Journal: Theranostics

Article Title: Deficiency of the X-inactivation escaping gene KDM5C in clear cell renal cell carcinoma promotes tumorigenicity by reprogramming glycogen metabolism and inhibiting ferroptosis

doi: 10.7150/thno.60233

Figure Lengend Snippet: X-inactivation escaping gene KDM5C harbors frame-shift mutation in ccRCC cell line with the highest glycogen level. A, Periodic acid-schiff (PAS) staining of a series of ccRCC cell lines. Scale bar, 20 µm. B, The glycogen level determination of indicated cell lines. C, The frame-shift mutation of KDM5C identified in RCC4 exome sequencing compared with HK2. D, The KDM5C and VHL protein level detected by Western Blot in various ccRCC cell lines. E, PAS staining of stable cell lines including RCC4-EV, RCC4-KDM5C, RCC4-H514A. Scale bar, 20 µm. F, Bar graph showing the intracellular glycogen content in indicated cells. G, Stacked bar graph showing the gender ratio of frequently mutated genes in KIRC. H, Representative hematoxylin and eosin (H&E) stained (left), KDM5C immunohistochemically stained (middle), and PAS stained (right) sections from the tumor (top) and adjacent normal tissue (bottom). Scale bar, 40 µm. Bar graph showing the statistical analysis of indicated tissues stained with KDM5C antibody. P values were calculated using two-tailed Student's t-test. Data represent means ± SD. Data are representative of three independent experiments.

Article Snippet: The human ccRCC cell lines (RCC4, ACHN, 786-O, 769-P, Caki-1 and A498) and human renal epithelial cell (HK-2) were obtained from ATCC and were grown in high-glucose Dulbecco's modified Eagle's medium (DMEM, Hyclon) supplemented with 10% fetal bovine serum (FBS, AusgeneX) in a humidified atmosphere containing 5% CO 2 at 37 °C.

Techniques: Mutagenesis, Staining, Sequencing, Western Blot, Stable Transfection, Two Tailed Test

Journal: Theranostics

Article Title: Deficiency of the X-inactivation escaping gene KDM5C in clear cell renal cell carcinoma promotes tumorigenicity by reprogramming glycogen metabolism and inhibiting ferroptosis

doi: 10.7150/thno.60233

Figure Lengend Snippet: kdm5c knock out elicit ccRCC-specific metabolic phenotype. A, Western blot analysis of the KDM5C protein level in renal tissues of wild-type and Kdm5c -/- mice. B-D, Hematoxylin and eosin (B), Ki67 immunohistochemical (C), and PAS (D) staining in the renal cortex from indicated mice, Scale bar, 20 µm. E, PAS staining of indicated 769-P cell lines. F-H, The intracellular glycogen content of indicated engineered 769-P (F), ACHN (G), A498 (H) cell lines. P values were calculated using paired t-test. Data represent means ± SD. Data are representative of three independent experiments.

Article Snippet: The human ccRCC cell lines (RCC4, ACHN, 786-O, 769-P, Caki-1 and A498) and human renal epithelial cell (HK-2) were obtained from ATCC and were grown in high-glucose Dulbecco's modified Eagle's medium (DMEM, Hyclon) supplemented with 10% fetal bovine serum (FBS, AusgeneX) in a humidified atmosphere containing 5% CO 2 at 37 °C.

Techniques: Knock-Out, Western Blot, Immunohistochemical staining, Staining

Journal: Theranostics

Article Title: Deficiency of the X-inactivation escaping gene KDM5C in clear cell renal cell carcinoma promotes tumorigenicity by reprogramming glycogen metabolism and inhibiting ferroptosis

doi: 10.7150/thno.60233

Figure Lengend Snippet: KDM5C suppresses the glucose flux through the pentose phosphate pathway. A, Schematic diagram for the conversion of 1,2- 13 C 2 -glucose into various metabolites. The KDM5C regulated genes involved in glycogen metabolism and PPP are indicated in blue. B-D, Control or KDM5C-overexpressing RCC4 cells were cultured with 1,2- 13 C 2 -glucose for 24 h (three replicates per group), and LC-MS was performed to determine (M)+2 G6P (B), (M)+2 6-PG (C), and (M)+1 R5P (D), respectively. E, Heat map of metabolomics showing the absolute intracellular levels of altered metabolites that were products of pentose phosphate pathway and glycolysis. Red, up-regulated; blue, down-regulated. F, The intracellular glucose-6-phosphate (G6P) determination of ccRCC cells. G-I, Quantification of intracellular G6P (G), NADPH (H) and glutathione (I) levels in RCC4 derived cell lines. J-L, Bar graphs showing intracellular G6P (J), NADPH (K) and glutathione (L) levels in indicated cells. P values were calculated using paired t-test. Data represent means ± SD. Data are representative of three independent experiments.

Article Snippet: The human ccRCC cell lines (RCC4, ACHN, 786-O, 769-P, Caki-1 and A498) and human renal epithelial cell (HK-2) were obtained from ATCC and were grown in high-glucose Dulbecco's modified Eagle's medium (DMEM, Hyclon) supplemented with 10% fetal bovine serum (FBS, AusgeneX) in a humidified atmosphere containing 5% CO 2 at 37 °C.

Techniques: Control, Cell Culture, Liquid Chromatography with Mass Spectroscopy, Derivative Assay

Journal: Theranostics

Article Title: Deficiency of the X-inactivation escaping gene KDM5C in clear cell renal cell carcinoma promotes tumorigenicity by reprogramming glycogen metabolism and inhibiting ferroptosis

doi: 10.7150/thno.60233

Figure Lengend Snippet: KDM5C inhibiting tumorigenicity largely by inhibiting ferroptosis. A, Percentage viability of ccRCC cell lines (ACHN, Caki-1, RCC4, 769-P, A498) following exposure to tert-Butyl hydroperoxide (TBHP) at indicated concentrations for 3 h. P -value was calculated by Gehan-Breslow-Wilcoxon test. B, Bar graph showing cell viability in indicated cells treated with 200 µM TBHP combined with 5 µM Z-VAD-FMK (Z-VAD), 2 µM Necrostain-1s (Nec-1s), 2 µM Ferrostatin-1 (Ferr-1) or 50 µM deferoxamine (DFO). C, Indicated cells were loaded with 500 nM CellROX Green and the ROS were analyzed by flow cytometry. The experiment was repeated in triplicate. D, Bar graph showing cell viability in indicated cells treated with 2.5 µM Erastin combined with 5 µM Z-VAD, 2 µM Nec-1s, 2 µM Ferr-1 or 50 µM DFO for 48 h. E, After C11-BODIPY staining in indicated cells, lipid peroxidation was assessed by flow cytometry. The experiment was repeated in triplicate. F, Soft agar assay of RCC4-EV, RCC4-KDM5C, RCC4-KDM5C-H514A with or without liproxstatin-1(Lip-1). G, Image of NOD/SCID mice xenograft tumors derived from RCC4-EV and RCC4-KDM5C cells were treated with 10 mg/kg liproxstatin-1 (Lip-1) or saline, respectively ( n = 6 per group). H, Hematoxylin and eosin (H&E), PAS, and immunohistochemical staining of tumor xenografts derived from RCC4-EV and RCC4-KDM5C cells. Scale bars, 20 µm. I and J, Representative images of transmission electron microscopy of indicated tumor xenografts. Blue arrows, glycogen (I); red arrows, mitochondria (J). Scale bars, 2 µm. P values were calculated using paired t-test (B, D, E). Data represent means ± SD.

Article Snippet: The human ccRCC cell lines (RCC4, ACHN, 786-O, 769-P, Caki-1 and A498) and human renal epithelial cell (HK-2) were obtained from ATCC and were grown in high-glucose Dulbecco's modified Eagle's medium (DMEM, Hyclon) supplemented with 10% fetal bovine serum (FBS, AusgeneX) in a humidified atmosphere containing 5% CO 2 at 37 °C.

Techniques: Flow Cytometry, Staining, Soft Agar Assay, Derivative Assay, Saline, Immunohistochemical staining, Transmission Assay, Electron Microscopy

Journal: Theranostics

Article Title: Deficiency of the X-inactivation escaping gene KDM5C in clear cell renal cell carcinoma promotes tumorigenicity by reprogramming glycogen metabolism and inhibiting ferroptosis

doi: 10.7150/thno.60233

Figure Lengend Snippet: Loss of KDM5C renders both aged kidney cell and ccRCC cells resistant to ferroptosis. A-B, Cell viability assay of indicated Caki-1 cells treated with 400 µM TBHP (A) or 12.5 µM Erastin (B). C-D, Cell viability was measured in KDM5C-knockdown ACHN cells treated with 200 µM TBHP (C) or 1 µM Erastin (D) combined with or without 20 µM glycogen phosphorylase inhibitor (GPI). E, Representative images showing colonies of the KDM5C-knockdown or control ACHN cells on soft agar. F, 4-HNE immunohistochemical staining of kidney tissue from wild-type C57 or kdm5c -/- mice at indicated month, scale bars, 20 µm. G, Representative image of BALB/c Nude mice xenograft tumors derived from Caki-1-shCtrl and Caki-1-shKDM5C cells (left), line chart represented variation of tumor volume(right), ( n = 6 per group). H, Hematoxylin and eosin (H&E), PAS, and 4-HNE immunohistochemical staining of tumor xenografts derived from Caki-1-shCtrl and Caki-1-shKDM5C cells. Scale bars, 20 µm. P values were calculated using paired t-test (A-D). Data represent means ± SD.

Article Snippet: The human ccRCC cell lines (RCC4, ACHN, 786-O, 769-P, Caki-1 and A498) and human renal epithelial cell (HK-2) were obtained from ATCC and were grown in high-glucose Dulbecco's modified Eagle's medium (DMEM, Hyclon) supplemented with 10% fetal bovine serum (FBS, AusgeneX) in a humidified atmosphere containing 5% CO 2 at 37 °C.

Techniques: Viability Assay, Knockdown, Control, Immunohistochemical staining, Staining, Derivative Assay

Journal: Theranostics

Article Title: Deficiency of the X-inactivation escaping gene KDM5C in clear cell renal cell carcinoma promotes tumorigenicity by reprogramming glycogen metabolism and inhibiting ferroptosis

doi: 10.7150/thno.60233

Figure Lengend Snippet: Clinically associated KDM5C mutants fail to reduce the glycogen level and suppress ferroptosis. A, Western blot analysis of KDM5C, H3 and H3K4me3 expression levels in indicated RCC4 cell lines. B, Bar graph showing the intracellular glycogen level in indicated cells. C, Cell viability of indicated cells treated with 2 µM Erastin for 48h. P values were calculated by paired t-test (B, C). Data represent means ± SD. D, A model of inactive KDM5C mutation and ccRCC onset. In females, there are two active alleles of KDM5C , and females are therefore protected from complete gene loss after a single alteration. In contrast, in males, one mutation can inactivate the only allele of KDM5C gene to elicit metabolic alterations and suppress ferroptosis, and males are therefore more likely to develop ccRCC.

Article Snippet: The human ccRCC cell lines (RCC4, ACHN, 786-O, 769-P, Caki-1 and A498) and human renal epithelial cell (HK-2) were obtained from ATCC and were grown in high-glucose Dulbecco's modified Eagle's medium (DMEM, Hyclon) supplemented with 10% fetal bovine serum (FBS, AusgeneX) in a humidified atmosphere containing 5% CO 2 at 37 °C.

Techniques: Western Blot, Expressing, Mutagenesis

Journal: bioRxiv

Article Title: Humanized nucleosomes reshape replication initiation and rDNA/nucleolar integrity in yeast

doi: 10.1101/2023.05.06.539710

Figure Lengend Snippet: ( A ) Representative electron microscopy images showing 10 nm nucleosome fibers. Left, wild-type yeast with native histones ( Saccharomyces cerevisiae, Sc, strain: BY4742; see also ). Right, histone-humanized ( Homo sapiens, Hs , strains: yDT92, yDT180 fibers; see also ). ( B ) Schematic representation of the nucleosome core particle (NCP) with dimensions in nm. ( C ) Bee swarm plots showing the average estimated NCP surface area (nm 2 ) in the wild-type ( Sc ) and histone-humanized strains ( Hs ). Median, S.D. and P values (*** P <0.0015; n.s. P > 0.05) were calculated using a two tailed t-test function (Table S1). ( D ) Boxplots quantifying the difference of the nucleosome fragment length in Hs relative to Sc (DNA fragment length analysis of MNase digested chromatin; data from 3 biological replicates: comparisons of lengths from monoup to penta-nucleosome fragments are shown by each dot Haase et al., co-submission).

Article Snippet: In this specific experiment, we used oligos mapping in the ETS and 18S sequences of the RDN37 repeat to generate by PCR a DNA probe (769-bp long), that was labelled using Klenow Fragment exo- (NEB, M0212L) with Digoxigenin-11-dUTP alkali-stable (Roche, 11093088910) at 37°C.

Techniques: Electron Microscopy, Two Tailed Test

Journal: bioRxiv

Article Title: Humanized nucleosomes reshape replication initiation and rDNA/nucleolar integrity in yeast

doi: 10.1101/2023.05.06.539710

Figure Lengend Snippet: ( A ) Schematic showing the organization of a ribosomal DNA repeat unit with rRNA genes ( 25S , 18S , 5.8S and 5S ) and regulatory sequences ( NTS1 and NTS2 silenced by SIR complex). ( B ) Diagram of RNA extractions from triplicates of Sc (yDT67) and Hs (yDT180 dad1 -E50D) strains and agarose gel used for rRNA quantifications in . ( C ) RT-qPCR bar plot used to estimate changes in the transcription of the NTS1/2 (“+” and “-“ DNA strands transcribed from the bidirectional E-promoter located in NTS1 ) and the rRNA precursor ( ETS ) relative to the control mRNA, ACT1 (see Table S3). ( D ) Total RNA-sequencing coverage tracks at the rDNA unit in Sc and Hs strains (see Table S4). y -axis normalized to read counts per million. ( E ) Growth curves in rich media of the “re-yeastified” strains with dad1- E50D mutation (without Hs histones, Sc : yMAH753-755; Hs histones-maintained, Hs + Sc : yMAH756-758). ( F ) rDNA read count of the “re-yeastified” strains in . ( G ) RNA gel of the “re-yeastified” strains, as descried in panel E ( Sc : yMAH753-755; Hs + Sc : yMAH756-758), relative to the wild-type Sc (yDT67) strain.

Article Snippet: In this specific experiment, we used oligos mapping in the ETS and 18S sequences of the RDN37 repeat to generate by PCR a DNA probe (769-bp long), that was labelled using Klenow Fragment exo- (NEB, M0212L) with Digoxigenin-11-dUTP alkali-stable (Roche, 11093088910) at 37°C.

Techniques: Agarose Gel Electrophoresis, Quantitative RT-PCR, Control, RNA Sequencing Assay, Mutagenesis

Journal: Aging (Albany NY)

Article Title: PLCL1 suppresses tumour progression by regulating AMPK/mTOR-mediated autophagy in renal cell carcinoma

doi: 10.18632/aging.205085

Figure Lengend Snippet: PLCL1 inhibits RCC cancer cell migration and invasion. ( A , B ) Representative RT–qPCR analysis of 786-O and ACHN cells transfected with lentiviral vector encoding PLCLA and lentivirus vector. ( C ) Representative RT–qPCR analysis of 769P cells transfected with PLCL1 siRNA or scrambled control. ( D – F ) CCK8 assays were utilized to determine the proliferation of 786-O, ACHN and 769P cell lines. ( G – I ) Wound healing assays were used to explore the role of PLCL1 in RCC cells, and quantification analyses of the results were performed at 0 and 24 hours. Scale bar: 100 μm. ( J – L ) 786-O, ACHN and 769P cell lines with different treatments were examined by transwell assay. Scale bar: 100 μm. Data are shown as the mean ± SE from three independent experiments. Student’s t test was performed to determine statistical significance between two groups. Scale bar: 100 μm. * P < 0.05; ** P < 0.01; *** P < 0.001.

Article Snippet: The normal human renal cell line HK2 and human renal cancer cell lines ACHN, 786-O, 769P and Caki-1 were purchased from the China Centre for Type Culture Collection (Wuhan, China) and The American Type Culture Collection (USA).

Techniques: Migration, Quantitative RT-PCR, Transfection, Plasmid Preparation, Control, Transwell Assay

Journal: Aging (Albany NY)

Article Title: PLCL1 suppresses tumour progression by regulating AMPK/mTOR-mediated autophagy in renal cell carcinoma

doi: 10.18632/aging.205085

Figure Lengend Snippet: PLCL1 induces apoptosis in RCC cells. ( A , B ) Flow cytometry analysis was utilized to measure apoptosis in 786-O and ACHN cells transfected with PLCL1 or vector. ( C , D ) 769P cells were transfected with siRNA-PLCL1 #1, shRNA-PLCL1 #2 or siRNA-scrambled, and proliferation was examined using Edu kits, nuclei stained blue by DAPI, green for positive cells. Scale bar: 50 μm. ( E – I ) Representative western blots and quantification analysis of Bax and Bcl2 in RCC cells with different treatments. GAPDH was used as a loading control. Data are shown as the mean ± SE from three independent experiments. Student’s t test was performed to determine statistical significance between two groups. * P < 0.05; ** P < 0.01; *** P < 0.001.

Article Snippet: The normal human renal cell line HK2 and human renal cancer cell lines ACHN, 786-O, 769P and Caki-1 were purchased from the China Centre for Type Culture Collection (Wuhan, China) and The American Type Culture Collection (USA).

Techniques: Flow Cytometry, Transfection, Plasmid Preparation, shRNA, Staining, Western Blot, Control

Journal: Aging (Albany NY)

Article Title: PLCL1 suppresses tumour progression by regulating AMPK/mTOR-mediated autophagy in renal cell carcinoma

doi: 10.18632/aging.205085

Figure Lengend Snippet: PLCL1 facilitates autophagy in RCC cells. ( A ) The heatmap of clustering analysis according to the sequencing of differentially expressed genes. ( B ) KEGG pathway enrichment analysis of the top five results. ( C – E ) Transmission electron microscopy indicating the formation of autophagosomes or autolysosomes in RCC cells. Red arrows: autophagosomes or autolysosomes. Scale bar: 1 μm. ( F ) 786-O and ACHN cells transfected with PLCL1 lentivirus and vector were subjected to immunofluorescence and quantitative analysis for LC3B. Scale bar, 20 μm. ( G ) Representative confocal images and quantitative analysis of LC3 dots in 769P cells transfected with siRNA-scramble, siRNA-PLCL1 #1, and shRNA-PLCL1 #2. Scale bar, 20 μm. Data are shown as the mean ± SE from three independent experiments. Student’s t test was performed to determine statistical significance between two groups. * P < 0.05.

Article Snippet: The normal human renal cell line HK2 and human renal cancer cell lines ACHN, 786-O, 769P and Caki-1 were purchased from the China Centre for Type Culture Collection (Wuhan, China) and The American Type Culture Collection (USA).

Techniques: Sequencing, Transmission Assay, Electron Microscopy, Transfection, Plasmid Preparation, Immunofluorescence, shRNA

Journal: Aging (Albany NY)

Article Title: PLCL1 suppresses tumour progression by regulating AMPK/mTOR-mediated autophagy in renal cell carcinoma

doi: 10.18632/aging.205085

Figure Lengend Snippet: PLCL1 promotes autophagic flux by regulating the AMPK/mTOR signalling pathway in RCC cells. ( A – C ) Expression of LC3B and p62 in RCC cells with different treatments was examined using western blotting with quantitative analysis. GAPDH was used as a loading control. ( D , E ) 786-O and 769P cells transfected with GFP-mRFP-LC3B adenovirus were analysed using immunofluorescence. Autolysosome (red dots) and autophagosome (yellow dots) formation are shown using confocal microscopy and were quantitively analysed. Scale bar, 20 μm. ( F – K ) Representative western blotting and quantitative analysis of AMPK/mTOR signalling pathway-related proteins. Data are shown as the mean ± SE from three independent experiments. Student’s t test was performed to determine statistical significance between two groups. * P < 0.05; ** P < 0.01; *** P < 0.001 versus vector or scramble group. # P < 0.05; # P < 0.01; # P < 0.001 versus cells overexpressing PLCLl or siRNA1.

Article Snippet: The normal human renal cell line HK2 and human renal cancer cell lines ACHN, 786-O, 769P and Caki-1 were purchased from the China Centre for Type Culture Collection (Wuhan, China) and The American Type Culture Collection (USA).

Techniques: Expressing, Western Blot, Control, Transfection, Immunofluorescence, Confocal Microscopy, Plasmid Preparation