Journal: Clinical and Translational Medicine

Article Title: Preclinical platforms to study therapeutic efficacy of human γδ T cells

doi: 10.1002/ctm2.814

Figure Lengend Snippet: Epigenetic modifiers increase γδ T cell treatment efficacy. (A) Dose–response curves of bicellular A375‐fibroblast spheroids, A2058‐fibroblast spheroids, patient derived melanoma spheroids and melanoma patient‐derived organoids (MPDOs) after treatment with Entinostat, Decitabine, JQ‐1, Tubastatin A, Valproic acid, Ricolinostat, Vorinostat and DMSO controls for 3 days. Values represent mean ± SEM, n = 4. (B) Expression of MICA/B, FasL, PD‐L1 and PD‐L2 in cells from A2058‐fibroblast spheroids was measured by FACS after Entinostat, Decitabine, JQ‐1, Tubastatin A, Valproic acid, Ricolinostat and Vorinostat treatment (5 μM) for 48 h. * p < 0.05. ** p < 0.01. (C) Western blot analysis of HDAC6, HDAC7, COX2, phospho‐STAT3, total STAT3, phosphor‐ERK, total ERK, phospho‐JNK, total JNK and GAPDH proteins in A2058‐fibroblast spheroids after Entinostat, Decitabine, JQ‐1, Tubastatin A, Valproic acid, Ricolinostat and Vorinostat treatment (5 μM) for 48 h (left panel). Densitometry quantification of the HDAC7, HDAC6 and COX2 (right panel). ** p < 0.01. (D) NKG2A, PD‐1, NKG2D, CXCR3. CD107a and intracellular IFN‐γ in γδ T cells was measured by FACS after Entinostat, Decitabine, JQ‐1, Tubastatin A, Valproic acid, Ricolinostat and Vorinostat treatment (5 μM) for 48 h. * p < 0.05. ** p < 0.01. (E) Cytotoxic function of γδ T cells against A2058‐fibro spheroids and MPDOs combined with 1 μM of Entinostat, Decitabine, JQ‐1, Tubastatin A, Valproic acid, Ricolinostat, Vorinostat and DMSO controls. n = 4. * p < 0.05. ** p < 0.01

Article Snippet: The membranes were incubated with specific primary antibodies at 4°C overnight and HRP‐conjugated secondary antibodies at room temperature for 2 h. The primary antibodies were including rabbit anti‐HDAC6 (#7558), HDAC7 (#10831), total STAT3 (#12640), total ERK (#4695), total JNK (#9258), phospho‐STAT3 (#9145), phosphor‐ERK (#4370), phospho‐JNK (#4668), COX2 (#12282) and GAPDH (Cell Signalling Technology, Beverly, MA, USA).

Techniques: Derivative Assay, Expressing, Western Blot

Journal: Cell Death & Disease

Article Title: High-fat diet induced cyclophilin B enhances STAT3/lncRNA-PVT1 feedforward loop and promotes growth and metastasis in colorectal cancer

doi: 10.1038/s41419-022-05328-0

Figure Lengend Snippet: A XTT assay of Caco-2 cells infected with Lenti-shPVT1, co-infected with either shCypB or STAT3 vectors and corresponding negative control as in Fig. S . Cell cycle analysis B and apoptosis analysis C using FACS in Caco-2 cells. D Expression of CypB, STAT3, and phosphorated STAT3 by immunoblots in Caco-2 cells upon IL-6 treatment (50 ng/mL) for 0–15 min. E qRT-PCR results of STAT3 downstream targets including c-Myc, CCND1, Bcl-2, Bcl-xl, Survivin, Twist, in Caco-2 cells infected with PVT1 shRNAs. F Representative images of IF analysis of CypB (green) and STAT3 (red) in Caco-2 cells infected with PVT1 shRNAs or control while being treated with IL-6 (50 ng/mL) for 15 min. Scale bars: 20 μm. Representative images of three independent experiments were shown. Right: Quantitation of STAT3 distribution. G, H Reciprocal co-IP assay showing interaction of CypB with endogenous STAT3 and phosphorated STAT3 in IL-6-treated Caco-2 cells, using beads coated with CypB antibody G and STAT3 antibody H or respective IgG. Bands were quantified and normalized as to CypB levels in G and STAT3, pSTAT3(Tyr705) and SOCS3 in H . Representative images of three independent experiments were shown. I Tagged STAT3 and truncates were constructed and transfected into Caco2 cells. After IL-6 treatment for 15 min, cell lysates were collected, precipitated with Flag antibody and immunoblotted with CypB antibody. The data are presented as the means ± SDs. * P < 0.05; **P < 0.01; N.S. not significant.

Article Snippet: Denatured proteins were separated on SDS-PAGE and transferred to membranes, followed by immunoblotting using antibodies of CypB (Abcam #ab16045), STAT3 (Cell Signaling Technology, #9139, #12640), pSTAT3 Tyr 705 (Cell Signaling Technology, #9145), SOCS3 (Cell Signaling Technology, #52113), β-actin (Sigma-Aldrich) and Histone H3 (Cell Signaling Technology, #4499).

Techniques: XTT Assay, Infection, Negative Control, Cell Cycle Assay, Expressing, Western Blot, Quantitative RT-PCR, Quantitation Assay, Co-Immunoprecipitation Assay, Construct, Transfection

Journal: Cell Death & Disease

Article Title: High-fat diet induced cyclophilin B enhances STAT3/lncRNA-PVT1 feedforward loop and promotes growth and metastasis in colorectal cancer

doi: 10.1038/s41419-022-05328-0

Figure Lengend Snippet: A HCT116 and Caco-2 cells were treated with IL-6 at concentration of 50 ng/mL for 0–48 h and expression of PVT1 were examined using qRT-PCR. GAPDH were used as internal control. B HCT116 and Caco-2 cells were infected with STAT3 shRNA and treated with IL-6 (50 ng/mL) or control for 24 h and expression of PVT1 were examined using qRT-PCR. C Serially truncated PVT1 promoter constructs were cloned to pGL3-luciferase reporter plasmids and transfected into HCT116 cells. 48 h after transfection, the relative luciferase activities were determined after IL-6 (50 ng/mL) treatment for 30 min. D Top: Schematic diagram showing the binding motifs of STAT3 in the PVT1 promoter. Bottom: Ch-IP targets the promoter of PVT1. E A Ch-IP assay demonstrated the direct binding of STAT3 to the PVT1 promoter in HCT116 cells. M: Marker. F qRT-PCR of the Ch-IP products validated the binding capacity of STAT3 to the PVT1 promoter. The data are presented as the means ± SDs. * P < 0.05; ** P < 0.01; N.S. not significant.

Article Snippet: Denatured proteins were separated on SDS-PAGE and transferred to membranes, followed by immunoblotting using antibodies of CypB (Abcam #ab16045), STAT3 (Cell Signaling Technology, #9139, #12640), pSTAT3 Tyr 705 (Cell Signaling Technology, #9145), SOCS3 (Cell Signaling Technology, #52113), β-actin (Sigma-Aldrich) and Histone H3 (Cell Signaling Technology, #4499).

Techniques: Concentration Assay, Expressing, Quantitative RT-PCR, Infection, shRNA, Construct, Clone Assay, Luciferase, Transfection, Binding Assay, Marker

Journal: Cell Death & Disease

Article Title: High-fat diet induced cyclophilin B enhances STAT3/lncRNA-PVT1 feedforward loop and promotes growth and metastasis in colorectal cancer

doi: 10.1038/s41419-022-05328-0

Figure Lengend Snippet: A – F Correlation of PVT1 and CypB with STAT3-downstream targets using TCGA data of COAD and READ by GEPIA database ( http://gepia.cancer-pku.cn/ ). Y-axis, transcripts per million (TPM) of STAT3 targes; X-axis, TPM of PVT1 and CypB. Expression of PVT1 and CypB were positively correlated with CCND1 A , CDK1 B , BAX C , BIRC5 D , SNAIL1 E , and CTNNB1 F . G The cases in TMA cohort were divided into two groups: high BMI (BMI ≥ 26) and low BMI (BMI <26). The representative images of high level of PVT1, nuclear CypB and pSTAT3 were shown in upper panel, while the low level of PVT1, nuclear CypB and pSTAT3 were shown lower panels. Scale Bars: 100 μm (main); 400 μm (inset). H The levels of BMI levels were positively correlated with expression of PVT1, CypB and pSTAT3 in CRC tissues. I Kaplan-Meier curves of patients with low vs high expression of PVT1 in TMA cohort. J PVT1 levels in TCGA data of COAD (left, P < 2e-12) and READ (right, P < 2e-12). K Kaplan-Meier curves of patients with low vs high expression of PVT1 in TCGA cohort of COAD (left, n = 286; P = 0.0134, HR = 2.077) and READ (right, P = 0.13). L Proposed working model in this study showing HFD-induced IL-6/STAT3 activation expedited by CypB, followed by STAT3 nuclear translocation and transcription of STAT3 targets, which increase cell proliferation, survival, and metastasis. A feedforward regulation of STAT3 promotes PVT1 transcription and its interaction with CypB, which reciprocally promotes STAT3 activation.

Article Snippet: Denatured proteins were separated on SDS-PAGE and transferred to membranes, followed by immunoblotting using antibodies of CypB (Abcam #ab16045), STAT3 (Cell Signaling Technology, #9139, #12640), pSTAT3 Tyr 705 (Cell Signaling Technology, #9145), SOCS3 (Cell Signaling Technology, #52113), β-actin (Sigma-Aldrich) and Histone H3 (Cell Signaling Technology, #4499).

Techniques: Expressing, Activation Assay, Translocation Assay

Journal: Communications Biology

Article Title: Involvement of sphingosine 1-phosphate signaling in insulin-like growth factor-II/mannose 6-phosphate receptor trafficking from endosome to the trans -Golgi network

doi: 10.1038/s42003-024-06828-9

Figure Lengend Snippet: HeLa cells stably expressing CD8-IGF-II/M6P receptor were incubated with 5 µM HACPT, 5 µM VPC23019 or DMSO as a vehicle in serum-depleted medium for 1 h ( A , B ) or transfected with control, SphK1-, SphK2-, or S1P 3 R-siRNA ( C – F ). After transfection, cells were cultured for 48 h at 37 °C and then incubated with anti-CD8α antibody (5 μg/mL) on ice for 30 min in DMEM without serum or bicarbonate. After two times wash with the same medium without the antibody, the cells were incubated for 15 min at 37 °C in serum- and bicarbonate-free medium. Cells were fixed, permeabilized, stained with an antibody against golgin-97, a TGN resident marker or an antibody against Rab5, an early endosome marker, and analyzed by confocal microscopy. Note that SphK1- or S1P 3 R-knockdown decreased the co-localization between CD8-IGF-II/M6P receptor and Golgin97 ( C , D ) while increased that between CD8-IGF-II/M6P receptor and Rab5 which were clearly seen in the inset of ( E ). The 30 cell images each were subjected for Pearson’s coefficient analysis ( B , D , F ). Results were expressed as the mean on scatter-dot plots (** p < 0.01 versus vehicle treatment in ( B ), versus control siRNA in ( D ) and ( F ); Welch’s t -test). Bars represent the mean ± s.e.m. The cell boundaries are indicated as dotted lines. Scale bars, 10 µm.

Article Snippet: Anti-CD8a antibody (Clone RPA-T8) was from BioLegend; rabbit monoclonal anti-CD8a antibody (clone 1B19) was from Merck; rabbit anti-golgin-97 antibody from Proteintech; mouse anti-golgin97 antibody (clone CDF4) from Molecular Probes; anti-Rab5 (clone C8B1), anti-Rab7 (clone D95F2) and anti-Rab11 (clone D4F5) antibodies from Cell Signaling Technologies; anti-DDDDK-tag antibody and anti-α-tubulin antibody from Medical & biological laboratories; anti-VPS35 (clone B-5), anti-CK2α (clone E-7) and anti-SphK1 (clone G-11) from Santa Cruz; rabbit anti-M6PR antibody (clone EPR6599) from Abcam; mouse anti-M6PR antibody (clone 2G11) from Fitzgerald; anti-β-actin antibody from WAKO.

Techniques: Stable Transfection, Expressing, Incubation, Transfection, Control, Cell Culture, Staining, Marker, Confocal Microscopy, Knockdown

Journal: Communications Biology

Article Title: Involvement of sphingosine 1-phosphate signaling in insulin-like growth factor-II/mannose 6-phosphate receptor trafficking from endosome to the trans -Golgi network

doi: 10.1038/s42003-024-06828-9

Figure Lengend Snippet: A HeLa cells were treated with 100 ng/mL PTX, 10 µM Gallein, 10 µM YM-254890, 5 µM VPC23019 or DMSO as a vehicle in serum-depleted DMEM for 24 h (in the case of PTX for overnight) at 37 °C were lysed in 1% Triton X-100 containing Tris buffer and subjected for cathepsin D activity assay. The mean activities were expressed as % of control and on scatter-dot plots (** p < 0.01; * p < 0.05 versus vehicle treatment; NS not significant versus vehicle treatment; Welch’s t -test). Bars represent mean ± s.e.m. B HeLa cells treated with various inhibitors were fixed, permeabilized, stained with an antibody against golgin-97 and analyzed by confocal microscopy. The cell boundaries are indicated as dotted lines. Scale bars, 10 µm. C Thirty cell images each were subjected to Pearson’s coefficient analysis (** p < 0.01 versus vehicle treatment; NS not significant versus vehicle treatment; Welch’s t -test). D HeLa cells stably expressing CD8-IGF-II/M6P receptor were transfected with cDNA encoding mCherry and wild type Gq (Gq WT) or dominant negative Gq (Gq(Q209L/D277N)) at a ratio of 1:3 and cultured for 24 h and incubated with anti-CD8α antibody on ice for 30 min in DMEM without serum or bicarbonate. After washing twice with the same medium, cells were incubated for 15 min at 37 °C in serum- and bicarbonate-free medium. Cells were fixed, permeabilized, stained with an antibody against golgin-97 and analyzed by confocal microscopy. The cell boundaries are indicated as dotted lines. Scale bars, 10 µm ( D ). The 30 cell images each were subjected for Pearson’s coefficient analysis. The cells expressing mCherry were considered to be transfected with Gq(WT) or Gq(Q209L/D277N) ( E ). Results were expressed as the mean on scatter-dot plots (** p < 0.01 versus Gq WT; Welch’s t -test). Bars represent the mean ± s.e.m.

Article Snippet: Anti-CD8a antibody (Clone RPA-T8) was from BioLegend; rabbit monoclonal anti-CD8a antibody (clone 1B19) was from Merck; rabbit anti-golgin-97 antibody from Proteintech; mouse anti-golgin97 antibody (clone CDF4) from Molecular Probes; anti-Rab5 (clone C8B1), anti-Rab7 (clone D95F2) and anti-Rab11 (clone D4F5) antibodies from Cell Signaling Technologies; anti-DDDDK-tag antibody and anti-α-tubulin antibody from Medical & biological laboratories; anti-VPS35 (clone B-5), anti-CK2α (clone E-7) and anti-SphK1 (clone G-11) from Santa Cruz; rabbit anti-M6PR antibody (clone EPR6599) from Abcam; mouse anti-M6PR antibody (clone 2G11) from Fitzgerald; anti-β-actin antibody from WAKO.

Techniques: Activity Assay, Control, Staining, Confocal Microscopy, Stable Transfection, Expressing, Transfection, Dominant Negative Mutation, Cell Culture, Incubation

Journal: Communications Biology

Article Title: Involvement of sphingosine 1-phosphate signaling in insulin-like growth factor-II/mannose 6-phosphate receptor trafficking from endosome to the trans -Golgi network

doi: 10.1038/s42003-024-06828-9

Figure Lengend Snippet: A HeLa cells stably expressing CD8-IGF-II/M6P receptor were transfected with cDNA encoding FLAG-PACS1 and cultured for 48 h under standard conditions and treated without (DMSO) or with 10 µM YM-254890 in serum-depleted medium for 1 h, lysed with lysis buffer containing protease inhibitor cocktail and immunoprecipitated by anti-FLAG beads, followed by immunoblotting analysis using ant-IGF-II/M6P receptor. HeLa cells stably expressing CD8-IGF-II/M6P receptor were transfected with FLAG-PACS1 in the absence ( B ) or presence ( C ) of constitutively active HA-Gq(Q209L), cultured for 48 h and treated with 5 µM VPC23019 or 20 µM apigenin in serum-depleted medium for 1 h. Cells were lysed and subjected to pull-down assay as in ( A ) and detected by anti-FLAG, anti-IGF-II/M6P receptor. The expression of HA-Gq(Q209L) was detected by anti-HA antibody in the lysates. HeLa cells stably expressing CD8-IGF-II/M6P receptor were transfected with control, S1P 3 R- or CK2α-siRNA and FLAG-PACS1 in the absence ( D ) or presence ( E ) of constitutively active HA-Gq(Q209L) and cultured for 48 h. Cells were lysed and subjected to pull-down assay as in ( B , C ) and detected by anti-FLAG or anti-IGF-II/M6P receptor antibody. The expression of HA-Gq(Q209L) was detected by anti-HA antibody in the lysates. The data presented are a typical representative of three independent experiments. F HeLa cells stably expressing CD8-IGF-II/M6P receptor were transfected with control or CK2α-siRNA. Cells were cultured for 48 h at 37 °C and retrograde transport of CD8-IGF-II/M6P receptor was assayed as Fig. (** p < 0.01 versus control siRNA; Welch’s t -test). G HeLa cells transfected with control or CK2α-siRNA were cultured for 48 h in DMEM, and for the last 3 h, cells were incubated in serum-free DMEM containing 100 µg/mL cycloheximide at 37 °C. Pro-cathepsin D in culture media and mature cathepsin in cell lysates were detected by immunoblotting as in Fig. . Cropped images from the experiment in Fig. were shown. Uncropped immunoblots were shown in the Supplementary Figure file. Three experiments were conducted, and the representative images are shown.

Article Snippet: Anti-CD8a antibody (Clone RPA-T8) was from BioLegend; rabbit monoclonal anti-CD8a antibody (clone 1B19) was from Merck; rabbit anti-golgin-97 antibody from Proteintech; mouse anti-golgin97 antibody (clone CDF4) from Molecular Probes; anti-Rab5 (clone C8B1), anti-Rab7 (clone D95F2) and anti-Rab11 (clone D4F5) antibodies from Cell Signaling Technologies; anti-DDDDK-tag antibody and anti-α-tubulin antibody from Medical & biological laboratories; anti-VPS35 (clone B-5), anti-CK2α (clone E-7) and anti-SphK1 (clone G-11) from Santa Cruz; rabbit anti-M6PR antibody (clone EPR6599) from Abcam; mouse anti-M6PR antibody (clone 2G11) from Fitzgerald; anti-β-actin antibody from WAKO.

Techniques: Stable Transfection, Expressing, Transfection, Cell Culture, Lysis, Protease Inhibitor, Immunoprecipitation, Western Blot, Pull Down Assay, Control, Incubation

Journal: Communications Biology

Article Title: Involvement of sphingosine 1-phosphate signaling in insulin-like growth factor-II/mannose 6-phosphate receptor trafficking from endosome to the trans -Golgi network

doi: 10.1038/s42003-024-06828-9

Figure Lengend Snippet: A HeLa cells were transfected with FLAG-PACS1 only (left two lanes), FLAG-PACS1 and control- or CK2α-siRNA (middle two lanes), or FLAG-PACS1 and GFP or GFP-CK2α (right two lanes). After incubation for 48 h cells were lysed and FLAG-PACS1 was immunoprecipitated by anti-FLAG beads. In an experiment, immunoprecipitants were treated with alkaline phosphatase for 30 min (indicated as Phosphatase). The phosphorylation of FLAG-PACS1 was detected by Zn 2+ -phos-tag-biotin and total FLAG-PACS1 was detected by anti-FLAG antibody. B FLAG-PACS1 or FLAG-PACS1(S278A) expressed in HeLa cells was immunoprecipitated and the phosphorylation level was analyzed as in ( A ). C HeLa cells transfected with FLAG-PACS1 were treated with 20 µM apigenin, 10 µM YM-254890, 5 µM HACPT, 5 µM VPC23019 or DMSO as vehicle for 1 h and the phosphorylation of FLAG-PACS1 was analyzed as in ( A ). D HeLa cells were transfected with FLAG-PACS1 in the absence or presence of constitutively active HA-Gq(Q209L), cultured for 48 h and treated with 5 µM VPC23019 or 20 µM apigenin in serum-depleted medium for 1 h. Cells were lysed and the phosphorylation of FLAG-PACS1 was analyzed as in ( A ). The expression of HA-Gq(Q209L) was detected by anti-HA antibody in the lysates. E HeLa cells stably expressing CD8-IGF-II/M6P receptor were transfected with cDNA encoding mCherry and wild type FLAG-PACS1(PACS1(WT)) or phospho-mimicking mutant of PACS1 (PACS1(S278D)) at a ratio of 1:3 and cultured for 48 h and incubated with 5 µM VPC23019 or DMSO as vehicle in serum-depleted medium for 1 h, then retrograde transport of CD8-IGF-II/M6P receptor was assayed as in Fig. . Results were expressed as the mean on scatter-dot plots (** p < 0.01; Welch’s t -test). Bars represent the mean ± s.e.m. The cell images and the expression level of FLAG-PACS1(WT) and FLAG-PACS1(S278D) were shown in Supplementary Fig. .

Article Snippet: Anti-CD8a antibody (Clone RPA-T8) was from BioLegend; rabbit monoclonal anti-CD8a antibody (clone 1B19) was from Merck; rabbit anti-golgin-97 antibody from Proteintech; mouse anti-golgin97 antibody (clone CDF4) from Molecular Probes; anti-Rab5 (clone C8B1), anti-Rab7 (clone D95F2) and anti-Rab11 (clone D4F5) antibodies from Cell Signaling Technologies; anti-DDDDK-tag antibody and anti-α-tubulin antibody from Medical & biological laboratories; anti-VPS35 (clone B-5), anti-CK2α (clone E-7) and anti-SphK1 (clone G-11) from Santa Cruz; rabbit anti-M6PR antibody (clone EPR6599) from Abcam; mouse anti-M6PR antibody (clone 2G11) from Fitzgerald; anti-β-actin antibody from WAKO.

Techniques: Transfection, Control, Incubation, Immunoprecipitation, Cell Culture, Expressing, Stable Transfection, Mutagenesis