Journal: Cells

Article Title: Patient-Derived Immortalized Limbal Epithelial Cells as In Vitro Models of Congenital Aniridia

doi: 10.3390/cells15050394

Figure Lengend Snippet: Marker expression profiles in limbal epithelial cell lines derived from a healthy control (ctrl-iLEC) and from patients with aniridia (AN1-iLEC and AN2-iLEC). ( A ) Relative mRNA expression levels of PAX6, ABCG2, TP63, ALDH1A1, FABP5, and FOSL2 in ctrl-iLEC, AN1-iLEC, and AN2-iLEC cell lines are shown. Expression values were normalized to reference genes (GUSB and TBP) and presented as fold change. Error bars represent standard deviation; each data point corresponds to an independent experiment. ( B ) Venn diagram depicting the distribution of differentially expressed genes (DEGs) among ctrl-iLEC, AN1-iLEC, and AN2-iLEC. DEGs were identified by calculating expression ratios between ctrl-iLEC and each aniridia cell line. ( C ) Heatmap illustrating RNA-Seq-based gene expression differences in DEGs between ctrl-iLEC and the two aniridia cell lines. Red indicates higher expression and blue indicates lower expression. RNA sequencing was performed from one biological replicate per cell line.

Article Snippet: With the exception of ABCG2, all antibodies targeted intracellular proteins; therefore, cells were fixed and permeabilized using the Transcription Factor Staining Buffer Set (Fix-Perm; Cat. No. 130-122-981, Miltenyi Biotec B.V. & Co. KG, Bergisch Gladbach, Germany), according to the manufacturer’s instructions.

Techniques: Marker, Expressing, Derivative Assay, Control, Standard Deviation, RNA Sequencing, Gene Expression

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: BMP6 as a therapeutic target for preeclampsia: enhancing trophoblast invasion and vascular mimicry

doi: 10.1007/s00018-025-06040-w

Figure Lengend Snippet: BMP6 signaling enhancement compensates for shallow trophoblast invasion in PE. An examination of clinical samples from PE patients and their gestational age-matched controls revealed that BMP6 was upregulated in preeclamptic placentas. BMP6 enhances trophoblast invasion via ID1-mediated upregulation of SERPINE2 and PlGF. Moreover, BMP6 also intensifies trophoblast vascular mimicry through the ID1-mediated upregulation of PlGF. Both canonical (p-SMAD1/5/9) and noncanonical (p-SMAD2/3) pathways participate in BMP6-induced ID1 expression. Our findings indicate that the increase in BMP6 signaling during late gestation could serve as a compensatory response to shallow trophoblast invasion in PE, which in light of BMP6 and its downstream targets could serve as diagnostic markers and therapeutic target applications in the clinical management of PE. This schematic diagram was created with Biorender.com

Article Snippet: Rat plasma was measured with a sFlt-1 ELISA kit (Proteintech, KE10069), a BMP6 ELISA Kit (Novus Biologicals, NBP2-69999, Centennial, CO, USA), and a PlGF ELISA kit (CUSABIO, CSB-E07400r, Wuhan, China).

Techniques: Expressing, Diagnostic Assay

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: BMP6 as a therapeutic target for preeclampsia: enhancing trophoblast invasion and vascular mimicry

doi: 10.1007/s00018-025-06040-w

Figure Lengend Snippet: ID1 mediates BMP6-induced SERPINE2 and PlGF upregulation in human trophoblasts. A-C , BMP6 upregulates SERPINE2 protein levels in trophoblasts. A , HTR8/SVneo cells were treated with different concentrations (0, 6.25, 12.5, 25, 50, or 100 ng/mL) of BMP6, and the SERPINE2 protein levels after 24 h of treatment were examined by Western blot analysis. The upper panel shows a representative Western blot image, and the lower panel shows the summarized quantitative results. B , SERPINE2 protein levels in HTR8/SVneo cells after treatment with vehicle (Ctrl) or 50 ng/mL BMP6 for different durations (24, 48, and 72 h). The upper panel shows a representative Western blot image, and the lower panel shows the summarized quantitative results. C , SERPINE2 protein levels in human primary EVTs. The left panel shows a representative Western blot image, and the right panel shows the summarized quantitative results. D-E , BMP6 promotes PlGF accumulation in the conditioned medium of trophoblasts. D , HTR8/SVneo cells were treated with or without 50 ng/mL BMP6 for 24–48 h. PlGF accumulation in conditioned medium was measured using ELISA. E , PlGF accumulation in conditioned medium was assayed by ELISA 48 h after BMP6 treatment in primary EVTs. F-J , ID1 mediates BMP6-induced SERPINE2 and PlGF upregulation in human trophoblasts. HTR8/SVneo cells were transfected for 48 h with 20 nM control non-targeting siRNA (si-Ctrl) or siRNA targeting ID1 (si- ID1 ) before treatment with or without 50 ng/mL BMP6. F , ID1 mRNA levels were examined by qPCR 6 h after BMP6 (50 ng/mL) treatment in HTR8/SVneo cells, with GAPDH used as the reference gene. G and H , SERPINE2 and ID1 protein levels in HTR8/SVneo cells ( G ) and human primary EVTs ( H ) after transfection with siRNA targeting ID1 , followed by treatment with or without BMP6 for 24 h, as assessed by Western blot. The left panel shows a representative Western blot image, and the right panel shows the summarized quantitative results. I , PGF mRNA levels were examined by qPCR 6 h after BMP6 treatment in HTR8/SVneo cells, with GAPDH used as the reference gene. J , PlGF accumulation in conditioned medium was assayed by ELISA 24 h after BMP6 treatment in HTR8/SVneo cells. K , SMAD4 mediates BMP6-induced upregulation of PlGF in human trophoblasts. HTR8/SVneo cells were transfected for 48 h with 20 nM control non-targeting siRNA (si-Ctrl) or siRNA targeting SMAD4 (si- SMAD4 ) before treatment with or without 50 ng/mL BMP6. PlGF accumulation in conditioned medium was assayed by ELISA 24 h after BMP6 treatment in HTR8/SVneo cells. The quantitative results are expressed as the means ± SEMs of at least three independent experiments. One-way ANOVA was used for analyses in A , and two-way ANOVA was used for comparisons in B-K . Groups without common letters are significantly different from each other ( P < 0.05). BMP6, bone morphogenetic protein 6; SERPINE2, serpin family E member 2; EVT, extravillous cytotrophoblast; Ctrl, control; PlGF, placental growth factor; ID1, inhibitor of DNA-binding 1

Article Snippet: Rat plasma was measured with a sFlt-1 ELISA kit (Proteintech, KE10069), a BMP6 ELISA Kit (Novus Biologicals, NBP2-69999, Centennial, CO, USA), and a PlGF ELISA kit (CUSABIO, CSB-E07400r, Wuhan, China).

Techniques: Western Blot, Enzyme-linked Immunosorbent Assay, Transfection, Control, Binding Assay

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: BMP6 as a therapeutic target for preeclampsia: enhancing trophoblast invasion and vascular mimicry

doi: 10.1007/s00018-025-06040-w

Figure Lengend Snippet: Both SERPINE2 and PlGF mediate BMP6-induced trophoblast invasion. A-J , HTR8/SVneo cells or primary EVTs were transfected for 48 h with 20 nM control nontargeting siRNA (si-Ctrl), 20 nM siRNA targeting SERPINE2 (si- SERPINE2 ) or PGF (si- PGF ) before treatment with or without 50 ng/mL BMP6 for 24 h. A and B , The protein levels of SERPINE2 in HTR8/SVneo cells ( A ) and human primary EVTs ( B ) after 24 h of BMP6 treatment. The upper panel shows a representative Western blot image, and the lower panel shows the summarized quantitative results. C , PlGF accumulation in conditioned medium with or without BMP6 treatment for 24 h was assayed by ELISA in HTR8/SVneo cells. D , PGF mRNA levels in human primary EVTs with or without BMP6 treatment for 6 h were examined by RT‒qPCR, with GAPDH as the reference gene. E - H , Transwell assays were employed to examine the invasiveness of HTR8/SVneo cells ( E and G ) and primary EVTs ( F and H ) with or without BMP6 treatment for 36 h. I and J , Endothelial-like tube formation assays were used to assess vascular mimicry of HTR8/SVneo cells with or without BMP6 treatment for 12 h. Representative images from the endothelial-like tube formation assay are displayed in the above panel; the summarized quantitative results are displayed in the lower panel. Scale bar, 100 μm. The quantitative results are expressed as the means ± SEMs of at least three independent experiments. Two-way ANOVA was used for data comparison. Groups without common letters are significantly different from each other ( P < 0.05). BMP6, bone morphogenetic protein 6; SERPINE2, serpin family E member 2; PGF, placental growth factor; Ctrl, control; EVT, extravillous cytotrophoblast

Article Snippet: Rat plasma was measured with a sFlt-1 ELISA kit (Proteintech, KE10069), a BMP6 ELISA Kit (Novus Biologicals, NBP2-69999, Centennial, CO, USA), and a PlGF ELISA kit (CUSABIO, CSB-E07400r, Wuhan, China).

Techniques: Transfection, Control, Western Blot, Enzyme-linked Immunosorbent Assay, Tube Formation Assay, Comparison

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: BMP6 as a therapeutic target for preeclampsia: enhancing trophoblast invasion and vascular mimicry

doi: 10.1007/s00018-025-06040-w

Figure Lengend Snippet: BMP6 is elevated in patients with PE and in PE model rats. A-C , BMP6 is elevated in patients with PE. A , RT‒qPCR analysis of BMP6 mRNA expression levels in the placentas of control women ( n = 10) and PE patients ( n = 10), with GAPDH as the reference gene. B , Western blot analysis of BMP6 protein expression levels in the placentas of control women ( n = 4) and PE patients ( n = 4). C , Spearman correlation analysis between the placental BMP6 mRNA levels and the value of log10 (SBP) of the corresponding patients. The gray area represents the 95% CI. Each dot represents one sample. D , The animal experimental protocol. E and F , BMP6 is elevated in the plasma of PE model rats. Rat plasma levels of BMP6 ( E ) and PlGF ( F ) in the Ad Fc + PBS group ( n = 4) and Ad Flt1 + PBS group ( n = 4). G-M , BMP6 is elevated in the placenta of PE model rats at G13. RNA-seq analysis of rat placentas at G13 in the Ad Fc + PBS group ( n = 3) and Ad Flt1 + PBS group ( n = 3). G , Heatmap depicting DEGs in the two groups. H , Dot plots of significantly enriched GO terms; the dot size represents the number of DEGs associated with a particular GO term. I , KEGG hierarchical network plot of pathways. J , GSEA-KEGG Ridge plot of pathways. K , GSEA plots of cytokine-cytokine receptor interaction pathway. L , Volcano plot of RNA-seq data showing DEGs between the Ad Fc + PBS group and the Ad Flt1 + PBS group. M , Circos graph displaying the coexpression networks of five genes in rat placenta samples. Each sector of the circle represents one gene, and its width indicates the total amount of co-occurrence that connects one gene to the other. The width of each link represents the total number of coexpressed genes among the linked genes. Student’s t-test was used for comparisons between two groups in A , B , E , and F . Groups without common letters are significantly different from each other ( P < 0.05). SD, Sprague–Dawley; BMP6, bone morphogenetic protein 6; PlGF, placental growth factor; PBS, phosphate-buffered saline; Ad Flt1, adenovirus expressing fms-like tyrosine kinase-1; Ad Fc, adenovirus-expressing control IgG2a Fc fragment; FC, fold change; Serpine2, serpin family E member 2; Id1, inhibitor of DNA-binding 1

Article Snippet: Rat plasma was measured with a sFlt-1 ELISA kit (Proteintech, KE10069), a BMP6 ELISA Kit (Novus Biologicals, NBP2-69999, Centennial, CO, USA), and a PlGF ELISA kit (CUSABIO, CSB-E07400r, Wuhan, China).

Techniques: Expressing, Control, Western Blot, Clinical Proteomics, RNA Sequencing, Saline, Binding Assay

Journal: bioRxiv

Article Title: Metformin induces lipogenic differentiation in myofibroblasts to reverse mouse and human lung fibrosis

doi: 10.1101/401265

Figure Lengend Snippet: (A) Schematic representation of the experimental setup. (B-D) qPCR analysis for the lipogenic marker genes PLIN2 and PPARg , as well as COL1A1 in human lung fibroblasts treated with metformin or vehicle. (E, F) Staining of lipid droplets in fibroblasts using LipidTOX (red). Nuclei were counterstained with DAPI (blue). (G-H) Gating strategy for detecting LipidTOX + cells by flow cytometry. (I) Quantification of LipidTOX + cells in response to metformin treatment. (J) Heatmap representation of the top 100 differentially expressed genes in fibroblasts following metformin treatment. Scale bars: (E, F) 25 µm. (B-D) Each data point within a given group corresponds to one patient. Vehicle-treated group: n=12, Metformin-treated group: n=11. (I) n=3 per group. ** P<0.01, *** P<0.001, **** P<0.0001.

Article Snippet: Anti-collagen 1 A1 (anti-COL1A1) antibodies (Rockland, 1:200) and goat anti-rabbit antibodies (Life Technologies, 1:500) were used for immunofluorescence.

Techniques: Marker, Staining, Flow Cytometry

Journal: bioRxiv

Article Title: Metformin induces lipogenic differentiation in myofibroblasts to reverse mouse and human lung fibrosis

doi: 10.1101/401265

Figure Lengend Snippet: (A) Schematic representation of the experimental setup. (B-D) qPCR analysis for PLIN2, PPARg and COL1A1 in human lung fibroblasts treated with TGFβ1 or vehicle for 72h. (E-H) Staining of TGFβ1- and vehicle-treated cells with LipidTOX (red), anti-ACTA2 antibodies (green) and DAPI (blue). (I-K) qPCR analysis for PLIN2, PPARg and COL1A1 in human lung fibroblasts treated with TGFβ1 or vehicle for 72 h, followed by treatment with metformin or vehicle for 72 h. (L-M) Staining of TGFβ1-and vehicle-treated cells with LipidTOX (red) and DAPI (blue) at the end of treatment (t=144 h). Scale bars: (E-H) and (L-M) 25 µm. (B-D, I-K) Each data point within a given group corresponds to one patient. (B-D) n=4 per group. (I-K) n=9-10 per group. * P<0.05, **P<0.01, ****P<0.0001.

Article Snippet: Anti-collagen 1 A1 (anti-COL1A1) antibodies (Rockland, 1:200) and goat anti-rabbit antibodies (Life Technologies, 1:500) were used for immunofluorescence.

Techniques: Staining

Journal: bioRxiv

Article Title: Metformin induces lipogenic differentiation in myofibroblasts to reverse mouse and human lung fibrosis

doi: 10.1101/401265

Figure Lengend Snippet: (A) Schematic representation of the experimental setup. (B-E) Bright-field imaging of PCLS treated with metformin or vehicle for five days. (F, G) Hematoxylin and eosin staining and COL1A1 immunostaining of PCLS prepared from a non-IPF donor lung. (H-M) Hematoxylin and eosin staining, Masson’s trichrome staining and COL1A1 immunostaining of PCLS prepared from an IPF lung and treated with metformin or vehicle for five days. (N, O) 3D-reconstruction of z-stacks of metformin- and vehicle-treated PCLS stained for COL1A1 (green) and lipid droplets (red). (P) Gating strategy for flow cytomety-based quantification of LipidTOX + cells that are negative for hematopoeitic (CD45), endothelial (CD31) and epithelial (EpCAM) cell markers. (Q) Quantification of flow cytometry measurements on metformin- and vehicle-treated cells. (R) Total collagen assay for metformin- and vehicle-treated cells. Scale bars: (B-E) 2 mm, (F) 500 µm, (G, L, M) 50 µm, (H-K) 200 µm. (Q, R) Each data point within a given group corresponds to one patient. (Q) n=4 per group. ® n=3 per group. * P<0.05, **P<0.01.

Article Snippet: Anti-collagen 1 A1 (anti-COL1A1) antibodies (Rockland, 1:200) and goat anti-rabbit antibodies (Life Technologies, 1:500) were used for immunofluorescence.

Techniques: Imaging, Staining, Immunostaining, Flow Cytometry, Collagen Assay

Journal: bioRxiv

Article Title: Metformin induces lipogenic differentiation in myofibroblasts to reverse mouse and human lung fibrosis

doi: 10.1101/401265

Figure Lengend Snippet: (A) Schematic representation of the Acta2-Cre-ERT2 and tdTomato flox construct. (B) Schematic representation of the timeline of the experiment. Bleomycin was administered intratracheally at day 0. Between days 5 and 14, mice were fed tamoxifen-containing pellets and starting at day 14, metformin (1.5 mg/mL) or vehicle was administered through drinking water. Mice were sacrificed at day 28. (C-F) Hematoxylin and eosin and Masson’s trichrome staining of metformin- and vehicle-treated lungs. (G) Quantification of fibrosis in metformin- and vehicle-treated lungs. (H, I) Immunofluorescence for COL1A1 (green). Endogenous tdTomato signal (red) and DAPI (blue) are also shown. (J) LipidTOX staining (green) and tdTomato + cells (red) are shown. The box in (J) is magnified in (K). Arrowheads indicate LipidTOX + tdTomato + cells. (L-S) Gating strategy (to detect CD45 - CD31 - EpCAM - tdTomato + and/or LipidTOX + cells) and quantification of various cell populations based on tdTomato and LipidTOX detection. Scale bars: (C-F) 1 mm, (H, I) 50 µm, (J) 25 µm. (G, Q-S) Each data point within a given group corresponds to one animal. n=5 per group. * P<0.05, **P<0.01. IF: Immunofluorescence, ns: Not significant.

Article Snippet: Anti-collagen 1 A1 (anti-COL1A1) antibodies (Rockland, 1:200) and goat anti-rabbit antibodies (Life Technologies, 1:500) were used for immunofluorescence.

Techniques: Construct, Staining, Immunofluorescence

Journal: bioRxiv

Article Title: Metformin induces lipogenic differentiation in myofibroblasts to reverse mouse and human lung fibrosis

doi: 10.1101/401265

Figure Lengend Snippet: (A) Schematic representation of the gain-of-function experimental setup for AMPK signaling. (B-E) qPCR analysis of PLIN2, PPARg , COL1A1 and BMP2 in IPF fibroblasts treated with AMPK agonist GSK621 or vehicle. (F) Schematic representation of the loss-of-function experimental setup for AMPK signaling. (G-I) qPCR analysis of PLIN2, PPARg and COL1A1 in IPF fibroblasts treated with AMPK siRNA or scramble siRNA. The decrease of AMPK protein levels at the time of analysis is shown in (J, K). (L-N) Staining of GSK621- and vehicle-treated cells with LipidTOX (red) and DAPI (blue). Metformin-treated cells were used as a positive control for lipid-droplet accumulation (M). Scale bars: (L-N) 25 µm. (B-E, G-I, K) Each data point corresponds to one patient. (B-E) Vehicle-treated group: n=7-8, GSK621-treated group: n=6-8. (G-I) n=4 per group. (K) n=3 per group. * P<0.05. ns: Not significant.

Article Snippet: Anti-collagen 1 A1 (anti-COL1A1) antibodies (Rockland, 1:200) and goat anti-rabbit antibodies (Life Technologies, 1:500) were used for immunofluorescence.

Techniques: Staining, Positive Control

Journal: bioRxiv

Article Title: Metformin induces lipogenic differentiation in myofibroblasts to reverse mouse and human lung fibrosis

doi: 10.1101/401265

Figure Lengend Snippet: (A) Schematic representation of the experimental setup. (B-D) qPCR analysis of PLIN2, PPARg and COL1A1 in IPF fibroblasts treated with rhBMP2 or vehicle. (E, F) Staining of rhBMP2- and vehicle-treated cells with LipidTOX (red) and DAPI (blue). (G) Western blot showing the induction of PPARγ phosphorylation in response to rhBMP2 treatment. Lanes 1-4 and lanes 5-8 were run in parallel on different gels under the same conditions. Quantification of the immunoblot is shown in the lower panel. (H) Western blot showing the opposing effects of metformin and TGFβ1 on PPARγ phosphorylation, and the ability of metformin to partially restore PPARγ phosphorylation in TGFβ1-treated cells. Lanes 1-12 and lanes 13-18 were run in parallel on different gels under the same conditions. Quantification of the immunoblot is shown in the lower panel. (I) Model for the antifibrotic mechanism of action of metformin in human lung fibrosis. Metformin activates AMPK signaling in myofibroblasts, leading to suppression of collagen production, and induces lipogenic differentiation via an AMPK-independent mechanism involving BMP2 release and PPARγ activation. Arising lipofibroblasts are known to support type 2 alveolar epithelial stem cells in the lung. Scale bars: (E-F) 50 µm. (B-D, G, H) Each data point corresponds to one patient. (B-D) n=10-11 per group. (G) n=4 per group. (H) n=3 per group. * P<0.05, ns: Not significant.

Article Snippet: Anti-collagen 1 A1 (anti-COL1A1) antibodies (Rockland, 1:200) and goat anti-rabbit antibodies (Life Technologies, 1:500) were used for immunofluorescence.

Techniques: Staining, Western Blot, Activation Assay

Journal: Methods and Protocols

Article Title: Capillary Western Immunoassay Optimization of Estrogen Related Factors in Human Subcutaneous Adipose Tissue

doi: 10.3390/mps5020034

Figure Lengend Snippet: All electropherograms report lysate concentrations of 0.5 and 1.0 mg/mL with 1:10 and 1:50 antibody concentrations. These detection antibodies were not close to their target protein’s predicted molecular weight and were, thus, not considered to be specifically binding target proteins of interest: ( A ) HSD17ß1 ( B ) HSD17ß2 ( C ) HSD17ß4 ( D ) HSD17ß7 and ( E ) SULTe1.

Article Snippet: Control lysates tested include those for: HSD17ß1 (human placental whole cell lysate; NB820-59248, Novus Biologicals), HSD17ß2 (human embryonic kidney cells; NBL1-11726, Novus Biologicals), HSD17ß4 (human embryonic kidney cells; NBL1-11728, Novus Biologicals), HSD17ß7 (human embryonic kidney cells; NBP2-07042, Novus Biologicals) and SULTe1 (human embryonic kidney cells; ab94289, Abcam).

Techniques: Molecular Weight, Binding Assay

Journal: Methods and Protocols

Article Title: Capillary Western Immunoassay Optimization of Estrogen Related Factors in Human Subcutaneous Adipose Tissue

doi: 10.3390/mps5020034

Figure Lengend Snippet: All electropherograms report results from over-expresser lysates at concentrations of 0.5 and 0.8 mg/mL ( A – D ) except for SULTe1 which was tested at: 1/50, 1/100, 1/200, 1/400, 1/800. Empty vector negative controls are also displayed on each electropherogram. Lysates were paired with antibody concentrations of 1:10 and 1:50. Data are listed as follows: ( A ) HSD17ß1, ( B ) HSD17ß2, ( C ) HSD17ß4, ( D ) HSD17ß7, and ( E ) SULTe1.

Article Snippet: Control lysates tested include those for: HSD17ß1 (human placental whole cell lysate; NB820-59248, Novus Biologicals), HSD17ß2 (human embryonic kidney cells; NBL1-11726, Novus Biologicals), HSD17ß4 (human embryonic kidney cells; NBL1-11728, Novus Biologicals), HSD17ß7 (human embryonic kidney cells; NBP2-07042, Novus Biologicals) and SULTe1 (human embryonic kidney cells; ab94289, Abcam).

Techniques: Plasmid Preparation

Journal: Methods and Protocols

Article Title: Capillary Western Immunoassay Optimization of Estrogen Related Factors in Human Subcutaneous Adipose Tissue

doi: 10.3390/mps5020034

Figure Lengend Snippet: All electropherograms report data for high fat (75 SAT lysate:25 over-expresser lysate) and low fat (25 SAT lysate:75 over-expresser lysate) mixtures with 1:10 antibody concentrations in duplicate: ( A ) HSD17ß1 ( B ) HSD17ß2 ( C ) HSD17ß4 and ( D ) SULTe1.

Article Snippet: Control lysates tested include those for: HSD17ß1 (human placental whole cell lysate; NB820-59248, Novus Biologicals), HSD17ß2 (human embryonic kidney cells; NBL1-11726, Novus Biologicals), HSD17ß4 (human embryonic kidney cells; NBL1-11728, Novus Biologicals), HSD17ß7 (human embryonic kidney cells; NBP2-07042, Novus Biologicals) and SULTe1 (human embryonic kidney cells; ab94289, Abcam).

Techniques:

Journal: Methods and Protocols

Article Title: Capillary Western Immunoassay Optimization of Estrogen Related Factors in Human Subcutaneous Adipose Tissue

doi: 10.3390/mps5020034

Figure Lengend Snippet: Summary of expected and observed molecular weights for all 11 antibodies tested in each experiment.

Article Snippet: Control lysates tested include those for: HSD17ß1 (human placental whole cell lysate; NB820-59248, Novus Biologicals), HSD17ß2 (human embryonic kidney cells; NBL1-11726, Novus Biologicals), HSD17ß4 (human embryonic kidney cells; NBL1-11728, Novus Biologicals), HSD17ß7 (human embryonic kidney cells; NBP2-07042, Novus Biologicals) and SULTe1 (human embryonic kidney cells; ab94289, Abcam).

Techniques: Extraction, Positive Control

Journal: Cell death discovery

Article Title: Plac8-ERK pathway modulation of monocyte function in sepsis.

doi: 10.1038/s41420-024-02012-4

Figure Lengend Snippet: Fig. 1 Expression of plac8 and ERK activation in peripheral blood mononuclear cells of septic patients and normal individuals. A Flow cytometry analysis of peripheral blood CD14+ and CD16+ cell subsets. B Flow cytometry analysis of CD14+ and CD16+ cell subsets. C qRT- PCR analysis of gene expression. D ELISA analysis of cytokine expression. E Western blot analysis of protein expression. F Western blot analysis of protein expression. G Flow cytometry analysis of cell cycle. H Cell proliferation index PI = (S + G2M)/(G0/1 + S + G2M). * indicates P < 0.05 compared to the normal group. Data is presented as mean ± standard deviation. Data analysis was performed using an independent samples t-test, and the experiment was repeated three times.

Article Snippet: After blocking with 0.5% BSA, the membranes were incubated overnight with primary antibodies: rabbit anti-CD14 (ab182032, 1:1000, Abcam, UK), CD16 (Cat No. 16559-1-AP, 1:1000, Proteintech), plac8 (Cat No. 12284-1-AP, 1:200, Proteintech), ERK1/2 (ab17942, 1:1000, Abcam, UK), p-ERK1/2 (ab223500, 1:1000, Abcam, UK), and GAPDH (ab8245, 1:1000, Abcam, UK).

Techniques: Expressing, Activation Assay, Flow Cytometry, Quantitative RT-PCR, Gene Expression, Enzyme-linked Immunosorbent Assay, Western Blot, Standard Deviation

Journal: Cell death discovery

Article Title: Plac8-ERK pathway modulation of monocyte function in sepsis.

doi: 10.1038/s41420-024-02012-4

Figure Lengend Snippet: Fig. 2 Effects of plac8 on peripheral blood mononuclear cell proliferation in septic patients. A qRT-PCR analysis of gene expression. B Western blot analysis of protein band. C Western blot analysis of protein expression. D ELISA analysis of cytokine expression. E Cell proliferation measured by CCK-8 assay, cell proliferation (%) = [OD (treatment group) −OD (blank group)] / [OD (control group) −OD (blank group)] × 100%. * indicates P < 0.05 compared to si-Plac8-NC or Plac8-NC group. Data is presented as mean ± standard deviation. An independent samples t-test was used to compare two groups, and two-way ANOVA was used to compare at different time points. The experiment was repeated three times.

Article Snippet: After blocking with 0.5% BSA, the membranes were incubated overnight with primary antibodies: rabbit anti-CD14 (ab182032, 1:1000, Abcam, UK), CD16 (Cat No. 16559-1-AP, 1:1000, Proteintech), plac8 (Cat No. 12284-1-AP, 1:200, Proteintech), ERK1/2 (ab17942, 1:1000, Abcam, UK), p-ERK1/2 (ab223500, 1:1000, Abcam, UK), and GAPDH (ab8245, 1:1000, Abcam, UK).

Techniques: Quantitative RT-PCR, Gene Expression, Western Blot, Expressing, Enzyme-linked Immunosorbent Assay, CCK-8 Assay, Control, Standard Deviation

Journal: Cell death discovery

Article Title: Plac8-ERK pathway modulation of monocyte function in sepsis.

doi: 10.1038/s41420-024-02012-4

Figure Lengend Snippet: Fig. 5 Effects of plac8 on monocyte cell survival and proliferation in the mouse model. A Flow cytometry analysis of CD14+ and CD16+ cell subsets. B Flow cytometry analysis of peripheral blood CD14+ and CD16+ cell subsets. C Western blot analysis of protein band. D Western blot analysis of protein expression. E ELISA analysis of cytokine expression. F Detection of cell proliferation. G Flow cytometry analysis of cell cycle. H Cell proliferation index PI. * indicates P < 0.05 compared to si-Plac8-NC or Plac8-NC group. Data is presented as mean ± standard deviation. Independent samples t-test was used to compare two groups, and two-way ANOVA was used for comparison at different time points. The experiment was repeated three times.

Article Snippet: After blocking with 0.5% BSA, the membranes were incubated overnight with primary antibodies: rabbit anti-CD14 (ab182032, 1:1000, Abcam, UK), CD16 (Cat No. 16559-1-AP, 1:1000, Proteintech), plac8 (Cat No. 12284-1-AP, 1:200, Proteintech), ERK1/2 (ab17942, 1:1000, Abcam, UK), p-ERK1/2 (ab223500, 1:1000, Abcam, UK), and GAPDH (ab8245, 1:1000, Abcam, UK).

Techniques: Flow Cytometry, Western Blot, Expressing, Enzyme-linked Immunosorbent Assay, Standard Deviation, Comparison

Journal: Cell death discovery

Article Title: Plac8-ERK pathway modulation of monocyte function in sepsis.

doi: 10.1038/s41420-024-02012-4

Figure Lengend Snippet: Fig. 6 The role and mechanism of Plac8-mediated ERK signaling pathway activation in the proliferation and activation of peripheral blood monocytes in septic patients.

Article Snippet: After blocking with 0.5% BSA, the membranes were incubated overnight with primary antibodies: rabbit anti-CD14 (ab182032, 1:1000, Abcam, UK), CD16 (Cat No. 16559-1-AP, 1:1000, Proteintech), plac8 (Cat No. 12284-1-AP, 1:200, Proteintech), ERK1/2 (ab17942, 1:1000, Abcam, UK), p-ERK1/2 (ab223500, 1:1000, Abcam, UK), and GAPDH (ab8245, 1:1000, Abcam, UK).

Techniques: Activation Assay