Journal: Human Mutation

Article Title: PLSCR3 Deficiency Triggers mtDNA‐Driven cGAS‐STING Activation to Potentiate Antitumor Immunity in Colorectal Cancer

doi: 10.1155/humu/8545428

Figure Lengend Snippet: PLSCR3 deficiency disrupts mitochondrial homeostasis in CRC cells. (A) Western blotting verified the reduction of PLSCR3 protein levels in HT29 cells. (B) RT‐qPCR analysis verified reduction of PLSCR3 mRNA levels normalized to GAPDH compared with negative control (NC) cells ( p < 0.0001, n = 3). (C) Representative JC‐1 staining (scale bar: 20 μ m) images showing red fluorescence (polarized mitochondria) and green fluorescence (depolarized mitochondria). (D) Quantification of the JC‐1 red/green fluorescence intensity ratio. Fluorescence intensity was quantified using ImageJ software as described in the section. (E) Real‐time oxygen consumption rate (OCR) profiles under sequential treatment with oligomycin, FCCP, and rotenone/Antimycin A. (F) Quantification of cytosolic mtDNA by qPCR using the D‐loop ratio in PLSCR3‐knockdown and control cells. Data are presented as mean ± SD from three independent experiments. Statistical significance was determined using an unpaired two‐tailed Student′s t ‐test for two‐group comparisons.

Article Snippet: The human colon carcinoma cell line HT29 (Cat. No. ATCC‐HTB‐38) and the mouse colon carcinoma cell line CT26.WT (Cat. No. CRL‐2638, ATCC, Virginia, United States) were maintained in RPMI 1640 medium (Cat. No. BC‐M‐017, Bio‐Channel, Jiangsu, China) supplemented with 10% fetal bovine serum.

Techniques: Western Blot, Quantitative RT-PCR, Negative Control, Staining, Fluorescence, Software, Knockdown, Control, Two Tailed Test

Journal: Human Mutation

Article Title: PLSCR3 Deficiency Triggers mtDNA‐Driven cGAS‐STING Activation to Potentiate Antitumor Immunity in Colorectal Cancer

doi: 10.1155/humu/8545428

Figure Lengend Snippet: PLSCR3 deficiency activates mtDNA‐associated cGAS‐STING signaling. (A) 2 ′ ‐3 ′ ‐cGAMP quantification by ELISA in PLSCR3 knockdown (KD_PLSCR3) HT29 cells and normal controls (NCs) treated with Scramble or dideoxycytidine (DDC) ( p < 0.0001; ns = not significant; n = 3). (B, C) KD_PLSCR3 cells exhibited an elevation in IFN β and CXCL10 production measured by ELISA compared to NC ( p < 0.0001, n = 3). CRISPR‐mediated STING knockout (STING sg) completely abrogated these effects. (D) Western blot analysis showed upregulation of phosphorylated STING and innate immune effectors (IFIH1, ISG60, and ISG15 protein) in STING‐competent KD_PLSCR3 cells relative to GAPDH loading controls. (E) Normalized mRNA level quantification by qPCR demonstrated significant induction of interferon‐stimulated genes (ISGs) in STING‐competent KD_PLSCR3 cells compared with the other groups. Data in panels A–C and E are presented as mean ± SD. Statistical significance was determined using one‐way ANOVA followed by Tukey′s multiple‐comparisons post hoc test for multiple‐group comparisons.

Article Snippet: The human colon carcinoma cell line HT29 (Cat. No. ATCC‐HTB‐38) and the mouse colon carcinoma cell line CT26.WT (Cat. No. CRL‐2638, ATCC, Virginia, United States) were maintained in RPMI 1640 medium (Cat. No. BC‐M‐017, Bio‐Channel, Jiangsu, China) supplemented with 10% fetal bovine serum.

Techniques: Enzyme-linked Immunosorbent Assay, Knockdown, CRISPR, Knock-Out, Western Blot

Journal: Human Mutation

Article Title: PLSCR3 Deficiency Triggers mtDNA‐Driven cGAS‐STING Activation to Potentiate Antitumor Immunity in Colorectal Cancer

doi: 10.1155/humu/8545428

Figure Lengend Snippet: PLSCR3 deficiency enhances the sensitivity to immune cell–mediated killing in CRC. (A) Schematic of coculture system: PLSCR3 knockdown (KD_PLSCR3) HT29 cells or control were cocultured with human cord blood–derived NK cells at an E:T ratio of 5:1. (B, C) Phase‐contrast microscopy (scale bar: 50 μ m) showing morphological changes. Morphological analysis demonstrated decreased cell number in KD_PLSCR3 cells in the coculture condition ( p < 0.0001, n = 5). (D) Mouse graft carcinoma with control or PLSCR3 knockout (PLSCR3 sg) CT26 cells were treated with or without anti‐PD‐1 antibodies ( α PD‐1). Tumor growth curves (mm 3 ) were recorded at Days 5, 10, 15, and 20 after tumor cell inoculation in immunocompetent BALB/c mice. (E) At Day 20, terminal tumor weights in the mouse graft carcinoma were measured. (F–H) Flow cytometry analysis of CD4+ and CD8+ tumor‐infiltrating T cells. The representative cell populations of CD4+ and CD8+ are shown. (I, J) Flow cytometry and ELISA analysis of Granzyme B (GZMB) expression ( n = 5). Data are presented as mean ± SD. Tumor growth curves were analyzed using two‐way repeated‐measures ANOVA with post hoc multiple‐comparison tests. For multiple‐group comparisons, including panels I and J, one‐way ANOVA followed by Tukey′s post hoc test was used.

Article Snippet: The human colon carcinoma cell line HT29 (Cat. No. ATCC‐HTB‐38) and the mouse colon carcinoma cell line CT26.WT (Cat. No. CRL‐2638, ATCC, Virginia, United States) were maintained in RPMI 1640 medium (Cat. No. BC‐M‐017, Bio‐Channel, Jiangsu, China) supplemented with 10% fetal bovine serum.

Techniques: Knockdown, Control, Derivative Assay, Microscopy, Knock-Out, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Expressing, Comparison

Journal: Foods

Article Title: Valorization and Functional Enhancement of Mature Assam Tea Leaves Through Indigenous Filamentous Fungi-Based Fermentation for Functional Drink Development

doi: 10.3390/foods15091562

Figure Lengend Snippet: The cytotoxicity of Caco-2 cells ( A ) and HT-29 cells ( B ) after treatment with co-fermented mature tea leaves samples for 72 h. Different lowercase letters indicate significant differences among fermentation times within the same treatment ( p < 0.05).

Article Snippet: Human colon carcinoma cell line (Caco-2) and Human colon adenocarcinoma cell line (HT-29) obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA) were used to carry out the cell cytotoxicity test.

Techniques:

Journal: Aging Cell

Article Title: Decreased Glucose Metabolism and Declined Chaperones Are Unique Features Required for the Survival of Senescent Fibroblasts and Pyruvate Dehydrogenase Is a Potent Senolytic Target

doi: 10.1111/acel.70434

Figure Lengend Snippet: Inhibiting PDH, GLS1 and Hsp90 by the combination of CPI‐613+BPTES+17‐AAG gave rise to enhanced senolysis on senescent fibroblasts as well as the therapy‐induced senescent tumor cells. (A, B) The effects of CPI‐613+BPTES+17‐AAG combination treatment on proliferating (A) and senescent (B) BJ cells. For the dose of each compound in use, see the results 2.7 section for more details. ** p < 0.01 by Student's t ‐test. (C, D) The effects of CPI‐613+BPTES+17‐AAG combination treatment on proliferating and Dox‐induced senescent lung adenocarcinoma A549 cells. *** p < 0.001 by Student's t ‐test. (E, F) The effects of CPI‐613+BPTES+17‐AAG combination treatment on proliferating and Dox‐induced senescent cervical carcinoma HeLa cells. *** p < 0.001 by Student's t ‐test. (G) The morphological changes of senescent BJ induced by IR, senescent A549 and HeLa cells induced by Dox at the indicated time of CPI‐613+BPTES+17‐AAG treatment under the light microscopy. The senescent cells without treatment were stained with SA‐β‐gal. Cells were imaged at magnification 200×. (H) The schematic summarization of our findings. The activities of TCA cycle and chaperones are reduced in DNA damage induced senescent cells. Co‐inhibiting Hsp90 and TCA cycle with 17‐AAG+CPI‐613+BPTES combination leads to enhanced selective elimination of senescent cells, hinting TCA cycle and glutaminolysis are novel and potent targets for senolysis.

Article Snippet: Human fibroblasts BJ (RRID: CVCL_3653; ATCC Cat#CRL‐2522), IMR‐90 (RRID: CVCL_0347; ATCC Cat#CCL‐186), WI‐38 (RRID: CVCL_0579; ATCC Cat#CCL‐75) and human cancer cell lines HeLa (RRID: CVCL_0030; ATCC Cat#CRM‐CCL‐2), A549 (RRID: CVCL_0023; ATCC Cat#CRM‐CCL‐185), and U2OS (RRID: CVCL_0042; ATCC Cat#HTB‐96) were purchased from American Type Culture Collection (ATCC) and cultured in Dulbecco's modified Eagle's medium (DMEM, Gibco) supplemented with 10% fetal bovine serum (FBS, Invitrogen) and 1% penicillin/streptomycin at 37°C under 5% CO 2 .

Techniques: Light Microscopy, Staining

Journal: Aging Cell

Article Title: Decreased Glucose Metabolism and Declined Chaperones Are Unique Features Required for the Survival of Senescent Fibroblasts and Pyruvate Dehydrogenase Is a Potent Senolytic Target

doi: 10.1111/acel.70434

Figure Lengend Snippet: Distinct proteomic and transcriptional signatures of metabolic enzymes and chaperones between senescent fibroblasts and the therapy‐induced senescent tumor cells. (A) The abundance of glycolysis‐related enzymes PFKP, ALDOA, PKM2, TCA cycle‐related PDHA, and glutaminolysis‐related GLS1 was decreased significantly in senescent BJ and IMR‐90 cells compared to their proliferating counterparts. (B) The protein levels of chaperones TCP1, Hsp70, and Hsp90 were decreased significantly in senescent BJ and IMR‐90 cells. (C) The abundance of those glycolysis‐related enzymes remained unchanged or even elevated in Dox‐induced senescent A549, HeLa, and U2OS tumor cells compared to their proliferating counterparts. (D) The abundance of these chaperone proteins in Dox‐induced senescent A549, HeLa, and U2OS tumor cells remained nearly unchanged. The relative abundance of each protein was quantified by signal density scanning on Western blots and normalized to the signal of β‐Actin or β‐tubulin. * p < 0.05, ** p < 0.01 tested by Student's t ‐test.

Article Snippet: Human fibroblasts BJ (RRID: CVCL_3653; ATCC Cat#CRL‐2522), IMR‐90 (RRID: CVCL_0347; ATCC Cat#CCL‐186), WI‐38 (RRID: CVCL_0579; ATCC Cat#CCL‐75) and human cancer cell lines HeLa (RRID: CVCL_0030; ATCC Cat#CRM‐CCL‐2), A549 (RRID: CVCL_0023; ATCC Cat#CRM‐CCL‐185), and U2OS (RRID: CVCL_0042; ATCC Cat#HTB‐96) were purchased from American Type Culture Collection (ATCC) and cultured in Dulbecco's modified Eagle's medium (DMEM, Gibco) supplemented with 10% fetal bovine serum (FBS, Invitrogen) and 1% penicillin/streptomycin at 37°C under 5% CO 2 .

Techniques: Western Blot