Journal: Cell death and differentiation

Article Title: Aberrant paracrine signalling for bone remodelling underlies the mutant histone-driven giant cell tumour of bone.

doi: 10.1038/s41418-022-01031-x

Figure Lengend Snippet: Fig. 5 SCUBE3 regulates osteoclast formation. A Representative photomicrographs on day 9 of osteoclasts generated from monocytes; treatment with rSCUBE3 results in fewer osteoclasts identifed by TRAP and vitronectin receptor (VNR) staining. B Dose-dependent inhibition of the total number of TRAP-positive osteoclasts by rSCUBE3; number relative to day 9 control cultures; 5 osteoclast preparations. C Quantification of number of nuclei per osteoclast following treatment with 1.25 μg/ml rSCUBE3, expressed as a proportion of OCs exhibiting 3–5 or ≥6 nuclei over the total number of OCs; 5 osteoclast preparations. D Number of osteoclasts generated in the presence of conditioned medium (CM) from differentiated (day 6) hFOBs in the presence or absence of 1.25 μg/ml rSCUBE3 (relative number to control H3.3WT CM); 20 osteoclast preparations. Data represent the mean ± SEM. B, C: 2-way RM ANOVA. D: 1-way ANOVA.

Article Snippet: Effects of rSCUBE3 were investigated by treatment of osteoclast cultures with 0.25–1.25 μg/ml recombinant human rSCUBE3 protein (Cat # 7730-SC, R&D Systems, Abingdon, UK) in the presence of RANKL and M-CSF; all the reagents were replaced at each media change for osteoclasts.

Techniques: Generated, Staining, Inhibition, Control

Journal: eNeuro

Article Title: Chronic hM4Di-DREADD-Mediated Chemogenetic Inhibition of Forebrain Excitatory Neurons in Postnatal or Juvenile Life Does Not Alter Adult Mood-Related Behavior

doi: 10.1523/ENEURO.0381-21.2021

Figure Lengend Snippet: Selective expression of hM4Di-DREADD in CaMKIIα-positive forebrain excitatory neurons in CaMKIIα-tTA::TRE-hM4Di bigenic mice. A , Shown are representative confocal images indicating expression of the HA-tagged hM4Di-DREADD in the hippocampus as identified by HA/CaMKIIα double immunofluorescence. B , C , HA-tagged hM4Di-DREADD expression was not observed in either PV-positive inhibitory interneurons ( B ) or GFAP-positive astrocytes ( C ). D , HA-tagged hM4Di-DREADD in the cortex was also observed in the CaMKIIα-positive neurons as identified with HA/CaMKIIα double immunofluorescence. E , F , Immunofluorescence experiments indicate the absence of expression of HA-tagged hM4Di-DREADD in subcortical brain regions, namely the periaqueductal gray ( E ) and pallidum ( F ). G , Shown is a schematic of the experimental paradigm for harvesting cortex and hippocampus at P7 for Western blotting analysis. H , I , HA expression was clearly noted in the cortex ( H ) as well as the hippocampus ( I ) in Western blots from CaMKIIα-tTA::TRE-hM4Di bigenic pups (P7). J , Shown are representative Western blots for c-Fos along with their respective actin loading controls at P7 half an hour after vehicle (Veh) or CNO treatment for cortex (top panel) and hippocampus (bottom panel). K , L , Quantitative densitometry indicated a significant reduction in c-Fos protein levels in the cortex ( K ) as well as the hippocampus ( L ) of PNCNO-treated bigenic pups at P7 compared with their vehicle-treated controls. M , Shown is a schematic of the experimental paradigm for harvesting cortex and hippocampus at P35 in the juvenile window for Western blotting analysis. N , O , HA expression was noted in the cortex ( N ) as well as the hippocampus ( O ) of CaMKIIα-tTA::TRE-hM4Di bigenic juvenile mice (P35). P , Shown are representative Western blots for c-Fos along with their respective actin loading controls at P35 half an hour after vehicle (Veh) or CNO treatment for cortex (top panel) and hippocampus (bottom panel). Q , R , Quantitative densitometry indicated a significant reduction in c-Fos protein levels in the cortex ( Q ) but not in the hippocampus ( R ) of JCNO-treated bigenic mice at P35 compared with their vehicle-treated controls. All immunofluorescence experiments and Western blotting experiments were performed on n = 3–5/group. Results are expressed as the mean ± SEM. * p < 0.05, compared with vehicle-treated controls using the two-tailed, unpaired Student’s t test.

Article Snippet: For postnatal drug treatments, bigenic CaMKIIα-tTA::TRE-hM4Di mouse pups were orally administered either CNO (catalog #4963, Tocris Bioscience; 5 mg/kg in 5% sucrose solution containing 1% DMSO) or vehicle (5% sucrose solution containing 1% DMSO) for 13 d, commencing from P2 to P14.

Techniques: Expressing, Immunofluorescence, Western Blot, Two Tailed Test

Journal: Signal Transduction and Targeted Therapy

Article Title: Dual targeting PD-L1 and 4-1BB to overcome dendritic cell-mediated lenalidomide resistance in follicular lymphoma

doi: 10.1038/s41392-024-02105-7

Figure Lengend Snippet: Altered dendritic cell maturation and function were induced by PU.1 and contributed to lenalidomide resistance in FL. a Gene ontology terms enriched in genes differentially expressed in samples with relapse/progression patients. The P values for the dysregulated pathways were represented by the color of the points, as determined by RNA sequencing. The point size indicates the gene number in the gene set. b Summary radar plot, with 0.05 radial intervals depicting normalized average scores of immune subsets in samples according to relapse/progression. c Deconvolution of bulk RNA-seq using CibersortX by the gene signatures defined in single-cell analysis depicting normalized average scores of DC subsets in samples according to relapse/progression. d Volcano plot of the distribution of all differentially expressed genes involved in cDC1. The cDC1 absolute cell numbers ( e ), maturation ( f , g ), and endocytic activity ( h ) detected by flow cytometry in samples of SpCas9-scramble transfected SC1 co-culture system and SpCas9-PU.1-transfected SC1 co-culture system, as well as pGMLV-vector transfected DOHH2 co-culture system and pGMLV-PU.1 transfected DOHH2 co-culture system. Mean with SD was presented ( n = 3). i Quantification of typical autophagosomes observed by transmission electron microscopy was performed in the SpCas9-scramble and SpCas9-PU.1 transfected SC1 co-culture system, as well as the pGMLV-vector and pGMLV-PU.1 transfected DOHH2 co-culture system. Cells were counted from five randomly selected fields and analyzed statistically. Mean with SD was presented ( n = 5). j Representative images of immunohistochemical analysis of autophagic cells in tumor samples from FL patients, comparing the cDC1 low group ( n = 13) and cDC1 high group ( n = 16)

Article Snippet: The human FL cell line SC1 and DOHH2 were purchased from DSMZ Cell Lines Bank (Braunschweig, Germany).

Techniques: RNA Sequencing, Single-cell Analysis, Activity Assay, Flow Cytometry, Transfection, Co-Culture Assay, Plasmid Preparation, Transmission Assay, Electron Microscopy, Immunohistochemical staining

Journal: Signal Transduction and Targeted Therapy

Article Title: Dual targeting PD-L1 and 4-1BB to overcome dendritic cell-mediated lenalidomide resistance in follicular lymphoma

doi: 10.1038/s41392-024-02105-7

Figure Lengend Snippet: PU.1 mediated lenalidomide resistance in vitro. a Cell growth in SpCas9-PU.1-transfected SC1 (left) and pGMLV-PU.1-transfected DOHH2 (right) co-culture systems, treated with or without lenalidomide assessed by MTT assay. Mean with SD was presented ( n = 5). b cDC1 absolute cell numbers in the SpCas9-PU.1 transfected SC1 co-culture system (left) and pGMLV-PU.1 transfected DOHH2 co-culture system (right) treated with or without lenalidomide assessed by flow cytometry. Mean with SD was presented ( n = 3). cDC1 maturation in the SpCas9-PU.1 transfected SC1 co-culture system ( c ) and pGMLV-PU.1 transfected DOHH2 co-culture system ( d ) treated with or without lenalidomide assessed by flow cytometry. Mean with SD was presented ( n = 3). e cDC1 endocytic activity in the SpCas9-PU.1 transfected SC1 co-culture system and pGMLV-PU.1 transfected DOHH2 co-culture system treated with or without lenalidomide assessed by flow cytometry. Mean with SD was presented ( n = 3). LC3B expression in the SpCas9-PU.1 transfected SC1 co-culture system ( f ) and pGMLV-PU.1 transfected DOHH2 co-culture system ( g ) treated with or without lenalidomide assessed by flow cytometry. Mean with SD was presented ( n = 3). Typical autophagosomes in the SpCas9-PU.1 transfected SC1 co-culture system ( h ) and pGMLV-PU.1 transfected DOHH2 co-culture system ( i ) treated with or without lenalidomide, were assessed using a transmission electron microscope. Five random fields were selected for cell counting and statistical analysis. Mean with SD was presented ( n = 5)

Article Snippet: The human FL cell line SC1 and DOHH2 were purchased from DSMZ Cell Lines Bank (Braunschweig, Germany).

Techniques: In Vitro, Transfection, Co-Culture Assay, MTT Assay, Flow Cytometry, Activity Assay, Expressing, Transmission Assay, Microscopy, Cell Counting

Journal: Signal Transduction and Targeted Therapy

Article Title: Dual targeting PD-L1 and 4-1BB to overcome dendritic cell-mediated lenalidomide resistance in follicular lymphoma

doi: 10.1038/s41392-024-02105-7

Figure Lengend Snippet: PU.1 modulated PD-1/PD-L1 and 4-1BB/4-1BBL-mediated FL cell interaction with cDC1. a Immune checkpoints with differential expression between the cDC1 high and low groups identified by RNA sequencing. b Correlation between PU.1 and PD-L1, as well as PU.1 and 4-1BBL expression calculated by Pearson correlation coefficient analysis. c Relative luciferase activities in PD-L1 WT or PD-L1 MUT transfected HEK-293T cells, SC1 cells, DOHH2 cells were detected. Mean with SD was presented ( n = 3). d Relative luciferase activities in 4-1BBL WT or 4-1BBL MUT transfected HEK-293T cells, SC1 cells, DOHH2 cells were detected. Mean with SD was presented ( n = 3). e Western blot of PU.1, PD-L1, and 4-1BBL expression in the SpCas9-PU.1 transfected SC1 cells and pGMLV-PU.1 transfected DOHH2 cells

Article Snippet: The human FL cell line SC1 and DOHH2 were purchased from DSMZ Cell Lines Bank (Braunschweig, Germany).

Techniques: Quantitative Proteomics, RNA Sequencing, Expressing, Luciferase, Transfection, Western Blot

Journal: Signal Transduction and Targeted Therapy

Article Title: Dual targeting PD-L1 and 4-1BB to overcome dendritic cell-mediated lenalidomide resistance in follicular lymphoma

doi: 10.1038/s41392-024-02105-7

Figure Lengend Snippet: Dual targeting PD-L1 and 4-1BB counteracted PU.1-mediated cDC1 alterations in vitro . a Cell growth of the SpCas9-PU.1-transfected SC1 co-culture system, treated with or without the bi-specific PD-L1/4-1BB antibody assessed by MTT assay. Mean with SD was presented ( n = 5). b cDC1 absolute cell numbers in the SpCas9-PU.1 transfected SC1 co-culture system treated with or without bi-specific PD-L1/4-1BB antibody assessed by flow cytometry. Mean with SD was presented ( n = 3). c cDC1 maturation in the SpCas9-PU.1 transfected SC1 co-culture system treated with or without bi-specific PD-L1/4-1BB antibody assessed by flow cytometry. Mean with SD was presented ( n = 3). d cDC1 endocytic activity in the SpCas9-PU.1 transfected SC1 co-culture system treated with or without bi-specific PD-L1/4-1BB antibody assessed by flow cytometry. Mean with SD was presented ( n = 3). e LC3B expression in the SpCas9-PU.1 transfected SC1 co-culture system treated with or without bi-specific PD-L1/4-1BB antibody. Mean with SD was presented ( n = 3). f Typical autophagosomes in the SpCas9-PU.1-transfected SC1 co-culture system, with or without bi-specific PD-L1/4-1BB antibody treatment assessed by transmission electron microscope. Cells were counted from randomly selected fields ( n = 5). Mean with SD was presented

Article Snippet: The human FL cell line SC1 and DOHH2 were purchased from DSMZ Cell Lines Bank (Braunschweig, Germany).

Techniques: In Vitro, Transfection, Co-Culture Assay, MTT Assay, Flow Cytometry, Activity Assay, Expressing, Transmission Assay, Microscopy

Journal: Arthritis & rheumatology (Hoboken, N.J.)

Article Title: Monocarboxylate transporter 4, associated with the acidification of synovial fluid, is a novel therapeutic target for inflammatory arthritis.

doi: 10.1002/art.39270

Figure Lengend Snippet: Figure 2. Monocarboxylate transporter 4 (MCT4) mRNA and protein are up-regulated in rheumatoid arthritis synovial fibroblasts (RASFs). A, Lev- els of mRNA transcripts of Na1/H1 exchanger 1 (NHE-1), vacuolar-type H1ATPase (V-ATPase), MCT1, and MCT4 relative to that of b-actin in nor- mal SFs, 4 osteoarthritis SF (OASF) lines, and 4 RASF lines. B, Western blot analysis of MCT4 and b-actin protein in normal SFs, OASF lines, and RASF lines. C, Quantification depiction of the findings shown in B. Shown is MCT4 protein expression relative to b-actin protein levels in the 4 OASF lines and the 4 RASF lines. Values in A and C are the mean 6 SD. * 5 P , 0.05; ** 5 P , 0.01 by Student’s t-test. D, RA synovial tissue specimens incubated with rabbit anti-MCT4 antibodies (left) and with control normal rabbit serum (middle), and OA synovial tissue specimens incubated with anti-MCT4 antibodies (right). Boxed areas at the top are shown at higher magnification at the bottom. Results are representative of 8 RA synovial tis- sue specimens and 5 OA synovial tissue specimens. Bars 5 100 mm (top); 10 mm (bottom). Original magnification 3 100 (top); 3 400 (bottom).

Article Snippet: A rabbit anti-human MCT4 antibody (1:100) or a rabbit anti-mouse MCT4 antibody (1:100; Santa Cruz Biotechnology) served as the primary antibody, and normal rabbit serum (1:100; Wako) served as the control.

Techniques: Western Blot, Expressing, Incubation, Control

Journal: Arthritis & rheumatology (Hoboken, N.J.)

Article Title: Monocarboxylate transporter 4, associated with the acidification of synovial fluid, is a novel therapeutic target for inflammatory arthritis.

doi: 10.1002/art.39270

Figure Lengend Snippet: Figure 3. Knockdown of monocarboxylate transporter 4 (MCT4) with small interfering RNA (siRNA). A, Rheumatoid arthritis syno- vial fibroblast (RASF) lines were transfected with MCT4 siRNA or with control siRNA (mock). MCT4 siRNA transfection caused the number of MCT4 mRNA transcripts to fall relative to the number of b-actin mRNA transcripts. Values are the mean 6 SD of 3 RASF lines. ** 5 P , 0.01 by Student’s unpaired t-test. B, Western blot analysis of MCT4 and b-actin showed that MCT4 siRNA transfec- tion abrogated MCT4 expression in RASFs. Results are representa- tive of 3 independent experiments.

Article Snippet: A rabbit anti-human MCT4 antibody (1:100) or a rabbit anti-mouse MCT4 antibody (1:100; Santa Cruz Biotechnology) served as the primary antibody, and normal rabbit serum (1:100; Wako) served as the control.

Techniques: Knockdown, Small Interfering RNA, Transfection, Control, Western Blot, Expressing

Journal: Arthritis & rheumatology (Hoboken, N.J.)

Article Title: Monocarboxylate transporter 4, associated with the acidification of synovial fluid, is a novel therapeutic target for inflammatory arthritis.

doi: 10.1002/art.39270

Figure Lengend Snippet: Figure 4. Knockdown of monocarboxylate transporter 4 (MCT4) inhibits growth of rheumatoid arthritis synovial fibroblasts (RASFs). A, Num- bers of RASFs and osteoarthritis SFs (OASFs) after transfection with MCT4 small interfering RNA (siRNA) or control siRNA (mock). B, Flow cytometry data showing dilution of 5,6-carboxyfluorescein succinimidyl ester (CFSE) in MCT4 siRNA– and control siRNA–transfected RASFs on day 3. Shown are representative histograms (left) and percentages of divided RASFs (n 5 3 cell lines) (right). C, Flow cytometric analysis of annexin V and propidium iodide (PI) staining of RASFs and OASFs transfected with MCT4 siRNA or control siRNA, and of RASFs treated with cycloheximide (CHX; final concentration 100 mg/ml) as a positive control. Shown are representative dot plots (top) and percentages of PI- negative/annexin V–positive early apoptotic cells (bottom left) and PI-positive/annexin V–positive late apoptotic cells (bottom right). D, Left, Intracellular pH and L-lactate concentrations in RASFs transfected with MCT4 siRNA or control siRNA. Right, Extracellular pH and L-lactate concentrations in medium of RASFs transfected with MCT4 siRNA or control siRNA. Values are the mean 6 SD. * 5 P , 0.05; ** 5 P , 0.01 versus control siRNA–transfected RASFs, by Student’s t-test.

Article Snippet: A rabbit anti-human MCT4 antibody (1:100) or a rabbit anti-mouse MCT4 antibody (1:100; Santa Cruz Biotechnology) served as the primary antibody, and normal rabbit serum (1:100; Wako) served as the control.

Techniques: Knockdown, Transfection, Small Interfering RNA, Control, Flow Cytometry, Staining, Concentration Assay, Positive Control

Journal: Arthritis & rheumatology (Hoboken, N.J.)

Article Title: Monocarboxylate transporter 4, associated with the acidification of synovial fluid, is a novel therapeutic target for inflammatory arthritis.

doi: 10.1002/art.39270

Figure Lengend Snippet: Figure 5. Knockdown of monocarboxylate transporter 4 (MCT4) induces intrinsic apoptosis of rheumatoid arthritis synovial fibroblasts (RASFs). A, Western blot analysis of RASF lines transfected with MCT4 small interfering RNA (siRNA) or control siRNA (mock) to determine caspase 3, caspase 9, and caspase 8 protein expression. B, Levels of cleaved caspase 3 and caspase 9 expression relative to pro- caspase 3 and procaspase 9 expression levels, respectively, for all 3 RASF lines. Values are the mean 6 SD. * 5 P , 0.05 versus control siRNA–transfected RASFs, by Student’s t-test.

Article Snippet: A rabbit anti-human MCT4 antibody (1:100) or a rabbit anti-mouse MCT4 antibody (1:100; Santa Cruz Biotechnology) served as the primary antibody, and normal rabbit serum (1:100; Wako) served as the control.

Techniques: Knockdown, Western Blot, Transfection, Small Interfering RNA, Control, Expressing

Journal: Arthritis & rheumatology (Hoboken, N.J.)

Article Title: Monocarboxylate transporter 4, associated with the acidification of synovial fluid, is a novel therapeutic target for inflammatory arthritis.

doi: 10.1002/art.39270

Figure Lengend Snippet: Figure 6. Knockdown of monocarboxylate transporter 4 (MCT4) reduces severity of collagen-induced arthritis (CIA) in mice. A, Synovial tissue from the knee joint of a mouse with CIA and a control naive mouse after incubation with a rabbit anti-MCT4 antibody. Bars 5 100 mm. B, Left, Expression of 6-FAM in the knee joint 24 hours after electroporation, as observed under a fluorescence stereomicroscope. Right, Brightfield image of the same joint. C, Synovial tissue from the left knee joints of mice with CIA after electrotransfer of MCT4-specific small interfering RNA (siRNA) or control siRNA (mock) and incubation with a rabbit anti-MCT4 antibody. Bars 5 100 mm. D, Top, Hematoxylin and eosin (H&E)–stained synovial tissue from the knee joints of mice with CIA 72 hours after electrotransfer of MCT4-specific siRNA or control siRNA. Bars 5 100 mm. Middle, Histologic scores (n 5 10 mice per group). Values are the mean 6 SD. * 5 P , 0.05 by Mann-Whitney U test. Bottom, TUNEL assay performed in synovial tissue from the knee joints of mice with CIA transfected with MCT4-specific siRNA or control siRNA. Bars 5 10 mm.

Article Snippet: A rabbit anti-human MCT4 antibody (1:100) or a rabbit anti-mouse MCT4 antibody (1:100; Santa Cruz Biotechnology) served as the primary antibody, and normal rabbit serum (1:100; Wako) served as the control.

Techniques: Knockdown, Control, Incubation, Expressing, Electroporation, Fluorescence, Electrotransfer, Small Interfering RNA, Staining, MANN-WHITNEY, TUNEL Assay, Transfection