Additional file 1 of Diacerein versus non-steroidal anti-inflammatory drugs in the treatment of knee osteoarthritis: a meta-analysis

Additional file 1: Search strategies

Data_Sheet_2_Comprehensive Analysis of the Clinical and Biological Significances of Endoplasmic Reticulum Stress in Diffuse Gliomas.pdf

BackgroundAs a critical organelle for protein and lipid synthesis, the dysfunction of endoplasmic reticulum has a significant impact on multiple biological processes of cells. Thus, in this study, we constructed an ER stress-related risk signature to investigate the functional roles of ER stress in gliomas.MethodsA total of 626 samples from TCGA RNA-seq dataset (training cohort) and 310 samples from CGGA RNA-seq dataset (validation cohort) were enrolled in this study. Clinical information and genomic profiles were also obtained. The ER stress signature was developed by the LASSO regression model. The prognostic value of the risk signature was evaluated by Cox regression, Kaplan-Meier and ROC Curve analyses. Bioinformatics analysis and experiment in vitro were performed to explore the biological implication of this signature.ResultsWe found that the ER stress-related signature was tightly associated with major clinicopathological features and genomic alterations of gliomas. Kaplan-Meier curve and Cox regression analysis indicated that ER stress activation was an independent prognostic factor for patients with glioma. Besides, we also constructed an individualized prognosis prediction model through Nomogram and ROC Curve analysis. Bioinformatics analysis suggested that ER stress activation also promoted the malignant progression of glioma and participated in the regulation of tumor immune microenvironment, especially the infiltration of macrophages in M2 phase. These results were further validated in IHC analysis and cell biology experiments.ConclusionThe ER stress activation had a high prognostic value and could serve as a promising target for developing individualized treatment of glioma.</p

Data_Sheet_1_Comprehensive Analysis of the Clinical and Biological Significances of Endoplasmic Reticulum Stress in Diffuse Gliomas.PDF

BackgroundAs a critical organelle for protein and lipid synthesis, the dysfunction of endoplasmic reticulum has a significant impact on multiple biological processes of cells. Thus, in this study, we constructed an ER stress-related risk signature to investigate the functional roles of ER stress in gliomas.MethodsA total of 626 samples from TCGA RNA-seq dataset (training cohort) and 310 samples from CGGA RNA-seq dataset (validation cohort) were enrolled in this study. Clinical information and genomic profiles were also obtained. The ER stress signature was developed by the LASSO regression model. The prognostic value of the risk signature was evaluated by Cox regression, Kaplan-Meier and ROC Curve analyses. Bioinformatics analysis and experiment in vitro were performed to explore the biological implication of this signature.ResultsWe found that the ER stress-related signature was tightly associated with major clinicopathological features and genomic alterations of gliomas. Kaplan-Meier curve and Cox regression analysis indicated that ER stress activation was an independent prognostic factor for patients with glioma. Besides, we also constructed an individualized prognosis prediction model through Nomogram and ROC Curve analysis. Bioinformatics analysis suggested that ER stress activation also promoted the malignant progression of glioma and participated in the regulation of tumor immune microenvironment, especially the infiltration of macrophages in M2 phase. These results were further validated in IHC analysis and cell biology experiments.ConclusionThe ER stress activation had a high prognostic value and could serve as a promising target for developing individualized treatment of glioma.</p

Additional file 2 of Diacerein versus non-steroidal anti-inflammatory drugs in the treatment of knee osteoarthritis: a meta-analysis

Additional file 2: Results of fixed effects model

CRISPR rescues and possible expansion of ICs in Islet>Sp6/7/8.

(A) CryBG>GFP expression in Islet (left) and Foxg (right) CRISPR larvae is rescued by co-electroporation with Islet intron 1 + bpFOG>Flag::Islet-rescue or Foxg>Foxg-rescue constructs, respectively, thanks to silent point mutations disrupting the sgRNA target binding sites. (B) Expression of C11.360>GFP is rescued in Sp6/7/8 CRISPR larvae upon co-electroporation with an Islet intron 1 + bpFOG>Sp6/7/8-rescue construct. (C) Example of expanded IC reporter (C11.360>Unc-76::GFP, green) in larvae (20 hpf/20 °C, ~st. 29) electroporated with Islet intron 1 + -473/-9>Sp6/7/8, as determined by perfect overlap with the Islet intron 1 + -473/-9>H2B::mCherry reporter (pink). See text for more details. See S1 File for exact sequences and detailed electroporation recipes. See S4 Data for the data underlying the graphs and for statistical test details. (TIF)</p

Novel genetic markers label distinct cell types of the papillae.

(A) GFP reporter plasmid (green) constructed using the cis-regulatory sequences from the KH.L96.43 gene labels basal cells in between and surrounding the protruding papillae labeled by Foxg reporter plasmid (pink). (B) TGFB>GFP reporter (green) labels PNs, the axons of which make contacts with BTN axons labeled by a BTN-specific Islet reporter (pink), at 23.5 h hpf, approximately corresponding to Hotta stage 30. (C) A KH.C4.78 reporter (C4.78>GFP) also labels PNs, which are also labeled by Foxg>H2B::mCherry (mCh) reporter (pink nuclei). (D) Lack of overlap between expression of C4.78>GFP (green) and a papilla-specific Islet reporter plasmid (pink nuclei) showing that PNs do not arise from Islet+ cells. (E, F) Co-electroporation of C11.360>GFP (green) with H2B::mCherry reporter plasmids (pink nuclei) indicates these cells come from Foxg-expressing cells that also express Islet. (G) C11.360>mCherry reporter (pink) labels centrally located ICs adjacent to ACCs labeled by CryBG>LacZ reporter (green). (H, I) L141.36>GFP reporter (green) labels OCs that arise from Foxg+ cells (pink nuclei) but do not express Islet (pink nuclei). (J) ICs and OCs are distinct cells as there is no overlap between C11.360 (green) and L141.36 (pink) reporter plasmid expression. (K) Ciona intestinalis (Type B) larva ICs labeled with a reporter plasmid made from the corresponding cis-regulatory sequence of the C. intestinalis Chr11.1038 gene, orthologous to C. robusta KH.C11.360. (L) C. intestinalis larva OCs labeled by a Chr7.130 reporter, corresponding to the C. robusta ortholog KH.L141.36. (M) Summary of the main marker genes and corresponding reporter plasmids used in this study to label different subsets of papilla progenitors and their derivative cell types. All GFP and mCherry reporters fused to the Unc-76 tag, unless specified (see Methods and supplement for details). Weaker Foxg -2863/-3 promoter used in panel A, all other Foxg reporters used the improved Foxg -2863/+54 sequence instead. All Islet reporters shown correspond to the Islet intron 1 + bpFOG>H2B::mCherry plasmid. White channel shows either DAPI (nuclei) and/or larva outline in brightfield, depending on the panel. All C. robusta raised at 20 °C to 18 hpf (roughly st. 28) except: panel B (23.5 hpf, ~st. 30); panels C–F (17 hpf, ~st. 27); panels H–J (20 hpf, ~st. 29). C. intestinalis raised at 18 °C to 20–22 hpf (Hotta stage 28). ACC, axial columnar cell; BTN, bipolar tail neuron; hpf, hours post-fertilization; IC, inner collocyte; OC, outer collocyte; PN, papilla neuron.</p

Validation of sgRNAs for CRISPR/Cas9-mediated mutagenesis.

Gene loci diagrams for the 4 transcription factor-encoding genes investigated in this study: Sp6/7/8, Foxg, Islet, and Pou4. Plots underneath each gene show validation by Illumina sequencing (“Next-generation sequencing” or NGS) of amplicons, performed as “Amplicon-EZ” service by Azenta. Mutagenesis efficacies are calculated by this service, and histograms of mapped reads show specificity of indels elicited by each sgRNA. Negative control amplicons are amplified from samples that were electroporated with no sgRNA, U6>Control sgRNA, or sgRNAs targeting unrelated amplicon regions. Note different y axis scales for each plot. Asterisks in Villin exon 5 and Tuba3 amplicon plots indicate naturally occurring indels. Precise calculation of mutagenesis efficacy for Villin.5.105 and Tuba3.3.24 sgRNAs was not given due to these natural indels. (TIF)</p

Development of the papillae of Ciona.

(A) Diagram showing the early cell lineages that give rise to the papillae. The papillae invariantly derive from Foxc+ cells in the anterior neural plate, more specifically the anterior daughter cells of “Row 6” of the neural plate, which activate Foxg downstream of Foxc. Foxg is also activated in the posterior daughter cells of “Row 5,” which go on to give rise to part of the OSP. Numbers in each cell indicate their invariant identity according to the Conklin cell lineage nomenclature. Black bars indicate sibling cells born from the same mother cell. (B) Diagram of what is currently known about the later lineage and fates of the Foxg+ “Anterior Row 6” cells shown in panel A. As the cells divide mediolaterally, some cells up-regulate Sp6/7/8 and down-regulate Foxg (gray cells). Those cells that maintain Foxg expression turn on Islet and coalesce as 3 clusters of cells (pink with green outline): 1 medial, more ventral cluster, and 2 left/right, more dorsal clusters. Later, these 3 clusters organize the territory into the 3 protruding papillae of the larva, which contains several cell types described in detail by TEM [9]. Dashed cell outlines indicate uncertain number/provenance of cells. A-P: anterior-posterior. D-V: dorsal-ventral. Lineages and gene networks are based mostly on: [21,22,86,87]. OSP, oral siphon primordium; TEM, transmission electron microscopy.</p

Genetic perturbations of metamorphosis.

(A) Ciona robusta juvenile undergoing metamorphosis, showing the retracted tail and rotated anterior-posterior body axis (dashed lines). PNs in the former papilla (now substrate attachment stolon, or holdfast) labeled by TGFB>Unc-76::GFP (green). Animal counterstained with DAPI (blue). (B) Scoring of Foxc>H2B::mCherry+ individuals showing tail retraction and/or body rotation at 48 hpf/20 °C in various papilla territory-specific (using Foxc>Cas9) CRISPR-based gene knockouts. Experiments were performed and scored in duplicate and percentages averaged, except for Foxg CRISPR for which a third replicate was performed (see S9 Fig). Number scored individuals in each replicate indicated underneath. “Tailed juveniles” have undergone body rotation but not tail retraction, whereas normally body rotation follows tail retraction. The sgRNA plasmids used for each condition were as follows- Control: U6>Control; Pou4: U6>Pou4.3.21 + U6>Pou4.4.106; Islet: U6>Islet.2; Foxg: U6>Foxg.1.116 + U6>Foxg.5.419; Sp6/7/8: U6>Sp6/7/8.4.29 + U6>Sp6/7/8.8.117. (C) Plot showing lack of any discernable metamorphosis defect after eliminating ACCs using Islet intron 1 + bpFOG>Sp6/7/8 (images not shown). Only Islet intron 1 + bpFOG>H2B::mCherry+ individuals were scored. Experiment was performed and scored in duplicate and averaged (n = 100 each duplicate). ACC specification was scored using the CryBG>Unc-76::GFP reporter. (D) Example of “tailed juveniles” at 47 hpf/20 °C compared to a larva in which no tail retraction or body rotation has occurred, elicited by tissue-specific Foxg CRISPR (Foxc>Cas9 + U6>Foxg.1.116 + U6>Foxg5.419). See S9 Fig for scoring. All error bars denote upper and lower limits. **** p p S4 Data for the data underlying the graphs and for statistical test details. ACC, axial columnar cell; PN, papilla neuron; sgRNA, single-chain guide RNA.</p

Islet is also required for papilla morphogenesis.

(A) Papilla shape is shortened and blunt at the apical end upon tissue-specific CRISPR/Cas9-mediated mutagenesis of Islet. Embryos were electroporated with Islet intron 1 + -473/-9>Unc-76::GFP and Foxc>Cas9. Islet CRISPR was performed using U6>Islet.2 sgRNA plasmid and the negative control used U6>Control. Larvae were imaged at 20 hpf/20 °C (~st. 28). Right: Scoring of percentage of GFP+ larvae classified as having normal “protruding” or blunt papillae, as represented to the left. Experiment was performed and scored in duplicate, using 2 different GFP fusions: Unc-76::GFP and DcxΔC::GFP [88]. Replicate 1: n = 100 for either condition; replicate 2: n = 55 for either condition **** p Islet intron 1 +-473/-9>Unc-76::GFP+) lengths along apical-basal axis in negative control and Islet CRISPR larvae at 18 hpf/20 °C (~st. 28). Both Islet.1 and Islet.2 sgRNAs used in combination. Statistical significance tested by unpaired t test (two-tailed). See S8 Fig for duplicate experiment. (C) In situ mRNA hybridization of Villin, showing expression in Foxg+/Islet+ central papilla cells at 10 hpf/20 °C (st. 21, left) and at larval stage (~st. 27, right). (D) Villin -1978/-1>Unc-76::GFP showing expression in the papilla territory of electroporated larvae (~st. 28), strongest in the central cells. (E) Villin -1978/-1>Unc-76::GFP in st. 28 larvae is down-regulated by tissue-specific CRISPR/Cas9 mutagenesis of Islet (Foxc>Cas9 + U6>Islet.1 + U6>Islet.2, see text for details). (F) Quantification of effect of Islet CRISPR (as in panel E) on Villin -1978/-1>Unc 76::GFP/Foxc>H2B::mCherry mean fluorescence intensity ratios in ROIs defined by the mCherry+ nuclei (see Methods for details). Significance determined by Mann–Whitney test (two-tailed). (G) Villin reporter is up-regulated in st. 28 larvae by overexpressing Islet (Foxc>Islet, see text for details). (H) GFP/mCherry ratio quantification done in identical manner as in F, but comparing Islet overexpression (as in panel G) and control lacZ larvae. (I) Quantification of ACC lengths measured in negative control and papilla-specific Villin CRISPR larvae at 17 hpf/20 °C (~st. 27). Significance tested by unpaired t test (two-tailed). Although no statistically significant difference between control and CRISPR larvae was observed in this replicate, average ACC length was significantly shorter in the CRISPR condition in an additional replicate (S8 Fig). (J) mRNA in situ hybridization for Tuba3, showing enrichment in the central cells of the papillae in st. 27 larvae. (K) Tuba3>Unc-76::GFP reporter plasmid is broadly expressed in the papillae of st. 27 larvae but stronger in central cells. (L) Papilla-specific CRISPR knockout of Tuba3 does not result in decrease of average ACC apical-basal cell length compared to negative control CRISPR using U6>Control sgRNA instead. Significance tested by unpaired t test (two-tailed). ns = not significant. All large bars indicate medians and smaller bars indicate interquartile ranges. See S3 and S4 Data for the data underlying the graphs and for statistical test details. ACC, axial columnar cell; ROI, region of interest; sgRNA, single-chain guide RNA.</p