Journal: bioRxiv

Article Title: Haploinsufficiency of the essential gene RpS12 causes defects in erythropoiesis and hematopoietic stem cell maintenance

doi: 10.1101/2021.05.04.442585

Figure Lengend Snippet: (A) Diagram of the WT, Flox and KO alleles of RpS12 generated in this study indicating the position of Snord100 and Snora33 (snoRNA),Cas9 gRNAs target locations, and primers used for genotyping. The homology arms starting sites are indicated and the ends fall outside of the RpS12 locus. To identify the first transformants, two pair of primers were used for PCR amplification: F2/R2 and F3/R3. F2 and R3 fall outside of the sequence covered by the homology arms, to ensure the inserts are on the correct location. The presence of LoxP sites was confirmed by Sanger sequencing using primers F1 and F4 for F2/R2 fragments, and with F3 and R3 for F3/R3 fragments. To determine excision of exon 2 and 3 by Cre recombination primers F1 and R1 were used, which generate a 900bp fragment in RpS12 + and a 300bp fragment in RpS12 KO (B) .

Article Snippet: The following primary antibodies were used: RpS12 (Proteintech; polyclonal), β-actin (Cell Signaling; 13E5), eIF2α (Cell signaling, 5324T), phospho-eIF2α (Thermo Scientific; Ser52; polyclonal).

Techniques: Generated, Amplification, Sequencing

Journal: bioRxiv

Article Title: Haploinsufficiency of the essential gene RpS12 causes defects in erythropoiesis and hematopoietic stem cell maintenance

doi: 10.1101/2021.05.04.442585

Figure Lengend Snippet: (A) Conditional RpS12 flox transgenic knock-in has two loxP sites flanking exons 2 and 3, that are removed by Cre recombinase activity to generate RpS12 KO . (B) Post-natal growth curve of RpS12 KO/+ and RpS12 +/+ littermates (+/+ n=8 and KO/+ n=11 pups). (C) Picture of 5-day-old RpS12 KO/+ and RpS12 +/+ littermates. (D) Representative picture of “kinked” tail in RpS12 KO/+ mouse. (E) Representative picture of the anterior footpad hyperpigmentation in RpS12 KO/+ . (F) Quantification of the percentage of mice presenting hydrocephalus per litter (n=27 litters, 2-way ANOVA p=0.0035). (G) Kaplan-Meier survival curves of RpS12 KO/+ and RpS12 +/+ littermates starting at day 5 of age (+/+ n=39 and KO/+ n=60, log-rank Mantel-Cox test p=0.012). (H) Embryo genotype segregation from crosses between RpS12 KO/+ male and female. Graph represents percentage of developed embryos and the table shows the total numbers (E%=expected percentages, E#=expected numbers, O=observed numbers). (I) Representative pictures of E13.5 embryos with their placentas. (J) E13.5 embryo weights (n=10 on each genotype, unpaired t-test p=0.0420).

Article Snippet: The following primary antibodies were used: RpS12 (Proteintech; polyclonal), β-actin (Cell Signaling; 13E5), eIF2α (Cell signaling, 5324T), phospho-eIF2α (Thermo Scientific; Ser52; polyclonal).

Techniques: Transgenic Assay, Knock-In, Activity Assay

Journal: bioRxiv

Article Title: Haploinsufficiency of the essential gene RpS12 causes defects in erythropoiesis and hematopoietic stem cell maintenance

doi: 10.1101/2021.05.04.442585

Figure Lengend Snippet: (A) Non-competitive BM transplant strategy testing the long-term reconstituting activity of RpS12 KO/+ HSCs. 10 6 bone marrow cells from RpS12 KO/+ or RpS12 flox/flox samples (CD45.2+) were transplanted into lethally irradiated B6.SJL (CD45.1+) mice, peripheral blood chimerism was determined every 4 weeks. (B) Kaplan-Meier survival curves of mice transplanted with BM cells from RpS12 KO/+ and control RpS12 flox/+ or RpS12 flox/flox mice (control n=20 and KO/+ n=20 transplanted mice, combination of 2 independent non-competitive transplants with 1 donor per genotype transplanted into 10 host mice each). (C) Frequency of recipient mice with long-term (20-weeks) multi-lineage reconstitution (≥0.5% in all three macrophages, B, and T cells)(control n=20 and KO/+ n=20 transplanted mice, combination of 2 independent non-competitive transplants). (D-G) Peripheral blood donor derived (D) total chimerism and (E-G) multi-lineage chimerism in non-competitively transplanted whole bone marrow (CD45.2+) recipients (flox/flox n=10 and KO/+ n=10). (H) Schematic representation of the competitive bone marrow transplant. 5×10 5 cells from RpS12 KO/+ or RpS12 flox/+ donor bone marrow (CD45.2+) mixed with 5×10 5 competitor bone marrow cells from B6.SJL (CD45.1+) mice were injected into lethally irradiated B6.SJL (CD45.1+) mice. Chimerism in peripheral blood was determined every 4 weeks and bone marrow chimerism was analyzed at 20 weeks after transplant. (I) Total bone marrow chimerism and (J) HSCs donor-derived (CD45.2+) chimerism in the recipient bone marrow (Flox/+ n=10 and KO/+ n=10 competitive-transplanted mice). (K-N) Donor derived peripheral blood chimerism of competitively transplanted RpS12 KO/+ or RpS12 flox/+ bone marrow cells as described in H . Non-competitive transplants were performed twice, using different controls: RpS12 flox/+ or RpS12 flox/flox . The competitive transplant was performed once, using RpS12 flox/+ mice as a control group Statistical analysis: data represent mean +/-SEM, unpaired t-tests were performed to assess significance among populations between genotypes *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001

Article Snippet: The following primary antibodies were used: RpS12 (Proteintech; polyclonal), β-actin (Cell Signaling; 13E5), eIF2α (Cell signaling, 5324T), phospho-eIF2α (Thermo Scientific; Ser52; polyclonal).

Techniques: Activity Assay, Irradiation, Control, Derivative Assay, Injection

Journal: bioRxiv

Article Title: Haploinsufficiency of the essential gene RpS12 causes defects in erythropoiesis and hematopoietic stem cell maintenance

doi: 10.1101/2021.05.04.442585

Figure Lengend Snippet: (A) Representative images of RpS12 +/- and littermate E13.5 fetal livers. (B) Quantification of total number of cells per liver, normalized to embryo weight (+/+ n=9 and KO/+ n=8). (C) Representative flow cytometry gating of erythropoietic populations using Ter119 and CD71 markers of fetal liver samples from E13.5 embryos. (E) Representative flow cytometry gating of Lin- (top) and LSK (bottom) populations in E13.5 fetal livers. (F,G,H) LT-HSCs, ST-HSCs and indicated progenitor populations represented as percentages of the Lin- population in E13.5 fetal livers. (D,F,G,H) Biological samples are +/+ n=8 and KO/+ n=4. Statistical analysis: quantifications represent mean +/-SEM, unpaired t-tests were performed to established significance among populations between genotypes *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001

Article Snippet: The following primary antibodies were used: RpS12 (Proteintech; polyclonal), β-actin (Cell Signaling; 13E5), eIF2α (Cell signaling, 5324T), phospho-eIF2α (Thermo Scientific; Ser52; polyclonal).

Techniques: Flow Cytometry

Journal: bioRxiv

Article Title: Haploinsufficiency of the essential gene RpS12 causes defects in erythropoiesis and hematopoietic stem cell maintenance

doi: 10.1101/2021.05.04.442585

Figure Lengend Snippet: (A) Representative flow cytometry gating of HSCs (Flk2 - CD48 - LSK) cell cycle stages (G0, G1, S/G2/M) distribution determined by DNA (Hoechst) and Ki67 levels. (B) Cell cycle stages distribution in HSCs and in indicated progenitor populations. Asterisks correspond to p values assessing significant differences in each cell cycle stage between RpS12 KO/+ and RpS12 +/+ mice (6-8-weeks-old littermates, +/+ n=4 and KO/+ n=3). (C) Representative flow cytometry histogram showing OPP intensity in RpS12 KO/+ (green) and RpS12 +/+ (grey) HSCs. (D, E) Median OPP intensity of the indicated bone marrow populations (6-8-weeks-old littermates, +/+ n=4 and KO/+ n=3). This analysis was repeated in 6-7-month-old mice with similar results. (F) Representative images of bone marrow cytospins showing high number of apoptotic cells (arrows) in RpS12 KO/+ samples. (G) Representative flow cytometry gating of LIN- population showing apoptotic populations as determined by AnexinV and PI staining. (H, I) Percentage of apoptotic (AnnexinV+) cells in LSK (Lin - cKit + Sca1 + ) and Myeloid progenitor (MPROG; Lin - cKit + Sca1 - ) populations (6-8-weeks-old littermates, +/+ n=4 and KO/+ n=3). Statistical analysis: quantifications represent mean+/-SEM, two-way ANOVA (B-F) and unpaired t-tests were performed to established significance among populations between genotypes *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001

Article Snippet: The following primary antibodies were used: RpS12 (Proteintech; polyclonal), β-actin (Cell Signaling; 13E5), eIF2α (Cell signaling, 5324T), phospho-eIF2α (Thermo Scientific; Ser52; polyclonal).

Techniques: Flow Cytometry, Staining

Journal: bioRxiv

Article Title: Sialin2 Functions as a Mammalian Nitrate Sensor to Sustain Mitochondrial Homeostasis

doi: 10.1101/2025.05.04.652104

Figure Lengend Snippet: Nitrate-Sialin2 signaling promotes mitochondrial biogenesis and functional homeostasis via LKB1-AMPK activation a, Immunoblot analysis of NT-PGC1α, TFAM, mitochondrial-encoded proteins (MT- ND5, CYTB, MT-CO2, and ATP8), and Sialin2 in control and sg SLC17A5 HEK293T cells reconstituted with Sialin (KRI/AAA) or Sialin2 and treated with nitrate (4 mM, 4 h). Representative images of n = 3 independent experiments were shown. See full quantitation in Extended Data Fig. 12a–d. b, c, IF staining images (left) and quantification (right) of Cox IV (mitochondria; b) and JC-1 (mitochondrial membrane potential; c) in control and sg SLC17A5 NRK cells reconstituted with Sialin (KRI/AAA) or Sialin2 and treated with nitrate (4 mM, 4 h). N = 30 cells from representative experiments of three repeats. d, e, ATP production (d) and mitochondrial DNA (mtDNA) copy number (e) in control and sg SLC17A5 NRK cells reconstituted with Sialin (KRI/AAA) or Sialin2 and treated with nitrate (4 mM, 4 h). N = 3. f, Mitochondrial ROS (mtROS) production in control and sg SLC17A5 NRK cells reconstituted with Sialin (KRI/AAA) or Sialin2 and treated with nitrate (4 mM, 4 h). Production normalized to control. N = 3 from three independent experiments, each in triplicate. g, Proliferation (%EdU positive cells) in control and sg SLC17A5 NRK cells reconstituted with Sialin (KRI/AAA) or Sialin2 and treated with nitrate (4 mM, 4 h). N = 30 cells from representative experiments of three repeats. See EdU images in Extended Data Fig. 12m. h, Immunoblot analysis of NT-PGC1α, TFAM, mitochondrial-encoded proteins (MT- ND5, CYTB, MT-CO2, and ATP8), Flag, and Sialin2 in control and sg SLC17A5 HEK293T cells reconstituted with vector, Flag-LKB1, Flag-LKB1-M, or Flag-LKB1- M (D194A). Representative images of n = 3 independent experiments were shown. See full quantitation in Extended Data Fig. 13a–d. i, j, IF staining images (left) and quantification (right) of Cox IV (i) and JC-1 (j) in control and sg SLC17A5 NRK cells reconstituted with vector, Flag-LKB1, Flag-LKB1- M, or Flag-LKB1-M (D194A). N = 30 cells from representative experiments of three repeats. k, l, ATP production (k) and mtDNA copy number (l) in control and sg SLC17A5 NRK cells reconstituted with vector, Flag-LKB1, Flag-LKB1-M, or Flag-LKB1-M (D194A). N = 3. m, mtROS production in control and sg SLC17A5 NRK cells reconstituted with vector, Flag-LKB1, Flag-LKB1-M, or Flag-LKB1-M (D194A). Production normalized to control. N = 3 from three independent experiments, each in triplicate. n, Proliferation (%EdU positive cells) in control and sg SLC17A5 NRK cells reconstituted with vector, Flag-LKB1, Flag-LKB1-M, or Flag-LKB1-M (D194A). N = 30 cells from representative experiments of three repeats. o, Schematic illustration of nitrate triggers CTSB-mediated cleavage of Sialin at residues K256/R257/I258 to generate Sialin2, which translocates to mitochondria, acts as the nitrate sensor, recruits LKB1 to activate mitochondrial AMPK phosphorylation, and drives mitochondrial biogenesis to sustain mitochondrial function and cellular homeostasis. For all panels, data are represented as mean ± SD, P value denotes t -test. Scale bar: 10 μm.

Article Snippet: Monoclonal antibodies against CTSB (Santa Cruz Biotechnology, clone H-5, #sc- 365558), Tomm20 (Cell Signaling, clone D8T4N, #42406), LAMP1 (Invitrogen, clone LY1C6, #MA1-164), pAMPK T172 (Cell Signaling, clone 40H9, #2535), AMPK (Cell Signaling, clone D5A2, #5831), LKB1 (Santa Cruz Biotechnology, clone G-12, #sc- 374300), CAMKK2 (Cell Signaling, clone 6G9, #50049), Cox IV (Cell Signaling, clone 3E11, #4850), PGC1α (Santa Cruz Biotechnology, clone D-5, #sc-518025), HA- tag (Cell Signaling, clone C29F4, #3724; clone 6E2, #2367), Flag-tag (Cell Signaling, clone D6W5B, #14793; clone 9A3, #8146), GAPDH (Cell Signaling, clone 14C10, #2118), ACTB (Proteintech, clone 2D4H5, #66009), HSP90 (Cell Signaling, clone C45G5, #4877), and polyclonal antibodies against Sialin (Invitrogen, #PA5-30517), pAMPK T172 (Invitrogen, #PA5-37821), LKB1 (Proteintech, #10746), MT-ND5 (Proteintech, #55410), CYTB (Proteintech, #55090), MT-CO2 (Proteintech, #55070), ATP8 (Proteintech, #26723), TFAM (Proteintech, #22586), GFP-tag (Abcam, #ab290), and mCherry (Abcam, #167453) were used in this study.

Techniques: Functional Assay, Activation Assay, Western Blot, Control, Quantitation Assay, Staining, Membrane, Plasmid Preparation, Phospho-proteomics