Journal: American journal of physiology. Lung cellular and molecular physiology

Article Title: Pim1 kinase activity preserves airway epithelial integrity upon house dust mite exposure.

doi: 10.1152/ajplung.00043.2015

Figure Lengend Snippet: Fig. 2. Inhibition of Pim1 kinase impairs barrier function in the presence of HDM in primary bronchial epithelial cells. Primary bronchial epithelial cells (PBECs) derived from bronchial brushes of healthy control subjects were cultured and the low frequency electrical resistance across a monolayer of PBECS was measured by ECIS for up to 12 h. PBECs were stimulated with 100 g/ml HDM (A), treated with 5 M Pim1 kinase inhibitor (PKI) (B), and stimulated with HDM upon pretreatment for 1 h with PKI (C). Normalized resistance relative to the resistance of the monolayer cultures at the last measurement before stimulation is shown. Experiments were repeated 4 times with PBECs from independent donors and means SE are shown. *P 0.05.

Article Snippet: Immunoblotting was performed for Pim1 kinase (1:1,500; OriGene Technologies, Rockville, MD) and -actin (1:20,000; SigmaAldrich) conform standard procedures.

Techniques: Inhibition, Derivative Assay, Control, Cell Culture

Journal: American journal of physiology. Lung cellular and molecular physiology

Article Title: Pim1 kinase activity preserves airway epithelial integrity upon house dust mite exposure.

doi: 10.1152/ajplung.00043.2015

Figure Lengend Snippet: Fig. 6. Pim1 kinase activity does not affects HDM-induced local airway inflammation. Female FVB/Nrcl mice deficient (KO) or proficient (WT) for Pim1 kinase were treated intranasally with 20 l HDM extract (2.5 mg total weight/ml) or PBS twice a week for 5 wk and 24 h after the last HDM exposure lungs were collected for histological analysis. Hemotoxylin and eosin (HE) staining was performed on lung sections of PBS-treated WT mice (A), HDM-treated WT mice (B), PBS-treated Pim1 KO mice (C), and HDM-treated Pim1 KO mice (D). Periodic acid-Schiff (PAS) staining was performed on lung sections of PBS-treated WT mice (E), HDM-treated WT mice (F), PBS-treated Pim1 KO mice (G), and HDM-treated Pim1 KO mice (H). Representative staining of 3–5 mice in each experimental groups are shown.

Article Snippet: Immunoblotting was performed for Pim1 kinase (1:1,500; OriGene Technologies, Rockville, MD) and -actin (1:20,000; SigmaAldrich) conform standard procedures.

Techniques: Activity Assay, Staining

Journal: Cellular and molecular gastroenterology and hepatology

Article Title: PIM1-Induced Cytoplasmic Expression of RBMY Mediates Hepatocellular Carcinoma Metastasis.

doi: 10.1016/j.jcmgh.2022.09.014

Figure Lengend Snippet: Figure 5. Regulation of RBMY oncogenicity by its upstream kinases. (A) AlamarBlue cell viability assay evaluated the survival and proliferation ability of the indicated transfectants. Cell viability represented by the measured absorbance at 570 nm was plotted as the means ± SDs after normalization to the corresponding values at 0 hours. *P < .005 vs the GFPþHA control was determined by the Student t test. (B) 3D spheroid invasion assay of indicated transfectants. The invasiveness of spheroids grown in a 3D matrix was determined by the formation and enrichment of spindle-like protrusions (black arrows). Representative micrographs acquired before and after the formation of protrusions from spheroid bodies are shown. The mean percentage of the invaded spheroids was calculated and is shown in a bar graph. The error bars indicate the SDs of 3 wells in 2 independent experiments, and significance was evaluated by the Student t test. (C) A wound healing assay showed the migration efficacy of the indicated transfectants. A representative image of each group before (0 h) and after (48 h) cell migration is displayed. Cells expressing GFP and HA served as controls. The migration distances were measured and are reported as the means ± SDs (Student t test). The data shown are representative of 3 independent experiments. (D) Transwell invasion assay to determine the invasive potential of the indicated transfectants. The numbers of invaded cells are presented as the means ± SDs of 4 independent experiments, and significance was evaluated by the Student t test. (E) Scratch wound healing assay showed the migratory abilities of SNU-387 cells cotransfected with plasmids indicated. The migration distances were depicted as the means ± SDs (Student t test). (F) Transwell invasion assay on the indicated transfectants. The number of invaded cells was plotted as means ± SDs and evaluated by the Student t test for statistical significance. (G) Scratch wound healing and Transwell invasion assays on cells transfected twice with siCtrl/siRBMY, then once with HA/HA-PIM1 expression plasmids. Migration distance (means ± SDs) and number of invaded cells (means ± SDs) were analyzed 48 hours postseeding, and differences between groups were estimated by the Student t test.

Article Snippet: Cultured cells plated on 8-well chamber slides (Millipore, Billerica, MA) were fixed with 4% paraformaldehyde Table 1.Detailed Information of Antibodies Used in IHC and IFA Analysis Antibody (clone/catalog number) Manufacturer Host species Dilution and incubation conditions RBMY (SRGY clone 1) Homemade Mouse 5 , 4 C, Overnight (O/N) RBMY1A1 (GTX110098) GeneTex Rabbit 150 , 4 C, O/N Her Par-1 (OCH1E5, ab75677) Abcam Mouse 1 , 4 C, O/N PIM1 (TA354029) OriGene Rabbit 150 , 4 C, O/N COX4 (GTX101499) GeneTex Rabbit 100 , 4 C, O/N c-MET (8198) Cell Signalling Technology Rabbit 100 , 4 C, O/N Fibronectin (GTX112794) GeneTex Rabbit 100 , 4 C, O/N E-cadherin (ab1416) Abcam Mouse 50 , 4 C, O/N Snail1 (sc-271977) Santa Cruz Biotechnology Mouse 50 , 37 C, 2 h GRP94 (GTX103232) GeneTex Rabbit 100 , 4 C, O/N Naþ/Kþ-ATPase a (sc-48345) Santa Cruz Biotechnology Mouse 500 , 4 C, O/N GOLPH2 (GTX107702) GeneTex Rabbit 100 , 4 C, O/N CK19 (TA313117) OriGene Rabbit 100 , 4 C, O/N HNF4a (C11F12) Cell Signalling Technology Rabbit 100 , 4 C, O/N HA.11 epitope tag (901501) BioLegend Mouse 100 , 4 C, O/N for 12 minutes, except those used for Snail1 staining, which were fixed with ice-cold methanol at -20oC for 5 minutes.

Techniques: Viability Assay, Control, Invasion Assay, Wound Healing Assay, Migration, Expressing, Transwell Invasion Assay, Transfection

Journal: Cellular and molecular gastroenterology and hepatology

Article Title: PIM1-Induced Cytoplasmic Expression of RBMY Mediates Hepatocellular Carcinoma Metastasis.

doi: 10.1016/j.jcmgh.2022.09.014

Figure Lengend Snippet: Figure 6. Increasing cytoplasmic accumulation of RBMY by PIM1. (A) Subcellular fractionation coupled with Western blot assay on Huh-7 cells co-expressed with proteins indicated. Detection of Lamin B1 and glyceraldehyde-3-phosphate dehy- drogenase (GAPDH) was performed as for controls of the purities of nuclear (Nuc) and cytoplasmic (Cyt) fractions, respectively. Bar plot represents the densitometry analysis of GFP-RBMY-p. The densities of GFP-RBMY-p in nuclear and cytoplasmic fractions were normalized to the intensities of their corresponding markers, Lamin B1 and GAPDH, respectively, and then divided by the total nuclear þ cytoplasmic densities of GFP-RBMY-p to obtain a percentage value. (B) Fluorescence imaging distinguished the impact of the indicated kinases on the distribution of GFP-RBMY in Huh-7 cells. The arrowheads and arrows indicate the RBMY-Nþ and RBMY-Cþ/Mix patterns, respectively. (C) Comparison of the subcellular distribution of GFP-RBMY upon co-expression with HA-PIM1, HA-MEK2, or HA-CK1ε kinases in HCC cell lines. The microscopic signals of GFP-RBMY- Mix/Cþ and Nþ were plotted as a mean percentages of the cell population (±SDs). (D) Fluorescence microscopy analysis showed the Nþ (arrowhead) and Cþ/Mix (arrow) patterns of GFP-RBMY in Huh-7 cells upon treatment of kinases inhibitors. (E) Western blot analysis exposed the expression level of GFP-RBMY in nuclear and cytoplasmic fractions of HCC cells treated with dimethyl sulfoxide (DMSO) solvent control or kinase inhibitors. Cells were incubated with the indicated inhibitors (30 mmol/ L per well) after attachment. Transfection of GFP-RBMY was performed 24 hours post-treatment. Cells then were refreshed with medium containing the indicated inhibitors and cultured for another 24 hours before subcellular fractionation. Quantitative densitometry analysis was accomplished as mentioned previously. DAPI, 40,6-diamidino-2-phenylindole; RBMY-p, phosphory- lated RBMY.

Article Snippet: Cultured cells plated on 8-well chamber slides (Millipore, Billerica, MA) were fixed with 4% paraformaldehyde Table 1.Detailed Information of Antibodies Used in IHC and IFA Analysis Antibody (clone/catalog number) Manufacturer Host species Dilution and incubation conditions RBMY (SRGY clone 1) Homemade Mouse 5 , 4 C, Overnight (O/N) RBMY1A1 (GTX110098) GeneTex Rabbit 150 , 4 C, O/N Her Par-1 (OCH1E5, ab75677) Abcam Mouse 1 , 4 C, O/N PIM1 (TA354029) OriGene Rabbit 150 , 4 C, O/N COX4 (GTX101499) GeneTex Rabbit 100 , 4 C, O/N c-MET (8198) Cell Signalling Technology Rabbit 100 , 4 C, O/N Fibronectin (GTX112794) GeneTex Rabbit 100 , 4 C, O/N E-cadherin (ab1416) Abcam Mouse 50 , 4 C, O/N Snail1 (sc-271977) Santa Cruz Biotechnology Mouse 50 , 37 C, 2 h GRP94 (GTX103232) GeneTex Rabbit 100 , 4 C, O/N Naþ/Kþ-ATPase a (sc-48345) Santa Cruz Biotechnology Mouse 500 , 4 C, O/N GOLPH2 (GTX107702) GeneTex Rabbit 100 , 4 C, O/N CK19 (TA313117) OriGene Rabbit 100 , 4 C, O/N HNF4a (C11F12) Cell Signalling Technology Rabbit 100 , 4 C, O/N HA.11 epitope tag (901501) BioLegend Mouse 100 , 4 C, O/N for 12 minutes, except those used for Snail1 staining, which were fixed with ice-cold methanol at -20oC for 5 minutes.

Techniques: Fractionation, Western Blot, Fluorescence, Imaging, Comparison, Expressing, Microscopy, Solvent, Control, Incubation, Transfection, Cell Culture

Journal: Cellular and molecular gastroenterology and hepatology

Article Title: PIM1-Induced Cytoplasmic Expression of RBMY Mediates Hepatocellular Carcinoma Metastasis.

doi: 10.1016/j.jcmgh.2022.09.014

Figure Lengend Snippet: Figure 7. Tumorigenic expression of PIM1 and RBMY in primary HCC tissues. (A) PIM1-specific IHC on paraffin- embedded tissue sections of primary HCC. PIM1- Cþ–expressing tissue showed PIM1-positive signal only in the cyto- plasm. PIM1-Mix pattern comprised cells express- ing both nuclear and cytoplasmic PIM1 (arrow- heads) and also PIM1-Cþ (arrows). (B) PIM1 and RBMY double-stained IFA was accomplished on pri- mary HCC tissues using anti-PIM1 and SRGY clone 1 antibodies. Yellow ar- rows, PIM1-Mix/RBMY- Nþ; white arrows, PIM1- Mix/RBMY-Mix; and white arrowheads, PIM1-Cþ/ RBMY-Cþ. (C) PIM1- and RBMY-specific IHC on serial paraffin-embedded tissue sections of primary HCC. BV, blood vessel; DAPI, 40,6-diamidino-2- phenylindole.

Article Snippet: Cultured cells plated on 8-well chamber slides (Millipore, Billerica, MA) were fixed with 4% paraformaldehyde Table 1.Detailed Information of Antibodies Used in IHC and IFA Analysis Antibody (clone/catalog number) Manufacturer Host species Dilution and incubation conditions RBMY (SRGY clone 1) Homemade Mouse 5 , 4 C, Overnight (O/N) RBMY1A1 (GTX110098) GeneTex Rabbit 150 , 4 C, O/N Her Par-1 (OCH1E5, ab75677) Abcam Mouse 1 , 4 C, O/N PIM1 (TA354029) OriGene Rabbit 150 , 4 C, O/N COX4 (GTX101499) GeneTex Rabbit 100 , 4 C, O/N c-MET (8198) Cell Signalling Technology Rabbit 100 , 4 C, O/N Fibronectin (GTX112794) GeneTex Rabbit 100 , 4 C, O/N E-cadherin (ab1416) Abcam Mouse 50 , 4 C, O/N Snail1 (sc-271977) Santa Cruz Biotechnology Mouse 50 , 37 C, 2 h GRP94 (GTX103232) GeneTex Rabbit 100 , 4 C, O/N Naþ/Kþ-ATPase a (sc-48345) Santa Cruz Biotechnology Mouse 500 , 4 C, O/N GOLPH2 (GTX107702) GeneTex Rabbit 100 , 4 C, O/N CK19 (TA313117) OriGene Rabbit 100 , 4 C, O/N HNF4a (C11F12) Cell Signalling Technology Rabbit 100 , 4 C, O/N HA.11 epitope tag (901501) BioLegend Mouse 100 , 4 C, O/N for 12 minutes, except those used for Snail1 staining, which were fixed with ice-cold methanol at -20oC for 5 minutes.

Techniques: Expressing, Staining

Journal: Cellular and molecular gastroenterology and hepatology

Article Title: PIM1-Induced Cytoplasmic Expression of RBMY Mediates Hepatocellular Carcinoma Metastasis.

doi: 10.1016/j.jcmgh.2022.09.014

Figure Lengend Snippet: Figure 8. PIM1-associated cytoplasmic expression of RBMY is a risk factor for poor prognosis in HCC. (A) Subcellular fractionation and Western blot were performed to analyze the levels of PIM1 and RBMY in the nuclear and cytoplasmic fractions of primary HCC tissues (N ¼ 42). Lamin A/C and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were used as the controls for the nuclear and cytoplasmic fractions, respectively, to confirm the purity and loading amount. (B and C) A significant positive correlation between PIM1 and RBMY in patients with HCC. (A) Densitometry analysis was conducted to measure the nuclear and cytoplasmic intensities of PIM1 p34 and RBMY and standardized with the intensities of Lamin A/C and GAPDH, respectively. (B) Linear correlations were established between the nuclear and cytoplasmic fractions of PIM1 p34 and RBMY. (C) The levels of nuclear and cytoplasmic RBMY were compared among cases expressing PIM1- (N ¼ 2), PIM1- Mix (N ¼ 21), and PIM1-Cþ (N ¼ 19). Data were analyzed by the Mann–Whitney U test for statistical significance. Kaplan–Meier analysis with the log-rank test was performed to estimate the (D) 5-year metastasis-free survival rate and the (E) 5-year survival rate after metastasis according to the expression patterns of PIM1 and RBMY in (D) primary tumors and (E) metastases of HCC tissues. (F) Fluorescence microscopy analysis of PLC/PRF/5 cells cotransfected with AsRed2-PIM1 and GFP-RBMY plasmids. 40,6-diamidino-2-phenylindole (DAPI) indicates nuclear staining. White arrows point to cells expressing PIM1-Mix/RBMY-Cþ. Yellow arrowheads specify cells showing PIM1-Mix/RBMY-Mix. White arrowheads show cells expressing PIM1-Cþ/RBMY- Nþ. Yellow arrows indicate cell expressing RBMY-Nþ, but the absence of PIM1. (G) Double IFA staining of primary tumors and paired metastases of HCC. White arrows, PIM1-Mix/RBMY-Cþ or PIM-Cþ/RBMY-Mix; white arrowheads, PIM1-Cþ/RBMY- Cþ. The yellow arrows show the dissociation of the PIM1-RBMY complex. A, alive; BV, blood vessel; C, cytoplasm; D, death; M, metastasis; N, nucleus.

Article Snippet: Cultured cells plated on 8-well chamber slides (Millipore, Billerica, MA) were fixed with 4% paraformaldehyde Table 1.Detailed Information of Antibodies Used in IHC and IFA Analysis Antibody (clone/catalog number) Manufacturer Host species Dilution and incubation conditions RBMY (SRGY clone 1) Homemade Mouse 5 , 4 C, Overnight (O/N) RBMY1A1 (GTX110098) GeneTex Rabbit 150 , 4 C, O/N Her Par-1 (OCH1E5, ab75677) Abcam Mouse 1 , 4 C, O/N PIM1 (TA354029) OriGene Rabbit 150 , 4 C, O/N COX4 (GTX101499) GeneTex Rabbit 100 , 4 C, O/N c-MET (8198) Cell Signalling Technology Rabbit 100 , 4 C, O/N Fibronectin (GTX112794) GeneTex Rabbit 100 , 4 C, O/N E-cadherin (ab1416) Abcam Mouse 50 , 4 C, O/N Snail1 (sc-271977) Santa Cruz Biotechnology Mouse 50 , 37 C, 2 h GRP94 (GTX103232) GeneTex Rabbit 100 , 4 C, O/N Naþ/Kþ-ATPase a (sc-48345) Santa Cruz Biotechnology Mouse 500 , 4 C, O/N GOLPH2 (GTX107702) GeneTex Rabbit 100 , 4 C, O/N CK19 (TA313117) OriGene Rabbit 100 , 4 C, O/N HNF4a (C11F12) Cell Signalling Technology Rabbit 100 , 4 C, O/N HA.11 epitope tag (901501) BioLegend Mouse 100 , 4 C, O/N for 12 minutes, except those used for Snail1 staining, which were fixed with ice-cold methanol at -20oC for 5 minutes.

Techniques: Expressing, Fractionation, Western Blot, MANN-WHITNEY, Fluorescence, Microscopy, Staining

Journal: Cellular and molecular gastroenterology and hepatology

Article Title: PIM1-Induced Cytoplasmic Expression of RBMY Mediates Hepatocellular Carcinoma Metastasis.

doi: 10.1016/j.jcmgh.2022.09.014

Figure Lengend Snippet: Figure 10. The PIM1–RBMY axis controls the translational activation of EMT. (A) Immunoblotting was performed to determine the levels of EMT proteins upon depletion of PIM1 and RBMY by siPIM1 and siRBMY, as well as restoration with HA-PIM1 and GFP-RBMY. The numbers under the blots of Snail1 and ZEB1 show the band densities of these proteins relative to a-tubulin and are presented as ratios with respect to the value of siCtrl, which is set as 1. (B) A schematic domain structure of RBMY is depicted. The mutation sites of NESm and NLSm are specially marked in pink characters and blue characters, respectively, showing the substitution of amino acids (a.a.) by alanine in comparison with the original sequence of RBMY-WT. Fluorescence imaging showed the distribution patterns of GFP-RBMY WT, NESm, and NLSm in Huh-7 cells. White arrow- heads, white arrows, and yellow arrows indicate Nþ, Cþ, and Mix patterns of RBMY, respectively. Immunoblotting was performed to assess the expression of EMT proteins in cells overexpressing GFP-RBMY and its mutants with/without HA- PIM1 co-expression. (C) IFA showed the distribution of Snail1 and E-cadherin in Huh-7 cells overexpressing GFP-RBMY and GFP-NLSm. The white arrows, yellow arrows, and white arrowhead indicate GFP-RBMY-Mix, GFP-RBMY-Cþ, and GFP-RBMY-Nþ cells, respectively. (D) Immunoblotting showed the expressions of GFP-RBMY-WT/mutants and EMT proteins upon SMI-4a treatment. Dimethyl sulfoxide (DMSO) served as the solvent control. (E) Immunoblotting evaluated the expres- sions of RBMY and EMT proteins in response to the knockout of PIM1. PLC/PRF/5 cells were transfected 3 times with sgPIM1 (clones 6, 4, and 3) and sgCtrl at 24-hour intervals. Part of these sgRNAs-expressed PLC/PRF/5 cells were transfected once with HA-RBMY (lower panel). All cells were harvested for measuring the levels of endogenous RBMY (upper) and HA-RBMY (lower), as well as PIM1 and EMT proteins, 24 hours after the last transfection. (F) Schematic diagram summarizing the findings of this study showing how PIM1 and RBMY are involved in the expression of EMT proteins. DAPI, 40,6-diamidino-2- phenylindole; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

Article Snippet: Cultured cells plated on 8-well chamber slides (Millipore, Billerica, MA) were fixed with 4% paraformaldehyde Table 1.Detailed Information of Antibodies Used in IHC and IFA Analysis Antibody (clone/catalog number) Manufacturer Host species Dilution and incubation conditions RBMY (SRGY clone 1) Homemade Mouse 5 , 4 C, Overnight (O/N) RBMY1A1 (GTX110098) GeneTex Rabbit 150 , 4 C, O/N Her Par-1 (OCH1E5, ab75677) Abcam Mouse 1 , 4 C, O/N PIM1 (TA354029) OriGene Rabbit 150 , 4 C, O/N COX4 (GTX101499) GeneTex Rabbit 100 , 4 C, O/N c-MET (8198) Cell Signalling Technology Rabbit 100 , 4 C, O/N Fibronectin (GTX112794) GeneTex Rabbit 100 , 4 C, O/N E-cadherin (ab1416) Abcam Mouse 50 , 4 C, O/N Snail1 (sc-271977) Santa Cruz Biotechnology Mouse 50 , 37 C, 2 h GRP94 (GTX103232) GeneTex Rabbit 100 , 4 C, O/N Naþ/Kþ-ATPase a (sc-48345) Santa Cruz Biotechnology Mouse 500 , 4 C, O/N GOLPH2 (GTX107702) GeneTex Rabbit 100 , 4 C, O/N CK19 (TA313117) OriGene Rabbit 100 , 4 C, O/N HNF4a (C11F12) Cell Signalling Technology Rabbit 100 , 4 C, O/N HA.11 epitope tag (901501) BioLegend Mouse 100 , 4 C, O/N for 12 minutes, except those used for Snail1 staining, which were fixed with ice-cold methanol at -20oC for 5 minutes.

Techniques: Activation Assay, Western Blot, Mutagenesis, Comparison, Sequencing, Fluorescence, Imaging, Expressing, Solvent, Control, Knock-Out, Transfection, Clone Assay

Journal: Cellular and molecular gastroenterology and hepatology

Article Title: PIM1-Induced Cytoplasmic Expression of RBMY Mediates Hepatocellular Carcinoma Metastasis.

doi: 10.1016/j.jcmgh.2022.09.014

Figure Lengend Snippet: Figure 11. Positive feedback stabilization exists between PIM1 and RBMY. A CHX-chase assay followed by Western blot analysis was conducted to estimate the turnover rates of (A) HA-PIM1 upon GFP-RBMY co-expression and (B) RBMY depletion by siRBMY. Cells cotransfected with HA-PIM1 and (A) either GFP control or GFP-RBMY and (B) either siRBMY or siCtrl were harvested at the times indicated after CHX (300 mg/mL) treatment. The percentage of protein remaining was plotted by quantifying the HA-PIM1 intensities before normalization to their respective glyceraldehyde-3-phosphate dehydrogenase (GAPDH) signals. (C) CHX-chase assay assessing the half-life of GFP-RBMY upon HA-PIM1 overexpression was performed. Cell lysates harvested at the times indicated were analyzed by immunoblotting, and the percentage of protein remaining was calculated as described earlier. (D) Western blot analysis showed the absence of endogenous RBMY and PIM1 p34 in the HCC cell line SNU-387 derived from a female patient, as compared with the male HCC line PLC/PRF/5. (E) A CHX-chase assay was performed in SNU-387 cells to determine the half-life of HA-PIM1 in the presence or absence of GFP-RBMY. (F) Immuno- blotting of PIM1 and RBMY expressions in male (N ¼ 4) and female (N ¼ 6) HCC tissue samples. Frozen tissues (20 mg) were homogenized in 400 mL RIPA lysis buffer containing 0.5% sodium dodecyl sulfate. After sonication, the protein lysates were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis using Bolt 4%–20% Bis-Tris Plus gels, transferred onto a polyvinylidene difluoride membrane, and subsequently probed with antibodies recognizing the indicated proteins. (G) A CHX-chase assay was achieved before subcellular fractionation of cells cotransfected with HA-PIM1 and GFP-RBMY. The fractions were analyzed by Western blot. Protein degradation was estimated by quantifying the nuclear and cytoplasmic GFP- RBMY intensities before normalization to their respective markers, Lamin A/C and GAPDH. (H) CHX-chase assay determined the half-life of HA-PIM1 upon co-expression of GFP-RBMY-WT and its mutants.

Article Snippet: Cultured cells plated on 8-well chamber slides (Millipore, Billerica, MA) were fixed with 4% paraformaldehyde Table 1.Detailed Information of Antibodies Used in IHC and IFA Analysis Antibody (clone/catalog number) Manufacturer Host species Dilution and incubation conditions RBMY (SRGY clone 1) Homemade Mouse 5 , 4 C, Overnight (O/N) RBMY1A1 (GTX110098) GeneTex Rabbit 150 , 4 C, O/N Her Par-1 (OCH1E5, ab75677) Abcam Mouse 1 , 4 C, O/N PIM1 (TA354029) OriGene Rabbit 150 , 4 C, O/N COX4 (GTX101499) GeneTex Rabbit 100 , 4 C, O/N c-MET (8198) Cell Signalling Technology Rabbit 100 , 4 C, O/N Fibronectin (GTX112794) GeneTex Rabbit 100 , 4 C, O/N E-cadherin (ab1416) Abcam Mouse 50 , 4 C, O/N Snail1 (sc-271977) Santa Cruz Biotechnology Mouse 50 , 37 C, 2 h GRP94 (GTX103232) GeneTex Rabbit 100 , 4 C, O/N Naþ/Kþ-ATPase a (sc-48345) Santa Cruz Biotechnology Mouse 500 , 4 C, O/N GOLPH2 (GTX107702) GeneTex Rabbit 100 , 4 C, O/N CK19 (TA313117) OriGene Rabbit 100 , 4 C, O/N HNF4a (C11F12) Cell Signalling Technology Rabbit 100 , 4 C, O/N HA.11 epitope tag (901501) BioLegend Mouse 100 , 4 C, O/N for 12 minutes, except those used for Snail1 staining, which were fixed with ice-cold methanol at -20oC for 5 minutes.

Techniques: Western Blot, Expressing, Control, Over Expression, Derivative Assay, Lysis, Sonication, Polyacrylamide Gel Electrophoresis, Membrane, Fractionation

Journal: Cellular and molecular gastroenterology and hepatology

Article Title: PIM1-Induced Cytoplasmic Expression of RBMY Mediates Hepatocellular Carcinoma Metastasis.

doi: 10.1016/j.jcmgh.2022.09.014

Figure Lengend Snippet: Figure 12. PIM1 fosters the mitochondrial translocation of RBMY to regulate mitochondrial integrity and dynamics. Subcytoplasmic localization of RBMY was analyzed by double-stained IFA probing for the colocalization of RBMY and organelle markers in (A) GFP-RBMY–transfected Huh-7 cells and (B) primary HCC tissue. Glucose-regulated protein of 94 kDa (GRP94), Golgi phosphoprotein 2 (GOLPH2), and COX4 are markers of endoplasmic reticulum (ER), Golgi, and mitochondria, respectively. (C and D) IFA showed the distribution of GFP-RBMY in Huh-7 cells transfected 3 times with siCtrl or siPIM1. (D) The percentage of the cell population (means ± SDs, Student t test) was determined with respect to the different subcellular distribution patterns of GFP-RBMY, which are indicated as patterns I–IV. (E) Immunoblotting showed the submitochondrial localization of PIM1 and RBMY. Mitochondria isolated from Huh-7 cells 24 hours post-transfection were subjected to pro- teinase K and/or Triton X-100 treatment and analyzed by Western blot. Mock indicates the basal levels of mitochondrial proteins. Bcl2 and COX4 represent OMM and IMM proteins, respectively. (F) Mitochondrial levels of apoptosis-related proteins (Bcl2 and Bad) upon overexpression of HA-PIM1 and GFP-RBMY. After subcellular fractionation, the isolated cytoplasmic and mitochondrial fractions were analyzed by Western blot. COX4 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were used to indicate the purity of the mitochondrial and cytoplasmic fractions, respectively. (G) Fluorescence staining of the mitochondria, cytoplasm, and nucleus of live PLC/PRF/5 cells with PIM1 or RBMY depletion using Mito-ID Green, CytoPainter Red, and Hoechst 33342, respectively. The white arrows indicate mitochondrial fusion. (H) Immunoblotting of PLC/PRF/5 cells transfected 3 times with the indicated siRNAs and once with HA-PIM1 or GFP-RBMY. A hypothetical model of the mechanism by which RBMY regulates mitochondrial dynamics is presented. (I) Western blot analyzed the Drp1 levels of the indicated cells expressing GFP-RBMY WT or mutants together with HA-PIM1 (upper) and kinase inhibitor SMI-4a (lower). Cells were treated with SMI-4a (30 mmol/L) or solvent control (dimethyl sulfoxide [DMSO]) 24 hours before transfecting with GFP-RBMY-WT or mutants. These cells were continuously supplied with SMI-4a and harvested after 24 hours post-transfection. DAPI, 40,6- diamidino-2-phenylindole.

Article Snippet: Cultured cells plated on 8-well chamber slides (Millipore, Billerica, MA) were fixed with 4% paraformaldehyde Table 1.Detailed Information of Antibodies Used in IHC and IFA Analysis Antibody (clone/catalog number) Manufacturer Host species Dilution and incubation conditions RBMY (SRGY clone 1) Homemade Mouse 5 , 4 C, Overnight (O/N) RBMY1A1 (GTX110098) GeneTex Rabbit 150 , 4 C, O/N Her Par-1 (OCH1E5, ab75677) Abcam Mouse 1 , 4 C, O/N PIM1 (TA354029) OriGene Rabbit 150 , 4 C, O/N COX4 (GTX101499) GeneTex Rabbit 100 , 4 C, O/N c-MET (8198) Cell Signalling Technology Rabbit 100 , 4 C, O/N Fibronectin (GTX112794) GeneTex Rabbit 100 , 4 C, O/N E-cadherin (ab1416) Abcam Mouse 50 , 4 C, O/N Snail1 (sc-271977) Santa Cruz Biotechnology Mouse 50 , 37 C, 2 h GRP94 (GTX103232) GeneTex Rabbit 100 , 4 C, O/N Naþ/Kþ-ATPase a (sc-48345) Santa Cruz Biotechnology Mouse 500 , 4 C, O/N GOLPH2 (GTX107702) GeneTex Rabbit 100 , 4 C, O/N CK19 (TA313117) OriGene Rabbit 100 , 4 C, O/N HNF4a (C11F12) Cell Signalling Technology Rabbit 100 , 4 C, O/N HA.11 epitope tag (901501) BioLegend Mouse 100 , 4 C, O/N for 12 minutes, except those used for Snail1 staining, which were fixed with ice-cold methanol at -20oC for 5 minutes.

Techniques: Translocation Assay, Staining, Transfection, Western Blot, Isolation, Over Expression, Fractionation, Fluorescence, Expressing, Solvent, Control

Journal: Cellular and molecular gastroenterology and hepatology

Article Title: PIM1-Induced Cytoplasmic Expression of RBMY Mediates Hepatocellular Carcinoma Metastasis.

doi: 10.1016/j.jcmgh.2022.09.014

Figure Lengend Snippet: Figure 13. The PIM1–RBMY axis increases the mitochondrial motility. Bar plots showing the (A) luminescence signal of mitochondrial ATP in Huh-7 cells and the (B) fluorescence signal of mtROS in PLC/PRF/5 cells upon transfection with the indicated plasmids. The results shown are the means ± SDs from quadruplicate samples, and a representative data from 3 separate experiments is shown. The significance of differences was analyzed by the Student t test. (C) Flow cytometry analysis of mtROS in PLC/PRF/5 cells. Stable lines expressing HA-PIM1 and/or HA-RBMY were treated with antimycin A in the absence or presence of NAC. Cells were incubated with mtROS detection reagent before staining with SYTOX blue dead cell dye. For each sample, 10,000 cells were gated. The mtROS level was estimated in PE channel, and the live/dead cells were detected using V450 channel. (D) Mitochondrial locomotion in live Huh-7 transfectants. Representative fluorescence images of nonmotile and motile cells are shown. The percentage of these cells was depicted as means ± SDs and evaluated by the Student t test. (E) Traveling distance of mitochondria from the nuclear front in Huh-7 transfectants was plotted (means ± SDs, Student t test). Representative pictures showed staining of mitochondria, cytoplasm, and nucleus in live Huh-7 cells over- expressed with proteins indicated. Orange and blue arrows indicate nonmotile and motile cells, respectively.

Article Snippet: Cultured cells plated on 8-well chamber slides (Millipore, Billerica, MA) were fixed with 4% paraformaldehyde Table 1.Detailed Information of Antibodies Used in IHC and IFA Analysis Antibody (clone/catalog number) Manufacturer Host species Dilution and incubation conditions RBMY (SRGY clone 1) Homemade Mouse 5 , 4 C, Overnight (O/N) RBMY1A1 (GTX110098) GeneTex Rabbit 150 , 4 C, O/N Her Par-1 (OCH1E5, ab75677) Abcam Mouse 1 , 4 C, O/N PIM1 (TA354029) OriGene Rabbit 150 , 4 C, O/N COX4 (GTX101499) GeneTex Rabbit 100 , 4 C, O/N c-MET (8198) Cell Signalling Technology Rabbit 100 , 4 C, O/N Fibronectin (GTX112794) GeneTex Rabbit 100 , 4 C, O/N E-cadherin (ab1416) Abcam Mouse 50 , 4 C, O/N Snail1 (sc-271977) Santa Cruz Biotechnology Mouse 50 , 37 C, 2 h GRP94 (GTX103232) GeneTex Rabbit 100 , 4 C, O/N Naþ/Kþ-ATPase a (sc-48345) Santa Cruz Biotechnology Mouse 500 , 4 C, O/N GOLPH2 (GTX107702) GeneTex Rabbit 100 , 4 C, O/N CK19 (TA313117) OriGene Rabbit 100 , 4 C, O/N HNF4a (C11F12) Cell Signalling Technology Rabbit 100 , 4 C, O/N HA.11 epitope tag (901501) BioLegend Mouse 100 , 4 C, O/N for 12 minutes, except those used for Snail1 staining, which were fixed with ice-cold methanol at -20oC for 5 minutes.

Techniques: Transfection, Flow Cytometry, Expressing, Incubation, Staining

Journal: Cellular and molecular gastroenterology and hepatology

Article Title: PIM1-Induced Cytoplasmic Expression of RBMY Mediates Hepatocellular Carcinoma Metastasis.

doi: 10.1016/j.jcmgh.2022.09.014

Figure Lengend Snippet: Figure 14. ROS genera- tion is crucial for PIM1/ HA-RBMY-mediated mito- chondrial motility and cellular migration/inva- sion. (A) PLC/PRF/5 cells stably expressing HA-PIM1 and/or HA-RBMY were treated with 30 mmol/L NAC and 1 PBS (mock) as sol- vent control. The distribu- tion of mitochondria of these live cells were visual- ized by staining with Mito- ID Green. Staining of 40,6- diamidino-2-phenylindole (DAPI) indicates the nu- cleus. Bar plots (means ± SDs) showed the distance of mitochondrial move- ment. The statistical signifi- cance was evaluated by the Student t test. (B) Scratch wound healing assay showed the migratory abil- ity of cells indicated in the absence and presence of NAC. The migration dis- tance was calculated and presented as means ± SDs. The Student t test was applied for the statistical analysis of the results. (C) Transwell invasion assay assessed the invasive po- tential ofcellsindicated with or without NAC treatment. The number of invaded cells was counted(means ± SDs) and the result between groups was compared by the Student t test. *P < .05, **P < .005, and ***P < .0005.

Article Snippet: Cultured cells plated on 8-well chamber slides (Millipore, Billerica, MA) were fixed with 4% paraformaldehyde Table 1.Detailed Information of Antibodies Used in IHC and IFA Analysis Antibody (clone/catalog number) Manufacturer Host species Dilution and incubation conditions RBMY (SRGY clone 1) Homemade Mouse 5 , 4 C, Overnight (O/N) RBMY1A1 (GTX110098) GeneTex Rabbit 150 , 4 C, O/N Her Par-1 (OCH1E5, ab75677) Abcam Mouse 1 , 4 C, O/N PIM1 (TA354029) OriGene Rabbit 150 , 4 C, O/N COX4 (GTX101499) GeneTex Rabbit 100 , 4 C, O/N c-MET (8198) Cell Signalling Technology Rabbit 100 , 4 C, O/N Fibronectin (GTX112794) GeneTex Rabbit 100 , 4 C, O/N E-cadherin (ab1416) Abcam Mouse 50 , 4 C, O/N Snail1 (sc-271977) Santa Cruz Biotechnology Mouse 50 , 37 C, 2 h GRP94 (GTX103232) GeneTex Rabbit 100 , 4 C, O/N Naþ/Kþ-ATPase a (sc-48345) Santa Cruz Biotechnology Mouse 500 , 4 C, O/N GOLPH2 (GTX107702) GeneTex Rabbit 100 , 4 C, O/N CK19 (TA313117) OriGene Rabbit 100 , 4 C, O/N HNF4a (C11F12) Cell Signalling Technology Rabbit 100 , 4 C, O/N HA.11 epitope tag (901501) BioLegend Mouse 100 , 4 C, O/N for 12 minutes, except those used for Snail1 staining, which were fixed with ice-cold methanol at -20oC for 5 minutes.

Techniques: Migration, Stable Transfection, Expressing, Control, Staining, Wound Healing Assay, Transwell Invasion Assay

Journal: Cellular and molecular gastroenterology and hepatology

Article Title: PIM1-Induced Cytoplasmic Expression of RBMY Mediates Hepatocellular Carcinoma Metastasis.

doi: 10.1016/j.jcmgh.2022.09.014

Figure Lengend Snippet: Figure 15. PIM1 and RBMY prime HCC cells for lung metastasis in a mouse model. (A) A schematic illustration of the establishment of the liver cancer lung metastasis model is shown. (B) Double-stained IFA with anti-HA and anti-human RBMY (SRGY clone 1) antibodies was performed to assess the expression of transfected HA-PIM1 and/or GFP-RBMY WT/mutants in the liver tissue sections. A representative figure from each group is shown. (C) The number of pulmonary metastatic nodules and the percentage incidence of lung metastasis in each group were plotted (means ± SEMs, Student t test ). (D) H&E staining assessed the number of pulmonary HCC metastases (black arrows). A representative image from each group is presented. (E) Double IFA staining of PIM1 and RBMY in liver tissues of HA-PIM1þGFP-NLSm mice. Positive immunoreactivity for PIM1-Mix/ RBMY-Cþ is indicated by white arrows. (F) IHC staining of the indicated proteins in serial lung sections from HA-PIM1þGFP- NLSm mice. The black arrows indicate the metastatic nodule in serial sections. BV, blood vessel; DAPI, 40,6-diamidino-2- phenylindole.

Article Snippet: Cultured cells plated on 8-well chamber slides (Millipore, Billerica, MA) were fixed with 4% paraformaldehyde Table 1.Detailed Information of Antibodies Used in IHC and IFA Analysis Antibody (clone/catalog number) Manufacturer Host species Dilution and incubation conditions RBMY (SRGY clone 1) Homemade Mouse 5 , 4 C, Overnight (O/N) RBMY1A1 (GTX110098) GeneTex Rabbit 150 , 4 C, O/N Her Par-1 (OCH1E5, ab75677) Abcam Mouse 1 , 4 C, O/N PIM1 (TA354029) OriGene Rabbit 150 , 4 C, O/N COX4 (GTX101499) GeneTex Rabbit 100 , 4 C, O/N c-MET (8198) Cell Signalling Technology Rabbit 100 , 4 C, O/N Fibronectin (GTX112794) GeneTex Rabbit 100 , 4 C, O/N E-cadherin (ab1416) Abcam Mouse 50 , 4 C, O/N Snail1 (sc-271977) Santa Cruz Biotechnology Mouse 50 , 37 C, 2 h GRP94 (GTX103232) GeneTex Rabbit 100 , 4 C, O/N Naþ/Kþ-ATPase a (sc-48345) Santa Cruz Biotechnology Mouse 500 , 4 C, O/N GOLPH2 (GTX107702) GeneTex Rabbit 100 , 4 C, O/N CK19 (TA313117) OriGene Rabbit 100 , 4 C, O/N HNF4a (C11F12) Cell Signalling Technology Rabbit 100 , 4 C, O/N HA.11 epitope tag (901501) BioLegend Mouse 100 , 4 C, O/N for 12 minutes, except those used for Snail1 staining, which were fixed with ice-cold methanol at -20oC for 5 minutes.

Techniques: Staining, Expressing, Transfection, Immunohistochemistry

Journal: Cellular and molecular gastroenterology and hepatology

Article Title: PIM1-Induced Cytoplasmic Expression of RBMY Mediates Hepatocellular Carcinoma Metastasis.

doi: 10.1016/j.jcmgh.2022.09.014

Figure Lengend Snippet: Figure 16. Increasing mitochondrial mass and fission is essential for PIM1 and RBMY-mediated distance metastasis of HCC. (A) Macroscopic views of livers and lungs harvested from the indicated mice. The black arrows show hepatic and pulmonary nodules. No metastatic tumors were found in the lungs of any control vector and HA-PIM1þGFP-NESm mice. (B) Immunoblotting of the protein lysates harvested from liver tissues of the indicated mice after death. A cartoon diagram illustrating that PIM1/RBMY-Cþ and PIM1/RBMY-Nþ impact different mechanisms is shown. (C) Double IFA staining of COX4 and Naþ/Kþ adenosine triphosphatase (ATPase), a plasma membrane marker, in liver tissue sections from the indicated mice. The white arrows show cells with mitochondria-surrounding nuclei. The yellow arrows indicate cells with increased mito- chondrial mass and increased mitochondrial trafficking to the leading edge. DAPI, 40,6-diamidino-2-phenylindole.

Article Snippet: Cultured cells plated on 8-well chamber slides (Millipore, Billerica, MA) were fixed with 4% paraformaldehyde Table 1.Detailed Information of Antibodies Used in IHC and IFA Analysis Antibody (clone/catalog number) Manufacturer Host species Dilution and incubation conditions RBMY (SRGY clone 1) Homemade Mouse 5 , 4 C, Overnight (O/N) RBMY1A1 (GTX110098) GeneTex Rabbit 150 , 4 C, O/N Her Par-1 (OCH1E5, ab75677) Abcam Mouse 1 , 4 C, O/N PIM1 (TA354029) OriGene Rabbit 150 , 4 C, O/N COX4 (GTX101499) GeneTex Rabbit 100 , 4 C, O/N c-MET (8198) Cell Signalling Technology Rabbit 100 , 4 C, O/N Fibronectin (GTX112794) GeneTex Rabbit 100 , 4 C, O/N E-cadherin (ab1416) Abcam Mouse 50 , 4 C, O/N Snail1 (sc-271977) Santa Cruz Biotechnology Mouse 50 , 37 C, 2 h GRP94 (GTX103232) GeneTex Rabbit 100 , 4 C, O/N Naþ/Kþ-ATPase a (sc-48345) Santa Cruz Biotechnology Mouse 500 , 4 C, O/N GOLPH2 (GTX107702) GeneTex Rabbit 100 , 4 C, O/N CK19 (TA313117) OriGene Rabbit 100 , 4 C, O/N HNF4a (C11F12) Cell Signalling Technology Rabbit 100 , 4 C, O/N HA.11 epitope tag (901501) BioLegend Mouse 100 , 4 C, O/N for 12 minutes, except those used for Snail1 staining, which were fixed with ice-cold methanol at -20oC for 5 minutes.

Techniques: Control, Plasmid Preparation, Western Blot, Staining, Clinical Proteomics, Membrane, Marker

Journal: Cellular and molecular gastroenterology and hepatology

Article Title: PIM1-Induced Cytoplasmic Expression of RBMY Mediates Hepatocellular Carcinoma Metastasis.

doi: 10.1016/j.jcmgh.2022.09.014

Figure Lengend Snippet: Figure 17. Metastasis of cells expressing HA-PIM1 and GFP-RBMY-WT/mutants in a tail-vein injection mouse model. (A) Schematic diagram shows the steps by which a tail-vein injection metastasis mouse model was generated. (B) Western blot monitored the levels of EMT and mitochondrial proteins achieved on PLC/PRF/5 stable lines expressing proteins indicated before subjecting to tail-vein injection. (C) Number of lung metastatic masses and percentage of the metastatic occurrence in each group were counted and reported as means ± SEMs (Student t test). (D) H&E staining was performed on the lung tissues resected from mice indicated, and a representative image from each group is shown. Black arrowheads show pulmonary nodules. (E) Representative macroscopic views of lungs excised from each group at 10 weeks postinjection. Black arrowheads denote pulmonary nodules. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

Article Snippet: Cultured cells plated on 8-well chamber slides (Millipore, Billerica, MA) were fixed with 4% paraformaldehyde Table 1.Detailed Information of Antibodies Used in IHC and IFA Analysis Antibody (clone/catalog number) Manufacturer Host species Dilution and incubation conditions RBMY (SRGY clone 1) Homemade Mouse 5 , 4 C, Overnight (O/N) RBMY1A1 (GTX110098) GeneTex Rabbit 150 , 4 C, O/N Her Par-1 (OCH1E5, ab75677) Abcam Mouse 1 , 4 C, O/N PIM1 (TA354029) OriGene Rabbit 150 , 4 C, O/N COX4 (GTX101499) GeneTex Rabbit 100 , 4 C, O/N c-MET (8198) Cell Signalling Technology Rabbit 100 , 4 C, O/N Fibronectin (GTX112794) GeneTex Rabbit 100 , 4 C, O/N E-cadherin (ab1416) Abcam Mouse 50 , 4 C, O/N Snail1 (sc-271977) Santa Cruz Biotechnology Mouse 50 , 37 C, 2 h GRP94 (GTX103232) GeneTex Rabbit 100 , 4 C, O/N Naþ/Kþ-ATPase a (sc-48345) Santa Cruz Biotechnology Mouse 500 , 4 C, O/N GOLPH2 (GTX107702) GeneTex Rabbit 100 , 4 C, O/N CK19 (TA313117) OriGene Rabbit 100 , 4 C, O/N HNF4a (C11F12) Cell Signalling Technology Rabbit 100 , 4 C, O/N HA.11 epitope tag (901501) BioLegend Mouse 100 , 4 C, O/N for 12 minutes, except those used for Snail1 staining, which were fixed with ice-cold methanol at -20oC for 5 minutes.

Techniques: Expressing, Injection, Generated, Western Blot, Staining

Journal: BMC Cancer

Article Title: Synergistic apoptosis induction in myelodysplastic syndrome cells by azacitidine and PIM-2 inhibitors via nuclear factor-kappa B pathway inhibition

doi: 10.1186/s12885-025-14657-0

Figure Lengend Snippet: Synergistic inhibition of proliferation of MDS cells by azacitidine and SMI-16a. a The IC 50 values of azacitidine and SMI-16a against MDS cells were determined to be 20 nM and 25 µM, respectively. b Combination therapy with azacitidine and SMI-16a significantly suppressed cell proliferation compared to monotherapy. c Combination effects were evaluated using the CalcuSyn software, with combination index (CI) < 1 indicating synergy, CI = 1 indicating additivity, and CI > 1 indicating antagonism

Article Snippet: Azacitidine and SMI-16a (a PIM-2 inhibitor) were supplied by MedChemExpress (Monmouth Junction, NJ, USA) and stored as stock solutions at − 80 °C.

Techniques: Inhibition, Software

Journal: BMC Cancer

Article Title: Synergistic apoptosis induction in myelodysplastic syndrome cells by azacitidine and PIM-2 inhibitors via nuclear factor-kappa B pathway inhibition

doi: 10.1186/s12885-025-14657-0

Figure Lengend Snippet: Combination of azacitidine and SMI-16a induces cell apoptosis. A Apoptosis was assessed using propidium iodide (PI) and annexin V staining followed by flow cytometry after 48 h of treatment. B TUNEL assay results for MDS cell lines, including quantitative analysis of TUNEL-positive cells. C Western blot analysis showing upregulation of the apoptosis-associated proteins PARP and cleaved caspase-3. * indicates a significant difference compared with the control ( P < 0.05) and ** indicates a significant difference compared with monotherapy ( P < 0.05)

Article Snippet: Azacitidine and SMI-16a (a PIM-2 inhibitor) were supplied by MedChemExpress (Monmouth Junction, NJ, USA) and stored as stock solutions at − 80 °C.

Techniques: Staining, Flow Cytometry, TUNEL Assay, Western Blot, Control

Journal: BMC Cancer

Article Title: Synergistic apoptosis induction in myelodysplastic syndrome cells by azacitidine and PIM-2 inhibitors via nuclear factor-kappa B pathway inhibition

doi: 10.1186/s12885-025-14657-0

Figure Lengend Snippet: Inhibition of NF-κB pathway-related protein expression by azacitidine and SMI-16a. a Western blot analysis demonstrating downregulation of phosphorylated p65 (p-p65) and phosphorylated IκB (p-IκB) expression. b Western blot analysis showing downregulation of survivin and Bcl-2 expression

Article Snippet: Azacitidine and SMI-16a (a PIM-2 inhibitor) were supplied by MedChemExpress (Monmouth Junction, NJ, USA) and stored as stock solutions at − 80 °C.

Techniques: Inhibition, Expressing, Western Blot

Journal: BMC Cancer

Article Title: Synergistic apoptosis induction in myelodysplastic syndrome cells by azacitidine and PIM-2 inhibitors via nuclear factor-kappa B pathway inhibition

doi: 10.1186/s12885-025-14657-0

Figure Lengend Snippet: Azacitidine and SMI-16a induce apoptosis in primary CD34 + cells. a Schematic diagram of drug treatment of primary CD34 + cells. The illustration was created with BioRender.com. b Induction of apoptosis in primary CD34 + cells by azacitidine and SMI-16a. Primary CD34 + cells obtained from patients with high-risk MDS and AML were treated with azacitidine and SMI-16a for 24 h. The combination of these two drugs induced apoptosis in CD34 + cells

Article Snippet: Azacitidine and SMI-16a (a PIM-2 inhibitor) were supplied by MedChemExpress (Monmouth Junction, NJ, USA) and stored as stock solutions at − 80 °C.

Techniques:

Journal: bioRxiv

Article Title: Proteasomal Inhibition Triggers Viral Oncoprotein Degradation via Autophagy-Lysosomal Pathway

doi: 10.1101/780171

Figure Lengend Snippet: (A) ∼10 x 10 6 HEK293 cells transfected with either empty vector (pEGFP-C1) or GFP-tagged EBNA3C expression plasmid, either left untreated (DMSO control), or treated with 20 µM MG132 alone or MG132 plus 50 µM chloroquine (CQ). (B) HEK293 cells stably transfected with pTripz-mCherry-Sh-Beclin1 construct expressing sh-Beclin 1 under doxycycline (Dox) inducible promoter were further transfected either empty vector (pA3M) or myc-tagged EBNA3C expressing construct. 36 h post-transfection cells were either left untreated or treated with 20 µM MG132. (A-B) 4 h post-treatment cells were harvested, washed with 1 x PBS, lysed in RIPA buffer and subjected for western blot analyses for the indicated antibodies. (C) ∼5 x 10 4 HEK293 cells stably expressing sh-Beclin 1 with or without doxycycline treatment were transfected with GFP-tagged EBNA3C expressing plasmid using Lipofectamine 3000. 24 h post transfection cells were either left untreated (DMSO control) or treated with 20 µM MG132 for another 4h and subjected to live cell confocal analysis after staining the nucleus with Hochest 33342. Doxycycline treatment induces both mCherry and sh-RNA expression. Scale bars, 5 µm. (D) A colony formation assay was conducted as described in the “Materials and Methods” section using a similar experimental setup in doxycycline inducible sh-Beclin1 stably expressing HEK293 cells transiently transfected with GFP-EBNA3C construct.

Article Snippet: Ltd., India). pEGFP-C1 and pDsRED-Monomer-N1 plasmids were obtained from Clontech Laboratories, Inc. mCherry-tagged p62 expressing plasmid was a kind gift from Edward M. Campbell (Loyola University Chicago, IL, USA). pBabePuro-GFP-LC3 construct was a kind gift from Jayanta Debnath (Addgene plasmid# 22405). pTripz-mCherry-Sh-Beclin1 construct (Clone ID: V2THS_23692) was obtained from GE Healthcare Dharmacon Inc. (Lafayette, CO, USA). pGipz-Sh-Control and pGipz-Sh-EBNA3C were previously described.

Techniques: Transfection, Plasmid Preparation, Expressing, Stable Transfection, Construct, Western Blot, Staining, RNA Expression, Colony Assay

Journal: bioRxiv

Article Title: Proteasomal Inhibition Triggers Viral Oncoprotein Degradation via Autophagy-Lysosomal Pathway

doi: 10.1101/780171

Figure Lengend Snippet: Due to uncontrolled cell proliferation, cancer cells often encounter excess mis-/un-folded protein aggregates, which are subsequently labeled with either K48-linked or K63-linked polyubiquitination for proteolytic degradation. While K48-linked ubiquitin chains are targeted for the proteasomal pathway, K63-linked directs autophagy mediated protein degradation. Upon proteasomal inhibition, EBV oncoprotein EBNA3C is predominantly tagged with K63-linked polyubiquitin chains, translocated to cytoplasm by an unknown mechanism and degraded through autophagy-lysosomal pathway. The N-terminal domain plays a central role in EBNA3C’s degradation through autophagy mechanism by participating within the p62-LC3B complex. Suppression of autophagy pathway (Beclin1 knockdown or chloroquine, CQ treatment) reverses EBNA3C degradation in response to proteasomal inhibition. Additionally, proteasomal inhibitors (such as MG132) induce both autophagy and viral gene transcription that eventually activate viral lytic replication. The results provide foundation to exploit proteasome inhibitors as potential therapeutic approach for EBV associated B-cell lymphomas, where EBNA3C is expressed, typically diagnosed in immunocompromised individuals.

Article Snippet: Ltd., India). pEGFP-C1 and pDsRED-Monomer-N1 plasmids were obtained from Clontech Laboratories, Inc. mCherry-tagged p62 expressing plasmid was a kind gift from Edward M. Campbell (Loyola University Chicago, IL, USA). pBabePuro-GFP-LC3 construct was a kind gift from Jayanta Debnath (Addgene plasmid# 22405). pTripz-mCherry-Sh-Beclin1 construct (Clone ID: V2THS_23692) was obtained from GE Healthcare Dharmacon Inc. (Lafayette, CO, USA). pGipz-Sh-Control and pGipz-Sh-EBNA3C were previously described.

Techniques: Labeling, Inhibition

Journal: mBio

Article Title: PIM1 orchestrates sepsis-associated inflammatory imbalance in CD4 + T cell subsets via cholesterol metabolism

doi: 10.1128/mbio.01680-25

Figure Lengend Snippet: PIM1 expression is altered in sepsis-associated CD4 + T cells by transcriptome meta-analysis. ( A ) Workflow diagram of the transcriptome meta-analysis. ( B ) KEGG analysis of DEGs. ( C ) Metabolic pathway analysis of metabolism-related differential genes. ( D ) Heatmap of the top 10 DEGs.

Article Snippet: After blocking, the cells were incubated with anti-PIM1 antibody (1:200, NOVUS, USA) for 1 h at 4°C, followed by incubation with the Goat anti-Rabbit IgG (H + L) conjugated with Alexa Fluor Plus 488 secondary antibody (Thermo Fisher Scientific, USA).

Techniques: Expressing

Journal: mBio

Article Title: PIM1 orchestrates sepsis-associated inflammatory imbalance in CD4 + T cell subsets via cholesterol metabolism

doi: 10.1128/mbio.01680-25

Figure Lengend Snippet: PIM1 expression is correlated with the disease severity of sepsis. ( A, B, and C ) Percentage ( P = 0.0002) and MFI ( P = 0.0022) of PIM1 protein expression on CD4 + T cells from sepsis patients and healthy donors. Healthy donor ( n = 28) and sepsis ( n = 29). ( D and E ) Percentage ( P = 0.0021) and MFI ( P = 0.0412) of PIM1 protein expression on PBMCs from sepsis patients and healthy donors. Healthy donor ( n = 18) and sepsis ( n = 20). ( F and G ) Correlation analysis between PIM1 protein expression and SOFA score ( n = 29, P = 0.0457) and APACHE II score ( n = 29, P = 0.0111). ( H and I ) Correlation analysis of PIM1 protein expression with plasma CRP ( n = 29, P = 0.8830) and PCT concentrations ( n = 29, P = 0.4843). Statistical significance was determined using the Mann-Whitney U test. Data are represented as mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: After blocking, the cells were incubated with anti-PIM1 antibody (1:200, NOVUS, USA) for 1 h at 4°C, followed by incubation with the Goat anti-Rabbit IgG (H + L) conjugated with Alexa Fluor Plus 488 secondary antibody (Thermo Fisher Scientific, USA).

Techniques: Expressing, Clinical Proteomics, MANN-WHITNEY

Journal: mBio

Article Title: PIM1 orchestrates sepsis-associated inflammatory imbalance in CD4 + T cell subsets via cholesterol metabolism

doi: 10.1128/mbio.01680-25

Figure Lengend Snippet: PIM1 is involved in regulating Th1, Th17, and Treg subsets imbalance. ( A ) The gating strategy for CD4 + T cells. ( B and C ) The change of CD4 + IFN-γ + Th1 cells expression level after inhibiting PIM1 kinase activity ( n = 10, Control vs AZD1208 5 µM, P = 0.0039; Control vs AZD1208 10 µM, P = 0.0020). ( D and E ) Changes in the expression level of CD4 + IL-17A + Th17 cells after inhibiting PIM1 kinase activity ( n = 7, Control vs AZD1208 5 µM, P = 0.0156; Control vs AZD1208 10 µM, P = 0.0313). ( F and G ) Changes in expression level of CD4 + CD25 + FoxP3 + Treg cells after inhibiting of PIM1 kinase activity ( n = 10, Control vs AZD1208 5 µM, P = 0.5566; Control vs AZD1208 10 µM, P = 0.0137). P value was calculated by the Wilcoxon signed-rank test. Data are plotted as mean ± SEM. * P < 0.05, ** P < 0.01.

Article Snippet: After blocking, the cells were incubated with anti-PIM1 antibody (1:200, NOVUS, USA) for 1 h at 4°C, followed by incubation with the Goat anti-Rabbit IgG (H + L) conjugated with Alexa Fluor Plus 488 secondary antibody (Thermo Fisher Scientific, USA).

Techniques: Expressing, Activity Assay, Control

Journal: mBio

Article Title: PIM1 orchestrates sepsis-associated inflammatory imbalance in CD4 + T cell subsets via cholesterol metabolism

doi: 10.1128/mbio.01680-25

Figure Lengend Snippet: Bulk RNA sequencing analysis of CD4 + T cells after inhibition of PIM1 kinase activity. ( A and B ) KEGG analysis of DEGs. ( C ) GO analysis of DEGs. ( D ) Volcanic maps of DEGs. ( E ) ABCG1 mRNA expression in CD4 + T cells after inhibiting PIM1 kinase activity ( n = 6, P = 0.0313). ( F ) MFI of ABCG1 protein expression on CD4 + T cells after PIM1 siRNA transfection ( n = 7, siCtrl vs siPIM1#1, P = 0.0313; siCtrl and siPIM1#2, P = 0.0313). ( G ) ABCG1 mRNA expression ( P value = 0.0112) of CD4 + T cells in sepsis and healthy donors. Healthy Donor ( n = 12) and sepsis ( n = 10). For panels E and F , P value was calculated by the Wilcoxon signed rank test. For panel G , P value was calculated by Mann-Whitney U test. Data are plotted as mean ± SEM. * P < 0.05.

Article Snippet: After blocking, the cells were incubated with anti-PIM1 antibody (1:200, NOVUS, USA) for 1 h at 4°C, followed by incubation with the Goat anti-Rabbit IgG (H + L) conjugated with Alexa Fluor Plus 488 secondary antibody (Thermo Fisher Scientific, USA).

Techniques: RNA Sequencing, Inhibition, Activity Assay, Expressing, Transfection, MANN-WHITNEY

Journal: mBio

Article Title: PIM1 orchestrates sepsis-associated inflammatory imbalance in CD4 + T cell subsets via cholesterol metabolism

doi: 10.1128/mbio.01680-25

Figure Lengend Snippet: PIM1 regulates cholesterol metabolism in CD4 + T cells. ( A ) Gating strategy for filipin III expression in CD4 + T cells. ( B and C ) Intracellular cholesterol staining with filipin III in CD4 + T cells following inhibition of PIM1 kinase activity ( n = 6, P = 0.0313). ( D ) Gating strategy for filipin III and cholera toxin subunit B (CTB) expression in CD4 + T cells following PIM1 siRNA transfection or PIM1 kinase inhibition in CD3 + T cells. ( E and F ) Intracellular cholesterol staining with filipin III in CD4 + T cells after PIM1 siRNA transfection ( n = 10, siCtrl vs siPIM1#1, P = 0.0371; siCtrl vs siPIM1#2, P = 0.0059). ( G and H ) Lipid rafts detection by AF488-conjugated CTB on CD4 + T cells following PIM1 kinase inhibition ( n = 13, P = 0.0010). Statistical significance was determined using the Wilcoxon signed-rank test. Data are represented as mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: After blocking, the cells were incubated with anti-PIM1 antibody (1:200, NOVUS, USA) for 1 h at 4°C, followed by incubation with the Goat anti-Rabbit IgG (H + L) conjugated with Alexa Fluor Plus 488 secondary antibody (Thermo Fisher Scientific, USA).

Techniques: Expressing, Staining, Inhibition, Activity Assay, Transfection

Journal: mBio

Article Title: PIM1 orchestrates sepsis-associated inflammatory imbalance in CD4 + T cell subsets via cholesterol metabolism

doi: 10.1128/mbio.01680-25

Figure Lengend Snippet: PIM1 induces the inflammatory imbalance in CD4 + T cells via cholesterol-dependent mechanisms. ( A ) The gating strategy for CD4 + T cells. ( B and C ) Expression of CD4 + IFN-γ + Th1 cells following PIM1 kinase inhibition and cholesterol supplementation ( n = 9, Control vs AZD1208, P = 0.0039; AZD1208 vs AZD1208+ Cholesterol, P = 0.0273). ( D and E ) Expression of CD4 + IL-17A + Th17 cells following PIM1 kinase inhibition and cholesterol supplementation ( n = 7, Control vs AZD1208, P = 0.0156; AZD1208 vs AZD1208+ Cholesterol, P = 0.0156). ( F and G ) Expression of CD4 + CD25 + FoxP3 + Treg cells following PIM1 kinase inhibition and cholesterol supplementation ( n = 11, Control vs AZD1208, P = 0.0010; AZD1208 vs AZD1208+ Cholesterol, P = 0.0186). Statistical significance was assessed using the Wilcoxon signed-rank test. Data are presented as mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001, ns: not significant.

Article Snippet: After blocking, the cells were incubated with anti-PIM1 antibody (1:200, NOVUS, USA) for 1 h at 4°C, followed by incubation with the Goat anti-Rabbit IgG (H + L) conjugated with Alexa Fluor Plus 488 secondary antibody (Thermo Fisher Scientific, USA).

Techniques: Expressing, Inhibition, Control