exposed for 24, 48, 72, 96 h to increasing concentration of Metformin (0–1000 µM); cell proliferation was evaluated by MTS assay and expressed as ratio with respect to controls. Metformin significantly reduced cell proliferation in a dose-dependent manner. In most experiments the inhibitory effect of Metformin on both Large and Small duct-type iCCA started at 5–10 µM. * p < 0.05 versus controls. Data represent mean ± SD of N = 5 independent experiments.

Article Snippet: Primary cell cultures were fixed in 1:1 Acetone/Methanol (10 min at room temperature), incubated in 20% fetal bovine serum (FBS) in Dulbecco’s Phosphate Buffered Saline (DPBS) for 30 min, and then incubated for 1 h at room temperature with primary antibodies anti Human Vimentin RV202 (sc-32322, Santa Cruz Biotechnology Dallas, Texas, USA), Human Cytokeratin-19 (sc-6278, Santa Cruz Biotechnology), Human Vimentin (AlexaFluor 488 conjugated, goat IgG, IC8104G, R&D System, Minneapolis, MN, USA), Human Cytokeratin-19 (AlexaFluor 594 clone A53-B/A2, 628,504, Biolegend San Diego, CA, USA).

Techniques: Immunofluorescence, Staining, Concentration Assay, MTS Assay

Journal: Scientific Reports

Article Title: Metformin exerts anti-cancerogenic effects and reverses epithelial-to-mesenchymal transition trait in primary human intrahepatic cholangiocarcinoma cells

doi: 10.1038/s41598-021-81172-0

Figure Lengend Snippet: Effect of Metformin on gene expression of mesenchymal, EMT, epithelial markers, AMPK and FOXO3 in iCCA primary cells and nuclear translocation of FOXO3 in iCCA primary cell cultures. ( A ) The gene expression of mesenchymal and EMT markers (Vimentin, SNAIL1, SNAIL2, TWIST1) and epithelial markers (E-Cadherin, Cytokeratin-19) was analysed by RT-qPCR in Large and Small duct-type iCCA primary cultures exposed to Metformin 10 µM for 48 and 96 h and normalized to the expression of GAPDH (housekeeping gene). In both iCCA subtypes, the gene expression of Vimentin was significantly decreased by Metformin only after 96 h while the expression of SNAIL1, SNAIL2 and TWIST1 decreased after 48 and 96 h. In contrast, the gene expression of the epithelial marker E-Cadherin significantly increased after 48 and 96 h in both Large and Small duct-type iCCA while Cytokeratin-19 increased only in Large duct-type iCCA after 96 h of exposure to Metformin. Data represent mean ± SD of N = 5 independent experiments. * p < 0.05. ( B ) The gene expression of AMPK and FOXO3, two signalling molecules involved in the mechanism of action of Metformin, was analysed by RT-qPCR and normalized to the expression of GAPDH (housekeeping gene). After 48 and 96 h, Metformin 10 µM significantly induced the gene expression of AMPK and FOXO3 in both Large and Small duct-type iCCA primary cultures. Data represent mean ± SD of N = 5 independent experiments. * p < 0.05. ( C ) IF analysis of FOXO3 in Large and Small duct-type iCCA primary cultures exposed for 96 h to Metformin 10 µM. Migration of FOXO3 from the cytoplasm to the nucleus is evident and occurred in 64 ± 10% of the cells. Nuclei were stained with DAPI. Magnification 20 × and 10 × ; representative images of N = 3 independent experiments.

Article Snippet: Primary cell cultures were fixed in 1:1 Acetone/Methanol (10 min at room temperature), incubated in 20% fetal bovine serum (FBS) in Dulbecco’s Phosphate Buffered Saline (DPBS) for 30 min, and then incubated for 1 h at room temperature with primary antibodies anti Human Vimentin RV202 (sc-32322, Santa Cruz Biotechnology Dallas, Texas, USA), Human Cytokeratin-19 (sc-6278, Santa Cruz Biotechnology), Human Vimentin (AlexaFluor 488 conjugated, goat IgG, IC8104G, R&D System, Minneapolis, MN, USA), Human Cytokeratin-19 (AlexaFluor 594 clone A53-B/A2, 628,504, Biolegend San Diego, CA, USA).

Techniques: Gene Expression, Translocation Assay, Quantitative RT-PCR, Expressing, Marker, Migration, Staining

Journal: Scientific Reports

Article Title: Metformin exerts anti-cancerogenic effects and reverses epithelial-to-mesenchymal transition trait in primary human intrahepatic cholangiocarcinoma cells

doi: 10.1038/s41598-021-81172-0

Figure Lengend Snippet: Effect of prolonged treatment (12 and 57 days) of iCCA primary cells with Metformin on the gene expression of mesenchymal, EMT and epithelial markers. The gene expression of mesenchymal and EMT markers (Vimentin, SNAIL1, SNAIL2, TWIST1) and epithelial markers (E-Cadherin, Cytokeratin-19) was analysed by RT-qPCR in Large duct-type iCCA ( A ) and Small duct-type iCCA ( B ) primary cultures exposed to Metformin 10 µM for 12 or 57 days and normalized to the expression of GAPDH (housekeeping gene). The mesenchymal and EMT markers, Vimentin, SNAIL1, SNAIL2 and TWIST1, were markedly down-regulated with respect to controls; in some case almost completely suppressed (i.e. Vimentin and SNAIL2 in Large duct-type iCCA and SNAIL2 in Small duct-type iCCA). The epithelial markers, E-Cadherin and Cytokeratin-19 were markedly upregulated in both Large and Small duct-type iCCA. Data represent mean ± SD of N = 5 independent experiments; * p < 0.05 versus controls.

Article Snippet: Primary cell cultures were fixed in 1:1 Acetone/Methanol (10 min at room temperature), incubated in 20% fetal bovine serum (FBS) in Dulbecco’s Phosphate Buffered Saline (DPBS) for 30 min, and then incubated for 1 h at room temperature with primary antibodies anti Human Vimentin RV202 (sc-32322, Santa Cruz Biotechnology Dallas, Texas, USA), Human Cytokeratin-19 (sc-6278, Santa Cruz Biotechnology), Human Vimentin (AlexaFluor 488 conjugated, goat IgG, IC8104G, R&D System, Minneapolis, MN, USA), Human Cytokeratin-19 (AlexaFluor 594 clone A53-B/A2, 628,504, Biolegend San Diego, CA, USA).

Techniques: Gene Expression, Quantitative RT-PCR, Expressing

Journal: Scientific Reports

Article Title: Metformin exerts anti-cancerogenic effects and reverses epithelial-to-mesenchymal transition trait in primary human intrahepatic cholangiocarcinoma cells

doi: 10.1038/s41598-021-81172-0

Figure Lengend Snippet: IF and western blot analysis of Vimentin and Cytokeratin-19 in primary cultures of Large duct-type iCCA treated for 12 or 57 days with Metformin. ( A ) Vimentin and Cytokeratin-19 were analysed by IF (left panels), in Large duct-type iCCA primary cultures treated with Metformin 10 µM for 12 and 57 days. Metformin induced a marked IF positivity for Cytokeratin-19 and a marked decrease of Vimentin expression that were not observed after 48–96 h of treatment. By western blot (right panels, cropped gels/blots are displayed), the protein expression of Vimentin decreased while that of Cytokeratin-19 increased after prolonged (12 and 57 days) treatment with Metformin 10 µM. * p < 0.05 versus controls. Data represent mean ± SD of N = 3 independent experiments. ( B ) In contrast with Large duct-type iCCA, in the Small duct-type iCCA cells the Cytokeratin-19 expression was not observed neither by IF not by western blot (not detectable, ND). However, by western blot analysis a decreased level of Vimentin was observed. * p < 0.05 versus controls. Mean ± SD of N = 3 independent experiments.

Article Snippet: Primary cell cultures were fixed in 1:1 Acetone/Methanol (10 min at room temperature), incubated in 20% fetal bovine serum (FBS) in Dulbecco’s Phosphate Buffered Saline (DPBS) for 30 min, and then incubated for 1 h at room temperature with primary antibodies anti Human Vimentin RV202 (sc-32322, Santa Cruz Biotechnology Dallas, Texas, USA), Human Cytokeratin-19 (sc-6278, Santa Cruz Biotechnology), Human Vimentin (AlexaFluor 488 conjugated, goat IgG, IC8104G, R&D System, Minneapolis, MN, USA), Human Cytokeratin-19 (AlexaFluor 594 clone A53-B/A2, 628,504, Biolegend San Diego, CA, USA).

Techniques: Western Blot, Expressing