Journal: Advanced Science

Article Title: PIM1 Attenuates Innate Immunity to Foster Coronavirus Replication through Ubiquitin Ligase β‐TrCP‐Mediated IFNAR1 Degradation

doi: 10.1002/advs.202503487

Figure Lengend Snippet: PIM1 was induced by HCoV infection and promoted HCoV replication. a–c) PIM1 mRNA levels in posterior pharyngeal wall cells from SARS‐CoV‐2 positive (n = 55) and negative (n = 46) subjects; RD cells transfected with poly I:C (n=4); HEK293T cells transfected with SARS‐CoV‐2 non‐structural, structural, and accessory proteins for 48 h (n = 3). d–f) OC43 viral genome RNA (n = 4), protein, and viral titer (n = 3) levels in PIM1‐knockdown HEK293T cells post infection. HEK293T cells were transfected with PIM1‐specific siRNAs for 24 h, then challenged with OC43 at an MOI of 1 for 24 h. g,h) Viral genome RNA g) (n = 3) and protein levels h) in HEK293T cells transfected with indicated amounts of PIM1 plasmids for 24 h, followed by OC43 infection at an MOI of 1 for 48 h. i) Viral titration in HEK293T cells were transfected with PIM1/PIM1 K67M plasmids for 24 h, followed by OC43 infection at an MOI of 1 for 24 h (n = 3). j–m) Viral genome RNA (n=3) and protein levels in virus‐challenged RD cells pretreated with PIM1 inhibitors. RD cells were pre‐treated with CX‐6528 or PIM1 inhibitor 2 for 2 h, followed by OC43 challenge at an MOI of 0.05 for 48 h. n) The intracellular viral genome RNA level of 229E after PIM1 modulation, followed by 48 h.p.i (n = 3). Statistical analyses were conducted with Student's t‐test. * : p < 0.05, ** : p < 0.01. Data are shown as mean ±SD. All mRNA or viral genome RNA levels were determined by RT‐qPCR. Proteins were measured by western blot. Viral titration was determined by TCID50. The densities of all proteins of interest were quantified using ImageJ and normalized to the respective control indicated beneath the bands.

Article Snippet: PIM1 inhibitors were purchased from MCE (CX6528: HY‐18095; AZD‐1208: HY‐15604; SGI‐1776: HY‐13287; PIM1 inhibitor 2: [ ] HY‐147785).

Techniques: Infection, Transfection, Knockdown, Titration, Virus, Quantitative RT-PCR, Western Blot, Control

Journal: Advanced Science

Article Title: PIM1 Attenuates Innate Immunity to Foster Coronavirus Replication through Ubiquitin Ligase β‐TrCP‐Mediated IFNAR1 Degradation

doi: 10.1002/advs.202503487

Figure Lengend Snippet: PIM1 decreased IFNAR1 protein levels to promote OC43 replication. a) Luciferase assay of ISRE element activity after PIM1/K67M overexpression (n = 3). b) Immunoblots of lysates from HEK293T cells transfected with vector/PIM1 plasmids for 48 h. c) Western blot for IFNAR1 in HEK293T cells transfected with vector/WT‐PIM1/PIM1‐K67M plasmids for 48 h. d) IFNAR1 expression level in PIM1 knockdown cells. e) Immunoblots analysis of RD cells infected with OC43 at an MOI of 10 for the indicated time. f) Expression of IFNAR1 in RD cells transfected with siPIM1 RNA, followed by OC43 infection. g–j) Western blot for IFNAR1 expression in HEK293T or HepG2 cells treated with PIM1 inhibitors (CX‐6528, SGI‐1776, and AZD‐1208) at the indicated concentration for 48 h. k) The IFNAR1 expression in RD cells pre‐treated with PIM1 inhibitor 2 for 2 h, followed by OC43 infection for 48h. l)The intracellular OC43 genomic RNA in HEK293T cells transfected with vector/PIM1/PIM1 with IFNAR1 followed by the 24 h challenging of OC43 at an MOI of 0.1. Statistical analyses were conducted with Student's t‐test. * : p < 0.05, ** : p < 0.01. Data are depicted as mean ±SD. The densities of all proteins of interest were quantified using ImageJ and normalized to the respective control indicated beneath the bands.

Article Snippet: PIM1 inhibitors were purchased from MCE (CX6528: HY‐18095; AZD‐1208: HY‐15604; SGI‐1776: HY‐13287; PIM1 inhibitor 2: [ ] HY‐147785).

Techniques: Luciferase, Activity Assay, Over Expression, Western Blot, Transfection, Plasmid Preparation, Expressing, Knockdown, Infection, Concentration Assay, Control

Journal: Advanced Science

Article Title: PIM1 Attenuates Innate Immunity to Foster Coronavirus Replication through Ubiquitin Ligase β‐TrCP‐Mediated IFNAR1 Degradation

doi: 10.1002/advs.202503487

Figure Lengend Snippet: PIM1 promoted IFNAR1 degradation via ubiquitination. a) IFNAR1 mRNA level in HEK293T cells transfected with vector/PIM1 plasmids for 48 h. b) The mRNA level of IFNAR1 in RD cells transfected with scramble/PIM1‐specific siRNA for 48 h. c) The mRNA level of IFNAR1 in RD cells after challenging with/without OC43 at an MOI of 1 or 10. d) IFNAR1‐overexpressed HEK293T cells were transfected with scramble/PIM1‐specific siRNA, followed by 50 µ m of CHX treatment for the indicated periods. IFNAR1 protein level were determined by western blot. e) IFNAR1 expression level after PIM1/K67M overexpression followed by MG132 or NH 4 Cl treatment. f) Flow Cytometry of cell surface IFNAR1 after PIM1 overexpression, stained with IFNAR1‐PE (n = 3). g) IFNAR1 expression in cell membrane or cytosol fractions after PIM1/K67M overexpression. h) Immunoblots of IFNAR1 in HEK293T cells transfected with WT‐IFNAR1 or mutated IFNAR1 (S535A, S539A) with/without PIM1. i) Western blot analysis for ubiquitination and IFNAR1 in HEK293T cells co‐transfected with HA‐Ubiquitin, IFNAR1 and vector/PIM1/PIM1 K67M constructs. j) Immunoprecipitation of IFNAR1 and HA‐tagged ubiquitination from lysates of HEK293T cells co‐transfected with HA‐K48 ubi/HA‐K63 ubi, IFNAR1 and vector/PIM1 plasmids. Statistical analyses were conducted with Student's t‐test. * : p < 0.05, ** : p < 0.01. Data are depicted as mean ±SD. The densities of all proteins of interest were quantified using ImageJ and normalized to the respective control indicated beneath the bands.

Article Snippet: PIM1 inhibitors were purchased from MCE (CX6528: HY‐18095; AZD‐1208: HY‐15604; SGI‐1776: HY‐13287; PIM1 inhibitor 2: [ ] HY‐147785).

Techniques: Ubiquitin Proteomics, Transfection, Plasmid Preparation, Western Blot, Expressing, Over Expression, Flow Cytometry, Staining, Membrane, Construct, Immunoprecipitation, Control

Journal: Advanced Science

Article Title: PIM1 Attenuates Innate Immunity to Foster Coronavirus Replication through Ubiquitin Ligase β‐TrCP‐Mediated IFNAR1 Degradation

doi: 10.1002/advs.202503487

Figure Lengend Snippet: PIM1 promoted IFNAR1 degradation via β‐TrCP1. a) IFNAR1 protein level in IFNAR1‐expressed HEK293T cells transfected with diverse siRNAs. b) IFNAR1 protein level in IFANR1‐expressed HEK293T cells transfected with scramble/β‐TrCP1 specific siRNA with/without PIM1 plasmids. c) IFNAR1 protein level in IFNAR1‐expressed HEK293T cells with β‐TrCP1 and/or PIM1 siRNA knockdown. d) Viral genomic RNA of OC43 in RD cells transfected with scramble/β‐TrCP1 specific siRNA followed by infection at an MOI of 0.1 for 24 h (n = 4). e) Viral genomic RNA level of OC43 in HEK293T cells transfected with increasing amounts of β‐TrCP1 followed by the challenging of HCoV‐OC43 at an MOI of 0.1 for 24 h (n = 3). f) Viral genomic RNA level in HEK293T cells after β‐TrCP1 siRNA knockdown with or without ectopic expression of PIM1, followed by OC43 infection at an MOI of 0.1 for 24 h (n = 3). g) Viral genomic RNA level in RD cells treated with PIM1 inhibitor (CX6528=8 µ m ) with or without β‐TrCP1 inhibitor (GS143) at indicated concentration for 2 h, followed by OC43 infection at MOI of 0.01 for 72 h (n = 4). Statistical analyses were conducted with Student's t‐test. * : p <0.05, ** : p < 0.01. Data are shown as mean ±SD. The densities of all proteins of interest were quantified using ImageJ and normalized to the respective control indicated beneath the bands.

Article Snippet: PIM1 inhibitors were purchased from MCE (CX6528: HY‐18095; AZD‐1208: HY‐15604; SGI‐1776: HY‐13287; PIM1 inhibitor 2: [ ] HY‐147785).

Techniques: Transfection, Knockdown, Infection, Expressing, Concentration Assay, Control

Journal: Advanced Science

Article Title: PIM1 Attenuates Innate Immunity to Foster Coronavirus Replication through Ubiquitin Ligase β‐TrCP‐Mediated IFNAR1 Degradation

doi: 10.1002/advs.202503487

Figure Lengend Snippet: PIM1 promoted IFNAR1 and β‐TrCP1 interaction. a,b) Protein level of β‐TrCP1 in HEK293T cells transfected with vector/PIM1 construct or scramble/PIM1 specific siRNA. c) Immunoprecipitation of IFNAR1 and HA‐tagged β‐TrCP1 from lysates mixture of vector/PIM1/PIM1 mutant expressed HEK293T and IFNAR1 expressed HEK293T. d) IP blots for purified PIM1 post incubation with or without purified βTrCP1 in the IP buffer. e) Immunofluorescent assay of HEK293T cells co‐transfected with PIM1 and HA‐ β‐TrCP1 plasmids under 40× microscope. PIM1 was in Red and HA‐ β‐TrCP1 was in Green. Scale bar = 10 µ m . Line intensity profiles (indicated as white line) and Scatter plots (boxed ROI ) for PIM1 and β‐TrCP1 were analyzed by Image J software. Pearson's r = 0.62. f) Purified β‐TrCP1 incubated with/without recombinant PIM1 in a kinase assay buffer at 37 °C for 30 min. Lambda protein phosphatase (lambda PP) was added to one tube of the PIM1 and β‐TrCP1 mixture for another 30 min. The products were visualized post Phos‐tag PAGE separation, which separates the phosphorylated β‐TrCP1 as up‐shifted migration bands from the non‐phosphorylated ones. The densities of all proteins of interest were quantified using ImageJ and normalized to the respective control indicated beneath the bands.

Article Snippet: PIM1 inhibitors were purchased from MCE (CX6528: HY‐18095; AZD‐1208: HY‐15604; SGI‐1776: HY‐13287; PIM1 inhibitor 2: [ ] HY‐147785).

Techniques: Transfection, Plasmid Preparation, Construct, Immunoprecipitation, Mutagenesis, Purification, Incubation, Microscopy, Software, Recombinant, Kinase Assay, Migration, Control

Journal: Advanced Science

Article Title: PIM1 Attenuates Innate Immunity to Foster Coronavirus Replication through Ubiquitin Ligase β‐TrCP‐Mediated IFNAR1 Degradation

doi: 10.1002/advs.202503487

Figure Lengend Snippet: Phosphorylated β‐TrCP1 at S82 by PIM1 enhanced its interaction with IFNAR1. a) Predicted PIM1‐phosphorylation sites on β‐TrCP1. b) ISRE luciferase assays using HEK293T cells transfected with ISRE‐Luci and β‐TrCP1 / β‐TrCP1 mutants, followed by stimulation with 500U IFNα for 16 h (n = 3). c) Protein level of IFNAR1 in HEK293T cells ectopically expressed with WT‐ β‐TrCP1 / β‐TrCP1 S82A/ β‐TrCP1 S521A. d) Protein level of phosphorylated STAT1/2 (p‐STAT1/2) in HEK293T cells ectopically expressed with vector/WT‐ β‐TrCP1 / β‐TrCP1 S82A/ β‐TrCP1 S521A followed by the stimulation of IFNα for 16 h. e) Quantification of intracellular viral genomic RNA in HEK293T cells ectopically expressed with WT‐ β‐TrCP1 / β‐TrCP1 S82A/ β‐TrCP1 S521A followed by the infection with HCoV‐OC43 at an MOI of 1 for 36 h (n = 3). f) Western blot analysis of HEK293T cells transfected with WT/S82A/S521A β‐TrCP1 plasmids with/without PIM1. Blots of IP assay using p‐Ser/Thr antibody were shown in the bottom panel. g) Co‐IP assay of IFNAR1 from the cell lysate mixture of HEK293T cells transfected with IFNAR1/HA‐ β‐TrCP1 / β‐TrCP1 S82A/ β‐TrCP1 S521A separately.

Article Snippet: PIM1 inhibitors were purchased from MCE (CX6528: HY‐18095; AZD‐1208: HY‐15604; SGI‐1776: HY‐13287; PIM1 inhibitor 2: [ ] HY‐147785).

Techniques: Phospho-proteomics, Luciferase, Transfection, Plasmid Preparation, Infection, Western Blot, Co-Immunoprecipitation 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 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: The Journal of Clinical Investigation

Article Title: BETP degradation simultaneously targets acute myelogenous leukemic stem cells and the microenvironment

doi: 10.1172/JCI120654

Figure Lengend Snippet: (A) OCI-AML3 (normal or hypoxia-adapted) cells were cultured with or without a monolayer of NMSCs and treated with ARV-825 (50 nM) or cytarabine (1 μM) under either normoxic or hypoxic conditions for 72 hours, and apoptosis was assessed by annexin V assay using flow cytometry (n = 3). (B) OCI-AML3 cells were treated with ARV-825 (10 nM) for 12 or 24 hours and subjected to CyTOF, and a heatmap was generated using the publicly available Broad Institute Gene Pattern Heatmap viewer. OCI-AML3 cells treated for 24 hours were stained for CXCR4 with or without permeabilization to quantify the changes in intracellular or surface CXCR4 expression using (C) a PE-conjugated antibody for the flow-based assay (n = 3) and (D) a AF594-conjugated secondary antibody for confocal imaging (original magnification, ×40). (E) OCI-AML3 cells were treated with ARV-825 (10 nM) or plerixafor (100 nM) (positive control) for 24 hours and subjected to a migration assay 4 hours after incubation in media containing SDF-1 (100 ng). Surface expression of CXCR4 was measured by flow cytometry, and cell migration was measured by collecting the cells from the lower chamber containing SDF-1 following Beckman Vi-CELL counts (n = 3). Whole-cell lysates obtained from OCI-AML3 cells treated with or without ARV-825 in the presence of SDF-1 were processed for immunoblotting with the indicated antibodies. β-Actin served as a loading control. (F) OCI-AML3 cells were treated with ARV-825 (10 nM) for the indicated durations, and whole-cell lysates were analyzed with the indicated antibodies (β-actin served as a loading control). Numbers indicate normalized intensity. PIM1-overexpressing OCI-AML3 cells were treated with ARV-825 (10 nM) for 24 hours, and then (G) whole-cell lysates were analyzed by immunoblotting with the indicated antibodies (α-tubulin was used as a loading control). (H) Surface expression of CXCR4 was analyzed by flow cytometry followed by a migration assay (n = 3). **P < 0.01 and ***P < 0.001, by standard Student’s t test.

Article Snippet: Antibodies against BETP (product no. 13440), c-Myc (product no. 5605), cleaved PARP (product no. 9541), p-ERK1/2 (product no. 4370), and PIM1 (product no. 3247) were obtained from Cell Signaling Technology.

Techniques: Cell Culture, Annexin V Assay, Flow Cytometry, Generated, Staining, Expressing, Imaging, Positive Control, Migration, Incubation, Western Blot

Journal: Cancer Research

Article Title: Pim Kinases Promote Cell Cycle Progression by Phosphorylating and Down-regulating p27Kip1 at the Transcriptional and Posttranscriptional Levels

doi: 10.1158/0008-5472.can-08-0634

Figure Lengend Snippet: Figure 3. Pim-mediated p27Kip1 phosphorylation promotes its binding to 14-3-3 and nuclear export. A, HEK293T cells were transfected with empty pHM6 (, lanes 1 and 2) or pHM6-wt-Pim1S (+, lanes 3–8) together with empty pFLAG-CMV-2 (, lane 1) or pFLAG-CMV-2 encoding wt-p27kip1 (wt, lanes 2 and 3) or the indicated p27Kip1 mutants. After transfection for 24 h, the cells were harvested, lysed, and immunoprecipitated with an anti-FLAG agarose, as described in Materials and Methods. The immunoprecipitants (IP) and the cell lysates (Input) were subjected to immunoblot analysis with the indicated antibodies. B, HT1080 cells were transfected with pHM6-wt-Pim1S together with pFLAG-CMV-2 encoding wt-p27Kip1 (wt), T157A-p27Kip1 (T157A), T198A-p27Kip1 (T198A), or T157A/ T198A-p27Kip1 (T157A/T198A). After transfection for 24 h, HA-tagged Pim1S proteins were detected by staining with an anti-HA antibody following Oregon green–conjugated anti-rat antibody incubation. The FLAG-tagged p27Kip1 proteins were detected by staining with an anti-FLAG antibody, following treatment with Alexa Flour 568–conjugated anti-rabbit antibody. Nuclei were detected by staining with Hoechst 33342. The cells were washed and then visualized using a fluorescence microscope equipped with a CCD camera (top). The percentage of the cells that exhibited cytoplasmic p27Kip1 was determined by counting 200 transfectants that expressed both Pim1 and p27Kip1 protein in each cell (bottom). C, HEK293T cells were transfected with empty pc5FLAG alone () or pc5FLAG encoding PIm1S (+) together with pFLAG-CMV-2 encoding p27Kip1 (+). After transfection for 24 h, the cells were harvested. The cytoplasmic and nuclear fractions were separated, electrophoresed, and immunoblotted with the indicated antibodies. D, HEK293T cells were cotransfected with empty pHM6 alone () or pHM6 encoding wt-, T157A- or T198A-p27Kip1 together with pc5FLAG encoding none () or wt-Pim1S (+). After transfection for 36 h, the cells were stained with propidium iodide and analyzed with a flow cytometer.

Article Snippet: The membranes were incubated with antibodies to Pim1 (12H8; sc-13513), p27Kip1 (C-19; sc-528), p-p27Kip1 [(Ser10)-R; sc-12939-R], p-p27Kip1 [(Thr187)-R; sc-16324-R], GFP (B-2; sc-9996), actin (C-2-HRP; sc-8432 HRP), 14-3-3h (H-8; sc-1657), 14-3-3u (C-18; sc-7683; Santa Cruz Biotechnology), FoxO3a, phospho-FoxO1 (Thr24)/FoxO3a (Thr32), phospho-FoxO3a (Ser253), phospho- (Ser/Thr) Akt substrate (Cell Signaling Technology), human Topo IIh (BD Transduction Laboratories), and a-tubulin (Serotec).

Techniques: Phospho-proteomics, Binding Assay, Transfection, Immunoprecipitation, Western Blot, Staining, Incubation, Fluorescence, Microscopy, Flow Cytometry

Journal: Cancer Research

Article Title: Pim Kinases Promote Cell Cycle Progression by Phosphorylating and Down-regulating p27Kip1 at the Transcriptional and Posttranscriptional Levels

doi: 10.1158/0008-5472.can-08-0634

Figure Lengend Snippet: Figure 5. Pim kinases down-regulate p27Kip1 transcription by phosphorylating and inactivating FoxO3a. A, alignment of the amino acid sequence of the Pim consensus sequence with the primary sequences of human FoxO3a around T32 and S253 residues. Identical residues are denoted by white letters on black background (top). HEK293T cells were transfected with empty pHM6 alone () or pHM6 encoding wt-Pim1S (+) together with empty pc5FLAG alone () or pc5FLAG encoding wt-FoxO3a (wt) or AAA-FoxO3a (AAA). After transfection for 24 h, the whole cell lysates were electrophoresed and immunoblotted with the indicated antibodies. Asterisks, the background bands (bottom). B, HEK293T cells were transfected with empty pc5FLAG alone () or pc5FLAG encoding wt-Pim1S (+). After transfection for 24 h, the whole cell lysates were electrophoresed and immunoblotted with the indicated antibodies. C, HEK293T cells were transfected with empty pHM6 alone () or pHM6 encoding wt-Pim1S (+) together with pc5FLAG encoding none (Mock), wt- FoxO3a (wt), AAA-FoxO3a (AAA), or H212R-FoxO3a (H212R). Cells were also transfected with pGL4.10 vector containing p27Kip1 promoter and pGL4.74 as an internal control. After transfection for 24 h, luciferase activities were calculated as described in Materials and Methods. Columns, mean of experiments done in triplicate; bars, SD. D, cancer profiling Array II containing normalized cDNA from normal tissue (N) or tumor tissue (T) of four patients with prostate cancer was probed with 32P-labeled pim1 and p27Kip1 probes, as described in Materials and Methods. Images were acquired and subsequent data analysis was done using Typhoon 9410. The densitometry units for the pim1 and p27Kip1 probed array were normalized to the densitometry units for a control ubiquitin-probed array.

Article Snippet: The membranes were incubated with antibodies to Pim1 (12H8; sc-13513), p27Kip1 (C-19; sc-528), p-p27Kip1 [(Ser10)-R; sc-12939-R], p-p27Kip1 [(Thr187)-R; sc-16324-R], GFP (B-2; sc-9996), actin (C-2-HRP; sc-8432 HRP), 14-3-3h (H-8; sc-1657), 14-3-3u (C-18; sc-7683; Santa Cruz Biotechnology), FoxO3a, phospho-FoxO1 (Thr24)/FoxO3a (Thr32), phospho-FoxO3a (Ser253), phospho- (Ser/Thr) Akt substrate (Cell Signaling Technology), human Topo IIh (BD Transduction Laboratories), and a-tubulin (Serotec).

Techniques: Sequencing, Transfection, Plasmid Preparation, Control, Luciferase, Labeling, Ubiquitin Proteomics

Journal: bioRxiv

Article Title: Identification of hemicatenane-specific binding proteins by fractionation of Hela nuclei extracts

doi: 10.1101/844126

Figure Lengend Snippet: A Products of the interaction between radiolabeled HC (16 femtomoles) and proteins of the fraction A10 of the size exclusion chromatography (45 μL) were resolved by electrophoresis on a polyacrylamide gel (lanes 3, 5, 8, 10). 12 (lanes 2, 3, 7, 8) or 23 (lanes 4, 5, 9, 10) ng of λ DNA were added to reduce non specific binding. Sample without protein (lanes 1 and 6) or without HC (lanes 2, 4, 7, 9) were also loaded to serve as control for mass spectrometry analysis. Free DNA (dsMC or HC) and Protein-HC complexes are indicated. On the right panel, the gel pieces that were analyzed by mass spectrometry are shown as green rectangles and the name of the gel pieces of interest is indicated on the right side of the figure. B Same as in (A) but with fraction A11 of the size exclusion chromatography. No λ DNA was added in the reaction mixes. Lanes 1 and 4: HC without fraction A11; lanes 2 and 5: fraction A11 without HC; lanes 3 and 6: HC + fraction A11. On the right panel, the green rectangles represent the two gel pieces that were analyzed by mass spectrometry. The name of the gel piece of interest is indicated on the right side of the figure. C Fractions A10 and A11 were tested for their content of PSPC1, SND1, SFPQ and PTBP1 proteins by western blot. Lanes 1, 4, 7, 10: MW; the size of the proteins is given in kDa; lanes 2, 5, 8, 11: fraction A10; lanes 3, 6, 9, 12: fraction A11. The identity of the antibodies used to probe the faction is indicated above each panel. The arrow points to the protein of interest. D and E Three samples were prepared and centrifuged on a sucrose gradient: sample with HC, sample with fraction A10 and sample with fraction A10 mixed with HC. After centrifugation, 200 μL fractions were collected from the top to the bottom of the gradient. Number of the fraction increases from top to bottom. In (D), fractions 5 to 10 of the sucrose gradients were tested for their SND1 content by western blot (band indicated by an arrow on the right side of the panel). Lanes 1, 3, 5, 7, 9, 11: the fraction A10 was loaded on the sucrose gradient. Lanes 2, 4, 6, 8, 10, 12: the mixture (fraction A10 + HC) was loaded on the sucrose gradient. Bands on the membrane were quantified using Image J software and the relative intensity of each band is plotted as a function of fraction. The white bars correspond to the fraction A10 sample and the hatched bars to the (fraction A10 + HC) sample. In (E), fractions 5 to 10 of the sucrose gradients were tested for their radioactivity content. An aliquot of each fraction (0.5 μL) was spotted on a nitrocellulose membrane. When dried, the membrane was exposed on a 32 P-sensitive screen. After exposure, the screen was scanned. The radioactivity profile of the HC sample is compared with that of the (fraction A10 + HC) sample.

Article Snippet: Anti-SND1 and anti-PTPB1 antibodies were from St John’s Laboratory.

Techniques: Size-exclusion Chromatography, Electrophoresis, Binding Assay, Mass Spectrometry, Western Blot, Centrifugation, Software, Radioactivity

Journal: bioRxiv

Article Title: Identification of hemicatenane-specific binding proteins by fractionation of Hela nuclei extracts

doi: 10.1101/844126

Figure Lengend Snippet: A The organization of SND1 in domains is shown. The double arrow underneath the schematic representation corresponds to the proteins that were expressed in E. coli and purified. The name of the purified protein is indicated on the left side of the double arrow. B Interactions were performed in a final volume of 7.5 μL with 0.1 femtomole of radiolabeled DNA and the indicated amount of purified protein. Species were resolved by electrophoresis under native conditions. Three DNAs were tested for their binding to SND1-64: dsMC09 (lanes 1-5); dsMC10 (lane 6-10); HC (lanes 11-15). Concentrations of protein were as indicated (lanes 1, 6, 11: 0; lanes 2, 7, 12: 0.1 μM; lanes 3, 8, 13: 0.3 μM; lanes 4, 9, 14: 0.9 μM; lanes 5, 10, 15: 2.7 μM). Free DNAs and bound DNAs are indicated. C Interactions were performed in a final volume of 13.25 μL with 0.1 femtomole of radiolabeled DNA and the SDN1-110 at 70 nM. Species were resolved by electrophoresis under native conditions. Three DNAs were tested for their binding to SND1-110: dsMC09 (lanes 1 and 2); dsMC10 (lanes 3 and 4); HC (lanes 5 and 6). Free DNAs and bound DNAs are indicated. D Interactions were as described in (A). The DNA was C10ss, the single stranded circle obtained after nicking of the dsMC10 with Nt.BbvcI. 0.1 femtomole of C10ss was included in the reaction mixture and the concentrations of protein were as indicated (lane 1: 0; lane 2: 0.1 μM; lane 3: 0.3 μM; lane 4: 0.9 μM; lane 5: 2.7 μM). Free DNAs and bound DNAs are indicated. E The two curves (one for HC and one for C10ss) show the percentage of (SDN1-64)-DNA complexes as a function of protein concentration. Error bars correspond to the standard deviation. Percentages are the mean of three independent experiments. F Interactions were as described in (C). C10ss is the single stranded circle obtained after nicking of the dsMC10 with Nt.BbvcI. 0.1 femtomole of C10ss or HC was included in the reaction mixture and the concentration of protein was 70 nM. Free DNAs and bound DNAs are indicated. G The plot shows the percentage of (SDN1-110)-DNA complexes assembled at 70 nM SND1-110. Error bars correspond to the standard deviation. Percentages are the mean of three independent experiments. H The interaction between radiolabeled HC and SND1-64 was tested in the presence of OL21, an oligonucleotide long of 21 nucleotides. SDN1-64 was at 2.7 μM and HC at 14 pM. HC was premixed with increasing amount of OL21 (lane 1: no OL21, no SND1-64; lane 2: no OL21; lane 3: 0.2 nM OL21; lane 4: 0.7 nM OL21; lane 5: 2 nM OL21; lane 6: 7 nM OL21) before adding SND1-64. Free DNAs and bound DNAs are indicated. I The interaction between radiolabeled HC and SND1-110 was tested in the presence of OL21, an oligonucleotide long of 21 nucleotides. SDN1-110 was at 70 nM and HC at 7.5 pM. HC was premixed with increasing amount of OL21 (lane 1: no OL21, no SND1-110; lane 2: no OL21; lane 3: 0.2 nM OL21; lane 4: 0.7 nM OL21; lane 5: 2 nM OL21; lane 6: 7 nM OL21) before adding SND1-110. Species are separated by electrophoresis on a polyacrylamide gel. Free DNAs and bound DNAs are indicated. The plot shows the percentage of (SND1-110)-HC complexes as function of concentration of OL21. The standard deviation is calculated from two independent experiments.

Article Snippet: Anti-SND1 and anti-PTPB1 antibodies were from St John’s Laboratory.

Techniques: Purification, Electrophoresis, Binding Assay, Protein Concentration, Standard Deviation, Concentration Assay

Journal: bioRxiv

Article Title: N-Terminomic Changes of Neurons During Excitotoxicity Reveals Proteolytic Events Associated with Synaptic Dysfunctions and Potential Targets for Neuroprotection

doi: 10.1101/2022.03.13.484119

Figure Lengend Snippet: Ischemic stroke induced further proteolytic processing of CaMKIIβ in mouse brain cortex A . Schematic diagram depicting the cleavage sites in CaMKIIβ undergoing proteolytic processing in glutamate-treated neurons (+Glu) but not in neurons treated with glutamate and calpeptin (+Glu & Calp). M/L ratio : ratio of the neo-N-terminal peptide in the treated versus control neurons. N.D. : not detected. Arrow with dotted line : epitope of anti-CaMKIIβ antibody mapped to the C-terminal hub domain of CaMKIIβ. B. Differential cleavage of recombinant GST-CaMKIIβ and pT287 GST-CaMKIIβ by calpain-1. GST-CaMKIIβ with and without prior autophosphorylation at T287 (200 ng of GST-CaMKIIβ plus 100 μM ATP, 10 mM Mg2+, 1.5 Ca2+ and 5 μM CaM at 30 oC for 2 min) were incubated with calpain-1 (1 unit) in vitro for 45 min). The reaction mixtures were analyzed by Western blotting using the anti-CaMKIIβ antibody. C-term. ΔCaMKIIβ : C-terminal fragments of CaMKIIβ generated by calapin cleavage. Predicted fragments : Identities of C-term. ΔCaMKIIβ bands a and c predicted from N-terminomic findings shown in panel A . C. Western blot analysis of brain cortical lysates (20 μg of proteins) of sham-operated mice and mice at designated time points after ischemic stroke using the anti-CaMKIIβ antibody. Among the several C-term. ΔCaMKIIβ bands detected in ischemic stroke mouse brains, the mobility of one of them in SDS-PAGE (marked by a star) was similar to that of band a (marked by a star) in panel B .

Article Snippet: The following recombinant proteins were used: CaMKIIα (#PR4586C, Invitrogen), GST-CaMKIIβ (#02-110, Carna biosciences) and Calpain-1 (#208713-500UG, Calbiochem).

Techniques: Recombinant, Incubation, In Vitro, Western Blot, Generated, SDS Page

Journal: bioRxiv

Article Title: N-Terminomic Changes of Neurons During Excitotoxicity Reveals Proteolytic Events Associated with Synaptic Dysfunctions and Potential Targets for Neuroprotection

doi: 10.1101/2022.03.13.484119

Figure Lengend Snippet: A. Calpain cleavage sites of CaMKIIβ in excitotoxic neurons identified by TAILS and the corresponding predicted cleavage sites in CaMKIIα. ΔCaMKII short and ΔCaMKII long : N-terminal truncated fragments derived from cleavage at site 1 and site 2, respectively. B. Cleavage of unphosphorylated recombinant CaMKIIα (rCaMKIIα) and pT286-rCaMKIIα by calpain-1 in vitro generated truncated CaMKIIα (ΔCaMKIIα) fragments with molecular masses similar to those in ischemic stroke mouse brains. The pT286-rCaMKIIα was generated by incubating 200 μg of rCaMKIIα for 2 min at 30 °C in the presence of 10 μM ATP, 10 mM Mg 2+ , 1.5 mM Ca 2+ and 5 μM CaM. For the in vitro cleavage experiments, rCaMKIIα or pT286-rCaMKIIα was incubated with calpain-1 (1 unit) for 45 min. Reaction mixtures of the in vitro experiments were probed with the anti-Pan CaMKII and anti-CaMKIIα antibodies with epitopes mapped to the kinase domain. C. Western blots of lysates of brain cortex (20 μg of proteins) collected from wild type sham-operated mice and wild type mice and CaMKIIα KO mice at designated time points after photothrombotic stroke. The blots were probed with anti-CaMKIIα antibody ( left panel ) and anti-pan CaMKII antibody. One green star: Proteins in CaMKIIα KO brain lysates recognized by the anti-CaMKIIα antibody. These proteins were likely the intact and N-terminal proteolytic fragments of other CaMKII isoforms. Two green stars: A protein band cross-reacted with anti-CaMKIIα antibody but not with anti-pan CaMKII antibody. Three green stars: the molecular mass (∼35 kDa) of this long N-terminal fragment (ΔCaMKII long) differs from the predicted molecular mass (43.2 kDa) of ΔCaMKII long derived from CaMKIIβ. As this fragment was presented in the CaMKIIα KO brain lysate, it is likely derived from another CaMKII isoform such as CaMKIIδ expressed in brain cells. D. Western blot of lysates of brain cortex collected from wild type sham-operated mice and at designated time points after photothrombotic stroke treatment. The blot was probed with anti-pan CaMKII antibody ( left panel ). The abundance ratios of the intact CaMKIIα or CaMKIIβ versus actin and those of the short N terminal fragment of CaMKII (ΔCaMKII short) in brain cortical lysates of sham-operated and ischemic stroke mice ( right panels ). The p -values are represented as * for p -value < 0.05, ** for p -value < 0.01 and *** for p -value < 0.001. E . A model of differential cleavage of CaMKII oligomer by calpains in sham and ischemic stroke mouse brains. Under physiological conditions, calpains (black scissors) cleave CaMKIIα and CaMKIIβ homo- and hetero-oligomers at site 2 to generate ΔCaMKII long containing the kinase domain, the autoinhibitory/CaM binding motif and the linker motif. In ischemic stroke condition, calpains are over-activated and the CaMKII oligomers undergo autophosphorylation at Thr-286. The over-activated calpains (red scissors) cleave the autophosphorylated CaMKIIα and CaMKIIβ oligomers at sites 1 and 2, respectively to generate ΔCaMKII short and ΔCaMKII long fragments.

Article Snippet: The following recombinant proteins were used: CaMKIIα (#PR4586C, Invitrogen), GST-CaMKIIβ (#02-110, Carna biosciences) and Calpain-1 (#208713-500UG, Calbiochem).

Techniques: Derivative Assay, Recombinant, In Vitro, Generated, Incubation, Western Blot, Binding Assay

Journal: OncoTargets and Therapy

Article Title: PIM1 polymorphism and PIM1 expression as predisposing factors of esophageal squamous cell carcinoma in the Asian population

doi: 10.2147/ott.s103392

Figure Lengend Snippet: Figure 1 Immunohistochemical staining of PIM1 protein expression in esophageal squamous cell carcinoma tissues and adjacent normal tissues. Notes: (A) The expression of the PIM1 protein in adjacent normal tissues. (B) The expression of the PIM1 protein in esophageal squamous cell carcinoma tissues. Magnification, ×400.

Article Snippet: Rabbit-anti-human PIM1 polyclonal antibody (Boster Biotech Co., Ltd., Wuhan, People’s Republic of China) was added; the sections were incubated at 37°C for 1 hour and then incubated with a biotinylated secondary antibody.

Techniques: Immunohistochemical staining, Staining, Expressing